WO2005095619A1 - Plants with increased activity of multiple starch phosphorylating enzymes - Google Patents
Plants with increased activity of multiple starch phosphorylating enzymes Download PDFInfo
- Publication number
- WO2005095619A1 WO2005095619A1 PCT/EP2005/002457 EP2005002457W WO2005095619A1 WO 2005095619 A1 WO2005095619 A1 WO 2005095619A1 EP 2005002457 W EP2005002457 W EP 2005002457W WO 2005095619 A1 WO2005095619 A1 WO 2005095619A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- starch
- protein
- plants
- plant
- nucleic acid
- Prior art date
Links
Classifications
-
- 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
- C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
- C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
- C12N15/8245—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving modified carbohydrate or sugar alcohol metabolism, e.g. starch biosynthesis
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B31/00—Preparation of derivatives of starch
- C08B31/02—Esters
- C08B31/06—Esters of inorganic acids
- C08B31/066—Starch phosphates, e.g. phosphorylated starch
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
Definitions
- Plants with increased activity of multiple starch phosphorylating enzymes Plants with increased activity of multiple starch phosphorylating enzymes
- the present invention relates to plant cells and plants, which are genetically modified, wherein the genetic modification leads to an increase in the activity of a starch phosphorylating OK1 protein and a starch phosphorylating R1 protein in comparison with corresponding wild type plant cells or wild type plants that have not been genetically modified. Furthermore, the present invention relates to means and methods for the manufacture of such plant cells and plants. Plant cells and plants of this type synthesise a modified starch. The present invention therefore also relates to the starch synthesised by the plant cells and plants according to the invention, methods for the manufacture of this starch, and the manufacture of starch derivatives of this modified starch, as well as flours containing starches according to the invention.
- the present invention relates to nucleic acid molecules and vectors containing sequences which code for an OK1 protein and an R1 protein, as well as host cells which contain these nucleic acid molecules.
- the polysaccharide starch is made up of chemically uniform base components, the glucose molecules, but constitutes a complex mixture of different molecule forms, which exhibit differences with regard to the degree of polymerisation and branching, and therefore differ strongly from one another in their physical-chemical characteristics.
- amylose starch an essentially unbranched polymer made from alpha-1 ,4-glycosidically linked glucose units
- amylopectin starch a branched polymer, in which the branches come about by the occurrence of additional alpha-1 ,6-glycosidic links.
- a further essential difference between amylose and amylopectin lies in the molecular weight. While amylose, depending on the origin of the starch, has a molecular weight of 5x10 5 - 10 6 Da, that of the amylopectin lies between 10 7 and 10 8 Da.
- the two macromolecules can be differentiated by their molecular weight and their different physical-chemical characteristics, which can most easily be made visible by their different iodine bonding characteristics.
- Amylose has long been looked upon as a linear polymer, consisting of alpha-1 ,4- glycosidically linked alpha-D-glucose monomers. In more recent studies, however, the presence of alpha-1 ,6-glycosidic branching points (ca. 0.1%) has been shown (Hizukuri and Takagi, Carbohydr. Res. 134, (1984), 1-10; Takeda et al., Carbohydr. Res. 132, (1984), 83-92).
- the functional characteristics of the starch such as, for example, the solubility, the retrogradation behaviour, the water bonding capability, the film formation characteristics, the viscosity, the sticking characteristics, the freezing-thawing stability, the acid stability, the gelling strength, the starch granule size of the starches, and others are affected by the amylose/amylopectin ratio, the molecular weight, the pattern of the side chain distribution, the ion concentration, the lipid and protein content, the average starch granule size of the starch, the starch granule morphology, etc.
- the functional characteristics of starch are also affected by phosphate content, a non-carbon component of starch. Discrimination is made between phosphate which is covalently bound to the glucose molecules in the form of monoesters (designated below as starch phosphate) and phosphate in the form of phospholipids with the starch associated.
- starch phosphate The concentration of starch phosphate varies depending on plant type. Thus certain maize mutants synthesise a starch with an increased concentration of starch phosphate (waxy maize 0.002% and high-amylose maize 0.013%), while conventional maize species have only traces of starch phosphate. Small amounts of starch phosphate are likewise found in wheat (0.001%), while no starch phosphate has been shown in oats and sorghum. Likewise, less starch phosphate is found in rice mutants than in conventional rice species (0.013%).
- starch phosphate content relate to the respective dry weight of the starch and have been determined by Jane et al. (1996, Cereal Foods World 41 (11), 827-832).
- Starch phosphate can exists in the form of monoesters at the C-2, C-3 or C-6 position of the polymerised glucose monomers (Takeda and Hizukuri, 1971 , Starch/Starke 23, 267-272).
- the phosphate distribution of the phosphate in starch synthesised by plants thus generally shows that approximately 30% to 40% of the phosphate residues are covalently bound in the C-3 position and approximately 60% to 70% of the phosphate residues are covalently bound in the C-6 position of the glucose molecules (Blennow et al., Int. J. of Biological Macromolecules 27, 211-218). Blennow et al.
- starch phosphate that is bound in the C-6 position of the glucose molecules for various starches such as potato starch (between 7.8 and 33.5 nMol per mg of starch, depending on species), starch from various Curcuma species (between 1.8 and 63 nMol per mg), tapioca starch (2.5 nMol per mg of starch), rice starch (1.0 nMol per mg of starch), mung bean starch (3.5 nMol per mg of starch) and sorghum starch (0.9 nMol per mg of starch).
- starches such as potato starch (between 7.8 and 33.5 nMol per mg of starch, depending on species), starch from various Curcuma species (between 1.8 and 63 nMol per mg), tapioca starch (2.5 nMol per mg of starch), rice starch (1.0 nMol per mg of starch), mung bean starch (3.5 nMol per mg of starch) and
- the educts alpha-1 ,4-glucan (starch), adenosine triphosphate (ATP) and water are converted to the products glucan phosphate (starch phosphate), monophosphate and adenosine monophosphate.
- the gamma phosphate residue of the ATP is transferred to water, and the beta phosphate residue of the ATP is transferred to the glucan (starch).
- R1 transfers the beta-phosphate residue in vitro from ATP to the C-6- and the C-3 position of the glucose molecules of alpha-1 ,4- glucans.
- the ratio of C-6 phosphate to C-3 phosphate which is obtained through the in vitro reaction corresponds to the ratio which exists in starch isolated from plants (Ritte et al., 2002, PNAS 99, 7166-7171 ). As about 70% of the starch phosphate present in potato starch is bonded to the glucose monomers of starch in the C-6 position and about 30% in the C-3 position, this means that R1 preferably phosphorylates the C-6 position of the glucose molecules.
- R1 can phosphorylate alpha-1 ,4-glucans, which do not yet contain covalently bonded phosphate (Ritte et al., 2002, PNAS 99, 7166-7171), i.e. R1 is able to introduce phosphate de novo into alpha-1 ,4-glucans.
- Wheat plants which have an increased activity of an R1 protein through overexpression of an R1 gene from potatoes are described in WO 02 34923. These plants synthesise a starch with significant amounts of starch phosphate in the C-6 position of the glucose molecules in comparison with corresponding wild type plants in which no starch phosphate could be detected.
- the object of the present invention is therefore based on providing modified starches with increased phosphate content and/or changed phosphate distribution as well as plant cells and/or plants that synthesise such a modified starch, as well as methods and means for the production of said plants and/or plant cells.
- the present invention therefore relates to genetically modified plant cells and plants, characterised in that they have an increased activity of at least one OK1 protein and at least one R1 protein in comparison with corresponding wild type plant cells or wild type plants that have not been genetically modified.
- a first aspect of the present invention relates to a plant cell or plant, which is genetically modified, wherein the genetic modification leads to an increase in the activity of at least one OK1 protein and, simultaneously, at least one R1 protein, in comparison with corresponding wild type plant cells or wild type plants that have not been genetically modified.
- the genetic modification can be any genetic modification, which leads to an increase in the activity of at least one OK1 protein and (simultaneously) at least one R1 protein in genetically modified plant cells or genetically modified plants in comparison with corresponding wild type plant cells or wild type plants that have not been genetically modified.
- wild type plant cell means that the plant cells concerned were used as starting material for the manufacture of the plant cells according to the invention, i.e. their genetic information, apart from the introduced genetic modification, corresponds to that of a plant cell according to the invention.
- wild type plant means that the plants concerned were used as starting material for the manufacture of the plants according to the invention, i.e. their genetic information, apart from the introduced genetic modification, corresponds to that of a plant according to the invention.
- the term “corresponding” means that, in the comparison of several objects, the objects concerned that are compared with one another have been kept under the same conditions.
- the term “corresponding” in conjunction with wild type plant cell or wild type plant compared to genetically modified plant cells or plants means that the plant cells or plants, which are compared with one another, have been raised under the same cultivation conditions and that they have the same (cultivation) age.
- the term "increased activity of at least one OK1 protein” means an increase in the expression of endogenous genes, which code OK1 proteins and/or an increase in the quantity of OK1 protein in the cells and/or an increase in the enzymatic activity of OK1 proteins in the cells.
- the term "increased activity of at least one R1 protein” means an increase in the expression of endogenous genes, which code R1 proteins and/or an increase in the quantity of R1 protein in the cells and/or an increase in the enzymatic activity of R1 proteins in the cells.
- the increase in the expression can, for example, be determined by measuring the quantity of transcripts coding OK1 proteins or R1 proteins. This can take place through Northern blot analysis or RT-PCR.
- an increase preferably means an increase in the quantity of transcripts in comparison with corresponding cells that have not been genetically modified by at least 50%, in particular by at least 70%, preferably by at least 85% and particularly preferably by at least 100%.
- An increase in the quantity of transcripts coding an OK1 protein also means that plants or plant cells that have no detectable quantities of transcripts coding an OK1 protein have detectable quantities coding an OK1 protein after genetic modification according to the invention.
- An increase in the quantity of transcripts coding an R1 protein also means that plants or plant cells that have no detectable quantities of transcripts coding an R1 protein have detectable quantities of transcripts coding an R1 protein after genetic modification according to the invention.
- an increase in the amount of protein of an OK1 protein or an R1 protein, which results in an increased activity of these proteins in the plant cells concerned can, for example, be determined by immunological methods such as Western blot analysis, ELISA (Enzyme Linked Immuno Sorbent Assay) or RIA (Radio Immune Assay).
- an increase preferably means an increase in the amount of protein in comparison with corresponding cells that have not been genetically modified by at least 50%, in particular by at least 70%, preferably by at least 85% and particularly preferably by at least 100%.
- An increase in the amount of an OK1 protein also means that plants or plant cells that have no detectable activity of an OK1 protein have a detectable amount of an OK1 protein after genetic modification according to the invention.
- An increase in the amount of an R1 protein also means that plants or plant cells that have no detectable activity of an R1 protein have a detectable amount of an R1 protein after genetic modification according to the invention.
- the term "OK 1 protein” should be understood to mean a protein that transfers a phosphate residue from ATP to starch that is already phosphorylated (P-starch).
- Starches isolated from leaves of an Arabisopsis thaliana sex1-3 mutant have no detectable quantity of covalently bound phosphate residues and are not phosphorylated by an OK1 protein, i.e., an OK1 protein according to the invention requires starch that is already phosphorylated as a substrate for the transfer of additional phosphate residues.
- the beta phosphate residue of the ATP is transferred from an OK1 protein to the starch and the gamma phosphate residue of the ATP is transferred to water.
- AMP adenosine monophosphate
- An OK1 protein is therefore designated as [phosphorylated alpha-glucanj-water-dikinase ([P-glucan]- water-dikinase) or as [phosphorylated starch] water-dikinase. Therefore, OK1 proteins catalyse a reaction according to the following formula: P-glucan + ATP + H 2 O P-glucan-P + AMP + Pi
- an additional phosphate monoester bond in the C-6 position and/or in the C-3 position of a glucose molecule of the P-starch is formed on the P-starch phosphorylated by an OK1 protein.
- several additional phosphate monoester bonds in the C-3 position are formed during the phosphorylation of P-starch catalysed by an OK1 protein in comparison with phosphate monoester bonds in the C-6 position of the glucose molecules of the corresponding P-starch.
- Amino acid sequences that code OK1 proteins contain a phosphohistidine domain.
- Phosphohistidine domains are, for example, described by Tien-Shin Yu et al. (2001, Plant Cell 13, 1907-1918).
- Phosphohistidine domains from OK1 proteins coding amino acid sequences preferably contain two histidines.
- a phosphorylated OK1 protein is formed as an intermediate product, through which a phosphate residue of the ATP is covalently bound to an amino acid of the OK1 protein.
- the intermediate product is formed through autophosphorylation of the OK1 protein, i.e., the OK1 protein itself catalyses the reaction that leads to the intermediate product.
- a histidine residue of the amino acid sequence coding an OK1 protein is phosphorylated through the autophosphorylation, particularly preferably a histidine residue that is part of a phosphohistidine domain.
- OK1 proteins according to the invention have an increased bonding activity to P-starch in comparison with non-phosphorylated starch.
- a further advantage of the present invention is that, for a simultaneous cooperation of an OK1 protein with an R1 protein, higher amounts of phosphate are incorporated into the starch than when the respective proteins separated from one another in space or time phosphorylate starch or P-starch, respectively.
- R1 protein should be understood to mean a protein that transfers a phosphate residue from ATP to starch.
- Starches isolated from leaves of an Arabidopsis thaliana sex1-3 mutant have no detectable amount of covalently bound phosphate residues but are phosphorylated from an R1 protein.
- This means non-phosphorylated starch for example, isolated from leave of an Arabidopsis thaliana sex1-3 mutant, is used as a substrate in a phosphorylation reaction catalysed by an R1 protein.
- the beta phosphate residue of the ATP is transferred from an R1 protein to the starch and the gamma phosphate residue of the ATP is transferred to water.
- An R1 protein is therefore designated as [alpha-1 ,4-glucan]-water-dikinase or as starch- water-dikinase (E.C.: 2.7.9.4; Ritte et al., 2002, PNAS 99, 7166-7171).
- R1 protein is therefore designated as [alpha-1 ,4-glucan]-water-dikinase or as starch- water-dikinase (E.C.: 2.7.9.4; Ritte et al., 2002, PNAS 99, 7166-7171).
- a R1 protein By a R1 protein, approximately 60% to 70% of the phosphate residue is introduced in the C-6 position of the glucose molecules of starch, and approximately 30% to 40% of the phosphate residue is introduced in the C-3 position of the glucose molecules of starch (Ritte et al., 2002, PNAS 99, 7166- 7171).
- a phosphorylated R1 protein results as an intermediate product, through which a phosphate residue of the ATP is covalently bound to an amino acid of the R1 protein (Ritte et al., 2002, PNAS 99, 7166-7171 ).
- the intermediate product results through autophosphorylation of the R1 protein, i.e., the R1 protein itself catalyses the reaction that leads to the intermediate product.
- Amino acid sequences that code R1 proteins contain a phosphohistidine domain. Phosphohistidine domains are, for example, described by Tien-Shin Yu et al. (2001 , Plant Cell 13, 1907-1918).
- Phosphohistidine domains from R1 proteins coding amino acid sequences preferably contain one histidine.
- a histidine residue in the phosphohistidine domain of the amino acid sequence coding an R1 protein is phosphorylated (Mikkelsen et al., 2003, Biochemical Journal Intermediate Publication). Published on October 2003 as manuscript BJ20030999; Mikkelsen et al., 2004, Biochemical Journal 377, 525-532).
- Nucleic acid sequences and corresponding amino acid sequences coding an R1 protein are described from different species such as potato (WO 97 11188, GenBank Ace: AY027522, Y09533), wheat (WO 00 77229, US 6,462,256, GenBank Ace: AAN93923, GenBank Ace: AR236165), rice (GenBank Ace: AAR61445, GenBank Ace: AR400814), maize (GenBank Ace: AAR61444, GenBank Ace: AR400813), soybean (GenBank Ace: AAR61446, GenBank Ace: AR400815), citrus (GenBank Ace: AY094062) and Arabidopsis (GenBank Ace: AF312027).
- nucleic acid sequences and amino acid sequences coding R1 proteins are published by NCBI (http://www.ncbi.nlm.nih.gov/entrez/), among others, and are explicitly included in the description of the present application by mention of the references.
- the term "increased bonding activity” is to be understood as an increased affinity of a protein for a first substrate in comparison to a second substrate. That is to say, the amount of protein, which, under the same incubation conditions, bonds to a first substrate to a greater extent in comparison with a second substrate, exhibits increased bonding activity to the first substrate.
- starch phosphate is to be understood as phosphate groups covalently bound to the glucose molecules of starch.
- non-phosphorylated starch is to be understood as a starch that contains no detectable amount of starch phosphate.
- Various methods are described for the determination of the amount of starch phosphate.
- the methods described by Ritte et al. 2000, Starch/Starke 52, 179-185) can be used for the determination of the amount of starch phosphate.
- the determination of the amount of starch phosphate is carried out by means of 31 P-NMR according to the methods described by Kasemusuwan and Jane (1996, Cereal Chemistry 73, 702-707).
- phosphorylated starch or "P- starch” is to be understood as a starch that contains starch phosphate.
- an OK1 protein can be shown, for example, through in vitro incubation of an OK1 protein using ATP that contains a labeled phosphate residue (labeled ATP) in the beta position.
- ATP for which the phosphate residue is specifically labeled in the beta position, i.e., for which only the phosphate residue in the beta position bears a label.
- radioactively labeled ATP is particularly preferably used.
- ATP for which the phosphate residue is specifically radioactively labeled
- especially preferably used is ATP for which the phosphate residue is specifically labeled with 33 P in the beta position.
- OK1 protein with labeled ATP and starches that are not phosphorylated is incubated, no phosphate is transferred through OK1 to the starch.
- leaf starch of the Arabidopsis thaliana mutant sex1-3 is incubated in the presence of labeled ATP, then labeled phosphate covalently bound to the P-starch can be subsequently shown.
- starch from leaves of Arabidopsis thaliana is starch from leaves of Arabidopsis thaliana, particularly preferably by means of an enzymatically-phosphorylated starch of an R1 protein from Arabidopsis thaliana sex1-3 mutant (Ritte et al., 2002, PNAS 99, 7166-7171 ).
- Labeled phosphate residues can be shown that were assembled by an OK1 protein in P-starch, for example, through separation of the labeled P-starch (for example, through precipitation means ethanol, filtration, chromatographic methods, etc.) from the residue of the reaction mixture and subsequent detection of the labeled phosphate residue in the P-starch fraction.
- the labeled phosphate residues bound in the P-starch fraction can be shown, for example, through determination of the amount of the radioactivity existing in the P-starch fraction (for example, by means of a scintillation counter).
- R1 protein The activity of an R1 protein can be shown as described in the literature, for example (Mikkelsen et al., 2003, Biochemical Journal Intermediate Publication. Published on October 2003 as manuscript BJ20030999; Mikkelsen et al., 2004, Biochemical Journal 377, 525-532, Ritte et al., 2002, PNAS 99, 7166-7171 ).
- Which positions of the carbon atoms (C-2, C-3 or C-6) of the glucose monomers in P- starch are preferably phosphorylated by an OK1 protein can be determined, for example, by analysing the P-starches phosphorylated by a protein, as described by Ritte et al. (2002, PNAS 99, 7166-7171 ). For this purpose, a P-starch phosphorylated by a protein is hydrolysed using an acid, and subsequently analysed by means of anion exchange chromatography.
- the P-starch phosphorylated by an OK1 protein is analysed by means of NMR in order to establish which positions of the carbon atoms (C-2, C-3 or C-6) of the glucose monomers in the P-starch are phosphorylated.
- NMR nuclear magnetic resonance
- a particularly preferred method for identifying the C-atom positions of a glucose molecule of a starch, which are phosphorylated by a reaction catalysed by an OK1 protein, is described below under General Methods, Item 13.
- Which positions of the carbon atoms (C-2, C-3 or C-6) of the glucose monomers in starch are preferably phosphorylated by an R1 protein can be determined, for example, by analysing the starches phosphorylated by an R1 protein, as described by Ritte et al. (2002, PNAS 99, 7166-7171 ).
- a starch phosphorylated by a protein is hydrolysed using an acid, and subsequently analysed by means of anion exchange chromatography.
- the P-starch phosphorylated by an OK1 protein, or the starch phosphorylated by an R1 protein is analysed by means of NMR in order to establish which positions of the carbon atoms (C-2, C-3 or C-6) of the glucose monomers in the P-starch or the starch are phosphorylated.
- NMR nuclear magnetic resonance
- a particularly preferred method for identifying the C-atom positions of a glucose molecule of a starch, which are phosphorylated by a reaction catalysed by an OK1 protein or an R1 protein, is described below under General Methods, Item 13.
- a phosphorylated protein which is produced as an intermediate product in the phosphorylation of P-starch facilitated by an OK1 protein, can be demonstrated for an R1 protein as described, for example, by Ritte et al. (2002, PNAS 99, 7166-7171) or Mikkelsen et al. (2003, Biochemical Journal Intermediate Publication. Published on October 2003 as manuscript BJ20030999, Mikkelsen et al., 2004, Biochemical Journal 377, 525-532)
- an OK1 protein is first incubated in the absence of starch with labeled ATP, preferably with ATP specifically labeled in the beta phosphate position, particularly preferably with ATP specifically labeled with 33 P in the beta phosphate position.
- a reaction preparation 2 which instead of labeled ATP contains corresponding amounts of non- labeled ATP however, is incubated under otherwise identical conditions.
- non- labeled ATP is added to the reaction mixture 1 in excess, and a mixture of non- labeled ATP and labeled ATP (the same amount of labeled ATP as used previously in reaction mixture 1 and the same amount of non- labeled ATP as added to reaction mixture 1 in excess) is added to reaction mixture 2 and further incubated before P-starch is added to a Part A of reaction mixture 1 (Part 1A) or to a Part A of reaction mixture 2 (Part 2A) respectively.
- the reaction in the remaining Part 1B and Part 2B of the reaction mixture is stopped by denaturing the protein.
- Part B of the reaction mixture can be stopped by the methods known to the person skilled in the art, which lead to the denaturing of proteins, preferably by adding sodium lauryl sulphate (SDS).
- Part 1 A and Part 2A of the reaction mixture are incubated for at least a further 10 minutes before these reactions are also stopped.
- the starch present in Part A and Part B of the respective reaction mixture is separated from the remainder of the reaction mixture. If the respective starch is separated by centrifugation, for example, then, on completion of centrifugation, the starch of the respective Part A or Part B of the reaction mixture is to be found in the sedimented pellet, and the proteins in the respective reaction mixture are to be found in the supernatant of the respective centrifugation.
- the supernatant of Part 1A or 2A respectively and Part 1 B or 2B respectively of the reaction mixture can subsequently be analysed by denaturing acrylamide gel electrophoresis followed by autoradiography of the acrylamide gel obtained.
- the so-called "phospho- imaging" method for example, known to the person skilled in the art, can be used.
- the starch of the respective Part A of reaction mixtures 1 and 2 remaining in the respective sedimented pellet can be investigated, if necessary, after subsequent washing of the respective starches, for the presence of starch phosphate, which has a label corresponding to the labeled ATP used. If the starches of Part A of reaction mixture 1 contain labeled phosphate residues, and if the autoradiography of the centrifugation supernatant of Part B of reaction mixture 1 shows a significantly increased signal in the autoradiography compared with the centrifugation supernatant of Part A of reaction mixture 1 , then this shows that a phosphorylation of starch- facilitating protein is present as an autophosphorylated intermediate product.
- Parts A and B of reaction mixture 2 serve as a control and should therefore not exhibit a significantly increased signal for alpha-1 ,4-glucans labeled with e.g. 33 P in the sedimented pellet containing alpha-1 ,4-glucans.
- Possible methods for demonstrating a phosphorylated OK1 protein intermediate product are described below under General Methods, Item 12 and in Example 7.
- an OK1 protein has an increased bonding activity to a P-starch compared with non-phosphorylated starch can be demonstrated by incubating the OK1 protein with P-starch and non-phosphorylated starch in separate preparations.
- All non-phosphorylated starches are basically suitable for incubating OK1 proteins with non-phosphorylated starch.
- a non-phosphorylated plant starch particularly preferably wheat starch, and especially preferably granular leaf starch of an Arabidopsis thaliana mutant sex1-3 is used.
- Methods for isolating starch from plants are known to the person skilled in the art. All methods known to the person skilled in the art are basically suitable for isolating non-phosphorylated starch from appropriate plant species. Preferably, the methods for isolating non-phosphorylated starch described below are used (see General Methods Item 2). All starches that contain starch phosphate are basically suitable for incubating OK1 proteins with P-starch. Chemically phosphorylated starches can also be used for this purpose.
- P-starches are used for the incubation with OK1 proteins, particularly preferably a retrospectively enzymatically phosphorylated plant starch, especially preferably a retrospectively enzymatically phosphorylated plant starch, which has been isolated from a sex-1 mutant of Arabidopsis thaliana.
- OK1 proteins are incubated in separate preparations with P-starch (Preparation A) and with non-phosphorylated starch (Preparation B).
- Preparation A preparations with P-starch
- Preparation B non-phosphorylated starch
- the proteins which are not bonded to the related starches of Preparations A and B, are separated from the starches and from the proteins bonded to them.
- the bond between the proteins and the P-starch in Preparation A and the bond between the proteins and non-phosphorylated starch in Preparation B are subsequently removed, i.e. the respective proteins are dissolved.
- the dissolved proteins of Preparation A and Preparation B can then be separated from the starches concerned, which are present in the respective preparations.
- the isolated P-starch bonding proteins of Preparation A and the isolated non-phosphorylated starch bonding proteins of Preparation B can be separated with the help of methods known to the person skilled in the art such as, for example, gel filtration, chromatographic methods, electrophoresis, SDS acrylamide gel electrophoresis etc.
- Example 8 Methods, which can be used to demonstrate a preferred bonding of proteins to P-starch compared with non-phosphorylated starch, are described below in Example 8.
- the amino acid sequence shown in SEQ ID NO 2 codes an OK1 protein from Arabidopsis thaliana and the amino acid sequence shown under SEQ ID NO 4 codes an OK1 protein from Oryza sativa.
- amino acid sequences coding an OK1 protein have an identity of at least 60% with the sequence specified in SEQ ID NO 2 or SEQ ID NO 4, in particular of at least 70%, preferably of at least 80% and particularly preferably of at least 90% and especially preferably of at least 95%.
- the OK1 protein has a phosphohistidine domain.
- Amino acid sequences coding OK1 proteins contain a phosphohistidine domain that has an identity of at least 60% with the amino acid sequence of the phosphohistidine domain of the OK1 protein from Arabidopsis thaliana specified in SEQ ID NO 5, in particular of at least 70%, preferably of at least 80% and particularly preferably of at least 90% and especially preferably of af least 95%.
- a further embodiment of the present invention relates to a genetically modified plant cell according to the invention or a genetically modified plant according to the invention, wherein the genetic modification consists in the introduction of at least one foreign nucleic acid molecule into the genome of the plant cell or into the genome of the plant.
- the term “genetic modification” means the introduction of homologous and/or heterologous foreign nucleic acid molecules into the genome of a plant cell or into the genome of a plant, wherein said introduction of these molecules leads to an increase in the activity of an OK1 protein and to the increase of the activity of an R1 protein.
- the plant cells according to the invention or plants according to the invention are modified with regard to their genetic information by the introduction of a foreign nucleic acid molecule.
- the presence or the expression of a foreign nucleic acid molecule leads to a phenotypic change.
- phenotypic change means preferably a measurable change of one or more functions of the cells.
- the genetically modified plant cells according to the invention and the genetically modified plants according to the invention exhibit an increase of the activity of an OK1 protein and an increase of the activity of an R1 protein due to the presence or on the expression of the introduced nucleic acid molecule.
- the term "foreign nucleic acid molecule” is understood to mean such a molecule that either does not occur naturally in the corresponding wild type plant cells, or that does not occur naturally in the concrete spatial arrangement in wild type plant cells, or that is localised at a place in the genome of the wild type plant cell at which it does not occur naturally.
- the foreign nucleic acid molecule is a recombinant molecule, which consists of different elements, the combination or specific spatial arrangement of which does not occur naturally in plant cells.
- a foreign nucleic acid molecule can be any nucleic acid molecule that effects an increase in the activity of an OK1 protein and an R1 protein in the plant cell or plant.
- the term "genome” is to be understood to mean the totality of the genetic material present in a plant cell. It is known to the person skilled in the art that, as well as the cell nucleus, other compartments (e.g. plastids, mitochondrions) also contain genetic material.
- the plant cells according to the invention and the plants according to the invention are characterised in that at least one foreign nucleic acid molecule codes an OK1 protein, preferably an OK1 protein from Arabidopsis thaliana or an OK1 protein from Oryza sativa.
- the foreign nucleic acid molecule codes an OK1 protein with the amino acid sequence specified in SEQ ID NO 2 or SEQ ID NO 4.
- the plant cells according to the invention and the plants according to the invention are characterised in that at least one foreign nucleic acid molecule codes an R1 protein, preferably an R1 protein from potato or an OK1 protein from Oryza sativa.
- the foreign nucleic acid molecule codes an R1 protein from potato with the amino acid sequence specified in GenBank Ace: Y09533 (22-JUL- 2003 Rel. 76, Last updated, Version 2).
- GenBank Ace: Y09533 The nucleic acid molecules and amino acid sequences coding an R1 protein from potato (GenBank Ace: Y09533) are explicitly included in the description of the present application by mention of the references.
- the plant cells according to the invention and the plants according to the invention are characterised in that a first foreign nucleic acid molecule codes an R1 protein and a second foreign nucleic acid molecule codes an OK1 protein.
- the foreign nucleic acid molecules assembled for the genetic modification in the plant cell or plant can be a single nucleic acid molecule or multiple nucleic acid molecules. It can therefore be nucleic acid molecules that contain nucleic acid sequences that code OK1 proteins and nucleic acid sequences that code R1 proteins, as well as nucleic acid molecules for which the nucleic acid sequences coding OK1 proteins and the nucleic acid sequences coding R1 proteins occur in various nucleic acid molecules.
- the nucleic acid sequences coding an OK1 protein and the nucleic acid sequences coding an R1 protein can be contained simultaneously, for example, in a vector, plasmid or linear nucleic acid molecule, or be, however, constituents of two vectors, plasmids or linear nucleic acid molecules respectively separated from one another. If the nucleic acid sequences coding an OK1 protein and the nucleic acid sequences coding an R1 protein occur in two nucleic acid molecules separated from one another, then they can be introduced either simultaneously ("cotransformation") or also successively, i.e., consecutively (“supertransformation") into the genome of the plant cells or plant. The nucleic acid molecules separated from one another can also be introduced into various individual plant cells or plants of a species.
- Plant cells or plants can thereby be produced in which the activity of either at least one OK1 protein or at least one R1 protein is increased. Such plants can be produced by subsequently crossing the plants in which the activity of an OK1 protein is increased with plants in which the activity of an R1 protein is increased.
- a mutant cell or a mutant that is characterised in that it already has an increased activity of an OK1 protein or an increased activity of an R1 protein can be used instead of a wild type plant cell or wild type plant for introducing a foreign nucleic acid molecule.
- Mutants can be spontaneously (naturally) occurring mutants as well as those that were produced through the targeted use of mutagens (such as, for example, chemical agents, ionising radiation) or genetic engineering methods (for example T-DNA activation tagging, transposon activation tagging, in situ activation, in vivo mutagenesis).
- plant cells according to the invention and plants according to the invention can also be produced through the introduction of a foreign nucleic acid molecule, which leads to the increase of the activity of an R1 protein in a mutant cell or a mutant that already has an increased activity of an OK1 protein.
- Plant cells according to the invention or plants according to the invention can also be produced through the introduction of a foreign nucleic acid molecule, which leads to the increase of the activity of an OK1 protein in a mutant cell or a mutant that already has an increased activity of an R1 protein.
- Plant cells according to the invention or plants according to the invention can also be produced in which a mutant in which the activity of an OK1 protein is increased is crossed with a plant that has an increased activity of an R1 protein due to the introduction of a foreign nucleic acid molecule.
- plant cells according to the invention or plants according to the invention in which a mutant in which the activity of an R1 protein is increased is crossed with a plant that has an increased activity of an OK1 protein due to the introduction of a foreign nucleic acid molecule.
- a large number of techniques are available for the introduction of DNA into a plant host cell. These techniques include the transformation of plant cells with T-DNA using Agrobacterium tumefaciens or Agrobacterium rhizogenes as the transformation medium, the fusion of protoplasts, injection, the electroporation of DNA, the introduction of DNA by means of the biolistic approach as well as other possibilities.
- the use of agrobacteria-mediated transformation of plant cells has been intensively investigated and adequately described in EP 120516; Hoekema, IN: The Binary Plant Vector System Offsetdrukkerij Kanters B.V., Alblasserdam (1985), Chapter V; Fraley et al., Crit. Rev. Plant Sci. 4, 1-46 and by An et al. EMBO J. 4, (1985), 277-287.
- For the transformation of potato see Rocha-Sosa et al., EMBO J. 8, (1989), 29-33, for example.
- plant cells and plants which have been genetically modified by the introduction of an OK1 protein and/or an R1 protein, can be differentiated from wild type plant cells and wild type plants respectively in that they contain a foreign nucleic acid molecule, which does not occur naturally in wild type plant cells or wild type plants, or in that such a molecule is present integrated at a place in the genome of the plant cell according to the invention or in the genome of the plant according to the invention at which it does not occur in wild type plant cells or wild type plants, i.e. in a different genomic environment.
- plant cells according to the invention and plants according to the invention of this type differ from wild type plant cells and wild type plants respectively in that they contain at least one copy of the foreign nucleic acid molecule stably integrated within their genome, possibly in addition to naturally occurring copies of such a molecule in the wild type plant cells or wild type plants.
- the plant cells according to the invention and the plants according to the invention can be differentiated from wild type plant cells or wild type plants respectively in particular in that this additional copy or these additional copies is (are) localised at places in the genome at which it does not occur (or they do not occur) in wild type plant cells or wild type plants. This can be verified, for example, with the help of a Southern blot analysis.
- the plant cells according to the invention and the plants according to the invention can preferably be differentiated from wild type plant cells or wild type plants respectively by at least one of the following characteristics: If a foreign nucleic acid molecule that has been introduced is heterologous with respect to the plant cell or plant, then the plant cells according to the invention or plants according to the invention have transcripts of the introduced nucleic acid molecules. These can be verified, for example, by Northern blot analysis or by RT-PCR (Reverse Transcription Polymerase Chain Reaction).
- Plant cells according to the invention and plants according to the invention which express an antisense and/or an RNAi transcript, can be verified, for example, with the help of specific nucleic acid probes, which are complimentary to the RNA (occurring naturally in the plant cell), which is coding for the protein.
- the plant cells according to the invention and the plants according to the invention contain a protein, which is coded by an introduced nucleic acid molecule. This can be demonstrated by immunological methods, for example, in particular by a Western blot analysis.
- the plant cells according to the invention or plants according to the invention can be differentiated from wild type plant cells or wild type plants respectively due to the additional expression of the introduced foreign nucleic acid molecule, for example.
- the plant cells according to the invention and the plants according to the invention preferably contain transcripts of the foreign nucleic acid molecules. This can be demonstrated by Northern blot analysis, for example, or with the help of so-called quantitative PCR.
- the plant cells according to the invention and the plants according to the invention are transgenic plant cells or transgenic plants respectively.
- the present invention relates to plant cells according to the invention and plants according to the invention wherein the foreign nucleic acid molecule coding an OK1 protein is chosen from the group consisting of a) Nucleic acid molecules, which code a protein with the amino acid sequence given under SEQ ID NO 2 or SEQ ID NO 4; b) Nucleic acid molecules, which code a protein, which includes the amino acid sequence, which is coded by the insertion in plasmid A.t.-OKI -pGEM or the insertion in plasmid pMI50; c) Nucleic acid molecules, which code a protein, the sequence of which has an identity of at least 60% with the amino acid sequence given under SEQ ID NO 2 or SEQ ID NO 4; d) Nucleic acid molecules, which code a protein, the sequence of which has an identity of at least 60% with the amino acid sequence, which is coded by
- the nucleic acid sequence shown SEQ ID NO 1 is a cDNA sequence, which includes the coding region for an OK1 protein from Arabidopsis thaliana and the nucleic acid sequence shown SEQ ID NO 3 is a cDNA sequence, which includes the coding region for an OK1 protein from Oryza sativa.
- a plasmid (A.t.-OK1-pGEM) containing a cDNA which codes for a protein according to the invention (A.t.-OK1 ) from Arabidopsis thaliana was deposited on 08.03.2004 under the number DSM16264 and a plasmid (pM150) containing a cDNA which codes for further protein according to the invention (O.s.-OKI ) from Oryza sativa was deposited on 24.03.2004 under the number DSM 16302 under the Budapest Treaty at the German Collection of Microorganisms and Cell Cultures GmbH, Mascheroder Weg 1b, 38124 Braunschweig, Germany.
- the amino acid sequence shown under SEQ ID NO 2 can be derived from the coding region of the cDNA sequence integrated in plasmid A.t.-OK1-pGEM and codes for an OK1 protein from Arabidopsis thaliana.
- the amino acid sequence shown SEQ ID NO 4 can be derived from the coding region of the cDNA sequence integrated in plasmid pMI50 and codes for an OK1 protein from Oryza sativa.
- the present invention therefore also relates to nucleic acid molecules, which code a protein with the enzymatic activity of an OK1 protein, which includes the amino acid sequence, which is coded by the insertion in plasmid A.t.-OK1-pGEM or by the insertion in plasmid pMI50, wherein the coded protein has an identity of at least 70%, preferably of at least 80%, particularly preferably of at least 90% and especially preferably of 95% with the amino acid sequence, which can be derived from the insertion in A.t.-OKI- pGEM or pMI50.
- the present invention also relates to nucleic acid molecules, which code an OK1 protein and include the coding region of the nucleotide sequences shown under SEQ ID NO 1 or SEQ ID NO 3 or sequences, which are complimentary thereto, nucleic acid molecules, which include the coding region of the nucleotide sequence of the insertion contained in plasmid A.t.-OKI -pGEM or in plasmid pMI50 and nucleic acid molecules, which have an identity of at least 70%, preferably of at least 80%, particularly preferably of at least 90% and especially preferably of at least 95% with the said nucleic acid molecules.
- nucleic acid molecules according to the invention With the help of the sequence information of nucleic acid molecules according to the invention or with the help of a nucleic acid molecule according to the invention, it is now possible for the person skilled in the art to isolate homologous sequences from other plant species, preferably from starch-storing plants, preferably from plant species of the genus Oryza, in particular Oryza sativa or from Arabidopsis thaliana. This can be carried out, for example, with the help of conventional methods such as the examination of cDNA or genomic libraries with suitable hybridisation samples.
- homologous sequences can also be isolated with the help of (degenerated) oligonucleotides and the use of PCR-based methods.
- nucleic acid molecules according to the invention which hybridise with the sequence specified under SEQ ID NO 1 or under SEQ ID NO 3 and which code an OK1 protein.
- hybridising means hybridisation under conventional hybridisation conditions, preferably under stringent conditions such as, for example, are described in Sambrock et al., Molecular Cloning, A Laboratory Manual, 2nd edition (1989) Cold Spring Harbor Laboratory Press, Cold
- hybridising means hybridisation under the following conditions:
- 2xSSC 10xDenhardt solution (Ficoll 400+PEG+BSA; Ratio 1 :1 :1); 0.1% SDS; 5 mM EDTA; 50 mM Na2HPO4; 250 ⁇ g/ml herring sperm DNA; 50 ⁇ g/ml tRNA; or
- nucleic acid molecules which hybridise with the nucleic acid molecules according to the invention, can originate from any plant species, which codes an appropriate protein; preferably they originate from starch-storing plants, preferably from species of the (systematic) family Poacea, particularly preferably from species of the genus Oryza.
- Nucleic acid molecules, which hybridise with the molecules according to the invention can, for example, be isolated from genomic or from cDNA libraries. The identification and isolation of nucleic acid molecules of this type can be carried out using the nucleic acid molecules according to the invention or parts of these molecules or the reverse complements of these molecules, e.g.
- nucleic acid molecules which exactly or essentially have the nucleotide sequence specified under SEQ ID NO 1 or SEQ ID NO 3 or parts of these sequences, can be used as hybridisation samples.
- the fragments used as hybridisation samples can also be synthetic fragments or oligonucleotides, which have been manufactured using established synthesising techniques and the sequence of which corresponds essentially with that of a nucleic acid molecule according to the invention.
- an OK1 protein can take place, for example, as described above under General Methods Item 11.
- a preferred bonding affinity to P-starch in comparison with non-phosphorylated starch, and autophosphorylation of an OK1 protein can be demonstrated using the methods already described above and under General Methods Items 8 and 12.
- the molecules hybridising with the nucleic acid molecules according to the invention particularly include fragments, derivatives and allelic variants of the nucleic acid molecules according to the invention, which code an OK1 protein from plants, preferably from starch-storing plants, preferably from plant species of the (systematic) family Poacea, particularly preferably from species of the genus Oryza.
- the term "derivative" means that the sequences of these molecules differ at one or more positions from the sequences of the nucleic acid molecules described above and have a high degree of identity with these sequences.
- the deviation from the nucleic acid molecules described above can have come about, for example, due to deletion, addition, substitution, insertion or recombination.
- the term “identity” means a sequence identity over the whole length of the coding region of at least 60%, in particular an identity of at least 80%, preferably greater than 80%, particularly preferably greater than 90% and especially of at least 95%.
- identity is to be understood to mean the number of amino acids/nucleotides (identity) corresponding with other proteins/nucleic acids, expressed as a percentage. Identity is preferably determined by comparing with other proteins/nucleic acids the SEQ ID NO 2 or SEQ ID NO 4 for amino acids or SEQ ID NO 1 or SEQ ID NO 3 for nucleic acids with the help of computer programs.
- identity is to be determined in such a way that the number of amino acids, which have the shorter sequence in common with the longer sequence, determines the percentage quotient of the identity.
- identity is determined by means of the computer program ClustalW, which is well- known and available to the public (Thompson et al., Nucleic Acids Research 22 (1994), 4673-4680).
- ClustalW is made publicly available by Julie Thompson (Thompson@EMBL-Heidelberg.DE) and Toby Gibson (Gibson@EMBL- Heidelberg.DE), European Molecular Biology Laboratory, Meyerhofstrasse 1 , D 69117 Heidelberg, Germany.
- ClustalW can also be downloaded from different Internet sites, including the IGBMC (Institut de Genetique et de Biologie Moleisme et Cellulaire, B.P.163, 67404 lllkirch Cedex, France; ftp://ftp-igbmc.u-strasbg.fr/pub/) and the EBI (ftp://ftp.ebi.ac.uk/pub/software/) as well as from all mirrored Internet sites of the EBI (European Bioinformatics Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SD, UK).
- Version 1.8 of the ClustalW computer program is used to determine the identity between proteins according to the invention and other proteins.
- Version 1.8 of the ClustalW computer program is used to determine the identity between the nucleotide sequence of the nucleic acid molecules according to the invention, for example, and the nucleotide sequence of other nucleic acid molecules. In doing so, the following parameters must be set:
- nucleic acid molecules which are homologous to the molecules described above and constitute derivatives of these molecules, are generally variations of these molecules, which constitute modifications, which execute the same biological function.
- the variations can occur naturally, for example they can be sequences from other plant species, or they can be mutants, wherein these mutants may have occurred in a natural manner or have been introduced by objective mutagenesis.
- the variations can also be synthetically manufactured sequences.
- allelic variants can be both naturally occurring variants and also synthetically manufactured variants or variants produced by recombinant DNA techniques.
- Nucleic acid molecules which deviate from nucleic acid molecules according to the invention due to degeneration of the genetic code, constitute a special form of derivatives.
- the proteins coded from the different derivatives of nucleic acid molecules according to the invention have certain common characteristics. These can include, for example, biological activity, substrate specificity, molecular weight, immunological reactivity, conformation etc, as well as physical characteristics such as, for example, the running behaviour in gel electrophoresis, chromatographic behaviour, sedimentation coefficients, solubility, spectroscopic characteristics, stability; optimum pH, optimum temperature etc. Preferred characteristics of an OK1 protein have already been described in detail above and are to be applied here accordingly.
- the nucleic acid molecules according to the invention can be any nucleic acid molecules, in particular DNA or RNA molecules, for example cDNA, genomic DNA, mRNA etc. They can be naturally occurring molecules or molecules manufactured by genetic engineering or chemical synthesis methods. They can be single-stranded molecules, which either contain the coding or the non-coding strand, or double- stranded molecules.
- the present invention relates to plant cells according to the invention and plants according to the invention wherein the foreign nucleic acid molecule coding an R1 protein is chosen from the group consisting of a) Nucleic acid molecules, which code a protein with the amino acid sequence given under SEQ ID NO 7, SEQ ID NO 9, SEQ ID NO 11, SEQ ID NO 13, SEQ ID NO 15 or SEQ ID NO 17, b) Nucleic acid rnolecules, which include the nucleotide sequence shown under SEQ ID NO 6/ SEQ ID NO 8, SEQ ID NO 10, SEQ ID NO 12, SEQ ID NO 14 or SEQ ID NO 16, or a complimentary sequence; c) Nucleic acid molecules, the nucleotide sequence of which deviates from the sequence of the nucleic acid molecules identified under a) or b) due to the degeneration of the genetic code; d) Nucleic acid molecules, which have an identity of at least 70% with the nucleic acid sequences described under a) or b) and e) Nucleic acid molecules, which
- the present invention relates to plant cells according to the invention and plants according to the invention wherein the foreign nucleic acid molecule is chosen from the group consisting of a) T-DNA molecules, which lead to an increase in the expression of an OK1 gene and/or an R1 gene due to integration in the plant genome (T-DNA activation tagging); b) DNA molecules, which contain transposons, which lead to an increase in the expression of an OK1 gene and/or an R1 gene due to integration in the plant genome (transposon activation tagging); c) DNA molecules, which code an OK1 protein and/or an R1 protein and which are linked with regulatory sequences, which guarantee transcription in plant cells and lead to an increase in an OK1 protein and/or R1 protein activity in the cell, d) Nucleic acid molecules introduced by means of in vivo mutagenesis, which lead to a mutation or an insertion of a heterologous sequence in at least one endogenous gene coding an OK1 protein, wherein the mutation or insertion effects an increase in the
- nucleic acid molecules introduced by means of in vivo mutagenesis, which lead to a mutation or an insertion of a heterologous sequence in at least one endogenous gene coding an R1 protein, wherein the mutation or insertion effects an increase in the expression of a gene coding an R1 protein.
- plant cells according to the invention and plants according to the invention can also be manufactured by the use of so-called insertion mutagenesis (overview article: Thorneycroft et al., 2001 , Journal of experimental Botany 52 (361), 1593-1601 ).
- insertion mutagenesis is to be understood to mean particularly the insertion of transposons or so-called transfer DNA (T-DNA) into a gene or in the vicinity of a gene coding for an OK1 protein and/or coding for an R1 protein, whereby as a result of which the activity of an OK1 protein and/or an R1 protein in the cell concerned is increased.
- the transposons can be both those that occur naturally in the cell (endogenous transposons) and also those that do not occur naturally in said cell but are introduced into the cell (heterologous transposons) by means of genetic engineering methods, such as transformation of the cell, for example. Changing the expression of genes by means of transposons is known to the person skilled in the art. An overview of the use of endogenous and heterologous transposons as tools in plant biotechnology is presented in Ramachandran and Sundaresan (2001 , Plant Physiology and Biochemistry 39, 234-252).
- T-DNA insertion mutagenesis is based on the fact that certain sections (T-DNA) of Ti plasmids from Agrobacterium can integrate into the genome of plant cells.
- the place of integration in the plant chromosome is not defined, but can take place at any point. If the T-DNA integrates into a part of the chromosome or in the vicinity of a part of the chromosome, which constitutes a gene function, then this can lead to an increase in the gene expression and thus also to a change in the activity of a protein coded by the gene concerned.
- sequences inserted into the genome are distinguished by the fact that they contain sequences, which lead to an activation of regulatory sequences of an OK1 gene ("activation tagging").
- Plant cells and plants according to the invention can be produced by means of the so-called "activation tagging" method (see, for example, Walden et al., Plant J. (1991), 281-288; Walden et al., Plant Mol. Biol. 26 (1994), 1521-1528).
- activation tagging methods are based on activating endogenous promoters by means of enhancer sequences, such as the enhancer of the 35S RNA promoter of the cauliflower mosaic virus, or the octopine synthase enhancer.
- T-DNA activation tagging is to be understood to mean a T-DNA fragment, which contains enhancer sequences and which leads to an increase in the activity of at least one OK1 protein and/or at least one R1 protein by integration into the genome of a plant cell.
- transposon activation tagging is to be understood to mean a transposon, which contains enhancer sequences and which leads to an increase in the activity of at least one OK1 protein and/or at least one R1 protein by integration into the genome of a plant cell.
- the DNA molecules according to the invention which code an OK1 protein and/or an R1 protein, are linked with regulatory sequences, which initiate transcription in plant cells and lead to an increase in OK1 protein and/or R1 protein activity in the cell.
- the nucleic acid molecules according to the invention are present in "sense" orientation to the regulatory sequences.
- nucleic acid molecules according to the invention which code an OK1 protein and/or R1 protein
- these are preferably linked with regulatory DNA sequences, which guarantee transcription in plant cells.
- these include promoters.
- any promoter that is active in plant cells is eligible for expression.
- the promoter can be chosen so that expression takes place constitutively or only in a certain tissue, at a certain stage of the plant development or at a time determined by external influences.
- the promoter can be homologous or heterologous both with respect to the plant and with respect to the nucleic acid molecule.
- Suitable promoters are, for example, the promoter of the 35S RNA of the cauliflower mosaic virus and the ubiquitin promoter from maize for constitutive expression, the patatin promoter B33 (Rocha-Sosa et al., EMBO J. 8 (1989), 23-29) for tuber-specific expression in potatoes or a promoter, which only ensures expression 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.
- HMG promoter from wheat
- USP promoter the phaseolin promoter
- promoters of zein genes from maize Pedersen et al., Cell 29 (1982), 1015-1026; Quatroccio et al., Plant Mol. Biol. 15 (1990), 81-93), glutelin promoter (Leisy et al., Plant Mol. Biol. 14 (1990), 41-50; Zheng et al., Plant J. 4 (1993), 357-366; Yoshihara et al., FEBS Lett.
- promoters can also be used, which are only activated at a time determined by external influences (see for example WO 9307279). Promoters of heat-shock proteins, which allow simple induction, can be of particular interest here.
- seed-specific promoters can be used, such as the USP promoter from Vicia faba, which guarantees seed-specific expression in Vicia faba and other plants (Fiedler et al., Plant Mol. Biol. 22 (1993), 669-679; Baumlein et al., Mol. Gen. Genet. 225 (1991), 459-467).
- a termination sequence (polyadenylation signal) can be present, which is used for adding a poly-A tail to the transcript.
- a function in the stabilisation of the transcripts is ascribed to the poly-A tail. Elements of this type are described in the literature (cf. Gieien et al., EMBO J. 8 (1989), 23-29) and can be exchanged at will.
- Intron sequences can also be present between the promoter and the coding region. Such intron sequences can lead to stability of expression and to increased expression in plants (Callis et al., 1987, Genes Devel. 1 , 1183-1200; Luehrsen, and Walbot, 1991 , Mol. Gen. Genet. 225, 81-93; Rethmeier, et al., 1997; Plant Journal. 12(4):895- 899; Rose and Beliakoff, 2000, Plant Physiol. 122 (2), 535-542; Vasil et al., 1989, Plant Physiol. 91 , 1575-1579; XU et al., 2003, Science in China Series C Vol. 46 No. 6, 561-569).
- Suitable intron sequences are, for example, the first intron of the sh1 gene from maize, the first intron of the polyubiquitin gene 1 from maize, the first intron of the EPSPS gene from rice or one of the two first introns of the PAT1 gene from Arabidopsis.
- plant cells according to the invention and plants according to the invention can be manufactured by means of so-called “in situ activation”.
- the introduced genetic modification effects a change in the regulatory sequences of endogenous OK1 genes and/or R1 genes, which leads to an increased expression of OK1 genes and/or R1 genes.
- the activation of an OK1 gene and/or an R1 gene takes place by "in vivo" mutagenesis of a promoter or of enhancer sequences of an endogenous OK1 gene and/or an R1 gene.
- a promoter or an enhancer sequence for example, can be changed through mutagenesis in such a way that the mutation produced leads to an increased expression of an OK1 protein gene and/or R1 gene in plant cells according to the invention or plants according to the invention in comparison with the expression of an OK1 gene and/or an R1 gene in wild type plant cells or wild type plants.
- the mutation in a promoter or an enhancer sequence can also lead to OK1 genes and/or R1 genes in plant cells according to the invention or plants according to the invention being expressed at a time at which they would not be expressed in wild type plant cells or wild type plants.
- OK1 gene is to be understood to mean a nucleic acid rnolecule (cDNA, DNA) that codes an OK1 protein, preferably an ⁇ A' ' ⁇ OK1 protein from starch-storing plants, particularly preferably from Arabidopsis thaliana, especially preferably from rice.
- cDNA nucleic acid rnolecule
- R1 gene is to be understood to mean a nucleic acid molecule (cDNA, DNA) that codes an R1 protein, preferably an R1 protein from starch-storing plants, particularly preferably from Arabidopsis thaliana, especially preferably from rice.
- cDNA nucleic acid molecule
- RNA-DNA oligonucleotide (Chimeroplast) is introduced into plant cells through transformation of plants cells (Kipp, P.B. et al., Poster Session at the "5th International Congress of Plant Molecular Biology", 21st-27th September 1997, Singapore; R. A. Dixon and CJ.
- a part of the DNA components of the RNA-DNA oligonucleotide is homologous to a nucleic acid sequence of an endogenous OK1 gene and/or R1 gene, but, in comparison with the nucleic acid sequence of an endogenous OK1 gene and/or R1 gene, it has a mutation or contains a heterologous region, which is surrounded by the homologous regions.
- the. mutation or heterologous region contained in the DNA components of the RNA-DNA oligonucleotide can be transferred into the genome of a plant cell. This leads to an increase of the activity of one or more OK1 proteins.
- plant cells according to the invention and plants according to the invention synthesise a modified starch in comparison with starch of corresponding wild type plant cells or wild type plants that have not been genetically modified.
- the plant cells according to the invention and plants according to the invention synthesise a modified starch, which in its physical-chemical characteristics, in particular the starch phosphate content and/or phosphate distribution is changed in comparison with the starch synthesised in wild type plant cells or wild type plants, so that this is better suited for special applications.
- the phosphate distribution of starch synthesised by plants was also not previously possible due to the lack of available means.
- a change of the phosphate ratio of native starches is also now possible through the present invention through the preparation of enzymes with the function of OK1 proteins and the preparation of nucleic acid molecules that code an OK1 protein.
- the present invention therefore also includes plant cells according to the invention and plants according to the invention, which synthesise a modified starch, in comparison with corresponding wild type plant cells or wild type plans that have not been genetically modified.
- modified starch means that the starch has changed physical-chemical characteristics compared with non-modified starch obtainable from corresponding wild type plant cells or wild type plants.
- plant cells according to the invention or plants according to the invention synthesise a starch, which has an increased concentration of starch phosphate and/or a changed phosphate distribution in comparison with starch isolated from corresponding wild type plant cells or wild type plants.
- phosphate distribution is to be understood to mean the proportion of the starch phosphate bound to a glucose molecule in the C-2 position, C-3 position or C-6 position in terms of the total concentration of starch phosphate from starch.
- plant cells according to the invention or plants according to the invention synthesise a starch, which has an increased concentration of starch phosphate and/or a changed ratio of C-3 phosphate to C-6 phosphate in comparison with starch from wild type plants that have not been genetically modified.
- starches which have an increased proportion of starch phosphate bonded in the C-3 position compared with starch phosphate bonded in the C-6 position in comparison with starches from wild type plant cells that have not been genetically modified or wild type plants that have not been genetically modified.
- ratio of C-3 phosphate to C-6 phosphate is to be understood to mean the proportion of starch phosphate for which the starch phosphate of a starch bonded in the C-3 position or C-6 position respectively adds to the total of the starch phosphate of the starch concerned that is bonded in the C-3 position and in the C-6 position (C-3 position + C-6 position).
- Various methods are described for the determination of the amount of starch phosphate.
- the methods described by Ritte et al. 2000, Starch/Starke 52, 179-185) can be used for the determination of the amount of starch phosphate.
- the determination of the amount of starch phosphate is carried out by means of 31 P-NMR according to the methods described by Kasemusuwan and Jane (1996, Cereal Chemistry 73, 702-707).
- plants which contain the plant cells according to the invention, are also the subject matter of the invention. Plants of this type can be produced from plant cells according to the invention by regeneration.
- the plants according to the invention can be plants of any plant species, i.e. both monocotyledonous and dicotyledonous plants.
- they are useful plants, i.e. plants, which are cultivated by people for the purposes of food or for technical, in particular industrial, purposes.
- the plant according to the invention is a starch-storing plant.
- starch-storing plants means all plants with plant parts, which contain a storage starch, such as, for example, maize, rice, wheat, rye, oats, barley, cassava, potato, sago, mung bean, pea or sorghum.
- potato plant or “potato” means plant species of the genus Solanum, in particular tuber-producing species of the genus Solanum and especially Solanum tuberosum.
- the term "wheat plant” means plant species of the genus Triticum or plants resulting from crosses with plants of the genus Triticum, particularly plant species of the genus Triticum or plants resulting from crosses with plants of the genus Triticum, which are used in agriculture for commercial purposes, and particularly preferably Triticum aestivum.
- the term "maize plant” means plant species of the genus Zea, particularly plant species of the genus Zea, which are used in agriculture for commercial purposes, particularly preferably Zea grass.
- the present invention relates to starch-storing plants of the (systematic) family Poaceae according to the invention.
- plants of the (systematic) family Poaceae Preferably these are here maize- or wheat plants.
- the present invention also relates to propagation material of plants according to the invention containing a plant cell according to the invention.
- propagation material includes those constituents of the plant that are suitable for producing offspring by vegetative or sexual means. Cuttings, callus cultures, rhizomes or tubers, for example, are suitable for plant propagation. Other propagation material includes, for example, fruits, seeds, seedlings, protoplasts, cell cultures, etc.
- the propagation material is tubers and particularly preferably grains, which contain endosperms.
- the present invention relates to harvestable plant parts of plants according to the invention such as fruits, storage roots, roots, blooms, buds, shoots or stems, preferably seeds, grains or tubers, wherein these harvestable parts contain plant cells according to the invention.
- the present invention also relates to a method for the manufacture of a genetically modified plant according to the invention, wherein a) a plant cell is genetically modified, wherein the genetic modification leads to an increase in the enzymatic activity of an OK1 protein and an R1 protein in comparison with corresponding wild type plant cells that have not been genetically modified; b) a plant is regenerated from plant cells from step a); c) and, if necessary, further plants are produced with the help of the plants according to Step b).
- the genetic modification introduced into the plant cell according to Step a) can basically be any type of genetic modification, which leads to the increase of the activity of an OK1 protein and an R1 protein.
- the regeneration of the plants according to Step (b) can be carried out using methods known to the person skilled in the art (e.g. described in "Plant Cell Culture Protocols", 1999, edt. by R.D. Hall, Humana Press, ISBN 0-89603-549-2).
- the production of further plants according to Step (c) of the method according to the invention can be carried out, for example, by vegetative propagation (for example using cuttings, tubers or by means of callus culture and regeneration of whole plants) or by sexual propagation.
- sexual propagation preferably takes place under controlled conditions, i.e. selected plants with particular characteristics are crossed and propagated with one another. In this case, the selection is preferably carried out in such a way that further plants, which are produced in accordance with Step c),. exhibit the modification, which was introduced in Step a).
- the genetic modifications for the production of the plant cells according to the invention can take place simultaneously or in successive steps.
- the genetic modification can be any genetic modification, which leads to the increase of the activity of at least one OK1 protein and/or at least one R1 protein. It can come from wild type plants as well as wild type plant cells, in which no prior genetic modification for the reduction of the activity of at least one OK1 protein or at least one R1 protein has yet taken place, or from plant cells or plants that are already genetically modified, in which the activity of at least one OK1 protein or at least one R1 protein is already increased through a genetic modification.
- the genetic modification consists in the introduction of at least one foreign nucleic acid molecule into the genome of the plant cell, wherein the availability or the expression of foreign nucleic acid molecule(s) lead(s) to an increased activity of an OK1 protein and an R1 protein in the cell.
- the genetic modification consists in the introduction of at least one foreign nucleic acid molecule into the genome of the plant cell, wherein the foreign nucleic acid molecule(s) contain(s) a sequence coding an OK1 protein and/or an R1 protein.
- Step a) of the method according to the invention for the manufacture of a genetically modified plant according to the invention can involve a single nucleic acid molecule or multiple nucleic acid molecules.
- the embodiments provided above are to be correspondingly applied for the method according to the invention described here.
- the genetic modification in Step a) of the method consists in the introduction of a foreign nucleic acid molecule which contains at least one sequence coding R1 protein and at least one sequence coding OK1 protein.
- the genetic modification in Step a) of the method consists in the introduction of multiple foreign nucleic acid molecules, wherein at least one first nucleic acid molecule contains a sequence coding an R1 protein and at least a second nucleic acid molecule contains a sequence coding an OK1 protein.
- a mutant cell or a mutant characterised in that it already has an increased activity of an OK1 protein or an increased activity of an R1 protein can be used for introducing a foreign nucleic acid molecule for the implementation of the method according to the invention.
- the additional information provided above for the use of mutants for the manufacture of plant cells according to the invention or plant are to be correspondingly applied here.
- At least one foreign nucleic acid molecule is selected from the group consisting of a) Nucleic acid molecules, which code a protein with the amino acid sequence given under SEQ ID NO 2 or SEQ ID NO 4; b) Nucleic acid molecules, which code a protein, which includes the amino acid sequence, which is coded by the insertion in plasmid A.t.-OK1-pGEM or the insertion in plasmid pMI50; c) Nucleic acid molecules, which code a protein, the sequence of which has an identity of at least 60% with the amino acid sequence given under SEQ ID NO 2 or SEQ ID NO 4; d) Nucleic acid molecules, which code a protein, the sequence of which has an identity of at least 60% with the amino acid sequence, which is coded by the insertion in plasmid A.t.-OK1-pGEM or by the insertion in plasmid pMI50;
- an R1 protein from potato, wheat, rice, maize, soybean, citrus or Arabidopsis codes at least one foreign nucleic acid molecule.
- References for the identified nucleic acid sequences coding R1 proteins from the identified plants have already been further specified above.
- a further embodiment of the present invention also relates to a method for the manufacture of a genetically modified plant according to the invention, wherein a) a plant cell is genetically modified, wherein the genetic modification leads to an increase in the enzymatic activity of an OK1 protein in comparison with corresponding wild type plant cells that have not been genetically modified; b) a plant is regenerated from plant cells from Step a); c) if necessary, further plants are produced with the help of the plants according to Step b) and d) Plants obtained according to Step b) or c) are crossed with a plant, which has an increased level of enzymatic activity of an R1 protein, in comparison with corresponding wild type plant cells that have not been genetically modified.
- a further embodiment of the present invention relates to a method for the manufacture of a genetically modified plant according to the invention, wherein a) a plant cell is genetically modified, wherein the genetic modification leads to an increase in the enzymatic activity of an R1 protein in comparison with corresponding wild type plant cells that have not been genetically modified; b) a plant is regenerated from plant cells from Step a); c) if necessary, further plants are produced with the help of the plants according to Step b) and d) Plants obtained according to Step b) or c) are crossed with a plant, which has an increased level of enzymatic activity of an OK1 protein, in comparison with corresponding wild type plant cells that have not been genetically modified.
- the plants according to Step a) can be genetically modified as already described above.
- the regeneration of plans according to Step b) and the production of additional plants according to Step c) was also already further presented above.
- a plant that is crossed with plants or offspring of plants obtained in Step b) or c) according to Step d) of the two most recently identified embodiments can be any plant that has an increased activity of an OK1 protein or an R1 protein, in comparison to corresponding wild type plants.
- the increase of the activity of an OK1 protein or an R1 protein can be brought about through any modification that leads to an increase of the activity of the related protein in the corresponding plants.
- These plants can be mutants or plants modified by means of genetic engineering methods.
- Mutants can be spontaneously (naturally) occurring mutants as well as those that were produced through the targeted use of mutagens (such as, for example, chemical agents, ionising radiation) or genetic engineering methods (for example transposon activation tagging, T-DNA activation tagging, in vivo mutagenesis).
- mutagens such as, for example, chemical agents, ionising radiation
- genetic engineering methods for example transposon activation tagging, T-DNA activation tagging, in vivo mutagenesis.
- the plants produced through genetic engineering methods are mutants manufactured by means of insertion mutagenesis, particularly preferably genetically modified plants that express a foreign nucleic acid molecule, especially preferably genetically modified plants in which the foreign nucleic acid molecule codes an OK1 protein or an R1 protein.
- the method according to the invention is used for manufacturing a genetically modified plant according to the invention for producing starch-storing plants. In a further embodiment, the method according to the invention is used for manufacturing a genetically modified plant according to the invention for producing maize- or wheat plants according to the invention.
- the present invention relates to a method according to the invention for manufacturing a genetically modified plant according to the invention, wherein the genetically modified plant synthesises a modified starch in comparison with wild type plants that have not been genetically modified.
- the plants according to the invention synthesise a modified starch, which has an increased concentration of starch phosphate and/or a changed starch phosphate distribution in comparison with starch isolated from corresponding wild type plants.
- the plants according to the invention synthesise a modified starch, which has a changed ratio of C-3 phosphate to C-6 phosphate in comparison with starch from wild type plants that have not been genetically modified.
- a modified starch which has a changed ratio of C-3 phosphate to C-6 phosphate in comparison with starch from wild type plants that have not been genetically modified.
- starches which have an increased proportion of starch phosphate bonded in the C-3 position compared with starch phosphate bonded in the C-6 position in comparison with starches from wild type plant that have not been genetically modified.
- the present invention also relates to plants ohbtainable by the methods according to the invention.
- starch isolated from plant cells according to the invention and plants according to the invention which have an increased activity of an OK1 protein and an increased activity of an R1 protein, synthesise a modified starch.
- the increased amount of starch phosphate in starches according to the invention gives the starches surprising and advantageous characteristics.
- starches according to the invention support an increased proportion of charged groups that significantly affect the functional characteristics of the starch.
- Starch that supports the charged functional groups is particularly applicable in the paper industry, where it is used for the coating of paper. Paper that otherwise has good adhesive characteristics with charged molecules is particularly suitable for the absorption of dyestuffs, such as ink, print colors, etc., when coated.
- the present invention also relates to modified starches obtainable from plant cells according to the invention or plants according to the invention, from propagation material according ; t the invention or from harvestable plant parts according to the invention.
- the present invention relates to modified starch according to the invention from starch-storing plants, preferably from starch-storing plants of the (systematic) family Poaceae, particularly preferably from maize or wheat plants.
- the present invention relates to a method for the manufacture of a modified starch including the step of extracting the starch from a plant cell according to the invention or from a plant according to the invention, from propagation material according to the invention of such a plant and/or from harvestable pjant parts according to the invention of such a plant, preferably from starch-storing parts according to the invention of such a plant.
- a method also includes the step of harvesting the cultivated plants or plant parts and/or the propagation material of these plants before the extraction of the starch and, further, particularly preferably the step of cultivating plants according to the invention before harvesting.
- Methods for extracting starches from plants or from starch-storing parts of plants are known to the person skilled in the art.
- starch-storing parts is to be understood to mean such parts of a plant in which, in contrast to transitory leaf starch, starch is stored as a deposit for surviving for longer periods.
- Preferred starch-storing plant parts are, for example, tubers, storage roots and grains, particularly preferred are grains containing an endosperm, especially particularly preferred are grains containing an endosperm of maize or wheat plants.
- Modified starch obtainable by a method according to the invention for manufacturing modified starch is also the subject matter of the present invention.
- the modified starch according to the invention is native starch.
- native starch means that the starch is isolated from plants according to the invention, harvestable plant plants according to the invention, starch-storing parts according to the invention or propagation material of plants according to the invention by methods known to the person skilled in the art.
- starch can be changed by thermal, chemical, enzymatic or mechanical derivation, for example.
- Derived starches are particularly suitable for different applications in the foodstuffs and/or non- foodstuffs sector.
- the starches according to the invention are better suited as a starting substance for the manufacture of derived starches than conventional starches, as they have a higher proportion of reactive functional groups due to the higher starch phosphate content.
- the present invention therefore also relates to the manufacture of a derived starch, wherein modified starch according to the invention is derived retrospectively.
- derived starch is to be understood to mean a modified starch according to the invention, the characteristics of which have been changed after isolation from plant cells with the help of chemical, enzymatic, thermal or mechanical methods.
- the derived starch according to the invention is starch that has been treated with heat and/or acid.
- the derived starches are starch ethers, in particular starch alkyl ethers, O-allyl ethers, hydroxylalkyl ethers, O-carboxylmethyl ethers, nitrogen- containing starch ethers, phosphate-containing starch ethers or sulphur-containing starch ethers.
- the derived starches are crosslinked starches.
- the derived starches are starch graft polymers.
- the derived starches are oxidised starches.
- the derived starches are starch esters, in particular starch esters, which have been introduced into the starch using organic acids. Particulariy preferably these are phosphate, nitrate, sulphate, xanthate, acetate or citrate starches.
- the derived starches according to the invention are suitable for different applications in the pharmaceutical industry and in the foodstuffs and/or non-foodstuffs sector.
- Methods for manufacturing derived starches according to the invention are known to the person skilled in the art and are adequately described in the general literature. An overview on the manufacture of derived starches can be found, for example, in Orthoefer (in Corn, Chemistry and Technology, 1987, eds. Watson und Ramstad, Chapter 16, 479-499).
- Derived starch obtainable by the method according to the invention for manufacturing a derived starch is also the subject matter of the present invention.
- modified starches according to the invention for manufacturing derived starch is the subject matter of the present invention.
- Starch-storing parts of plants are often processed into flours. Examples of parts of plants from which flours are manufactured are, for example, tubers of potato plants and grains from cereal plants. The grains of these plants that contain endosperms are milled and sifted for the manufacture of flours from cereal plants.
- Starch is a principal component of endosperm. For other plants that contain no endosperm, but rather other starch-storing parts, such as tubers or roots, flour is commonly manufactured by reducing, drying and then milling the storage organs concerned.
- the starch of the endosperm or contained in starch-storing parts of plants is an important part of flour, which is manufactured from the plant parts concerned.
- the characteristics of flours are therefore also affected by the starch present in the flour concerned.
- Plant cells according to the invention and plants according to the invention synthesise a modified starch in comparison with corresponding wild type plant cells that have not been genetically modified or wild type plants that have not been genetically modified.
- Flours manufactured from plant cells according to the invention, plants according to the invention, propagation material according to the invention or harvestable parts according to the invention therefore have modified characteristics.
- the characteristics of flours can also be affected by mixing starch with flours or by mixing flours with different characteristics.
- a further subject of the present invention therefore relates to flours containing a starch according to the invention.
- a further subject of the present invention relates to flours that are manufactured from plant cells according to the invention, plants according to the invention, starch-storing parts of plants according to the invention, from propagation material according to the invention or from harvestable plant parts according to the invention.
- Preferred starch- storing parts of plants according to the invention are tubers, storage roots and a grain, which contains endosperm.
- Tubers from potato plants and grains preferably originate from plants of the (systematic) family Poaceae; grains particularly preferably originate from maize or wheat plants.
- the term "flour" means a powder obtained by milling plant parts. If necessary, plant parts are dried before milling and reduced and/or sifted after milling.
- Flours according to the invention are particularly distinguished by their increased water binding capacity due to the starch present in them that has a modified phosphate content and/or a modified phosphate distribution. This is, for example, desired for the processing of flours in the food industry for many applications, in particular in the manufacture of bakery products.
- a further subjecj pf i ⁇ e present invention is a method for the manufacture of flours, including the step o Hhe milling of plant cells according to the invention, plants according to the invention, of parts of plants according to the invention, starch-storing parts of plants according to the invention, propagation material according to the invention or harvestable material according to the invention.
- Flours can be manufactured through the milling of starch-storing parts of according to the invention. It is known to the person skilled in the art how to manufacture flours. Preferably, a method for the manufacture of flours also includes the step of harvesting the cultivated plants or plant parts and/or of the propagation material or the starch- storing parts of these plants before the milling and particularly preferably further the step of cultivating plants according to the invention before harvesting.
- Parts of plants is to be understood to mean all parts of a plant, which represent a complete plant as constituents in their totality. Parts of plants are, for example, shoots, leaves, rhizomes, roots, beetroots, tubers, pods, seeds or grains.
- a further subject of the present invention includes the method for the manufacture of flours, of a processing of plants according to the invention, of starch-storing parts of plants according to the invention, of propagation material according to the invention or of material according to the invention harvestable before milling.
- the processing can be, for example, a heating treatment and/or a drying.
- Heating treatment followed by a drying of the heat-treated material is used, for example, for the manufacture of flours from storage roots or tubers such as, for example, from potato tubers, before which the milling takes place.
- the reduction of plants according to the invention, of starch-storing parts of plants according to the invention, of propagation material according to the invention or of material according to the invention harvestable before milling can likewise represent a processing in terms of the present invention.
- the removal of plant tissue such as, for example, of husks of grains, before the milling also represents a processing before the milling in terms of the present invention.
- a further embodiment of the present invention includes the method for the manufacture of flours after the milling of a product of the grist processing.
- the grist can be, for example, sifted after the milling in order to manufacture, for example, various types of flours.
- a further subject of the present invention is the use of genetically modified plant cells according to the invention, plants according to the invention, of parts of plants according to the invention, starch-storing parts of plants according to the invention, propagation material according to the invention or harvestable material according to the invention for the manufacture of flours.
- the provided DNA molecules contain nucleic acid sequences which code an OK1 protein.
- a protein with the enzymatic activity of an OK1 protein was not previously known to the person skilled in the art.
- no DNA molecules can thus be provided, which allow plant cells according to the invention and plants according to the invention and the starch synthesised from them and the flours extracted from them to be produced.
- the present invention also relates to a recombinant nucleic acid molecule containing a nucleic acid sequence coding an OK1 protein and a nucleic acid sequence coding an R1 protein.
- the term "recombinant nucleic acid molecule” is to be understood to mean a nucleic acid molecule that contains both nucleic acid sequences coding an OK1 protein as well as nucleic acid sequences coding an R1 protein and in which the nucleic acid sequences coding an OK1 protein and an R1 protein are present in an arrangement as they are not present naturally in the genome of an organism.
- the recombinant nucleic acid molecule can still contain additional sequences, which are not naturally present in one such arrangement as they are present in recombinant nucleic acid molecules according to the invention.
- the said additional sequences can be any sequences, preferably regulatory sequences (promoters, termination signals, enhancers), particularly preferably regulatory sequences, which are active in plant tissue, particularly preferably regulatory sequences, which are active in starch-storing plant tissue.
- Methods for producing recombinant nucleic acid molecules according to the invention are known to the person skilled in the art and include genetic engineering methods such as, for example, the linking of nucleic acid molecules by ligation, genetic recombination or the resynthesis of nucleic acid molecules (see, for example, Sambrok et al., Molecular Cloning, A Laboratory Manual, 3 rd edition (2001) Cold Spring Harbour Laboratory Press, Cold Spring Harbour, NY. ISBN: 0879695773, Ausubel et al., Short Protocols in Molecular Biology, John Wiley & Sons; 5 th edition (2002), ISBN: 0471250929).
- a further embodiment of the present invention of recombinant nucleic acid molecules according to the invention comprises vectors, in particular plasmids, cosmids, viruses, bacteriophages and other common vectors in genetic engineering, which contain the nucleic acid molecules according to the invention described above.
- the nucleic acid molecules according to the invention contained in the vectors are linked with regulatory sequences, which initiate the expression in prokaryotic or eukaryotic cells.
- expression can mean transcription as well as transcription and translation.
- the nucleic acid molecules according to the invention can be present in "sense” orientation and/or in "antisense” orientation to the regulatory sequences.
- the recombinant nucleic acid molecules according to the invention can collectively remain under the control of a single regulatory element, or they can each have their own respective individual regulatory element.
- prokaryotic organisms e.g. E. coli
- eukaryotic organisms are adequately described in the literature, in particular those for expression in yeast such as Saccharomyces cerevisiae, for example.
- yeast such as Saccharomyces cerevisiae
- An overview of different expression systems for proteins and different host organisms can be found, for example, in Methods in Enzymology 153 (1987), 383-516 and in Bitter et al. (Methods in Enzymology 153 (1987), 516-544).
- a further subject of the present invention is a host cell, in particular a prokaryotic or eukaryotic cell, which is genetically modified with a recombinant nucleic acid molecule according to the invention and/or with a vector according to the invention, as well as cells, which originate from host cells of this type and which contain the genetic modification according to the invention.
- the invention relates to host cells, particularly prokaryotic or eukaryotic cells, which were transformed with a nucleic acid molecule according to the invention or a vector according to the invention, as well as host cells, which originate from host cells of this type and which contain the described nucleic acid molecules according to the invention or vectors.
- the host cells can be bacteria (e.g. E. coli, bacteria of the genus Agrobacterium in particular Agrobacterium tumefaciens or Agrobacterium rhizogenes) or fungus cells (e.g. yeast, in particular S. cerevisiae, Agaricus, in particular Agaricus bisporus, Aspergillus, Trichoderma), as well as plant or animal cells.
- bacteria e.g. E. coli, bacteria of the genus Agrobacterium in particular Agrobacterium tumefaciens or Agrobacterium rhizogenes
- fungus cells e.g. yeast, in particular S. cerevisiae, Agaricus, in particular Agaricus bisporus, Aspergillus, Trichoderma
- the term "transformed” means that the cells according to the invention are genetically modified with a nucleic acid molecule according to the invention inasmuch as they contain at least one nucleic acid molecule
- the host cells are preferably microorganisms. Within the framework of the present application, these are understood to mean all bacteria and all protista (e.g. fungi, in particular yeast and algae), as defined, for example, in Schlegel “Allgemeine Mikrobiologie” (Georg Thieme Verlag (1985), 1-2).
- protista e.g. fungi, in particular yeast and algae
- the host cells according to the invention are plant cells.
- these can be plant cells from any plant species, i.e. both monocotyledonous and dicotyledonous plants.
- these will be plant cells from useful agricultural plants, i.e. from plants, which are cultivated by people for the purposes of food or for technical, in particular industrial purposes.
- the invention relates preferably to plant cells and plants from starch-storing plants (maize, rice, wheat, rye, oat, barley, cassava, potato, sago, mung bean, pea or sorghum), preferably plant cells from plants of the (systematic) family Poacea, especially particularly preferred are plant cells from maize or wheat plants.
- compositions containing a recombinant nucleic acid molecule according to the invention, or a vector according to the invention are compositions containing a recombinant nucleic acid molecule according to the invention, or a vector according to the invention.
- compositions according to the invention containing a recombinant nucleic acid molecule according to the invention, or a vector according to the invention and a host cell It is particularly preferred if the host cell is a plant cell, and especially preferred if it is a cell of a maize or wheat plant.
- a further subject of the present invention relates to a composition containing a nucleic acid sequence coding an OK1 protein and a nucleic acid sequence coding an R1 protein.
- nucleic acid sequences coding an OK1 protein or coding an R1 protein can exist together in a single nucleic acid molecule, or in nucleic acid . ' XAX . . molecules separated from one another.
- compositions according to the invention relate to compositions, which can be used for producing host cells according to the invention, preferably for producing plant cells according to the invention.
- this concerns a composition containing nucleic acid sequences coding an OK1 protein and nucleic acid sequences coding an R1 protein, a recombinant nucleic acid molecule according to the invention or a vector according to the invention and a biolistic carrier, which is suitable for the introduction of nucleic acid molecules into a host cell.
- Preferred biolistic carriers are particles of tungsten, gold or synthetic materials.
- compositions according to the invention relate to compositions containing nucleic acid sequences coding an OK1 protein and nucleic acid sequences coding an R1 protein, a recombinant nucleic acid molecule according to the invention or a vector according to the invention and a plant cell and a synthetic cultivation medium.
- compositions also contain polyethylene glycol (PEG) in addition to plant cells and synthetic cultivation medium.
- PEG polyethylene glycol
- Synthetic cultivation media which are suitable for the cultivation and/or transformation of plant cells, are known to the person skilled in the art and are adequately described in the literature, for example. Many different synthetic cultivation media are also available for purchase in the specialised trade (e.g. DUCHEFA Biochemie B.V., Belgium).
- compositions according to the invention for the transformation of plant cells.
- SEQ ID NO 1 Nucleic acid sequence containing the coding region of an A.t.- OKI protein from Arabidopsis thaliana. This sequence is inserted in the vectors OK1-pGEM-T and OK1-pDESTTM17.
- SEQ ID NO 2 Amino acid sequence coding an A.t.-OK1 protein from Arabidopsis thaliana. This sequence can be derived from the nucleic acid sequence shown under SEQ ID NO 1.
- SEQ ID NO 3 Nucleic acid sequence containing the coding region of an O.s.- OKI protein from Oryza sativa. The sequence is inserted in vector pMI50.
- SEQ ID NO 4 Amino acid sequence coding an O.s.-OKI protein from Oryza sativa. This sequence can be derived from the nucleic acid sequence shown under SEQ ID NO 3.
- SEQ ID NO 5 Peptide sequence coding the phosphohistidine domain of the OK1 proteins from Arabidopsis thaliana and Oryza sativa.
- SEQ ID NO 6 Nucleic acid sequence containing the coding region of a C.r.-R1 protein from Citrus reticulata.
- SEQ ID NO 7 Amino acid sequence coding a C.r.-R1 protein from Citrus reticulata.
- SEQ ID NO 8 Nucleic acid sequence containing the coding region of an A.t.-RI protein from Arabidopsis thaliana.
- SEQ ID NO 9 Amino acid sequence coding an A.t.-R1 protein from Arabidopsis thaliana.
- SEQ ID NO 10 Nucleic acid sequence containing the coding region of an S.t.-R1 protein from Solanum tuberosum.
- SEQ ID NO 11 Amino acid sequence coding an S.L-R1 protein from Solanum tuberosum.
- SEQ ID NO 12 Nucleic acid sequence containing the coding region of an O.s.- R1 protein from Oryza sativa.
- SEQ ID NO 13 Amino acid sequence coding an O.S.-R1 protein from Oryza sativa.
- SEQ ID NO 14 Nucleic acid sequence containing the coding region of a G.m.-R1 protein from Glycine max.
- SEQ ID NO 15 Amino acid sequence coding the S.t.-R1 protein from Glycine max.
- SEQ ID NO 16 Nucleic acid sequence containing a coding region of a Z.m.-R1 protein from Zea mays.
- SEQ ID NO 17 Amino acid sequence coding a Z.m-R1 protein from Zea mays.
- Fig. 1 Denaturing acrylamide gel for identifying proteins from Arabidopsis thaliana, which preferably bond to non-phosphorylated starch in comparison with phosphorylated starch. Standard protein molecular weight marker is shown in trace "M”. Proteins obtained after incubation of control preparation C from Example 1 d) are shown in trace "-”. Protein extracts of Arabidopsis thaliana, obtained after incubation with non-phosphorylated starch, isolated from leaves of an Arabidopsis thaliana sex1-3 mutant (Preparation B, Example 1 d), are shown in trace "K”.
- Protein extracts of Arabidopsis thaliana obtained after incubation with non-phosphorylated starch, isolated from leaves of an Arabidopsis thaliana sex1-3 mutant, which was phosphorylated retrospectively in vitro with an R1 protein (Preparation A, Example 1 d), are shown in trace "P". On completion of electrophoresis, the acrylamide gel was stained with Coomassie Blue.
- Fig. 2 Demonstration of the autophosphorylation of the OK1 protein.
- Fig. 2 A) shows a denaturing (SDS) acrylamide gel on completion of electrophoresis stained with Coomassie Blue.
- Fig. 2 B) shows the autoradiography of a denaturing (SDS) acrylamide gel. The same amounts of the same samples were applied to each of the two gels.
- M Standard protein molecular weight marker
- R1 Sample from reaction vessel 1 according to Example 7 (after incubating an OK1 protein with ATP)
- R2 Sample from reaction vessel 2 according to Example 7 (after incubating an OK1 protein with ATP the protein was heated to 95°C)
- R3 Sample from reaction vessel 3 according to Example 7 (after incubating an OK1 protein with ATP the protein was incubated in 0.5 M HCI)
- R4 Sample from reaction vessel 4 according to Example 7 (after incubating an OK1 protein with ATP the protein was incubated in 0.5 M NaOH).
- Fig. 3 Demonstration of the starch-phosphorylating activity of an OK1 protein (see Example 6).
- OK1 protein was incubated with non-phosphorylating starch isolated from leaves of an Arabidopsis thaliana sex1-3 mutant (Preparation A) and starch isolated from leaves of an Arabidopsis thaliana sex1-3 mutant, which was phosphorylated retrospectively in vitro with an R1 protein (Preparation B).
- Preparation C is the same as Preparation B, except that this Preparation C was incubated without OK1 protein. Two independent tests were carried out for each preparation (A, B, C) (Test 1 and Test 2).
- Fig. 4 Comparison of the C-atom positions of glucose molecules of the starch, which was phosphorylated from an R1 protein and an OK1 protein respectively (see Example 9).
- OK1 protein (Preparation A) was incubated in the presence of ATP labeled with 33 P with starch isolated from leaves of an Arabidopsis thaliana sex1-3 mutant, which was phosphorylated retrospectively in vitro with an R1 protein.
- R1 protein (Preparation B) was incubated in the presence of ATP labeled with 33P with starch isolated from leaves of an Arabidopsis thaliana sex1-3 mutant. On completion of incubation, a total hydrolysis of the starch was carried out and the obtained hydrolysis products were separated by means of HPAE chromatography.
- glucose-6-phosphate and glucose-3- phosphate were added to the hydrolysis products before separation.
- the hydrolysis products separated by means of HPAE chromatography were collected in individual fractions.
- the fractions obtained were subsequently investigated for the presence of radioactively labeled phosphate.
- the amount of 33 P labeled phosphate measured in the individual fractions, measured in cpm (counts per minute), which was introduced into the hydrolysis products of the phosphorylated starch by the OK1 protein or the R1 protein, is shown graphically.
- Fig. 5 Demonstration of the autophosphorylation of the OK1 protein.
- Fig. 5 A) shows a Western blot.
- Fig. 5 B) shows the autoradiography of a denaturing (SDS) acrylar ijde gel. The same amounts of the same samples were applied to : ' 1XX' each of the i p' gels.
- the OK1 protein was incubated either with randomised radioactively labeled ATP or with ATP specifically radioactively labeled in the gamma position. On completion of incubation, the proteins were either heated to 30°C or 95°C, or incubated in 0.5 M NaOH or 0.5 M HCI respectively.
- Fig. 6 Demonstration of the transfer of the beta-phosphate residue of ATP to starch in a reaction catalysed by an OK1 protein.
- Either ATP specifically labeled with 33 P in the gamma position or randomised 33 P ATP was used to phosphorylate starch, which had been phosphorylated in vitro by means of an R1 protein and isolated from leaves of an Arabidopsis thaliana sex1-3 mutant, by means of an OK1 protein.
- No OK1 protein was added in any of the experiments designated as "control”. Each test preparation was tested twice, independently from one another. The results of both tests are shown.
- Fig. 7 Demonstration of the increase of the phosphorylation activity in phosphorylation reactions when R1 proteins and OK1 proteins are simultaneously involved in the reaction Presented is the incorporation of phosphate, starting from radioactively labeled ATP (randomised 33 P-ATP) in the concerned starches by measuring the radioactivity (cpm] in the different starches.
- wheat starch was incubated in native form with R1 protein (Preparation 1-2, Preparation 1-2) or OK1 protein (Preparation 2), or in the form of in vitro phosphorylated wheat starch with OK1 protein (Preparation 3).
- Preparation 4 contained native wheat starch that was incubated simultaneously with R1 protein and OK1 protein. Each preparation was carried out in three repeats. The total of the respective preparations 1-2 and preparations 3 is presented for comparison.
- Fig. 8 Demonstration of the increased activity through the cooperation of an R1 protein and an OK1 protein
- R1 purified R1
- Preparation 2 purified OK1 protein
- Preparation 3 the same phosphorylated wheat starch was incubated simultaneously with an R1 protein and an OK1 protein (Preparation 3).
- the amount of the phosphate bound to the starch in the respective reaction preparation was determined after completion of incubation by means of measurement in the scintillation counter. Also presented in the figure is the total for the reaction preparations for which only one of the two enzymes was introduced respectively for the phosphorylation reaction.
- Leaf material is frozen in liquid nitrogen immediately after harvesting and subsequently homogenised in the mortar under liquid nitrogen.
- the reduced leaf material is mixed with ca. 3.5-times the volume (referred to the weight of the leaf material used) of cold (4°C) binding buffer and broken down for 2 x 10 s with an Ultraturrax (maximum speed).
- the reduced leaf material is cooled on ice before the second treatment is carried out.
- the treated leaf material is then passed through a 100 ⁇ m nylon mesh and centrifuged for 20 min (50 ml centrifuge vessel, 20,000 xg, 4°C).
- the supernatant obtained following centrifugation according to Step a) is removed and its volume determined.
- ammonium sulphate is added continuously to the supernatant over a period of 30 minutes while stirring on ice down to a final concentration of 75% (weight/volume).
- the supernatant is subsequently incubated for a further hour on ice while stirring.
- the proteins precipitated from the supernatant are pellitised at 20,000 xg and 4°C for 10 min and the pellet subsequently absorbed in 5 ml of binding buffer, i.e. the proteins present in the pellet are dissolved.
- the dissolved proteins are desalted by means of a PD10 column filled with Sephadex G25 (Amersham Bioscience, Freiburg, Prod. No. columns: 17-0851-01 , Prod. No. Sephadex G25-M: 17-0033-01) at a temperature of 4°C, i.e. the ammonium sulphate used under Step b) for precipitation is also separated from the dissolved protein.
- the PD10 column is equilibrated with binding buffer before the proteins dissolved in accordance with Step b) are applied. For this purpose, 5 ml of binding buffer are spread over the column five times in each case. Subsequently, 2.5 ml of the protein solution obtained in accordance with Step b) are added to each column before proteins are eluted from the column with 3.5 ml of binding buffer.
- the protein concentration is determined with a Bradford assay (Biorad, Kunststoff, Prod.
- Binding buffer 50 mM HEPES/NaOH (or KOH ), PH 7.2 1 mM EDTA 2 mM Dithioerythritol (DTE) 2 mM Benzamidine 2 mM D-aminocaproic acid 0.5 mM PMSF 0.02 % Triton X-100
- Isolation of leaf starch a) Isolation of starch granules from plant tissues
- Leaf material is frozen immediately after harvesting in liquid nitrogen.
- the leaf material is homogenised in portions in the mortar under liquid nitrogen and absorbed into a total of ca. 2.5-times the volume (weight/volume) of starch buffer.
- this suspension is again homogenised in the Waring blender for 20 s at maximum speed.
- the homogenate is passed through a nylon mesh (100 ⁇ m mesh width) and centrifuged for 5 minutes at 1,000 xg. The supernatant with the soluble proteins is discarded.
- the pellet containing the starch obtained from Step a) is absorbed in starch buffer and successively passed through nylon meshes with different mesh widths (in the order 60 ⁇ m, 30 ⁇ m, 20 ⁇ m).
- the filtrate is centrifuged using a 10 ml Percoll cushion (95% (v/v) Percoll (Pharmacia, Uppsala, Sweden), 5% (v/v) 0.5M HEPES-KOH pH7.2) (Correx tube, 15 min, 2,000 xg).
- the sediment obtained after this centrifugation is resuspended once in starch buffer and centrifuged again (5 min, 1,000 xg,).
- starch granules are obtained, which contain proteins bonded to the starch.
- the proteins bonded to the surface of the starch granules are removed by incubating four times with 0.5 % SDS (sodium lauryl sulphate) for 10-15 minutes in each case at room temperature under agitation. Each washing step is followed by a centrifugation (5 min, 5,000 xg), in order to separate the starch granules from the respective wash buffer.
- SDS sodium lauryl sulphate
- the starch obtained from Step c), which has been freed from the proteins bonded to its surface, is subsequently removed by incubating four times with wash buffer for 10- 15 minutes in each case at room temperature under agitation. Each washing step is followed by a centrifugation (5 min, 1,000 xg), in order to separate the starch granules from the respective wash buffer. These purification steps serve mainly to remove the SDS used in the incubations in Step c).
- Step d) Determination of the concentration of isolated starch
- the amount of starch isolated in Step d) is determined photometrically. After suitable dilution, the optical density of the starch suspension is measured against a calibration curve at a wavelength of 600 nm. The linear range of the calibration curve is located between 0 and 0.3 extinction units.
- starch for example isolated from leaves of an Arabidopsis thaliana sex1-3 mutant, is dried under vacuum, weighed and absorbed in a defined volume of water. The suspension so obtained is diluted with water in several steps in a ratio of 1 to 1 in each case until a suspension of ca. 5 ⁇ g of starch per ml of water is obtained.
- the suspensions obtained by the individual dilution steps are measured in the photometer at a wavelength of 600 nm.
- the absorption values obtained for each suspension are plotted against the concentration of starch in the respective suspension.
- the calibration curve obtained should follow a linear mathematical function in the range from 0 ⁇ g starch per ml of water to 0.3 ⁇ g starch per ml of water.
- Wash buffer 50 mM HEPES/KOH, pH 7.2
- the cDNA coding a starch-phosphorylating protein can be amplified, for example, using mRNA or poly-A-plus-mRNA from plant tissues as a "template", by means of a polymerase chain reaction (PCR).
- PCR polymerase chain reaction
- a reverse transcriptase is first used for the manufacture of a cDNA strand, which is complementary to an mRNA, which codes a starch-phosphorylating protein, before the cDNA strand concerned is amplified by means of DNA polymerase.
- So-called "kits” containing substances, enzymes and instructions for carrying out PCR reactions are available for purchase (e.g. SuperScriptTM One-Step RT-PCR System, Invitrogen, Prod. No.: 10928-034).
- the amplified cDNA coding a starch-phosphorylating protein can subsequently be cloned in a bacterial expression vector, e.g. pDESTTM17 (Invitrogen).
- pDESTTM17 contains the T7 promoter, which is used to initiate the transcription of the T7-RNA- polymerase.
- the expression vector pDESTTM17 contains a Shine Dalgarno sequence in the 5 '-direction of the T7 promoter followed by a start codon (ATG) and by a so-called His tag. This His tag consists of six codons directly following one another, which each code the amino acid histidine and are located in the reading frame of the said start codon.
- a cDNA coding a starch-phosphorylating protein in pDESTTM17 is carried out in such a way that a franslational fusion occurs between the codons for the start codon, the His tag and the cDNA coding a starch- phosphorylating protein.
- a starch-phosphorylating protein is obtained, which contains additional amino acids containing the His tag on its N-terminus.
- other vectors which are suitable for expression in microorganisms, can also be used for the expression of a starch-phosphorylating protein.
- Expression vectors and associated expression strains are known to the person skilled in the art and are also available for purchase from the appropriate dealer in suitable combinations.
- Suitable expression strains are, for example, BL21 strains (Invitrogen Prod. No.: C6010-03, which chromosomally code a T7-RNA polymerase under the control of an IPTG-inducable promoter (lacZ)).
- Bacteria colonies resulting from the transformation can be investigated using methods known to the person skilled in the art to see whether they contain the required expression plasmid containing a cDNA coding the starch-phosphorylating protein. At the same time, expression clones are obtained.
- a main culture for the expression of a starch-phosphorylating protein is then produced.
- 1 liter Erlenmeyer flasks each containing 300 ml of TB medium, pre-heated to 30°C, and an antibiotic for selection on the presence of the expression plasmid are each seeded with 10 ml of an appropriate pre-culture and incubated at 30°C under agitation (250 rpm) until an optical density (measured at a wavelength of 600 nm; OD ⁇ oo) of ca. 0.8 is achieved.
- an expression plasmid in which the expression of the starch-phosphorylating protein is initiated by means of an inducable system (e.g. the expression vector pDESTTM17 in BL21 E. coli strains, inducable by means of IPTG), then on reaching an OD 6 oo of ca. 0.8, the inductor concerned (e.g. IPTG) is added to the main culture. After adding the inductor, the main culture is incubated at 30°C under agitation (250 rpm) until an OD ⁇ oo of ca. 1.8 is achieved. The main culture is then cooled for 30 minutes on ice before the cells of the main culture are separated from the culture medium by centrifugation (10 minutes at 4,000 xg and 4°C).
- an inducable system e.g. the expression vector pDESTTM17 in BL21 E. coli strains, inducable by means of IPTG
- the starch-phosphorylating protein expressed in E. coli cells is a fusion protein with a His tag
- purification can take place with the help of nickel ions, to which the His tag bonds with greater affinity.
- 25 ml of the filtrate obtained in Step d) is mixed with 1 ml Ni-agarose slurry (Qiagen, Prod. No.: 30210) and incubated for 1 hour on ice.
- the mixture of Ni-agarose slurry and filtrate is subsequently spread over a polystyrene column (Pierce, Prod. No.: 29920). The product, which runs through the column, is discarded.
- the column is next washed by adding 8 ml of lysis buffer, whereby the product, which runs through the column, is again discarded. Elution of the starch-phosphorylating protein then takes place by fractionated addition to the column of 1 ml E1 buffer twice, followed by 1 ml E2 buffer once and subsequently 1 ml E3 buffer five times.
- the product, which runs through the column which is produced by adding the individual fraction of the appropriate elution buffer (E1, E2, E3 buffer) to the column, is collected in separate fractions. Aliquots of these fractions are subsequently analysed by means of denaturing SDS acrylamide gel electrophoresis followed by Coomassie Blue colouring.
- the fractions, which contain the starch-phosphorylating protein in sufficient quantity and satisfactory purity, are purified and concentrated with the help of pressurised filtration at 4°C.
- Pressurised filtration can be carried out, for example, with the help of an Amicon cell (Amicon Ultrafiltration Cell, Model 8010, Prod. No.: 5121) using a Diaflo PM30 membrane (Millipore, Prod. No.: 13212) at 4°C.
- Amicon cell Amicon Ultrafiltration Cell, Model 8010, Prod. No.: 5121
- Diaflo PM30 membrane Microflo PM30 membrane
- Other methods known to the person skilled in the art can also be used for concentration however.
- Elution buffer E1 50 mM HEPES 300 mM NaCl 50 mM Imidazol pH 8.0 (adjust with NaOH)
- Elution buffer E2 50 mM HEPES 300 mM NaCl 75 mM Imidazol pH 8.0 (adjust with NaOH)
- Elution buffer E3 50 mM HEPES 300 mM NaCl 250 mM Imidazol pH 8.0 (adjust with NaOH) 5. Recombinant expression of an R1 protein
- R1 protein The recombinant expression of an R1 protein is described in the literature (Ritte et al., 2002, PNAS 99, 7166-7171; Mikkelsen et al., 2004, Biochemical Journal 377, 525- 532), but can also be carried out in accordance with the methods relating to the recombinant expression of a starch expression of a starch-phosphorylating protein described above under Item 3. General Methods.
- R1 protein The purification of an R1 protein is described in the literature (Ritte et al., 2002, PNAS 99, 7166-7171; Mikkelsen et al., 2004, Biochemical Journal 377, 525-532), but can also be carried out in accordance with the methods relating to the purification of a starch-phosphorylating protein described above under Item 4.
- Starch which does not contain starch phosphate (e.g., isolated from endosperm from maize or wheat plants respectively, or from leaves of Arabidopsis thaliana sex1-3 mutants with the help of the methods described above under Item 2, General Methods), is mixed with R1 buffer and with purified R1 protein (ca. 0.25 ⁇ g R1 protein per mg starch) in order to produce a starch content of 25 mg per ml.
- This reaction preparation is incubated overnight (ca. 15 h) at room temperature under agitation.
- R1 bonded to the starch present in the reaction preparation is removed on completion of the reaction by washing four times with ca. 800 ⁇ l 0.5 % SDS in each case.
- the SDS still present in the in vitro phosphorylated starch is removed by washing five times with 1 ml wash buffer in each case. All washing steps are carried out at room temperature for 10 to 15 minutes under agitation. Each washing step is followed by a centrifugation (2 min, 10,000 xg), in order to separate the starch granules from the respective SDS buffer.
- Wash buffer 50 mM HEPES/KOH, pH 7,2
- Ca. 50 mg P-starch or ca. 50 mg non-phosphorylated starch respectively are resuspended in separate preparations in ca. 800 ⁇ l protein extract in each case.
- the protein concentration of the protein extracts should be ca. 4 mg to 5 mg per ml in each case.
- the incubation of the P-starch or non-phosphorylated starch with protein extracts is carried out at room temperature for 15 minutes at 4°C under agitation. On completion of the incubation, the reaction preparations are centrifuged out using a Percoll cushion (4 ml) (15 minutes, 3500 rpm, 4°C).
- Each washing step is followed by a centrifugation (5 minutes, 8000 rpm, 4°C in a table centrifuge, Hettich EBA 12R) in order to separate the P-starch or non-phosphorylated starch respectively from the wash buffer.
- the P-starch protein complexes or non-phosphorylated starch protein complexes respectively obtained in Step a) are resuspended in ca. 150 ⁇ l SDS test buffer and incubated at room temperature for 15 minutes under agitation.
- the P-starch or non- phosphorylated starch respectively is subsequently removed from the dissolved proteins by centrifugation (1 minute, 13,000 rpm, room temperature, Eppendorf table centrifuge).
- the supernatant obtained after centrifugation is centrifuged again in order to remove any residues of P-starch or non-phosphorylated starch respectively (1 minute, 13,000 rpm, room temperature, Eppendorf table centrifuge) and removed.
- dissolved proteins, which bond to the P-starch or non-phosphorylated starch respectively are obtained.
- Percoll is dialysed overnight against a solution consisting of and 25 mM HEPES / KOH, pH 7.0
- General Methods relating to the bonding of proteins to P-starch or non-phosphorylated starch respectively are incubated for 5 minutes at 95°C in each case and subsequently separated with the help of denaturing polyacrylamide gel electrophoresis. In doing so, an equal volume is applied to the acrylamide gel in each case for the dissolved proteins obtained by bonding to P-starch and for those obtained by bonding to non-phosphorylated starch.
- the gel obtained on completion of electrophoresis is stained at least overnight with colloidal Comassie (Roth, Düsseldorf, Roti-Blue Rod. No.: A152.1) and subsequently de-stained in 30 % methanol, 5 % acetic acid, or in 25% methanol.
- Proteins which, after separation by means of acrylamide gel electrophoresis and subsequent visualisation by colouration (see above, Item 9. General Methods), exhibit an increased signal after bonding to P-starch in comparison with a corresponding signal after bonding to non-phosphorylated starch, have increased bonding activity with respect to P-starch in comparison with non-phosphorylated starch.
- Proteins, which have increased bonding activity with respect to P-starch in comparison with non- phosphorylated starch are excised from the acrylamide gel.
- Proteins identified in accordance with Step a) are digested with trypsin, and the peptides obtained are analysed by means of MALDI-TOF to determine the masses of the peptides obtained.
- Trypsin is a sequence-specific protease, i.e. trypsin only splits proteins at a specified position when the proteins concerned contain certain amino acid sequences. Trypsin always splits peptide bonds when the amino acids arginine and lysine follow one another starting from the N-terminus.
- Amino acid sequences which have the same peptide masses after theoretical and/or real trypsin digestion, are to be looked upon as being identical.
- the databases concerned contain both peptide masses of proteins, the function of which has already been shown, and also peptide masses of proteins, which up to now only exist hypothetically by derivation from amino acid sequences starting from nucleic acid sequences obtained in sequencing projects. The actual existence and the function of such hypothetical proteins has therefore seldom been shown and, if there is a function at all, then this is usually based only on predictions and not on an actual demonstration of the function.
- Bands containing proteins identified in accordance with Step a) are excised from the acrylamide gel; the excised acrylamide piece is reduced and destained by incubating for approximately half an hour at 37°C in ca. 1 ml 60% 50mM NH HCO 3 , 40% acetonitrile.
- the decolourising solution is subsequently removed and the remaining gel dried under vacuum (e.g. Speedvac).
- trypsin solution is added to digest the proteins contained in the gel piece concerned. Digestion takes place overnight at 37°C. After digestion, a little acetonitrile is added (until the acrylamide gel is stained white) and the preparation dried under vacuum (e.g. Speedvac).
- amino acid sequences which give the same masses after theoretical trypsin digestion.
- amino acid sequences can be identified, which code proteins, which preferably bond to phosphorylated alpha-1 ,4-glucans and/or which need P-alpha-1 ,4-glucans as a substrate.
- proteins to be investigated can be incubated with starch and radioactively labeled ATP.
- ca. 5 mg P-starch or ca. 5 mg non-phosphorylated starch is incubated with the protein to be investigated (0.01 ⁇ g to 5.0 ⁇ g per mg of added starch) in 500 ⁇ l phosphorylation buffer for 10 minutes to 30 minutes at room temperature under agitation.
- the reaction is subsequently stopped by the addition of SDS up to a concentration of 2% (weight/volume).
- the starch granules in the respective reaction mixture are centrifuged out (1 minute, 13,000 xg), and washed once with 900 ⁇ l of a 2 % SDS solution and four times each with 900 ⁇ l of a 2 mM ATP solution. Each washing step is carried out for 15 minutes at room temperature under agitation. After each washing step, the starch granules are separated from the respective wash buffer by centrifugation (1 min, 13,000 xg).
- reaction preparations which do not contain protein or contain inactivated protein, but which are otherwise treated in the same way as the reaction preparations described, should be processed as so-called controls.
- the starch granules obtained in accordance with Step a) can be investigated for the presence of radioactively labeled phosphate residues.
- the respective starch is resuspended in 100 ⁇ l of water and mixed with 3 ml of scintillation cocktail in each case (e.g. Ready SafeTM, BECKMANN Coulter) and subsequently analysed with the help of a scintillation counter (e.g. LS 6500 Multi-Purpose Scintillation Counter, BECKMANN COULTERTM).
- a protein is incubated in separate preparations, once with P-starch and once with non-phosphorylated starch, in accordance with the method described under a), then, by comparing the values for the presence of starch phosphate obtained according to Step b), it can be determined whether the protein concerned has incorporated more phosphate in P-starch in comparison with non-phosphorylated starch. In this way, proteins can also be identified, which can introduce phosphate into P-starch but not into non-phosphorylated starch. This means proteins can be identified, which already require phosphorylated starch as a substrate for a further phosphorylation reaction.
- Phosphorylation buffer 50 mM HEPES/KOH, pH 7.5 1 mM EDTA 6 mM MgC.2 0.01 to 0.5 mM ATP 0.2 to 2 ⁇ Ci per ml randomised 33 P-ATP (alternatively, ATP, which contains a phosphate residue, which is specifically labeled in the gamma position, can also be used)
- randomised ATP is to be understood to mean ATP which contains labeled phosphate residues in both the gamma position as well as in the beta position (Ritte et al. 2002, PNAS 99, 7166- 7171 ). Randomised ATP is also described in the scientific literature as beta/gamma ATP. A method for manufacturing randomised ATP is described in the following. i) Manufacture of randomised ATP The method described here for manufacturing randomised ATP with the help of enzyme catalysed reactions is based on the following reaction mechanisms:
- the reaction is subsequently stopped by incubating for 12 minutes at 95°C before the reaction preparation is purified up by means of centrifugal filtration using a Microcon YM 10 filter (Amicon, Millipore Prod. No. 42407) at 14,000 xg for at least 10 minutes.
- iii) Carrying out the 2nd reaction step Two ⁇ l pyruvate kinase (see below for manufacture of an appropriate solution) and 3 ⁇ l 50 mM PEP (phosphoenolpyruvate) are added to the filtrate obtained in Step ii). This reaction mixture is incubated for 45 minutes at 30°C before the reaction is stopped by incubating at 95°C for 12 minutes. The reaction mixture is subsequently centrifuged (2 minutes, 12,000 rpm in an Eppendorf table centrifuge). The supernatant containing randomised ATP obtained after centrifugation is removed, aliquoted and can be stored at -20°C.
- pyruvate kinase solution Fifteen ⁇ l pyruvate kinase (from rabbit muscle, Roche, Prod. No. 12815), 10 mg/ml, 200 units/mg at 25 °C) are centrifuged out, the supernatant discarded and the pellet absorbed in 27 ⁇ l pyruvate kinase buffer. iv) Buffers used Pyruvate kinase buffer: 50 mM HEPES/KOH pH 7,5 1 mM EDTA
- Randomising buffer 100 mM HEPES/KOH pH 7,5 1 mM EDTA 10 % Glycerol 5 mM MgCI 2 5 mM KCI 0.1 mM ATP 0.3 mM AMP
- proteins to be investigated can be incubated with radioactively labeled ATP.
- proteins to be investigated 50 ⁇ g to 100 ⁇ g
- 220 ⁇ l phosphorylation buffer see above, Item 12 d), General Methods
- the reaction is subsequently stopped by the addition of EDTA up to a final concentration of 0.11 M.
- Ca. 2 ⁇ g to 4 ⁇ g of protein are separated with the help of denaturing polyacrylamide gel electrophoresis (7.5% acrylamide gel).
- the gel obtained after polyacrylamide gel electrophoresis is subjected to autoradiography. Proteins, which exhibit a signal in the autoradiography, carry a radioactive phosphate residue.
- the neutralised supernatant is subsequently centrifuged over a 10 kDa filter. By measuring an aliquot of the filtrate obtained, the quantity of labeled phosphate in the filtrate is determined with the help of a scintillation counter, for example.
- b) Fractionation of the hydrolysis products and determination of the phosphorylated C-atom positions The neutralised filtrates of the hydrolysis products obtained by means of Step a) can be separated (when using radioactively labeled ATP about 3,000 cpm) with the help of high-pressure anion exchange chromatography (HPAE), for example.
- HPAE high-pressure anion exchange chromatography
- the neutralised filtrate can be diluted with H 2 O to obtain the volume required for HPAE.
- glucose-6-phosphate ca.
- the hydrolysis products eluted from the column are collected in individual fractions of 1 ml each.
- non-labeled glucose-3-phosphate (Ritte et al. 2002, PNAS 99, 7166-7171) and non-labeled glucose-6-phosphate (Sigma, Prod. No.: G7879) have been added to the injected samples of hydrolysis products as internal standards, the fractions, which contain either glucose-3-phosphate or glucose-6- phosphate, can be determined by means of pulsed amperometric detection.
- Immature embryos of maize plants of the line A188 were transformed in accordance with the methods described by Ishida et al. (1996, Nature Biotechnology 14, 745- 750).
- a) Determination of the C-6 phosphate content The positions C2, C3 and C6 of the glucose units can be phosphorylated in the starch.
- 50 mg of starch is hydrolised in 500 ⁇ l of 0.7 M HCI for 4 hours at 95°C. The preparations are subsequently centrifuged for 10 minutes at 15500 g, and the supematants are removed. Seven ⁇ l of the supematants are mixed with 193 ⁇ l of imidazole buffer (100 MM imidazole, pH 7.4; 5 mM MgCI 2 , 1 mM EDTA and 0.4 mM NAD).
- the measurement is carried out in the photometer at 340 nm.
- the enzyme reaction was started by adding 2 units of glucose-6-phosphate dehydrogenase (from Leuconostoc mesenteroides, Boehringer Mannheim).
- the change in absorption is directly proportional to the concentration of the G-6-P content of the starch.
- the determination of the total phosphate content takes place according to the method by Ames (Methods in Enzymology VIII, (1996), 115-118). Approximately 50 mg starch is mixed with 30 ⁇ l ethanolic magnesium nitrate solution and incinerated for three hours at 500°C in the muffle furnace. The residue is mixed with 300 ⁇ l 0.5 M hydrochloric acid and incubated 30 min at 60°C. An aliquot of 300 ⁇ l 0.5 M hydrochloric acid is subsequently refilled, added to a mixture of 100 ⁇ l of 10% ascorbic acid and 600 ⁇ l 0.42 % ammonium molybdate in 2 M sulfuric acid and incubated 20 minutes at 45°C.
- the glucans concerned can be separated by means of HPAE after total hydrolysis according to the method presented under General Methods 13.
- the amounts of glucose-6-phosphate and glucose-3-phosphate can be determined by the integration of the individual peak areas obtained after HPEA separation. By comparing the peak areas obtained for glucose-6-phosphate and glucose-3-phosphate in unknown samples with the peak areas that are obtained with known amounts of glucose-6-phosphate and glucose-3- phosphate after separation by means of HPEA, the amount of glucose-6-phosphate and glucose-3-phosphate can be determined in the sample to be investigated.
- Protein extracts were manufactured from approximately 7 g of leaves (fresh weight) of Arabidopsis thaliana (Ecotype Columbia, Col-O) in accordance with the method described under Item 1 , General Methods.
- Starch granules were isolated from approximately 20g (fresh weight) of leaves of a sex1-3 mutant of Arabidopsis thaliana in accordance with the method described under Item 2, General Methods.
- the proteins of Preparations A, B and C obtained in Step d) were separated by means of a 9% acrylamide gel under denaturing conditions (SDS) using the method described under Item 9, General Methods, and subsequently stained with Coomassie Blue.
- SDS denaturing conditions
- the stained gel is shown in Fig. 1. It can be clearly seen that a protein, which has a molecular weight of ca. 130 kDa in denaturing acrylamide gel referred to a protein standard marker (Trace M), preferably bonds to phosphorylated starch (Trace P) in comparison with non-phosphorylated starch (K).
- the band of the protein with a molecular weight of ca. 130 kDa identified in Step e) was excised from the gel.
- the protein was subsequently released from the acrylamide as described under General Methods 10 b), digested with trypsin and the peptide masses obtained determined by means of MALD-TOF-MS.
- the protein identified with this method was designated A.t.-OK1.
- Analysis of the amino acid sequence of the OK1 protein from Arabidopsis thaliana showed that this deviated from the sequence that was present in the database (NP 198009, NCBI).
- the amino acid sequence shown in SEQ ID No 2 codes the A.t.-OK1 protein.
- SEQ ID No 2 contains deviations when compared with the sequence in the database (Ace: NP 198009.1 , NCBI).
- the amino acids 519 to 523 (WRLCE) and 762 to 766 (VRARQ) contained in SEQ ID No 2 are not in the sequence, which is present in the database (ACC: NP 198009.1).
- the amino acid sequence shown in SEQ ID NO 2 also contains the additional amino acids 519 to 523 (WRLCE).
- the plasmid obtained is designated A.t.-OK1-pGEM, the cDNA sequence coding the A.t.-OK1 protein was determined and is shown under SEQ ID NO. 1.
- the sequence shown under SEQ ID NO 1 is not the same as the sequence, which is contained in the database. This has already been discussed for the amino acid sequence coding an A.t.-OKA.t.-OK1 protein.
- reaction preparation for the first strand synthesis contained the following substances:
- reaction preparation was incubated for 5 minutes at 75°C and subsequently cooled to room temperature.
- Step 868°C 10 minutes The reaction was first carried out in accordance with Steps 1 to 4. Ten repeats (cycles) were carried out between Step 4 and Step 2, the temperature of Step 3 being reduced by 0.67°C after each cycle. This was subsequently followed by the reaction in accordance with the conditions specified in Steps 5 to 8. Twenty-five repeats (cycles) were carried out between Step 7 and Step 5, the time of Step 7 being increased by 5 sec on each cycle. On completion of the reaction, the reaction was cooled to 4°C.
- the expression vector obtained was designated as A.t.-OKI -pDESTTM17.
- the cloning resulted in a franslational fusion of the cDNA coding the A.t-OK1 protein with the nucleotides present in the expression vector pDESTTM17.
- the nucleotides originating from the vector pDESTTM17, which are translationally fused with the cDNA coding the A.t.-OK1 protein, code 21 amino acids. These 21 amino acids include, amongst others, the start codon (ATG) and a so-called His tag (6 histidine residues directly after one another).
- A.t.-OK1 protein After translation of these translationally fused sequences, this results in an A.t.-OK1 protein, which has the additional 21 amino acids coded by nucleotides originating from the vector at its N-terminus.
- the recombinant A.t.-OKI protein resulting from this vector therefore contains 21 additional amino acids originating from the vector pDESTTM17 at its N-terminus. 4.
- the expression vector A.t.-OKI -pDESTTM17 obtained in accordance with Example 3 was transformed in the E. coli strain BL21 StarTM (DE3) (Invitrogen, Prod. No. C6010- 03). A description of this expression system has already been given above (see Item 3, General Methods). Bacteria clones, containing the vector A.t.-OK1-pDESTTM17, resulting from the transformation were next used to manufacture a preliminary culture, which was subsequently used for inoculating a main culture (see Item 3.c, General Methods). The preliminary culture and the main culture where each incubated at 30°C under agitation (250 rpm). When the main culture had reached an OD ⁇ oo of ca.
- the expression of the recombinant A.t.-OKI protein was induced by the addition of IPTG (isopropyl-beta-D-thiogalactopyranoside) until a final concentration of 1 mM was achieved.
- IPTG isopropyl-beta-D-thiogalactopyranoside
- the main culture was incubated at 30°C under agitation (250 rpm) until an OD ⁇ oo of ca. 1.8 was achieved.
- the main culture was then cooled for 30 minutes on ice before the cells of the main culture were separated from the culture medium by centrifugation (10 minutes at 4,000 xg and 4°C).
- the purification and concentration of the A.t.-OK1 protein from cells obtained in accordance with Example 4 was carried out using the method described under Item 4, General Methods.
- the starch-phosphorylating activity of the A.t.-OKI protein was demonstrated in accordance with the method described under Item 11, General Methods. In doing so, 5 ⁇ g of purified A.t.-OK1 protein manufactured in accordance with Example 5 was in each case incubated in a Preparation A with 5 mg of starch isolated from a sex1-3 mutant of Arabidopsis thaliana in accordance with Example 1 b) and in a Preparation B with 5 mg of starch obtained by enzymatic phosphorylation in accordance with Example 1 c), in each case in 500 ⁇ l of phosphorylation buffer containing 0.05 mM radioactively ( 33 P) labeled, randomised ATP (in total 1 ,130,00 cpm, ca.
- Preparation C was used as a control, which was the same as Preparation B, except that it contained no OK1 protein, but was otherwise treated in the same way as Preparations A and B. Two tests, which were independent from one another, were carried out for all preparations (A, B, C).
- the OK1 protein does not transfer phosphate groups from ATP to starch when non-phosphorylated starch is provided as a substrate, as the quota of phosphate groups transferred to non-phosphorylated starch by means of an OK1 protein, measured in cpm, does not exceed the quota of radioactively labeled phosphate groups in Preparation C (control). If, on the other hand, P-starch is provided as a substrate, the quota of radioactive phosphate groups, measured in cpm, which are transferred from ATP to P-starch, is significantly higher.
- the OK1 protein requires P-starch as a substrate and that non-phosphorylated starch is not accepted as a substrate by the OK1 protein. If the test described above is carried out with ATP specifically labeled in the gamma position with 33 P, then it is not possible to establish an incorporation of radioactively labeled phosphate in the starch. From this, it can be seen that the beta phosphate residue of ATP is transferred from an OK1 protein to starch. The results of such a test are shown in Fig. 6. 7. Demonstration of autophosphorylation
- reaction vessel 3 HCI was added to reaction vessel 3 up to a final concentration of 0.5 M
- NaOH was added to reaction vessel 4 up to a final concentration of 0.5 M.
- Reaction vessels 3 and 4 were each incubated for 25 minutes at 30°C. Subsequently, 50 ⁇ l in each case were removed from reaction vessels 1, 2, 3 and 4, mixed with SDS test buffer and separated by means of SDS acrylamide gel electrophoresis (7.5% acrylamide gel). For this purpose, samples from the reaction vessels were applied to each of two identical acrylamide gels. One of the gels obtained on completion of electrophoresis was subjected to autoradiography, while the second gel was stained with Coomassie Blue. In the gel stained with Coomassie Blue (see Fig.
- the bond between the phosphate residue and an amino acid of the OK1 protein will be relatively stable with respect to bases.
- the sample treated with acid contains approximately the same amounts of protein as the sample incubated at 30°C and at 95°C, and yet has a significantly lower signal in the autoradiography than the sample treated at 30°C, it must be assumed that acid incubation conditions also split the bond between a phosphate residue and an amino acid of the OK1 protein to a certain extent. An instability in the bond between a phosphate residue and an amino acid of the OK1 protein could therefore also be established in the tests carried out. At the same time, the instability with respect to acids is significantly less labeled than the instability with respect to heat.
- recombinantly expressed OK1 protein as described above, is incubated with ATP specifically labeled with 33P in the gamma position, then no autophosphorylation can be detected.
- Fig. 5 A) shows the amount of protein in the respective reaction preparation that can still be demonstrated by means of Western blot analysis after the appropriate incubation steps.
- Fig. 5 B) shows an autoradiography of protein from the individual reaction preparations. It can be seen that, when ATP specifically labeled in the gamma position is used, no autophosphorylation of the OK1 protein takes place, whereas, when randomised ATP is used, autophosphorylation can be demonstrated. This means that when an OK1 protein is autophosphorylated, the phosphate residue of the beta position of the ATP is covalently bound to an amino acid of the OK1 protein.
- Phosphorylated starch was manufactured in accordance with Item 7, General Methods. To do this, 5 mg of non-phosphorylated starch, isolated from leaves of a sex1-3 mutant of Arabidopsis thaliana were used in a Preparation A with 25 ⁇ g of purified A.t.-OK1 protein and, in a second Preparation B, 5 mg of in vitro phosphorylated starch originally isolated from leaves of a sex1-3 mutant of Arabidopsis thaliana were used with 5 ⁇ g of purified R1 protein. The reaction was carried out in 500 ⁇ l of phosphorylation buffer in each case, which, in each case contained 33 P labeled ATP (ca.
- the starch pellets obtained were resuspended 1 ml H 2 O in each case and 100 ⁇ l of each preparation were mixed after the addition of 3 ml of scintillation cocktail (Ready SafeTM, BECKMANN) and subsequently measured with the help of a scintillation counter (LS 6500 Multi- Purpose Scintillation Counter, BECKMANN COULTERTM).
- Preparations A, B and C obtained in accordance with Step a) were centrifuged again (5 minutes in an Eppendorf table centrifuge at 13,000 rpm), the pellets obtained resuspended in 90 ⁇ l 0.7 M HCI (Baker, for analysis) and subsequently incubated for 2 hours at 95°C.
- Preparations A, B and C were then centrifuged again (5 minutes in an Eppendorf table centrifuge at 13,000 rpm), and the supernatant transferred to a new reaction vessel.
- the neutralised hydrolysis products were placed on a 10 kDa Microcon filter, which had previously been rinsed twice with 200 ⁇ l H2O in each case, and centrifuged for ca. 25 minutes at 12,000 rpm in an Eppendorf table centrifuge. Ten ⁇ l were taken from the filtrate obtained (ca. 120 ⁇ l in each case) and, after the addition of 3 ml of scintillation cocktail (Ready SafeTM, BECKMANN), were measured with the help of a scintillation counter (LS 6500 Multi-Purpose Scintillation Counter, BECKMANN COULTERTM).
- Table 4 Measured amounts of radioactivity [cpm] in individual fractions of hydrolysis products obtained by hydrolysis of starch phosphorylated by means of an OK1 protein or R1 protein. The results are also shown graphically in Fig. 5.
- Phosphorylated wheat starch was manufactured in accordance with the method described under a). The amount of phosphate bound to starch amounted to 0.0048 mg of phosphate per mg of starch. That the randomised ATP was used was not considered for this determination, as also described under a).
- each of the preparations was carried out in two repeats in each case. A sample was stopped after 0, 10 and 30 minutes of incubation time for each reaction preparation, and the amount of phosphate incorporated under the respective reaction conditions was determined in accordance with the method described under General methods, Item 11. As a control, native wheat starch was incubated with R1 protein in the presence of ATP not radioactively labeled. Subsequently, the reaction mixture was added to randomised 33 P-ATP and buffer before the reaction was stopped.
- the specified measurement values were determined by subtracting the measurement values of the associated controls from the average value of two independent measurements.
- the amino acid sequence coding the O.s.-OKI protein shown under SEQ ID NO 4 has an identity of 57% with the amino acid sequence coding the A.t.-OKI protein shown under SEQ ID NO 2.
- the nucleic acid sequence coding the O.s.-OKI protein shown under SEQ ID NO 3 has an identity of 61% with the nucleic acid sequence coding the A.t.-OK1 protein shown under SEQ ID NO 1.
- the vector pMI50 contains a DNA fragment, which codes the complete OK1 protein from rice of the variety M202.
- the amplification of the DNA from rice was carried out in five sub-steps.
- pML123 (AAAACTCGAGGAGGATCAATGACGTCGCTGCGGCCCCTC) as a primer on RNA of immature rice seeds.
- the amplified DNA fragment was cloned in the vector pCR2.1 (Invitrogen catalogue number K2020-20).
- the obtained plasmid was designated as pML123.
- the amplified DNA fragment was cloned in the vector pCR2.1 (Invitrogen catalogue number K2020-20).
- the obtained plasmid was designated as pML119. 5.
- the part of the open reading frame from position 2777 to position 3621 was amplified with the help of polymerase chain reaction using the synthetic oligonucleotides Os_ok1-F3 (CATTTGGATCAATGGAGGATG) and Os_ok1-R2 (CTATGGCTGTGGCCTGCTTTGCA) as a primer on genomic DNA of rice.
- the amplified DNA fragment was cloned in the vector pCR2.1 (Invitrogen catalogue number K2020-20).
- the obtained plasmid was designated as pML122.
- the obtained plasmid was designated as pMI47.
- a 960 base pair long fragment containing the areas of vectors from pML120 and pML123 coding for OK1 was amplified by means of polymerase chain reaction.
- the amplified DNA fragment was cloned in the vector pCR2.1 (Invitrogen catalogue number K2020-20). The obtained plasmid was designated as pMI44.
- An 845 base pair long fragment of pML122 was reamplified for introducing an Xhol site after the stop codon with the primers Os_ok1-F3 (see above) and Os_ok1-R2Xho (AAAACTCGAGCTATGGCTGTGGCCTGCTTTGCA) and cloned in the vector pCR2.1 (Invitrogen catalogue number K2020-20).
- the obtained plasmid was designated as t pMI45.
- a 1671 base pair long fragment containing part of the open reading frame of OK1 was obtained from pML119 by digesting with the restriction enzymes Spel and Pst ⁇ . The fragment was cloned in pBluescript II SK+ (Genbank Ace: X52328). The obtained plasmid was designated as pMI46.
- a 1706 base pair long fragment containing part of the open reading frame of OK1 was excised with the restriction enzymes Spel and X ⁇ ol from pMI46 and cloned in the vector pMI45, which had been excised with the same restriction enzymes.
- the obtained plasmid was designated as pMI47.
- a 146 base pair long fragment containing part of the open reading frame of OK1 was excised with the restriction enzymes Afl ⁇ INot ⁇ from pMI43 and cloned in the vector pMI44, which had been excised with the same restriction enzymes.
- the obtained plasmid was designated as pMI49.
- a 1657 base pair long fragment containing part of the open reading frame of OK1 was excised with the restriction enzymes Not ⁇ and Na ⁇ from the vector pMI49 and cloned in the vector pMI47, which had been excised with the same restriction enzymes.
- the plasmid obtained was designated as pMI50 and contains the whole coding region of the OK1 protein identified in rice.
- A.t.-OK1 protein As an antigen, ca. 100 ⁇ g of purified A.t.-OK1 protein was separated by means of SDS gel electrophoresis, the protein bands containing the A.t.-OK1 protein excised and sent to the company EUROGENTEC S.A. (Belgium), which carried out the manufacture of the antibody under contract.
- the preimmune serums of rabbits were investigated to see whether they would already detect a protein from an A. t. total extract before immunisation with recombinant OK1.
- the preimmune serums of two rabbits detected no proteins in the range 100-150 kDa and were thus chosen for immunisation. 4 injections of 100 ⁇ g of protein (day 0, 14, 28, 56) were given to each rabbit.
- this plasmid contains a right and a left T-DNA border sequence. Between these T-DNA border sequences the plasmid contains a bar gene from Streptomyces hygroscopicus (White et al., 1990, NAR 18, 1062; EMBL Ace: X17220), which facilitates resistance against the herbicide glufosinate. The expression of the bar gene is initiated through the promoter of the actin gene from rice (McElroy et al., 1990, Plant Cell 2, 163.171).
- the 1 st intron of the actin gene from rice is inserted between the actin promoter and the sequence coding the bar protein (McElroy et al., 1990, Plant Cell 2, 163.171).
- the polyadenylation signal of the nopaline synthase gene from Agrobacterium tumefaciens follows after the sequence coding the bar protein (Depicker et al., 1982 J Mol. Appl. Gent. 1 , 561-573).
- the ubiquitin promoter from Zea mays was inserted into the plasmid pMZ12 between the left and right T-DNA border sequence (Christensen et al., 1992, Plant Mol.
- Premature embryos of maize plants were isolated ten days after pollination and transformed with the help of Agrobacterium tumefaciens containing the plasmid pHN3-146 as a cointegrate in accordance with the methods described by Ishida et al. (1996, Nature Biotechnology 14, 745-750). So-called TO plants resulting from this transformation were raised in the greenhouse.
- the plasmid plR96 was manufactured.
- the plasmid plR96 was obtained by cloning a synthetic piece of DNA consisting of the two oligonucleotides X1
- the plasmid obtained was excised with Sdal, the protruding 3'- ends smoothed with T4 DNA polymerase and a smoothed Hindlll / Sphl fragment from pBinAR (H ⁇ fgen and Willmitzer, 1990, Plant Science 66, 221-230) with a size of 197 base pairs, containing the termination signal of the octopine synthase gene from Agrobacterium tumefaciens, was inserted.
- the obtained plasmid was designated as plR96.
- pGSV71 is a derivative of the plasmid pGSV7, which derives from the intermediate vector pGSVI .
- pGSVI constitutes a derivative of pGSC1700, the construction of which has been described by Cornelissen and Vanderwiele (Nucleic Acid Research 17, (1989), 19-25).
- pGSVI was obtained from pGSC1700 by deletion of the carbenicillin resistance gene and deletion of the T-DNA sequences of the TL-DNA region of the plasmid pTiB6S3.
- pGSV7 contains the replication origin of the plasmid pBR322 (Bolivar et al., Gene 2, (1977), 95-113) as well as the replication origin of the Pseudomonas plasmid pVS1 (Itoh et al., Plasmid 11 , (1984), 206).
- pGSV7 also contains the selectable marker gene aadA, from the transposon Tn1331 from Klebsiella pneumoniae, which gives resistance against the antibiotics spectinomycin and streptomycin (Tolmasky, Plasmid 244 (3), (1990), 218-226; Tolmasky and Crosa, Plasmid 29(1), (1993), 31-40).
- the plasmid pGSV71 was obtained by cloning a chimeric bar gene between the border regions of pGSV7.
- the chimeric bar gene contains the promoter sequence of the cauliflower mosaic virus for initiating the transcription (Odell et al., Nature 313, (1985), 180), the bar gene from Streptomyces hygroscopicus (Thompson et al., 1987, EMBO J. 6, 2519-2523) and the 3'-untranslated area of the nopaline synthase gene of the T-DNA of pTiT37 for terminating the transcription and polyadenylation.
- the bar gene provides tolerance against the herbicide glufosinate ammonium.
- a 1986 base pair long fragment containing the promoter of the polyubiquitin gene from maize (EMBLK Ace: 94464, Christensen et al., 1992, Plant Mol. Biol.
- the obtained plasmid was designated as pSK-ubq.
- the plasmid A.t.-OKI -pGEM was excised with the restriction enzymes ⁇ sp120l, smoothed with T4-DNA-polymerase and excised with Sacl.
- the DNA fragment coding the OK1 protein from Arabidopsis thaliana was cloned in the plasmid pSK-ubq, which was excised with Smal and Sacl.
- the obtained plasmid was designated as pSK-ubq- ok1.
- a fragment which contained the ubiquitin promoter from maize and the total open reading frame for the A.t.-OK1 protein from Arabidopsis thaliana was isolated from the plasmid pSK-ubq-ok1. To do this, the plasmid was excised with the restriction enzyme Asp718 ⁇ , the ends refilled with T4 DNA polymerase and excised with Sdal. The fragment with a size of 5799 base pairs that was obtained was cloned in the plasmid plR96 excised with EcoRV and Psti. The obtained plasmid from this cloning was designated as pUbi-A.t.-OK1. '
- T1 plants that have an expression of the S.t.-R1 protein or of the A.t.-OKI protein
- seeds of the individual plants were harvested, and in each case ca. 30 seeds per plant were laid out again and cultivated in the greenhouse.
- Plants of this T1 generation were sprayed in the three-leaf stage with a solution containing 0.5% Basta ® .
- Only those groups of T1 plants for which ca. 25% of the 30 cultivated plants in each case died off after spraying with the Basta ® solution were followed further, because these plants are those for which the integration of the related T-DNA of the plasmid pHN3-146 or pUbi-A.t.-OK1 is present in a locus in the genome.
- Genomic DNA was isolated from leaf material from the ca.
- T1 plants that have an increased expression of an S.t.-R1 protein and that are homozygotic with respect to the integration of the T-DNA of the plasmid pHN3-146 according to the analysis described under g), and in which the integration exists at a locus in the genome of the plant, are crossed with T1 plants that have an increased expression of an A.t.-OK1 protein and are homozygotic with respect to the integration of the T-DNA of the plasmid pUbi-A.t.-OK1 , and in which the integration exists at a locus in the genome of the plant.
- the offspring of these crosses have both an increased expression of the S.t.-R1 protein as well as an increased expression of the A.t.-OKI protein.
- Starches were isolated from the grains of the related maize plants resulting from the crosses described under h).
- the starch from grains that have an increased expression of the S.t.-R1 protein and an increased expression of the A.t.-OK1 protein contains more phosphate covalently bound to the starch, than does starch isolated from untransformed wild type plants.
- Starch isolated from grains that have an increased expression of the S.t.-R1 protein and an increased expression of the A.t.-OK1 protein likewise contains more phosphate covalently bound to the starch than does starch isolated from plants that have only an increased expression of the S.t.-R1 protein or only an increased expression of the A.t.-OKI protein.
- the nos-terminator from Agrobacterium tumefaciens was amplified with the primers P9 (ACTTCTgCAgCggCCgCgATCgTTCAAACATTTggCAATAAAgTTTC) and P10 (TCTAAgCTTggCgCCgCTAgCAgATCTgATCTAgTAACATAgATgACACC) (25 cycles, 30 sec 94°C, 30 sec 58°C, 30 sec 72°C), digested with Hindlll and Pstl, and cloned in the plasmid pML4 excised with the same enzyme.
- the obtained plasmid was designated as pML4-nos.
- a 1986 base pair long fragment containing the promoter of the polyubiquitin gene from maize (Genbank Ace: 94464, Christensen et al., 1992, Plant Mol. Biol. 18: 675-689) and the first intron of the same gene shortened by digestion with Clal and religation was cloned.
- the obtained plasmid was designated as pML8.
- the total open reading frame of the OK1 from Arabidopsis thaliana was cloned in the plasmid pML8. For this purpose the corresponding fragment was excised with Bsp120/Notl from A.t.-OK10pGEM and ligated in sense orientation into the Notl site.
- a fragment for the transformation of wheat plants can be excised from the obtained vector pML8-A.t.-OK1 , which contains the promoter of the polyubiquitin gene from maize, the total open reading frame of OK1 from Arabidopsis thaliana and the /los-terminator from Agrobacterium tumefaciens.
- a plasmid was manufactured in which the DNA fragment, which coded for the complete R1 protein from potato, lies between two detection sites for the restriction enzyme Pac ⁇ .
- the Multiple Cloning Site from the plasmid pBluescript II SK+ was amplified with the help of the polymerase chain reaction and both oligonucleotides MCS1-1
- a Not ⁇ fragment was cloned in the vector pSK-Pac, which was obtained from the clone pRL2 (WO 9711188).
- the Not ⁇ fragment contains the total open reading frame for the R1 protein from potato.
- the obtained plasmid was designated as plR1.
- a Pac ⁇ fragment was cloned from plR1 in sense orientation to the promoter, which contains the total open reading frame coding for the R1 protein from potato.
- the obtained plasmid was designated as pML82.
- Grains of the respective plants were harvested for the analysis of the starch from various TA-R1-OK1 lines, and the C-6 phosphate content and the C-3 phosphate content of the isolated starch was analysed. It was possible to identify some plants for which the content of C-6 phosphate plus C-3 phosphate was clearly higher in comparison with the content of C-6 phosphate plus C-3 phosphate from starch isolated from grains of the lines TA-R1 or 40A-11-8.
- the plasmid plR94 was obtained by amplifying the promoter of the globulin gene from rice by means of a polymerase chain reaction (30 x 20 sec 94 °C, 20 sec 62 °C, 1 min 68 °C, 4 mM Mg2SO4) with the primers glb1-F2 (AAAACAATTGGCGCCTGGAGGGAGGAGA) and glb1-R1
- the plasmid plR115 was obtained by cloning a synthetic piece of DNA consisting of the two oligonucleotides X1
- the plasmid plR115 obtained was excised with S ⁇ fal, the protruding 3'-ends smoothed with T4 DNA polymerase and a Hindlll I Sphl fragment from pBinAR (H ⁇ fgen and Willmitzer, 1990, Plant Science 66, 221-230) with a size of 197 base pairs, smoothed by means of T4 DNA polymerase and containing the termination signal of the octopine synthase gene from Agrobacterium tumefaciens, was inserted.
- the obtained plasmid was designated as plR96.
- the plasmid plR103 was obtained in which a DNA fragment cloned from plR94 with a length of 986 base pairs containing the promoter of the globulin gene from rice was cloned in the plasmid plR96.
- pGSV71 is a derivative of the plasmid pGSV7, which derives from the intermediate vector pGSVI .
- pGSVI constitutes a derivative of pGSC1700, the construction of which has been described by Cornelissen and Vanderwiele (Nucleic Acid Research 17, (1989), 19-25).
- pGSVI was obtained from pGSC1700 by deletion of the carbenicillin resistance gene and deletion of the T-DNA sequences of the TL-DNA region of the plasmid pTiB6S3.
- pGSV7 contains the replication origin of the plasmid pBR322 (Bolivar et al., Gene 2, (1977), 95-113) as well as the replication origin of the Pseudomonas plasmid pVS1 (Itoh et al., Plasmid 11 , (1984), 206).
- pGSV7 also contains the selectable marker gene aadA, from the transposon Tn1331 from Klebsiella pneumoniae, which gives resistance against the antibiotics spectinomycin and streptomycin (Tolmasky, Plasmid 24 (3), (1990), 218-226; Tolmasky and Crosa, Plasmid 29(1), (1993), 31-40).
- the plasmid pGSV71 was obtained by cloning a chimeric bar gene between the border regions of pGSV7.
- the chimeric bar gene contains the promoter sequence of the cauliflower mosaic virus for initiating the transcription (Odell et al., Nature 313, (1985), 180), the bar gene from Streptomyces hygroscopicus (Thompson et al., Embo J. 6, (1987), 2519-2523) and the 3'-untranslated area of the nopaline synthase gene of the T-DNA of pTiT37 for terminating the transcription and polyadenylation.
- the bar gene provides tolerance against the herbicide glufosinate ammonium.
- the plasmid A.t.-OK1-pGEM was excised with the restriction enzymes Bsp120l, the ends smoothed with T4-DNA polymerase and excised with Sacl.
- the DNA fragment coding the OK1 protein from Arabidopsis thaliana was cloned in the vector plR103 excised with - ⁇ c/13611 and Xho ⁇ .
- the obtained plasmid was designated as pGlo-A.t.-OK1.
- Rice plants (variety M202) were transformed by means of Agrobacterium (containing the plasmid pGlo-A.t.-OK1 ) using the method described by Hiei et al. (1994, Plant Journal 6(2), 271-282).
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Molecular Biology (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Nutrition Science (AREA)
- Microbiology (AREA)
- Medicinal Chemistry (AREA)
- Cell Biology (AREA)
- Plant Pathology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Inorganic Chemistry (AREA)
- Polymers & Plastics (AREA)
- Materials Engineering (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Polysaccharides And Polysaccharide Derivatives (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Cereal-Derived Products (AREA)
- Grain Derivatives (AREA)
- Jellies, Jams, And Syrups (AREA)
Abstract
Description
Claims
Priority Applications (11)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU2005229364A AU2005229364B2 (en) | 2004-03-05 | 2005-03-04 | Plants with increased activity of multiple starch phosphorylating enzymes |
CA2558747A CA2558747C (en) | 2004-03-05 | 2005-03-04 | Plants with increased activity of multiple starch phosphorylating enzymes |
AT05715852T ATE455178T1 (en) | 2004-03-05 | 2005-03-04 | PLANTS WITH INCREASED ACTIVITY OF MULTIPLE STRENGTH PHOSPHORYLATING ENZYMES |
DE602005018904T DE602005018904D1 (en) | 2004-03-05 | 2005-03-04 | PLANTS WITH INCREASED ACTIVITY MULTIPLE PHOSPHORYLIERENZ ENZYME |
DK05715852.9T DK1725667T3 (en) | 2004-03-05 | 2005-03-04 | Plants with increased activity of multiple starch phosphorylating enzymes |
US10/591,432 US7932436B2 (en) | 2004-03-05 | 2005-03-04 | Plants with increased activity of multiple starch phosphorylating enzymes |
PL05715852T PL1725667T3 (en) | 2004-03-05 | 2005-03-04 | Plants with increased activity of multiple starch phosphorylating enzymes |
JP2007501245A JP5623002B2 (en) | 2004-03-05 | 2005-03-04 | Plants with increased activity of multiple starch phosphatases |
CN2005800070770A CN1930295B (en) | 2004-03-05 | 2005-03-04 | Plants with increased activity of multiple starch phosphorylating enzymes |
SI200530956T SI1725667T1 (en) | 2004-03-05 | 2005-03-04 | Plants with increased activity of multiple starch phosphorylating enzymes |
EP05715852A EP1725667B1 (en) | 2004-03-05 | 2005-03-04 | Plants with increased activity of multiple starch phosphorylating enzymes |
Applications Claiming Priority (8)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US55002104P | 2004-03-05 | 2004-03-05 | |
US60/550,021 | 2004-03-05 | ||
EP04090089 | 2004-03-05 | ||
EP04090089.6 | 2004-03-05 | ||
EP04090121.7 | 2004-03-29 | ||
EP04090121 | 2004-03-29 | ||
EP04090284.3 | 2004-07-21 | ||
EP04090284 | 2004-07-21 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2005095619A1 true WO2005095619A1 (en) | 2005-10-13 |
Family
ID=36607227
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/EP2005/002457 WO2005095619A1 (en) | 2004-03-05 | 2005-03-04 | Plants with increased activity of multiple starch phosphorylating enzymes |
Country Status (15)
Country | Link |
---|---|
US (1) | US7932436B2 (en) |
EP (1) | EP1725667B1 (en) |
JP (2) | JP5623002B2 (en) |
CN (1) | CN1930295B (en) |
AR (1) | AR048024A1 (en) |
AT (1) | ATE455178T1 (en) |
AU (1) | AU2005229364B2 (en) |
CA (1) | CA2558747C (en) |
DE (1) | DE602005018904D1 (en) |
DK (1) | DK1725667T3 (en) |
ES (1) | ES2338242T3 (en) |
PL (1) | PL1725667T3 (en) |
PT (1) | PT1725667E (en) |
SI (1) | SI1725667T1 (en) |
WO (1) | WO2005095619A1 (en) |
Cited By (179)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP1629102A2 (en) * | 2003-05-22 | 2006-03-01 | Syngenta Participations AG | Modified starch, uses, methods for production thereof |
WO2007134877A1 (en) * | 2006-05-24 | 2007-11-29 | Bayer Cropscience Ag | Use of modified wheat flour for reducing baking losses |
EP2039771A2 (en) | 2009-01-06 | 2009-03-25 | Bayer CropScience AG | Method for improved utilization of the production potential of transgenic plants |
EP2039770A2 (en) | 2009-01-06 | 2009-03-25 | Bayer CropScience AG | Method for improved utilization of the production potential of transgenic plants |
EP2039772A2 (en) | 2009-01-06 | 2009-03-25 | Bayer CropScience AG | Method for improved utilization of the production potential of transgenic plants introduction |
EP2072506A1 (en) | 2007-12-21 | 2009-06-24 | Bayer CropScience AG | Thiazolyloxyphenylamidine or thiadiazolyloxyphenylamidine und its use as fungicide |
EP2090168A1 (en) | 2008-02-12 | 2009-08-19 | Bayer CropScience AG | Method for improving plant growth |
EP2168434A1 (en) | 2008-08-02 | 2010-03-31 | Bayer CropScience AG | Use of azols to increase resistance of plants of parts of plants to abiotic stress |
EP2198709A1 (en) | 2008-12-19 | 2010-06-23 | Bayer CropScience AG | Method for treating resistant animal pests |
EP2201838A1 (en) | 2008-12-05 | 2010-06-30 | Bayer CropScience AG | Active ingredient-beneficial organism combinations with insecticide and acaricide properties |
EP2204094A1 (en) | 2008-12-29 | 2010-07-07 | Bayer CropScience AG | Method for improved utilization of the production potential of transgenic plants Introduction |
WO2010083955A2 (en) | 2009-01-23 | 2010-07-29 | Bayer Cropscience Aktiengesellschaft | Use of enaminocarboxylic compounds for fighting viruses transmitted by insects |
WO2010086311A1 (en) | 2009-01-28 | 2010-08-05 | Bayer Cropscience Ag | Fungicide n-cycloalkyl-n-bicyclicmethylene-carboxamide derivatives |
WO2010086095A1 (en) | 2009-01-29 | 2010-08-05 | Bayer Cropscience Ag | Method for improved utilization of the production potential of transgenic plants introduction |
EP2218717A1 (en) | 2009-02-17 | 2010-08-18 | Bayer CropScience AG | Fungicidal N-((HET)Arylethyl)thiocarboxamide derivatives |
WO2010094728A1 (en) | 2009-02-19 | 2010-08-26 | Bayer Cropscience Ag | Pesticide composition comprising a tetrazolyloxime derivative and a fungicide or an insecticide active substance |
WO2010094666A2 (en) | 2009-02-17 | 2010-08-26 | Bayer Cropscience Ag | Fungicidal n-(phenylcycloalkyl)carboxamide, n-(benzylcycloalkyl)carboxamide and thiocarboxamide derivatives |
EP2223602A1 (en) | 2009-02-23 | 2010-09-01 | Bayer CropScience AG | Method for improved utilisation of the production potential of genetically modified plants |
EP2232995A1 (en) | 2009-03-25 | 2010-09-29 | Bayer CropScience AG | Method for improved utilisation of the production potential of transgenic plants |
EP2239331A1 (en) | 2009-04-07 | 2010-10-13 | Bayer CropScience AG | Method for improved utilization of the production potential of transgenic plants |
EP2251331A1 (en) | 2009-05-15 | 2010-11-17 | Bayer CropScience AG | Fungicide pyrazole carboxamides derivatives |
EP2255626A1 (en) | 2009-05-27 | 2010-12-01 | Bayer CropScience AG | Use of succinate dehydrogenase inhibitors to increase resistance of plants or parts of plants to abiotic stress |
WO2011006603A2 (en) | 2009-07-16 | 2011-01-20 | Bayer Cropscience Ag | Synergistic active substance combinations containing phenyl triazoles |
WO2011015524A2 (en) | 2009-08-03 | 2011-02-10 | Bayer Cropscience Ag | Fungicide heterocycles derivatives |
EP2292094A1 (en) | 2009-09-02 | 2011-03-09 | Bayer CropScience AG | Active compound combinations |
WO2011080254A2 (en) | 2009-12-28 | 2011-07-07 | Bayer Cropscience Ag | Fungicide hydroximoyl-heterocycles derivatives |
WO2011080256A1 (en) | 2009-12-28 | 2011-07-07 | Bayer Cropscience Ag | Fungicide hydroximoyl-tetrazole derivatives |
WO2011080255A2 (en) | 2009-12-28 | 2011-07-07 | Bayer Cropscience Ag | Fungicide hydroximoyl-tetrazole derivatives |
EP2343280A1 (en) | 2009-12-10 | 2011-07-13 | Bayer CropScience AG | Fungicide quinoline derivatives |
WO2011089071A2 (en) | 2010-01-22 | 2011-07-28 | Bayer Cropscience Ag | Acaricide and/or insecticide active substance combinations |
WO2011107504A1 (en) | 2010-03-04 | 2011-09-09 | Bayer Cropscience Ag | Fluoroalkyl-substituted 2-amidobenzimidazoles and the use thereof for boosting stress tolerance in plants |
EP2374791A1 (en) | 2008-08-14 | 2011-10-12 | Bayer CropScience Aktiengesellschaft | Insecticidal 4-phenyl-1H pyrazoles |
WO2011124554A2 (en) | 2010-04-06 | 2011-10-13 | Bayer Cropscience Ag | Use of 4-phenylbutyric acid and/or the salts thereof for enhancing the stress tolerance of plants |
WO2011124553A2 (en) | 2010-04-09 | 2011-10-13 | Bayer Cropscience Ag | Use of derivatives of the (1-cyanocyclopropyl)phenylphosphinic acid, the esters thereof and/or the salts thereof for enhancing the tolerance of plants to abiotic stress |
WO2011134913A1 (en) | 2010-04-28 | 2011-11-03 | Bayer Cropscience Ag | Fungicide hydroximoyl-heterocycles derivatives |
WO2011134912A1 (en) | 2010-04-28 | 2011-11-03 | Bayer Cropscience Ag | Fungicide hydroximoyl-heterocycles derivatives |
WO2011134911A2 (en) | 2010-04-28 | 2011-11-03 | Bayer Cropscience Ag | Fungicide hydroximoyl-tetrazole derivatives |
WO2011151368A2 (en) | 2010-06-03 | 2011-12-08 | Bayer Cropscience Ag | Fungicide n-[(trisubstitutedsilyl)methyl]-carboxamide derivatives |
WO2011151370A1 (en) | 2010-06-03 | 2011-12-08 | Bayer Cropscience Ag | N-[(het)arylalkyl)] pyrazole (thio)carboxamides and their heterosubstituted analogues |
WO2011151369A1 (en) | 2010-06-03 | 2011-12-08 | Bayer Cropscience Ag | N-[(het)arylethyl)] pyrazole(thio)carboxamides and their heterosubstituted analogues |
WO2011154159A1 (en) | 2010-06-09 | 2011-12-15 | Bayer Bioscience N.V. | Methods and means to modify a plant genome at a nucleotide sequence commonly used in plant genome engineering |
WO2011154158A1 (en) | 2010-06-09 | 2011-12-15 | Bayer Bioscience N.V. | Methods and means to modify a plant genome at a nucleotide sequence commonly used in plant genome engineering |
US8080688B2 (en) | 2007-03-12 | 2011-12-20 | Bayer Cropscience Ag | 3, 4-disubstituted phenoxyphenylamidines and use thereof as fungicides |
WO2012010579A2 (en) | 2010-07-20 | 2012-01-26 | Bayer Cropscience Ag | Benzocycloalkenes as antifungal agents |
WO2012028578A1 (en) | 2010-09-03 | 2012-03-08 | Bayer Cropscience Ag | Substituted fused pyrimidinones and dihydropyrimidinones |
WO2012038480A2 (en) | 2010-09-22 | 2012-03-29 | Bayer Cropscience Ag | Use of biological or chemical control agents for controlling insects and nematodes in resistant crops |
WO2012045798A1 (en) | 2010-10-07 | 2012-04-12 | Bayer Cropscience Ag | Fungicide composition comprising a tetrazolyloxime derivative and a thiazolylpiperidine derivative |
WO2012052490A1 (en) | 2010-10-21 | 2012-04-26 | Bayer Cropscience Ag | N-benzyl heterocyclic carboxamides |
WO2012052489A1 (en) | 2010-10-21 | 2012-04-26 | Bayer Cropscience Ag | 1-(heterocyclic carbonyl) piperidines |
WO2012059497A1 (en) | 2010-11-02 | 2012-05-10 | Bayer Cropscience Ag | N-hetarylmethyl pyrazolylcarboxamides |
WO2012065947A1 (en) | 2010-11-15 | 2012-05-24 | Bayer Cropscience Ag | 5-halogenopyrazolecarboxamides |
WO2012065944A1 (en) | 2010-11-15 | 2012-05-24 | Bayer Cropscience Ag | N-aryl pyrazole(thio)carboxamides |
WO2012065945A1 (en) | 2010-11-15 | 2012-05-24 | Bayer Cropscience Ag | 5-halogenopyrazole(thio)carboxamides |
EP2460407A1 (en) | 2010-12-01 | 2012-06-06 | Bayer CropScience AG | Agent combinations comprising pyridylethyl benzamides and other agents |
EP2460406A1 (en) | 2010-12-01 | 2012-06-06 | Bayer CropScience AG | Use of fluopyram for controlling nematodes in nematode resistant crops |
WO2012072660A1 (en) | 2010-12-01 | 2012-06-07 | Bayer Cropscience Ag | Use of fluopyram for controlling nematodes in crops and for increasing yield |
WO2012089757A1 (en) | 2010-12-29 | 2012-07-05 | Bayer Cropscience Ag | Fungicide hydroximoyl-tetrazole derivatives |
WO2012089722A2 (en) | 2010-12-30 | 2012-07-05 | Bayer Cropscience Ag | Use of open-chain carboxylic acids, carbonic esters, carboxamides and carbonitriles of aryl, heteroaryl and benzylsulfonamide or the salts thereof for improving the stress tolerance in plants |
EP2474542A1 (en) | 2010-12-29 | 2012-07-11 | Bayer CropScience AG | Fungicide hydroximoyl-tetrazole derivatives |
US8252975B2 (en) | 2007-01-26 | 2012-08-28 | Bayer Cropscience Ag | Genetically modified plants which synthesize a low amylose starch with increased swelling power |
EP2494867A1 (en) | 2011-03-01 | 2012-09-05 | Bayer CropScience AG | Halogen-substituted compounds in combination with fungicides |
WO2012120105A1 (en) | 2011-03-10 | 2012-09-13 | Bayer Cropscience Ag | Use of lipochito-oligosaccharide compounds for safeguarding seed safety of treated seeds |
WO2012123434A1 (en) | 2011-03-14 | 2012-09-20 | Bayer Cropscience Ag | Fungicide hydroximoyl-tetrazole derivatives |
WO2012136581A1 (en) | 2011-04-08 | 2012-10-11 | Bayer Cropscience Ag | Fungicide hydroximoyl-tetrazole derivatives |
EP2511255A1 (en) | 2011-04-15 | 2012-10-17 | Bayer CropScience AG | Substituted prop-2-in-1-ol and prop-2-en-1-ol derivatives |
WO2012139892A1 (en) | 2011-04-15 | 2012-10-18 | Bayer Cropscience Ag | Substituted 5-(bicyclo[4.1.0]hept-3-en-2-yl)-penta-2,4-dienes and 5-(bicyclo[4.1.0]hept-3-en-2-yl)-pent-2-ene-4-ines as active agents against abiotic stress in plants |
WO2012139890A1 (en) | 2011-04-15 | 2012-10-18 | Bayer Cropscience Ag | Substituted 5-(cyclohex-2-en-1-yl)-penta-2,4-dienes and 5-(cyclohex-2-en-1-yl)-pent-2-en-4-ines as active agents against abiotic stress in plants |
WO2012139891A1 (en) | 2011-04-15 | 2012-10-18 | Bayer Cropscience Ag | Substituted vinyl and alkinyl cyclohexenols as active agents against abiotic stress in plants |
US8299302B2 (en) | 2007-03-12 | 2012-10-30 | Bayer Cropscience Ag | 4-Cycloalkyl or 4-substituted phenoxyphenylamidines and use thereof as fungicides |
WO2012168124A1 (en) | 2011-06-06 | 2012-12-13 | Bayer Cropscience Nv | Methods and means to modify a plant genome at a preselected site |
US8334237B2 (en) | 2007-03-12 | 2012-12-18 | Bayer Cropscience Ag | Substituted phenylamidines and the use thereof as fungicides |
WO2013004652A1 (en) | 2011-07-04 | 2013-01-10 | Bayer Intellectual Property Gmbh | Use of substituted isoquinolinones, isoquinolindiones, isoquinolintriones and dihydroisoquinolinones or in each case salts thereof as active agents against abiotic stress in plants |
WO2013020985A1 (en) | 2011-08-10 | 2013-02-14 | Bayer Intellectual Property Gmbh | Active compound combinations comprising specific tetramic acid derivatives |
EP2561759A1 (en) | 2011-08-26 | 2013-02-27 | Bayer Cropscience AG | Fluoroalkyl-substituted 2-amidobenzimidazoles and their effect on plant growth |
WO2013026836A1 (en) | 2011-08-22 | 2013-02-28 | Bayer Intellectual Property Gmbh | Fungicide hydroximoyl-tetrazole derivatives |
WO2013026740A2 (en) | 2011-08-22 | 2013-02-28 | Bayer Cropscience Nv | Methods and means to modify a plant genome |
US8394991B2 (en) | 2007-03-12 | 2013-03-12 | Bayer Cropscience Ag | Phenoxy substituted phenylamidine derivatives and their use as fungicides |
WO2013034621A1 (en) | 2011-09-09 | 2013-03-14 | Bayer Intellectual Property Gmbh | Acyl-homoserine lactone derivatives for improving plant yield |
WO2013037955A1 (en) | 2011-09-16 | 2013-03-21 | Bayer Intellectual Property Gmbh | Use of acylsulfonamides for improving plant yield |
WO2013037717A1 (en) | 2011-09-12 | 2013-03-21 | Bayer Intellectual Property Gmbh | Fungicidal 4-substituted-3-{phenyl[(heterocyclylmethoxy)imino]methyl}-1,2,4-oxadizol-5(4h)-one derivatives |
WO2013037956A1 (en) | 2011-09-16 | 2013-03-21 | Bayer Intellectual Property Gmbh | Use of 5-phenyl- or 5-benzyl-2 isoxazoline-3 carboxylates for improving plant yield |
WO2013037958A1 (en) | 2011-09-16 | 2013-03-21 | Bayer Intellectual Property Gmbh | Use of phenylpyrazolin-3-carboxylates for improving plant yield |
WO2013041602A1 (en) | 2011-09-23 | 2013-03-28 | Bayer Intellectual Property Gmbh | Use of 4-substituted 1-phenyl-pyrazole-3-carboxylic-acid derivatives as agents against abiotic plant stress |
WO2013050324A1 (en) | 2011-10-06 | 2013-04-11 | Bayer Intellectual Property Gmbh | Combination, containing 4-phenylbutyric acid (4-pba) or a salt thereof (component (a)) and one or more selected additional agronomically active compounds (component(s) (b)), that reduces abiotic plant stress |
WO2013050410A1 (en) | 2011-10-04 | 2013-04-11 | Bayer Intellectual Property Gmbh | RNAi FOR THE CONTROL OF FUNGI AND OOMYCETES BY INHIBITING SACCHAROPINE DEHYDROGENASE GENE |
WO2013075817A1 (en) | 2011-11-21 | 2013-05-30 | Bayer Intellectual Property Gmbh | Fungicide n-[(trisubstitutedsilyl)methyl]-carboxamide derivatives |
US8455480B2 (en) | 2007-09-26 | 2013-06-04 | Bayer Cropscience Ag | Active agent combinations having insecticidal and acaricidal properties |
WO2013079566A2 (en) | 2011-11-30 | 2013-06-06 | Bayer Intellectual Property Gmbh | Fungicidal n-bicycloalkyl and n-tricycloalkyl (thio)carboxamide derivatives |
WO2013092519A1 (en) | 2011-12-19 | 2013-06-27 | Bayer Cropscience Ag | Use of anthranilic acid diamide derivatives for pest control in transgenic crops |
WO2013098146A1 (en) | 2011-12-29 | 2013-07-04 | Bayer Intellectual Property Gmbh | Fungicidal 3-[(1,3-thiazol-4-ylmethoxyimino)(phenyl)methyl]-2-substituted-1,2,4-oxadiazol-5(2h)-one derivatives |
WO2013098147A1 (en) | 2011-12-29 | 2013-07-04 | Bayer Intellectual Property Gmbh | Fungicidal 3-[(pyridin-2-ylmethoxyimino)(phenyl)methyl]-2-substituted-1,2,4-oxadiazol-5(2h)-one derivatives |
US8487118B2 (en) | 2009-01-19 | 2013-07-16 | Bayer Cropscience Ag | Cyclic diones and their use as insecticides, acaricides and/or fungicides |
US8519003B2 (en) | 2007-03-12 | 2013-08-27 | Bayer Cropscience Ag | Phenoxyphenylamidines as fungicides |
WO2013124275A1 (en) | 2012-02-22 | 2013-08-29 | Bayer Cropscience Ag | Use of succinate dehydrogenase inhibitors (sdhis) for controlling wood diseases in grape. |
WO2013127704A1 (en) | 2012-02-27 | 2013-09-06 | Bayer Intellectual Property Gmbh | Active compound combinations containing a thiazoylisoxazoline and a fungicide |
WO2013139949A1 (en) | 2012-03-23 | 2013-09-26 | Bayer Intellectual Property Gmbh | Compositions comprising a strigolactame compound for enhanced plant growth and yield |
WO2013153143A1 (en) | 2012-04-12 | 2013-10-17 | Bayer Cropscience Ag | N-acyl- 2 - (cyclo) alkylpyrrolidines and piperidines useful as fungicides |
WO2013156559A1 (en) | 2012-04-20 | 2013-10-24 | Bayer Cropscience Ag | N-cycloalkyl-n-[(heterocyclylphenyl)methylene]-(thio)carboxamide derivatives |
WO2013156560A1 (en) | 2012-04-20 | 2013-10-24 | Bayer Cropscience Ag | N-cycloalkyl-n-[(trisubstitutedsilylphenyl)methylene]-(thio)carboxamide derivatives |
WO2013160230A1 (en) | 2012-04-23 | 2013-10-31 | Bayer Cropscience Nv | Targeted genome engineering in plants |
EP2662363A1 (en) | 2012-05-09 | 2013-11-13 | Bayer CropScience AG | 5-Halogenopyrazole biphenylcarboxamides |
EP2662364A1 (en) | 2012-05-09 | 2013-11-13 | Bayer CropScience AG | Pyrazole tetrahydronaphthyl carboxamides |
EP2662361A1 (en) | 2012-05-09 | 2013-11-13 | Bayer CropScience AG | Pyrazol indanyl carboxamides |
EP2662362A1 (en) | 2012-05-09 | 2013-11-13 | Bayer CropScience AG | Pyrazole indanyl carboxamides |
EP2662360A1 (en) | 2012-05-09 | 2013-11-13 | Bayer CropScience AG | 5-Halogenopyrazole indanyl carboxamides |
EP2662370A1 (en) | 2012-05-09 | 2013-11-13 | Bayer CropScience AG | 5-Halogenopyrazole benzofuranyl carboxamides |
WO2013167544A1 (en) | 2012-05-09 | 2013-11-14 | Bayer Cropscience Ag | 5-halogenopyrazole indanyl carboxamides |
WO2013167545A1 (en) | 2012-05-09 | 2013-11-14 | Bayer Cropscience Ag | Pyrazole indanyl carboxamides |
WO2013174836A1 (en) | 2012-05-22 | 2013-11-28 | Bayer Cropscience Ag | Active compounds combinations comprising a lipo-chitooligosaccharide derivative and a nematicide, insecticidal or fungicidal compound |
WO2014009322A1 (en) | 2012-07-11 | 2014-01-16 | Bayer Cropscience Ag | Use of fungicidal combinations for increasing the tolerance of a plant towards abiotic stress |
WO2014037340A1 (en) | 2012-09-05 | 2014-03-13 | Bayer Cropscience Ag | Use of substituted 2-amidobenzimidazoles, 2-amidobenzoxazoles and 2-amidobenzothiazoles or salts thereof as active substances against abiotic plant stress |
WO2014060520A1 (en) | 2012-10-19 | 2014-04-24 | Bayer Cropscience Ag | Method for treating plants against fungi resistant to fungicides using carboxamide or thiocarboxamide derivatives |
WO2014060502A1 (en) | 2012-10-19 | 2014-04-24 | Bayer Cropscience Ag | Active compound combinations comprising carboxamide derivatives |
WO2014060518A1 (en) | 2012-10-19 | 2014-04-24 | Bayer Cropscience Ag | Method of plant growth promotion using carboxamide derivatives |
WO2014060519A1 (en) | 2012-10-19 | 2014-04-24 | Bayer Cropscience Ag | Method for enhancing tolerance to abiotic stress in plants using carboxamide or thiocarboxamide derivatives |
WO2014078588A1 (en) * | 2012-11-14 | 2014-05-22 | Agrivida, Inc. | Methods and compositions for processing biomass with elevated levels of starch |
EP2735231A1 (en) | 2012-11-23 | 2014-05-28 | Bayer CropScience AG | Active compound combinations |
WO2014079957A1 (en) | 2012-11-23 | 2014-05-30 | Bayer Cropscience Ag | Selective inhibition of ethylene signal transduction |
WO2014083089A1 (en) | 2012-11-30 | 2014-06-05 | Bayer Cropscience Ag | Ternary fungicidal and pesticidal mixtures |
WO2014083088A2 (en) | 2012-11-30 | 2014-06-05 | Bayer Cropscience Ag | Binary fungicidal mixtures |
WO2014083033A1 (en) | 2012-11-30 | 2014-06-05 | Bayer Cropsience Ag | Binary fungicidal or pesticidal mixture |
WO2014083031A2 (en) | 2012-11-30 | 2014-06-05 | Bayer Cropscience Ag | Binary pesticidal and fungicidal mixtures |
WO2014082950A1 (en) | 2012-11-30 | 2014-06-05 | Bayer Cropscience Ag | Ternary fungicidal mixtures |
EP2740356A1 (en) | 2012-12-05 | 2014-06-11 | Bayer CropScience AG | Substituted (2Z)-5(1-Hydroxycyclohexyl)pent-2-en-4-inic acid derivatives |
EP2740720A1 (en) | 2012-12-05 | 2014-06-11 | Bayer CropScience AG | Substituted bicyclic and tricyclic pent-2-en-4-inic acid derivatives and their use for enhancing the stress tolerance in plants |
WO2014086751A1 (en) | 2012-12-05 | 2014-06-12 | Bayer Cropscience Ag | Use of substituted 1-(aryl ethynyl)-, 1-(heteroaryl ethynyl)-, 1-(heterocyclyl ethynyl)- and 1-(cyloalkenyl ethynyl)-cyclohexanols as active agents against abiotic plant stress |
WO2014090765A1 (en) | 2012-12-12 | 2014-06-19 | Bayer Cropscience Ag | Use of 1-[2-fluoro-4-methyl-5-(2,2,2-trifluoroethylsulfinyl)phenyl]-5-amino-3-trifluoromethyl)-1 h-1,2,4 tfia zole for controlling nematodes in nematode-resistant crops |
WO2014095826A1 (en) | 2012-12-18 | 2014-06-26 | Bayer Cropscience Ag | Binary fungicidal and bactericidal combinations |
WO2014095677A1 (en) | 2012-12-19 | 2014-06-26 | Bayer Cropscience Ag | Difluoromethyl-nicotinic- tetrahydronaphtyl carboxamides |
US8796175B2 (en) | 2008-08-29 | 2014-08-05 | Bayer Cropscience Ag | Method for enhancing plant intrinsic defense |
US8828906B2 (en) | 2009-03-25 | 2014-09-09 | Bayer Cropscience Ag | Active compound combinations having insecticidal and acaricidal properties |
US8828907B2 (en) | 2009-03-25 | 2014-09-09 | Bayer Cropscience Ag | Active ingredient combinations having insecticidal and acaricidal properties |
WO2014135608A1 (en) | 2013-03-07 | 2014-09-12 | Bayer Cropscience Ag | Fungicidal 3-{phenyl[(heterocyclylmethoxy)imino]methyl}-heterocycle derivatives |
US8835657B2 (en) | 2009-05-06 | 2014-09-16 | Bayer Cropscience Ag | Cyclopentanedione compounds and their use as insecticides, acaricides and/or fungicides |
US8846568B2 (en) | 2009-03-25 | 2014-09-30 | Bayer Cropscience Ag | Active compound combinations having insecticidal and acaricidal properties |
US8846567B2 (en) | 2009-03-25 | 2014-09-30 | Bayer Cropscience Ag | Active compound combinations having insecticidal and acaricidal properties |
WO2014161821A1 (en) | 2013-04-02 | 2014-10-09 | Bayer Cropscience Nv | Targeted genome engineering in eukaryotes |
WO2014167008A1 (en) | 2013-04-12 | 2014-10-16 | Bayer Cropscience Ag | Novel triazolinthione derivatives |
WO2014167009A1 (en) | 2013-04-12 | 2014-10-16 | Bayer Cropscience Ag | Novel triazole derivatives |
WO2014170364A1 (en) | 2013-04-19 | 2014-10-23 | Bayer Cropscience Ag | Binary insecticidal or pesticidal mixture |
WO2014170345A2 (en) | 2013-04-19 | 2014-10-23 | Bayer Cropscience Ag | Method for improved utilization of the production potential of transgenic plants |
WO2014177582A1 (en) | 2013-04-30 | 2014-11-06 | Bayer Cropscience Ag | N-(2-fluoro-2-phenethyl)carboxamides as nematicides and endoparasiticides |
WO2014177514A1 (en) | 2013-04-30 | 2014-11-06 | Bayer Cropscience Ag | Nematicidal n-substituted phenethylcarboxamides |
WO2014206953A1 (en) | 2013-06-26 | 2014-12-31 | Bayer Cropscience Ag | N-cycloalkyl-n-[(bicyclylphenyl)methylene]-(thio)carboxamide derivatives |
US8927583B2 (en) | 2006-12-22 | 2015-01-06 | Bayer Cropscience Ag | Pesticidal composition comprising a 2-pyrdilmethylbenzamide derivative and an insecticide compound |
WO2015004040A1 (en) | 2013-07-09 | 2015-01-15 | Bayer Cropscience Ag | Use of selected pyridone carboxamides or salts thereof as active substances against abiotic plant stress |
US9012360B2 (en) | 2009-03-25 | 2015-04-21 | Bayer Intellectual Property Gmbh | Synergistic combinations of active ingredients |
WO2015082587A1 (en) | 2013-12-05 | 2015-06-11 | Bayer Cropscience Ag | N-cycloalkyl-n-{[2-(1-substitutedcycloalkyl)phenyl]methylene}-(thio)carboxamide derivatives |
WO2015082586A1 (en) | 2013-12-05 | 2015-06-11 | Bayer Cropscience Ag | N-cycloalkyl-n-{[2-(1-substitutedcycloalkyl)phenyl]methylene}-(thio)carboxamide derivatives |
US9173394B2 (en) | 2007-09-26 | 2015-11-03 | Bayer Intellectual Property Gmbh | Active agent combinations having insecticidal and acaricidal properties |
US9199922B2 (en) | 2007-03-12 | 2015-12-01 | Bayer Intellectual Property Gmbh | Dihalophenoxyphenylamidines and use thereof as fungicides |
WO2016012362A1 (en) | 2014-07-22 | 2016-01-28 | Bayer Cropscience Aktiengesellschaft | Substituted cyano cycloalkyl penta-2,4-dienes, cyano cycloalkyl pent-2-en-4-ynes, cyano heterocyclyl penta-2,4-dienes and cyano heterocyclyl pent-2-en-4-ynes as active substances against abiotic plant stress |
US9249474B2 (en) | 2009-11-06 | 2016-02-02 | Agrivida, Inc. | Consolidated pretreatment and hydrolysis of plant biomass expressing cell wall degrading enzymes |
EP2997825A1 (en) | 2011-04-22 | 2016-03-23 | Bayer Intellectual Property GmbH | Active compound combinations comprising a (thio)carboxamide derivative and a fungicidal compound |
EP3000809A1 (en) | 2009-05-15 | 2016-03-30 | Bayer Intellectual Property GmbH | Fungicide pyrazole carboxamides derivatives |
WO2016096942A1 (en) | 2014-12-18 | 2016-06-23 | Bayer Cropscience Aktiengesellschaft | Use of selected pyridone carboxamides or salts thereof as active substances against abiotic plant stress |
WO2016166077A1 (en) | 2015-04-13 | 2016-10-20 | Bayer Cropscience Aktiengesellschaft | N-cycloalkyl-n-(biheterocyclyethylene)-(thio)carboxamide derivatives |
US9598700B2 (en) | 2010-06-25 | 2017-03-21 | Agrivida, Inc. | Methods and compositions for processing biomass with elevated levels of starch |
US9763451B2 (en) | 2008-12-29 | 2017-09-19 | Bayer Intellectual Property Gmbh | Method for improved use of the production potential of genetically modified plants |
WO2018019676A1 (en) | 2016-07-29 | 2018-02-01 | Bayer Cropscience Aktiengesellschaft | Active compound combinations and methods to protect the propagation material of plants |
WO2018054911A1 (en) | 2016-09-23 | 2018-03-29 | Bayer Cropscience Nv | Targeted genome optimization in plants |
WO2018054832A1 (en) | 2016-09-22 | 2018-03-29 | Bayer Cropscience Aktiengesellschaft | Novel triazole derivatives |
WO2018054829A1 (en) | 2016-09-22 | 2018-03-29 | Bayer Cropscience Aktiengesellschaft | Novel triazole derivatives and their use as fungicides |
WO2018077711A2 (en) | 2016-10-26 | 2018-05-03 | Bayer Cropscience Aktiengesellschaft | Use of pyraziflumid for controlling sclerotinia spp in seed treatment applications |
EP3332645A1 (en) | 2016-12-12 | 2018-06-13 | Bayer Cropscience AG | Use of substituted pyrimidine diones or their salts as agents to combat abiotic plant stress |
WO2018104392A1 (en) | 2016-12-08 | 2018-06-14 | Bayer Cropscience Aktiengesellschaft | Use of insecticides for controlling wireworms |
WO2018108627A1 (en) | 2016-12-12 | 2018-06-21 | Bayer Cropscience Aktiengesellschaft | Use of substituted indolinylmethyl sulfonamides, or the salts thereof for increasing the stress tolerance of plants |
DE102007045953B4 (en) | 2007-09-26 | 2018-07-05 | Bayer Intellectual Property Gmbh | Drug combinations with insecticidal and acaricidal properties |
DE102007045919B4 (en) | 2007-09-26 | 2018-07-05 | Bayer Intellectual Property Gmbh | Drug combinations with insecticidal and acaricidal properties |
DE102007045920B4 (en) | 2007-09-26 | 2018-07-05 | Bayer Intellectual Property Gmbh | Synergistic drug combinations |
US10137169B2 (en) | 2011-08-10 | 2018-11-27 | Follicum Ab | Compositions and uses thereof |
WO2019025153A1 (en) | 2017-07-31 | 2019-02-07 | Bayer Cropscience Aktiengesellschaft | Use of substituted n-sulfonyl-n'-aryl diaminoalkanes and n-sulfonyl-n'-heteroaryl diaminoalkanes or salts thereof for increasing the stress tolerance in plants |
WO2019060746A1 (en) | 2017-09-21 | 2019-03-28 | The Broad Institute, Inc. | Systems, methods, and compositions for targeted nucleic acid editing |
US10443068B2 (en) | 2010-06-25 | 2019-10-15 | Agrivida, Inc. | Plants with engineered endogenous genes |
WO2019233863A1 (en) | 2018-06-04 | 2019-12-12 | Bayer Aktiengesellschaft | Herbicidally active bicyclic benzoylpyrazoles |
WO2020131862A1 (en) | 2018-12-17 | 2020-06-25 | The Broad Institute, Inc. | Crispr-associated transposase systems and methods of use thereof |
US10968257B2 (en) | 2018-04-03 | 2021-04-06 | The Broad Institute, Inc. | Target recognition motifs and uses thereof |
US11180751B2 (en) | 2015-06-18 | 2021-11-23 | The Broad Institute, Inc. | CRISPR enzymes and systems |
US11591601B2 (en) | 2017-05-05 | 2023-02-28 | The Broad Institute, Inc. | Methods for identification and modification of lncRNA associated with target genotypes and phenotypes |
Families Citing this family (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
AR048025A1 (en) | 2004-03-05 | 2006-03-22 | Bayer Cropscience Gmbh | PLANTS WITH INCREASED ACTIVITY OF AN ALMIDON FOSFORILING ENZYME |
PL2028944T3 (en) * | 2006-05-24 | 2017-01-31 | Bayer Intellectual Property Gmbh | Composition for reducing baking losses |
CL2007003743A1 (en) * | 2006-12-22 | 2008-07-11 | Bayer Cropscience Ag | COMPOSITION THAT INCLUDES FENAMIDONA AND AN INSECTICIDE COMPOUND; AND METHOD TO CONTROL FITOPATOGENOS CULTURES AND INSECTS FACING OR PREVENTIVELY. |
EP1969931A1 (en) | 2007-03-12 | 2008-09-17 | Bayer CropScience Aktiengesellschaft | Fluoroalkyl phenylamidines and their use as fungicides |
EP2146975B1 (en) | 2007-04-19 | 2015-06-17 | Bayer Intellectual Property GmbH | Thiadiazolyl oxyphenyl amidines and the use thereof as a fungicide |
US20100285965A1 (en) * | 2007-10-02 | 2010-11-11 | Bayer Cropscience Ag | Methods of improving plant growth |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1996027674A1 (en) * | 1995-03-08 | 1996-09-12 | Hoechst Schering Agrevo Gmbh | Modified starch from plants, plants synthesizing this starch, and process for its preparation |
WO1997011188A1 (en) * | 1995-09-19 | 1997-03-27 | Planttec Biotechnologie Gmbh | Plants which synthesise a modified starch, process for the production thereof and modified starch |
WO2002034923A2 (en) * | 2000-10-23 | 2002-05-02 | Bayer Cropscience Gmbh | Monocotyledon plant cells and plants which synthesise modified starch |
Family Cites Families (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6307123B1 (en) * | 1998-05-18 | 2001-10-23 | Dekalb Genetics Corporation | Methods and compositions for transgene identification |
DE19836098A1 (en) * | 1998-07-31 | 2000-02-03 | Hoechst Schering Agrevo Gmbh | Plants that synthesize a modified starch, process for producing the plants, their use and the modified starch |
AU773808B2 (en) * | 1998-11-09 | 2004-06-10 | Bayer Cropscience Aktiengesellschaft | Nucleic acid molecules from rice and their use for the production of modified starch |
DE19926771A1 (en) | 1999-06-11 | 2000-12-14 | Aventis Cropscience Gmbh | Nucleic acid molecules from wheat, transgenic plant cells and plants and their use for the production of modified starch |
EP1368484A2 (en) | 2000-09-15 | 2003-12-10 | Syngenta Participations AG | Plant genes, the expression of which are altered by pathogen infection |
AU2001289843A1 (en) | 2001-08-28 | 2002-02-13 | Bayer Cropscience Ag | Polypeptides for identifying herbicidally active compounds |
JP2005185101A (en) * | 2002-05-30 | 2005-07-14 | National Institute Of Agrobiological Sciences | VEGETABLE FULL-LENGTH cDNA AND UTILIZATION THEREOF |
AR048026A1 (en) | 2004-03-05 | 2006-03-22 | Bayer Cropscience Gmbh | PROCEDURES FOR THE IDENTIFICATION OF PROTEINS WITH ENZYMATIC ACTIVITY FOSFORILADORA DE ALMIDON |
AR048025A1 (en) * | 2004-03-05 | 2006-03-22 | Bayer Cropscience Gmbh | PLANTS WITH INCREASED ACTIVITY OF AN ALMIDON FOSFORILING ENZYME |
-
2005
- 2005-03-03 AR ARP050100808A patent/AR048024A1/en not_active Application Discontinuation
- 2005-03-04 CN CN2005800070770A patent/CN1930295B/en not_active Expired - Fee Related
- 2005-03-04 PL PL05715852T patent/PL1725667T3/en unknown
- 2005-03-04 AT AT05715852T patent/ATE455178T1/en active
- 2005-03-04 AU AU2005229364A patent/AU2005229364B2/en not_active Ceased
- 2005-03-04 EP EP05715852A patent/EP1725667B1/en not_active Not-in-force
- 2005-03-04 US US10/591,432 patent/US7932436B2/en not_active Expired - Fee Related
- 2005-03-04 ES ES05715852T patent/ES2338242T3/en active Active
- 2005-03-04 DE DE602005018904T patent/DE602005018904D1/en active Active
- 2005-03-04 WO PCT/EP2005/002457 patent/WO2005095619A1/en active Application Filing
- 2005-03-04 SI SI200530956T patent/SI1725667T1/en unknown
- 2005-03-04 JP JP2007501245A patent/JP5623002B2/en not_active Expired - Fee Related
- 2005-03-04 PT PT05715852T patent/PT1725667E/en unknown
- 2005-03-04 DK DK05715852.9T patent/DK1725667T3/en active
- 2005-03-04 CA CA2558747A patent/CA2558747C/en not_active Expired - Fee Related
-
2012
- 2012-12-13 JP JP2012272442A patent/JP5812978B2/en not_active Expired - Fee Related
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1996027674A1 (en) * | 1995-03-08 | 1996-09-12 | Hoechst Schering Agrevo Gmbh | Modified starch from plants, plants synthesizing this starch, and process for its preparation |
WO1997011188A1 (en) * | 1995-09-19 | 1997-03-27 | Planttec Biotechnologie Gmbh | Plants which synthesise a modified starch, process for the production thereof and modified starch |
WO2002034923A2 (en) * | 2000-10-23 | 2002-05-02 | Bayer Cropscience Gmbh | Monocotyledon plant cells and plants which synthesise modified starch |
Non-Patent Citations (5)
Title |
---|
BAUNSGAARD LONE ET AL: "A novel isoform of glucan, water dikinase phosphorylates pre-phosphorylated alpha-glucans and is involved in starch degradation in Arabidopsis", PLANT JOURNAL, vol. 41, no. 4, February 2005 (2005-02-01), pages 595 - 605, XP002339143, ISSN: 0960-7412 * |
BLENNOW ANDREAS ET AL: "Starch phosphorylation: A new front line in starch research", TRENDS IN PLANT SCIENCE, vol. 7, no. 10, October 2002 (2002-10-01), pages 445 - 450, XP002339248, ISSN: 1360-1385 * |
KOETTING OLIVIER ET AL: "Identification of a novel enzyme required for starch metabolism in Arabidopsis leaves. The phosphoglucan, water dikinase", PLANT PHYSIOLOGY (ROCKVILLE), vol. 137, no. 1, January 2005 (2005-01-01), pages 242 - 252, XP002339144, ISSN: 0032-0889 * |
MIKKELSEN RENE ET AL: "Functional characterization of alpha-glucan, water dikinase, the starch phosphorylating enzyme.", BIOCHEMICAL JOURNAL, vol. 377, no. 2, 15 January 2004 (2004-01-15), pages 525 - 532, XP002339213, ISSN: 0264-6021 * |
RITTE GERHARD ET AL: "The starch-related R1 protein is an alpha -glucan, water dikinase", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA, NATIONAL ACADEMY OF SCIENCE. WASHINGTON, US, vol. 99, no. 10, 14 May 2002 (2002-05-14), pages 7166 - 7171, XP002200501, ISSN: 0027-8424 * |
Cited By (206)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP1629102A4 (en) * | 2003-05-22 | 2007-10-17 | Syngenta Participations Ag | Modified starch, uses, methods for production thereof |
EP1629102A2 (en) * | 2003-05-22 | 2006-03-01 | Syngenta Participations AG | Modified starch, uses, methods for production thereof |
WO2007134877A1 (en) * | 2006-05-24 | 2007-11-29 | Bayer Cropscience Ag | Use of modified wheat flour for reducing baking losses |
AU2007253587B2 (en) * | 2006-05-24 | 2013-02-21 | Basf Se | Use of modified wheat flour for reducing baking losses |
US8927583B2 (en) | 2006-12-22 | 2015-01-06 | Bayer Cropscience Ag | Pesticidal composition comprising a 2-pyrdilmethylbenzamide derivative and an insecticide compound |
US8252975B2 (en) | 2007-01-26 | 2012-08-28 | Bayer Cropscience Ag | Genetically modified plants which synthesize a low amylose starch with increased swelling power |
US8785692B2 (en) | 2007-03-12 | 2014-07-22 | Bayer Cropscience Ag | Substituted phenylamidines and the use thereof as fungicides |
US8334237B2 (en) | 2007-03-12 | 2012-12-18 | Bayer Cropscience Ag | Substituted phenylamidines and the use thereof as fungicides |
US8299302B2 (en) | 2007-03-12 | 2012-10-30 | Bayer Cropscience Ag | 4-Cycloalkyl or 4-substituted phenoxyphenylamidines and use thereof as fungicides |
US8080688B2 (en) | 2007-03-12 | 2011-12-20 | Bayer Cropscience Ag | 3, 4-disubstituted phenoxyphenylamidines and use thereof as fungicides |
US8748662B2 (en) | 2007-03-12 | 2014-06-10 | Bayer Cropscience Ag | 4-cycloalkyl or 4-aryl substituted phenoxyphenylamidines and use thereof as fungicides |
US9199922B2 (en) | 2007-03-12 | 2015-12-01 | Bayer Intellectual Property Gmbh | Dihalophenoxyphenylamidines and use thereof as fungicides |
US8394991B2 (en) | 2007-03-12 | 2013-03-12 | Bayer Cropscience Ag | Phenoxy substituted phenylamidine derivatives and their use as fungicides |
US8519003B2 (en) | 2007-03-12 | 2013-08-27 | Bayer Cropscience Ag | Phenoxyphenylamidines as fungicides |
US9173394B2 (en) | 2007-09-26 | 2015-11-03 | Bayer Intellectual Property Gmbh | Active agent combinations having insecticidal and acaricidal properties |
DE102007045920B4 (en) | 2007-09-26 | 2018-07-05 | Bayer Intellectual Property Gmbh | Synergistic drug combinations |
US8455480B2 (en) | 2007-09-26 | 2013-06-04 | Bayer Cropscience Ag | Active agent combinations having insecticidal and acaricidal properties |
DE102007045953B4 (en) | 2007-09-26 | 2018-07-05 | Bayer Intellectual Property Gmbh | Drug combinations with insecticidal and acaricidal properties |
DE102007045919B4 (en) | 2007-09-26 | 2018-07-05 | Bayer Intellectual Property Gmbh | Drug combinations with insecticidal and acaricidal properties |
EP2072506A1 (en) | 2007-12-21 | 2009-06-24 | Bayer CropScience AG | Thiazolyloxyphenylamidine or thiadiazolyloxyphenylamidine und its use as fungicide |
EP2090168A1 (en) | 2008-02-12 | 2009-08-19 | Bayer CropScience AG | Method for improving plant growth |
EP2168434A1 (en) | 2008-08-02 | 2010-03-31 | Bayer CropScience AG | Use of azols to increase resistance of plants of parts of plants to abiotic stress |
EP2374791A1 (en) | 2008-08-14 | 2011-10-12 | Bayer CropScience Aktiengesellschaft | Insecticidal 4-phenyl-1H pyrazoles |
US8796175B2 (en) | 2008-08-29 | 2014-08-05 | Bayer Cropscience Ag | Method for enhancing plant intrinsic defense |
EP2201838A1 (en) | 2008-12-05 | 2010-06-30 | Bayer CropScience AG | Active ingredient-beneficial organism combinations with insecticide and acaricide properties |
EP2198709A1 (en) | 2008-12-19 | 2010-06-23 | Bayer CropScience AG | Method for treating resistant animal pests |
US9763451B2 (en) | 2008-12-29 | 2017-09-19 | Bayer Intellectual Property Gmbh | Method for improved use of the production potential of genetically modified plants |
EP2204094A1 (en) | 2008-12-29 | 2010-07-07 | Bayer CropScience AG | Method for improved utilization of the production potential of transgenic plants Introduction |
WO2010075994A1 (en) | 2008-12-29 | 2010-07-08 | Bayer Cropscience Aktiengesellschaft | Treatment of transgenic crops with mixtures of fiproles and chloronicotinyls |
EP2039771A2 (en) | 2009-01-06 | 2009-03-25 | Bayer CropScience AG | Method for improved utilization of the production potential of transgenic plants |
EP2039772A2 (en) | 2009-01-06 | 2009-03-25 | Bayer CropScience AG | Method for improved utilization of the production potential of transgenic plants introduction |
EP2039770A2 (en) | 2009-01-06 | 2009-03-25 | Bayer CropScience AG | Method for improved utilization of the production potential of transgenic plants |
US8487118B2 (en) | 2009-01-19 | 2013-07-16 | Bayer Cropscience Ag | Cyclic diones and their use as insecticides, acaricides and/or fungicides |
EP2227951A1 (en) | 2009-01-23 | 2010-09-15 | Bayer CropScience AG | Application of enaminocarbonyl compounds for combating viruses transmitted by insects |
WO2010083955A2 (en) | 2009-01-23 | 2010-07-29 | Bayer Cropscience Aktiengesellschaft | Use of enaminocarboxylic compounds for fighting viruses transmitted by insects |
WO2010086311A1 (en) | 2009-01-28 | 2010-08-05 | Bayer Cropscience Ag | Fungicide n-cycloalkyl-n-bicyclicmethylene-carboxamide derivatives |
WO2010086095A1 (en) | 2009-01-29 | 2010-08-05 | Bayer Cropscience Ag | Method for improved utilization of the production potential of transgenic plants introduction |
EP2218717A1 (en) | 2009-02-17 | 2010-08-18 | Bayer CropScience AG | Fungicidal N-((HET)Arylethyl)thiocarboxamide derivatives |
WO2010094666A2 (en) | 2009-02-17 | 2010-08-26 | Bayer Cropscience Ag | Fungicidal n-(phenylcycloalkyl)carboxamide, n-(benzylcycloalkyl)carboxamide and thiocarboxamide derivatives |
WO2010094728A1 (en) | 2009-02-19 | 2010-08-26 | Bayer Cropscience Ag | Pesticide composition comprising a tetrazolyloxime derivative and a fungicide or an insecticide active substance |
EP2223602A1 (en) | 2009-02-23 | 2010-09-01 | Bayer CropScience AG | Method for improved utilisation of the production potential of genetically modified plants |
US9012360B2 (en) | 2009-03-25 | 2015-04-21 | Bayer Intellectual Property Gmbh | Synergistic combinations of active ingredients |
US8846567B2 (en) | 2009-03-25 | 2014-09-30 | Bayer Cropscience Ag | Active compound combinations having insecticidal and acaricidal properties |
US8846568B2 (en) | 2009-03-25 | 2014-09-30 | Bayer Cropscience Ag | Active compound combinations having insecticidal and acaricidal properties |
EP2232995A1 (en) | 2009-03-25 | 2010-09-29 | Bayer CropScience AG | Method for improved utilisation of the production potential of transgenic plants |
US8828907B2 (en) | 2009-03-25 | 2014-09-09 | Bayer Cropscience Ag | Active ingredient combinations having insecticidal and acaricidal properties |
US8828906B2 (en) | 2009-03-25 | 2014-09-09 | Bayer Cropscience Ag | Active compound combinations having insecticidal and acaricidal properties |
EP2239331A1 (en) | 2009-04-07 | 2010-10-13 | Bayer CropScience AG | Method for improved utilization of the production potential of transgenic plants |
US8835657B2 (en) | 2009-05-06 | 2014-09-16 | Bayer Cropscience Ag | Cyclopentanedione compounds and their use as insecticides, acaricides and/or fungicides |
EP3000809A1 (en) | 2009-05-15 | 2016-03-30 | Bayer Intellectual Property GmbH | Fungicide pyrazole carboxamides derivatives |
EP2251331A1 (en) | 2009-05-15 | 2010-11-17 | Bayer CropScience AG | Fungicide pyrazole carboxamides derivatives |
EP2255626A1 (en) | 2009-05-27 | 2010-12-01 | Bayer CropScience AG | Use of succinate dehydrogenase inhibitors to increase resistance of plants or parts of plants to abiotic stress |
WO2011006603A2 (en) | 2009-07-16 | 2011-01-20 | Bayer Cropscience Ag | Synergistic active substance combinations containing phenyl triazoles |
WO2011015524A2 (en) | 2009-08-03 | 2011-02-10 | Bayer Cropscience Ag | Fungicide heterocycles derivatives |
EP2292094A1 (en) | 2009-09-02 | 2011-03-09 | Bayer CropScience AG | Active compound combinations |
WO2011035834A1 (en) | 2009-09-02 | 2011-03-31 | Bayer Cropscience Ag | Active compound combinations |
US10006038B2 (en) | 2009-11-06 | 2018-06-26 | Agrivida, Inc. | Consolidated pretreatment and hydrolysis of plant biomass expressing cell wall degrading enzymes |
US9249474B2 (en) | 2009-11-06 | 2016-02-02 | Agrivida, Inc. | Consolidated pretreatment and hydrolysis of plant biomass expressing cell wall degrading enzymes |
EP2343280A1 (en) | 2009-12-10 | 2011-07-13 | Bayer CropScience AG | Fungicide quinoline derivatives |
WO2011080255A2 (en) | 2009-12-28 | 2011-07-07 | Bayer Cropscience Ag | Fungicide hydroximoyl-tetrazole derivatives |
WO2011080254A2 (en) | 2009-12-28 | 2011-07-07 | Bayer Cropscience Ag | Fungicide hydroximoyl-heterocycles derivatives |
WO2011080256A1 (en) | 2009-12-28 | 2011-07-07 | Bayer Cropscience Ag | Fungicide hydroximoyl-tetrazole derivatives |
US8722072B2 (en) | 2010-01-22 | 2014-05-13 | Bayer Intellectual Property Gmbh | Acaricidal and/or insecticidal active ingredient combinations |
WO2011089071A2 (en) | 2010-01-22 | 2011-07-28 | Bayer Cropscience Ag | Acaricide and/or insecticide active substance combinations |
WO2011107504A1 (en) | 2010-03-04 | 2011-09-09 | Bayer Cropscience Ag | Fluoroalkyl-substituted 2-amidobenzimidazoles and the use thereof for boosting stress tolerance in plants |
WO2011124554A2 (en) | 2010-04-06 | 2011-10-13 | Bayer Cropscience Ag | Use of 4-phenylbutyric acid and/or the salts thereof for enhancing the stress tolerance of plants |
WO2011124553A2 (en) | 2010-04-09 | 2011-10-13 | Bayer Cropscience Ag | Use of derivatives of the (1-cyanocyclopropyl)phenylphosphinic acid, the esters thereof and/or the salts thereof for enhancing the tolerance of plants to abiotic stress |
WO2011134913A1 (en) | 2010-04-28 | 2011-11-03 | Bayer Cropscience Ag | Fungicide hydroximoyl-heterocycles derivatives |
WO2011134911A2 (en) | 2010-04-28 | 2011-11-03 | Bayer Cropscience Ag | Fungicide hydroximoyl-tetrazole derivatives |
WO2011134912A1 (en) | 2010-04-28 | 2011-11-03 | Bayer Cropscience Ag | Fungicide hydroximoyl-heterocycles derivatives |
WO2011151369A1 (en) | 2010-06-03 | 2011-12-08 | Bayer Cropscience Ag | N-[(het)arylethyl)] pyrazole(thio)carboxamides and their heterosubstituted analogues |
WO2011151370A1 (en) | 2010-06-03 | 2011-12-08 | Bayer Cropscience Ag | N-[(het)arylalkyl)] pyrazole (thio)carboxamides and their heterosubstituted analogues |
WO2011151368A2 (en) | 2010-06-03 | 2011-12-08 | Bayer Cropscience Ag | Fungicide n-[(trisubstitutedsilyl)methyl]-carboxamide derivatives |
US9593317B2 (en) | 2010-06-09 | 2017-03-14 | Bayer Cropscience Nv | Methods and means to modify a plant genome at a nucleotide sequence commonly used in plant genome engineering |
US9574201B2 (en) | 2010-06-09 | 2017-02-21 | Bayer Cropscience Nv | Methods and means to modify a plant genome at a nucleotide sequence commonly used in plant genome engineering |
WO2011154158A1 (en) | 2010-06-09 | 2011-12-15 | Bayer Bioscience N.V. | Methods and means to modify a plant genome at a nucleotide sequence commonly used in plant genome engineering |
WO2011154159A1 (en) | 2010-06-09 | 2011-12-15 | Bayer Bioscience N.V. | Methods and means to modify a plant genome at a nucleotide sequence commonly used in plant genome engineering |
US10443068B2 (en) | 2010-06-25 | 2019-10-15 | Agrivida, Inc. | Plants with engineered endogenous genes |
US9598700B2 (en) | 2010-06-25 | 2017-03-21 | Agrivida, Inc. | Methods and compositions for processing biomass with elevated levels of starch |
WO2012010579A2 (en) | 2010-07-20 | 2012-01-26 | Bayer Cropscience Ag | Benzocycloalkenes as antifungal agents |
WO2012028578A1 (en) | 2010-09-03 | 2012-03-08 | Bayer Cropscience Ag | Substituted fused pyrimidinones and dihydropyrimidinones |
WO2012038480A2 (en) | 2010-09-22 | 2012-03-29 | Bayer Cropscience Ag | Use of biological or chemical control agents for controlling insects and nematodes in resistant crops |
WO2012038476A1 (en) | 2010-09-22 | 2012-03-29 | Bayer Cropscience Ag | Use of active ingredients for controlling nematodes in nematode-resistant crops |
WO2012045798A1 (en) | 2010-10-07 | 2012-04-12 | Bayer Cropscience Ag | Fungicide composition comprising a tetrazolyloxime derivative and a thiazolylpiperidine derivative |
WO2012052490A1 (en) | 2010-10-21 | 2012-04-26 | Bayer Cropscience Ag | N-benzyl heterocyclic carboxamides |
WO2012052489A1 (en) | 2010-10-21 | 2012-04-26 | Bayer Cropscience Ag | 1-(heterocyclic carbonyl) piperidines |
WO2012059497A1 (en) | 2010-11-02 | 2012-05-10 | Bayer Cropscience Ag | N-hetarylmethyl pyrazolylcarboxamides |
US9206137B2 (en) | 2010-11-15 | 2015-12-08 | Bayer Intellectual Property Gmbh | N-Aryl pyrazole(thio)carboxamides |
WO2012065945A1 (en) | 2010-11-15 | 2012-05-24 | Bayer Cropscience Ag | 5-halogenopyrazole(thio)carboxamides |
WO2012065944A1 (en) | 2010-11-15 | 2012-05-24 | Bayer Cropscience Ag | N-aryl pyrazole(thio)carboxamides |
WO2012065947A1 (en) | 2010-11-15 | 2012-05-24 | Bayer Cropscience Ag | 5-halogenopyrazolecarboxamides |
EP3103334A1 (en) | 2010-12-01 | 2016-12-14 | Bayer Intellectual Property GmbH | Agent combinations comprising pyridylethyl benzamides and other agents |
WO2012072696A1 (en) | 2010-12-01 | 2012-06-07 | Bayer Cropscience Ag | Active ingredient combinations comprising pyridylethylbenzamides and other active ingredients |
EP3103340A1 (en) | 2010-12-01 | 2016-12-14 | Bayer Intellectual Property GmbH | Agent combinations comprising pyridylethyl benzamides and other agents |
EP3103338A1 (en) | 2010-12-01 | 2016-12-14 | Bayer Intellectual Property GmbH | Agent combinations comprising pyridylethyl benzamides and other agents |
EP3092900A1 (en) | 2010-12-01 | 2016-11-16 | Bayer Intellectual Property GmbH | Active ingredient combinations comprising pyridylethylbenzamides and other active ingredients |
EP2460407A1 (en) | 2010-12-01 | 2012-06-06 | Bayer CropScience AG | Agent combinations comprising pyridylethyl benzamides and other agents |
EP2460406A1 (en) | 2010-12-01 | 2012-06-06 | Bayer CropScience AG | Use of fluopyram for controlling nematodes in nematode resistant crops |
WO2012072660A1 (en) | 2010-12-01 | 2012-06-07 | Bayer Cropscience Ag | Use of fluopyram for controlling nematodes in crops and for increasing yield |
EP3103339A1 (en) | 2010-12-01 | 2016-12-14 | Bayer Intellectual Property GmbH | Agent combinations comprising pyridylethyl benzamides and other agents |
WO2012089757A1 (en) | 2010-12-29 | 2012-07-05 | Bayer Cropscience Ag | Fungicide hydroximoyl-tetrazole derivatives |
EP2474542A1 (en) | 2010-12-29 | 2012-07-11 | Bayer CropScience AG | Fungicide hydroximoyl-tetrazole derivatives |
WO2012089722A2 (en) | 2010-12-30 | 2012-07-05 | Bayer Cropscience Ag | Use of open-chain carboxylic acids, carbonic esters, carboxamides and carbonitriles of aryl, heteroaryl and benzylsulfonamide or the salts thereof for improving the stress tolerance in plants |
WO2012089721A1 (en) | 2010-12-30 | 2012-07-05 | Bayer Cropscience Ag | Use of substituted spirocyclic sulfonamidocarboxylic acids, carboxylic esters thereof, carboxamides thereof and carbonitriles thereof or salts thereof for enhancement of stress tolerance in plants |
EP2494867A1 (en) | 2011-03-01 | 2012-09-05 | Bayer CropScience AG | Halogen-substituted compounds in combination with fungicides |
WO2012120105A1 (en) | 2011-03-10 | 2012-09-13 | Bayer Cropscience Ag | Use of lipochito-oligosaccharide compounds for safeguarding seed safety of treated seeds |
WO2012123434A1 (en) | 2011-03-14 | 2012-09-20 | Bayer Cropscience Ag | Fungicide hydroximoyl-tetrazole derivatives |
WO2012136581A1 (en) | 2011-04-08 | 2012-10-11 | Bayer Cropscience Ag | Fungicide hydroximoyl-tetrazole derivatives |
EP2511255A1 (en) | 2011-04-15 | 2012-10-17 | Bayer CropScience AG | Substituted prop-2-in-1-ol and prop-2-en-1-ol derivatives |
WO2012139892A1 (en) | 2011-04-15 | 2012-10-18 | Bayer Cropscience Ag | Substituted 5-(bicyclo[4.1.0]hept-3-en-2-yl)-penta-2,4-dienes and 5-(bicyclo[4.1.0]hept-3-en-2-yl)-pent-2-ene-4-ines as active agents against abiotic stress in plants |
WO2012139890A1 (en) | 2011-04-15 | 2012-10-18 | Bayer Cropscience Ag | Substituted 5-(cyclohex-2-en-1-yl)-penta-2,4-dienes and 5-(cyclohex-2-en-1-yl)-pent-2-en-4-ines as active agents against abiotic stress in plants |
WO2012139891A1 (en) | 2011-04-15 | 2012-10-18 | Bayer Cropscience Ag | Substituted vinyl and alkinyl cyclohexenols as active agents against abiotic stress in plants |
EP2997825A1 (en) | 2011-04-22 | 2016-03-23 | Bayer Intellectual Property GmbH | Active compound combinations comprising a (thio)carboxamide derivative and a fungicidal compound |
WO2012168124A1 (en) | 2011-06-06 | 2012-12-13 | Bayer Cropscience Nv | Methods and means to modify a plant genome at a preselected site |
WO2013004652A1 (en) | 2011-07-04 | 2013-01-10 | Bayer Intellectual Property Gmbh | Use of substituted isoquinolinones, isoquinolindiones, isoquinolintriones and dihydroisoquinolinones or in each case salts thereof as active agents against abiotic stress in plants |
US9265252B2 (en) | 2011-08-10 | 2016-02-23 | Bayer Intellectual Property Gmbh | Active compound combinations comprising specific tetramic acid derivatives |
US10517932B2 (en) | 2011-08-10 | 2019-12-31 | Follicum Ab | Compositions and uses thereof |
US10137169B2 (en) | 2011-08-10 | 2018-11-27 | Follicum Ab | Compositions and uses thereof |
WO2013020985A1 (en) | 2011-08-10 | 2013-02-14 | Bayer Intellectual Property Gmbh | Active compound combinations comprising specific tetramic acid derivatives |
US10538774B2 (en) | 2011-08-22 | 2020-01-21 | Basf Agricultural Solutions Seed, Us Llc | Methods and means to modify a plant genome |
US9670496B2 (en) | 2011-08-22 | 2017-06-06 | Bayer Cropscience N.V. | Methods and means to modify a plant genome |
WO2013026836A1 (en) | 2011-08-22 | 2013-02-28 | Bayer Intellectual Property Gmbh | Fungicide hydroximoyl-tetrazole derivatives |
WO2013026740A2 (en) | 2011-08-22 | 2013-02-28 | Bayer Cropscience Nv | Methods and means to modify a plant genome |
EP2561759A1 (en) | 2011-08-26 | 2013-02-27 | Bayer Cropscience AG | Fluoroalkyl-substituted 2-amidobenzimidazoles and their effect on plant growth |
WO2013034621A1 (en) | 2011-09-09 | 2013-03-14 | Bayer Intellectual Property Gmbh | Acyl-homoserine lactone derivatives for improving plant yield |
WO2013037717A1 (en) | 2011-09-12 | 2013-03-21 | Bayer Intellectual Property Gmbh | Fungicidal 4-substituted-3-{phenyl[(heterocyclylmethoxy)imino]methyl}-1,2,4-oxadizol-5(4h)-one derivatives |
WO2013037955A1 (en) | 2011-09-16 | 2013-03-21 | Bayer Intellectual Property Gmbh | Use of acylsulfonamides for improving plant yield |
WO2013037956A1 (en) | 2011-09-16 | 2013-03-21 | Bayer Intellectual Property Gmbh | Use of 5-phenyl- or 5-benzyl-2 isoxazoline-3 carboxylates for improving plant yield |
WO2013037958A1 (en) | 2011-09-16 | 2013-03-21 | Bayer Intellectual Property Gmbh | Use of phenylpyrazolin-3-carboxylates for improving plant yield |
WO2013041602A1 (en) | 2011-09-23 | 2013-03-28 | Bayer Intellectual Property Gmbh | Use of 4-substituted 1-phenyl-pyrazole-3-carboxylic-acid derivatives as agents against abiotic plant stress |
WO2013050410A1 (en) | 2011-10-04 | 2013-04-11 | Bayer Intellectual Property Gmbh | RNAi FOR THE CONTROL OF FUNGI AND OOMYCETES BY INHIBITING SACCHAROPINE DEHYDROGENASE GENE |
WO2013050324A1 (en) | 2011-10-06 | 2013-04-11 | Bayer Intellectual Property Gmbh | Combination, containing 4-phenylbutyric acid (4-pba) or a salt thereof (component (a)) and one or more selected additional agronomically active compounds (component(s) (b)), that reduces abiotic plant stress |
WO2013075817A1 (en) | 2011-11-21 | 2013-05-30 | Bayer Intellectual Property Gmbh | Fungicide n-[(trisubstitutedsilyl)methyl]-carboxamide derivatives |
WO2013079566A2 (en) | 2011-11-30 | 2013-06-06 | Bayer Intellectual Property Gmbh | Fungicidal n-bicycloalkyl and n-tricycloalkyl (thio)carboxamide derivatives |
WO2013092519A1 (en) | 2011-12-19 | 2013-06-27 | Bayer Cropscience Ag | Use of anthranilic acid diamide derivatives for pest control in transgenic crops |
WO2013098147A1 (en) | 2011-12-29 | 2013-07-04 | Bayer Intellectual Property Gmbh | Fungicidal 3-[(pyridin-2-ylmethoxyimino)(phenyl)methyl]-2-substituted-1,2,4-oxadiazol-5(2h)-one derivatives |
WO2013098146A1 (en) | 2011-12-29 | 2013-07-04 | Bayer Intellectual Property Gmbh | Fungicidal 3-[(1,3-thiazol-4-ylmethoxyimino)(phenyl)methyl]-2-substituted-1,2,4-oxadiazol-5(2h)-one derivatives |
WO2013124275A1 (en) | 2012-02-22 | 2013-08-29 | Bayer Cropscience Ag | Use of succinate dehydrogenase inhibitors (sdhis) for controlling wood diseases in grape. |
WO2013127704A1 (en) | 2012-02-27 | 2013-09-06 | Bayer Intellectual Property Gmbh | Active compound combinations containing a thiazoylisoxazoline and a fungicide |
WO2013139949A1 (en) | 2012-03-23 | 2013-09-26 | Bayer Intellectual Property Gmbh | Compositions comprising a strigolactame compound for enhanced plant growth and yield |
WO2013153143A1 (en) | 2012-04-12 | 2013-10-17 | Bayer Cropscience Ag | N-acyl- 2 - (cyclo) alkylpyrrolidines and piperidines useful as fungicides |
WO2013156560A1 (en) | 2012-04-20 | 2013-10-24 | Bayer Cropscience Ag | N-cycloalkyl-n-[(trisubstitutedsilylphenyl)methylene]-(thio)carboxamide derivatives |
WO2013156559A1 (en) | 2012-04-20 | 2013-10-24 | Bayer Cropscience Ag | N-cycloalkyl-n-[(heterocyclylphenyl)methylene]-(thio)carboxamide derivatives |
US11518997B2 (en) | 2012-04-23 | 2022-12-06 | BASF Agricultural Solutions Seed US LLC | Targeted genome engineering in plants |
WO2013160230A1 (en) | 2012-04-23 | 2013-10-31 | Bayer Cropscience Nv | Targeted genome engineering in plants |
EP2662363A1 (en) | 2012-05-09 | 2013-11-13 | Bayer CropScience AG | 5-Halogenopyrazole biphenylcarboxamides |
EP2662364A1 (en) | 2012-05-09 | 2013-11-13 | Bayer CropScience AG | Pyrazole tetrahydronaphthyl carboxamides |
EP2662362A1 (en) | 2012-05-09 | 2013-11-13 | Bayer CropScience AG | Pyrazole indanyl carboxamides |
EP2662360A1 (en) | 2012-05-09 | 2013-11-13 | Bayer CropScience AG | 5-Halogenopyrazole indanyl carboxamides |
EP2662370A1 (en) | 2012-05-09 | 2013-11-13 | Bayer CropScience AG | 5-Halogenopyrazole benzofuranyl carboxamides |
WO2013167544A1 (en) | 2012-05-09 | 2013-11-14 | Bayer Cropscience Ag | 5-halogenopyrazole indanyl carboxamides |
WO2013167545A1 (en) | 2012-05-09 | 2013-11-14 | Bayer Cropscience Ag | Pyrazole indanyl carboxamides |
EP2662361A1 (en) | 2012-05-09 | 2013-11-13 | Bayer CropScience AG | Pyrazol indanyl carboxamides |
WO2013174836A1 (en) | 2012-05-22 | 2013-11-28 | Bayer Cropscience Ag | Active compounds combinations comprising a lipo-chitooligosaccharide derivative and a nematicide, insecticidal or fungicidal compound |
WO2014009322A1 (en) | 2012-07-11 | 2014-01-16 | Bayer Cropscience Ag | Use of fungicidal combinations for increasing the tolerance of a plant towards abiotic stress |
WO2014037340A1 (en) | 2012-09-05 | 2014-03-13 | Bayer Cropscience Ag | Use of substituted 2-amidobenzimidazoles, 2-amidobenzoxazoles and 2-amidobenzothiazoles or salts thereof as active substances against abiotic plant stress |
WO2014060520A1 (en) | 2012-10-19 | 2014-04-24 | Bayer Cropscience Ag | Method for treating plants against fungi resistant to fungicides using carboxamide or thiocarboxamide derivatives |
WO2014060502A1 (en) | 2012-10-19 | 2014-04-24 | Bayer Cropscience Ag | Active compound combinations comprising carboxamide derivatives |
WO2014060518A1 (en) | 2012-10-19 | 2014-04-24 | Bayer Cropscience Ag | Method of plant growth promotion using carboxamide derivatives |
WO2014060519A1 (en) | 2012-10-19 | 2014-04-24 | Bayer Cropscience Ag | Method for enhancing tolerance to abiotic stress in plants using carboxamide or thiocarboxamide derivatives |
WO2014078588A1 (en) * | 2012-11-14 | 2014-05-22 | Agrivida, Inc. | Methods and compositions for processing biomass with elevated levels of starch |
WO2014079789A1 (en) | 2012-11-23 | 2014-05-30 | Bayer Cropscience Ag | Active compound combinations |
WO2014079957A1 (en) | 2012-11-23 | 2014-05-30 | Bayer Cropscience Ag | Selective inhibition of ethylene signal transduction |
EP2735231A1 (en) | 2012-11-23 | 2014-05-28 | Bayer CropScience AG | Active compound combinations |
WO2014083088A2 (en) | 2012-11-30 | 2014-06-05 | Bayer Cropscience Ag | Binary fungicidal mixtures |
WO2014083031A2 (en) | 2012-11-30 | 2014-06-05 | Bayer Cropscience Ag | Binary pesticidal and fungicidal mixtures |
WO2014083089A1 (en) | 2012-11-30 | 2014-06-05 | Bayer Cropscience Ag | Ternary fungicidal and pesticidal mixtures |
WO2014083033A1 (en) | 2012-11-30 | 2014-06-05 | Bayer Cropsience Ag | Binary fungicidal or pesticidal mixture |
WO2014082950A1 (en) | 2012-11-30 | 2014-06-05 | Bayer Cropscience Ag | Ternary fungicidal mixtures |
EP2740720A1 (en) | 2012-12-05 | 2014-06-11 | Bayer CropScience AG | Substituted bicyclic and tricyclic pent-2-en-4-inic acid derivatives and their use for enhancing the stress tolerance in plants |
WO2014086751A1 (en) | 2012-12-05 | 2014-06-12 | Bayer Cropscience Ag | Use of substituted 1-(aryl ethynyl)-, 1-(heteroaryl ethynyl)-, 1-(heterocyclyl ethynyl)- and 1-(cyloalkenyl ethynyl)-cyclohexanols as active agents against abiotic plant stress |
EP2740356A1 (en) | 2012-12-05 | 2014-06-11 | Bayer CropScience AG | Substituted (2Z)-5(1-Hydroxycyclohexyl)pent-2-en-4-inic acid derivatives |
WO2014090765A1 (en) | 2012-12-12 | 2014-06-19 | Bayer Cropscience Ag | Use of 1-[2-fluoro-4-methyl-5-(2,2,2-trifluoroethylsulfinyl)phenyl]-5-amino-3-trifluoromethyl)-1 h-1,2,4 tfia zole for controlling nematodes in nematode-resistant crops |
WO2014095826A1 (en) | 2012-12-18 | 2014-06-26 | Bayer Cropscience Ag | Binary fungicidal and bactericidal combinations |
WO2014095677A1 (en) | 2012-12-19 | 2014-06-26 | Bayer Cropscience Ag | Difluoromethyl-nicotinic- tetrahydronaphtyl carboxamides |
WO2014135608A1 (en) | 2013-03-07 | 2014-09-12 | Bayer Cropscience Ag | Fungicidal 3-{phenyl[(heterocyclylmethoxy)imino]methyl}-heterocycle derivatives |
WO2014161821A1 (en) | 2013-04-02 | 2014-10-09 | Bayer Cropscience Nv | Targeted genome engineering in eukaryotes |
WO2014167009A1 (en) | 2013-04-12 | 2014-10-16 | Bayer Cropscience Ag | Novel triazole derivatives |
WO2014167008A1 (en) | 2013-04-12 | 2014-10-16 | Bayer Cropscience Ag | Novel triazolinthione derivatives |
WO2014170345A2 (en) | 2013-04-19 | 2014-10-23 | Bayer Cropscience Ag | Method for improved utilization of the production potential of transgenic plants |
WO2014170364A1 (en) | 2013-04-19 | 2014-10-23 | Bayer Cropscience Ag | Binary insecticidal or pesticidal mixture |
WO2014177514A1 (en) | 2013-04-30 | 2014-11-06 | Bayer Cropscience Ag | Nematicidal n-substituted phenethylcarboxamides |
WO2014177582A1 (en) | 2013-04-30 | 2014-11-06 | Bayer Cropscience Ag | N-(2-fluoro-2-phenethyl)carboxamides as nematicides and endoparasiticides |
WO2014206953A1 (en) | 2013-06-26 | 2014-12-31 | Bayer Cropscience Ag | N-cycloalkyl-n-[(bicyclylphenyl)methylene]-(thio)carboxamide derivatives |
WO2015004040A1 (en) | 2013-07-09 | 2015-01-15 | Bayer Cropscience Ag | Use of selected pyridone carboxamides or salts thereof as active substances against abiotic plant stress |
WO2015082587A1 (en) | 2013-12-05 | 2015-06-11 | Bayer Cropscience Ag | N-cycloalkyl-n-{[2-(1-substitutedcycloalkyl)phenyl]methylene}-(thio)carboxamide derivatives |
WO2015082586A1 (en) | 2013-12-05 | 2015-06-11 | Bayer Cropscience Ag | N-cycloalkyl-n-{[2-(1-substitutedcycloalkyl)phenyl]methylene}-(thio)carboxamide derivatives |
WO2016012362A1 (en) | 2014-07-22 | 2016-01-28 | Bayer Cropscience Aktiengesellschaft | Substituted cyano cycloalkyl penta-2,4-dienes, cyano cycloalkyl pent-2-en-4-ynes, cyano heterocyclyl penta-2,4-dienes and cyano heterocyclyl pent-2-en-4-ynes as active substances against abiotic plant stress |
WO2016096942A1 (en) | 2014-12-18 | 2016-06-23 | Bayer Cropscience Aktiengesellschaft | Use of selected pyridone carboxamides or salts thereof as active substances against abiotic plant stress |
WO2016166077A1 (en) | 2015-04-13 | 2016-10-20 | Bayer Cropscience Aktiengesellschaft | N-cycloalkyl-n-(biheterocyclyethylene)-(thio)carboxamide derivatives |
US11180751B2 (en) | 2015-06-18 | 2021-11-23 | The Broad Institute, Inc. | CRISPR enzymes and systems |
WO2018019676A1 (en) | 2016-07-29 | 2018-02-01 | Bayer Cropscience Aktiengesellschaft | Active compound combinations and methods to protect the propagation material of plants |
WO2018054832A1 (en) | 2016-09-22 | 2018-03-29 | Bayer Cropscience Aktiengesellschaft | Novel triazole derivatives |
WO2018054829A1 (en) | 2016-09-22 | 2018-03-29 | Bayer Cropscience Aktiengesellschaft | Novel triazole derivatives and their use as fungicides |
WO2018054911A1 (en) | 2016-09-23 | 2018-03-29 | Bayer Cropscience Nv | Targeted genome optimization in plants |
WO2018077711A2 (en) | 2016-10-26 | 2018-05-03 | Bayer Cropscience Aktiengesellschaft | Use of pyraziflumid for controlling sclerotinia spp in seed treatment applications |
WO2018104392A1 (en) | 2016-12-08 | 2018-06-14 | Bayer Cropscience Aktiengesellschaft | Use of insecticides for controlling wireworms |
WO2018108627A1 (en) | 2016-12-12 | 2018-06-21 | Bayer Cropscience Aktiengesellschaft | Use of substituted indolinylmethyl sulfonamides, or the salts thereof for increasing the stress tolerance of plants |
EP3332645A1 (en) | 2016-12-12 | 2018-06-13 | Bayer Cropscience AG | Use of substituted pyrimidine diones or their salts as agents to combat abiotic plant stress |
US11591601B2 (en) | 2017-05-05 | 2023-02-28 | The Broad Institute, Inc. | Methods for identification and modification of lncRNA associated with target genotypes and phenotypes |
WO2019025153A1 (en) | 2017-07-31 | 2019-02-07 | Bayer Cropscience Aktiengesellschaft | Use of substituted n-sulfonyl-n'-aryl diaminoalkanes and n-sulfonyl-n'-heteroaryl diaminoalkanes or salts thereof for increasing the stress tolerance in plants |
WO2019060746A1 (en) | 2017-09-21 | 2019-03-28 | The Broad Institute, Inc. | Systems, methods, and compositions for targeted nucleic acid editing |
US10968257B2 (en) | 2018-04-03 | 2021-04-06 | The Broad Institute, Inc. | Target recognition motifs and uses thereof |
US11999767B2 (en) | 2018-04-03 | 2024-06-04 | The Broad Institute, Inc. | Target recognition motifs and uses thereof |
WO2019233863A1 (en) | 2018-06-04 | 2019-12-12 | Bayer Aktiengesellschaft | Herbicidally active bicyclic benzoylpyrazoles |
WO2020131862A1 (en) | 2018-12-17 | 2020-06-25 | The Broad Institute, Inc. | Crispr-associated transposase systems and methods of use thereof |
Also Published As
Publication number | Publication date |
---|---|
PT1725667E (en) | 2010-03-18 |
AU2005229364A1 (en) | 2005-10-13 |
CN1930295A (en) | 2007-03-14 |
US7932436B2 (en) | 2011-04-26 |
JP5812978B2 (en) | 2015-11-17 |
ES2338242T3 (en) | 2010-05-05 |
PL1725667T3 (en) | 2010-06-30 |
AR048024A1 (en) | 2006-03-22 |
US20070163003A1 (en) | 2007-07-12 |
AU2005229364B2 (en) | 2011-04-07 |
SI1725667T1 (en) | 2010-05-31 |
JP2013066481A (en) | 2013-04-18 |
CA2558747C (en) | 2017-06-20 |
ATE455178T1 (en) | 2010-01-15 |
CN1930295B (en) | 2013-11-27 |
DE602005018904D1 (en) | 2010-03-04 |
EP1725667A1 (en) | 2006-11-29 |
JP2007525987A (en) | 2007-09-13 |
CA2558747A1 (en) | 2005-10-13 |
JP5623002B2 (en) | 2014-11-12 |
DK1725667T3 (en) | 2010-05-10 |
EP1725667B1 (en) | 2010-01-13 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
AU2005229364B2 (en) | Plants with increased activity of multiple starch phosphorylating enzymes | |
US9327285B2 (en) | Plants with increased activity of a starch phosphorylating enzyme | |
EP1725666B1 (en) | Plants with reduced activity of the starch phosphorylating enzyme phosphoglucan, water dikinase | |
EP1786908B1 (en) | Plants with increased plastidic activity of r3 starch-phosphorylating enzyme | |
EP1483390A2 (en) | Transgenic plants synthesising high amylose starch |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AK | Designated states |
Kind code of ref document: A1 Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BW BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE EG ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NA NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SM SY TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW |
|
AL | Designated countries for regional patents |
Kind code of ref document: A1 Designated state(s): GM KE LS MW MZ NA SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IS IT LT LU MC NL PL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG |
|
121 | Ep: the epo has been informed by wipo that ep was designated in this application | ||
DPEN | Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed from 20040101) | ||
WWE | Wipo information: entry into national phase |
Ref document number: 2005229364 Country of ref document: AU |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2558747 Country of ref document: CA |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2007163003 Country of ref document: US Ref document number: 10591432 Country of ref document: US |
|
WWE | Wipo information: entry into national phase |
Ref document number: 200580007077.0 Country of ref document: CN Ref document number: 2007501245 Country of ref document: JP |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
WWW | Wipo information: withdrawn in national office |
Country of ref document: DE |
|
ENP | Entry into the national phase |
Ref document number: 2005229364 Country of ref document: AU Date of ref document: 20050304 Kind code of ref document: A |
|
WWP | Wipo information: published in national office |
Ref document number: 2005229364 Country of ref document: AU |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2005715852 Country of ref document: EP |
|
WWP | Wipo information: published in national office |
Ref document number: 2005715852 Country of ref document: EP |
|
WWP | Wipo information: published in national office |
Ref document number: 10591432 Country of ref document: US |