Classifications

• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
  • A01H3/00—Processes for modifying phenotypes, e.g. symbiosis with bacteria
  • A01H3/04—Processes for modifying phenotypes, e.g. symbiosis with bacteria by treatment with chemicals
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
  • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
  • C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
  • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
  • A01H1/02—Methods or apparatus for hybridisation; Artificial pollination ; Fertility
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
  • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
  • A01H1/04—Processes of selection involving genotypic or phenotypic markers; Methods of using phenotypic markers for selection
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N25/00—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests
  • A01N25/32—Ingredients for reducing the noxious effect of the active substances to organisms other than pests, e.g. toxicity reducing compositions, self-destructing compositions
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
  • A01N37/18—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing the group —CO—N<, e.g. carboxylic acid amides or imides; Thio analogues thereof
  • A01N37/28—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing the group —CO—N<, e.g. carboxylic acid amides or imides; Thio analogues thereof containing the group; Thio analogues thereof
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N41/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a sulfur atom bound to a hetero atom
  • A01N41/02—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a sulfur atom bound to a hetero atom containing a sulfur-to-oxygen double bond
  • A01N41/04—Sulfonic acids; Derivatives thereof
  • A01N41/06—Sulfonic acid amides
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
  • A01N43/48—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with two nitrogen atoms as the only ring hetero atoms
  • A01N43/56—1,2-Diazoles; Hydrogenated 1,2-diazoles
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
  • A01N43/72—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with nitrogen atoms and oxygen or sulfur atoms as ring hetero atoms
  • A01N43/80—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with nitrogen atoms and oxygen or sulfur atoms as ring hetero atoms five-membered rings with one nitrogen atom and either one oxygen atom or one sulfur atom in positions 1,2
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N61/00—Biocides, pest repellants or attractants, or plant growth regulators containing substances of unknown or undetermined composition, e.g. substances characterised only by the mode of action
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/158—Expression markers

Definitions

• the invention relates to a method of finding compounds which increase the tolerance of plants to abiotic stress factors acting on this plant, such as, for example, temperature (such as chill, frost or heat), water (such as dryness, drought or anoxia), or the chemical load (such as lack of or excess of mineral salts, heavy metals, gaseous noxious substances) by increasing the expression of plant-endogenous proteins, and to the use of these compounds for increasing the stress defense in plants to abiotic stress factors.
• abiotic stress factors acting on this plant such as, for example, temperature (such as chill, frost or heat), water (such as dryness, drought or anoxia), or the chemical load (such as lack of or excess of mineral salts, heavy metals, gaseous noxious substances) by increasing the expression of plant-endogenous proteins, and to the use of these compounds for increasing the stress defense in plants to abiotic stress factors.
• abiotic stress for example chill, heat, drought, salt
• Some of them belong to signal transduction chains (for example transcription factors, kinases, phosphatases) or they bring about a physiological response of the plant cell (for example ion transport, detoxification of reactive oxygen species).
• the signal chain genes of the abiotic stress reaction include, inter alia, transcription factors of classes DREB and CBF (Jaglo-Ottosen et al., 1998, Science 280: 104-106).
• Phosphatases of the ATPK and MP2C type are involved in the salt stress reaction.
• HSFs Heat shock factors
• HSPs heat shock proteins
• antioxidants such as, for example, naphthols and xanthins for increasing the abiotic stress tolerance in plants has also already been described (Bergmann et al., DD-277832, Bergmann et al., DD-277835).
• the molecular causes of the anti-stress effect of the substances are largely unknown.
• cDNA complementary DNA
• RNA complementary DNA
• cDNA can correspond either to the total length of the RNA or else only constitute a partial-sequence of the RNA which acts as template.
• cluster analysis means the summary of the individual data obtained by means of a computer program developed for this purpose, where groups of genes which code for proteins with a similar function, or else genes with a similar expression pattern, are shown in a conclusive fashion. This results in a hierarchic minimization of the complex data pattern which can be shown in the form of a dendrogram.
• the cluster analysis makes possible the classifying assessment of the data sets obtained, which markedly exceeds a mere accumulation of unrelated data.
• DNA chip and “DNA microarray”, which are used synonymously in this context, refer to a support whose matrix consists for example of glass or nylon and whose matrix has DNA fragments fixed to it, where the attachment of the DNA can be effected for example by (a) a photolithographic method (DNA is synthetized directly on the support of the array), (b) a microspotting method (extraneously synthesized oligonucleotides or PCR products are applied to the support and bounded covalently), or (c) by a microspraying method (extraneously synthesized oligonucleotides or PCR products are sprayed onto the support without touching, using an ink-jet printer) (R.
• a photolithographic method DNA is synthetized directly on the support of the array
• a microspotting method extraneously synthesized oligonucleotides or PCR products are applied to the support and bounded covalently
• a microspraying method extraneously synthe
• a DNA chip which represents genomic sequences of an organism is referred to as a “genomic DNA chip”.
• the evaluation of the data obtained with these “DNA chips” is referred to as “DNA chip analysis”.
• DNA chip hybridization means the pairing of two single-stranded, complementary nucleic acid molecules, where one of the base-pairing molecule partners is located on the DNA chip as DNA (deoxyribonucleic acid) in preferably covalently bonded form, while the other is in solution in the form of the RNA (ribonucleic acid) or the corresponding cDNA (complementary DNA).
• DNA deoxyribonucleic acid
• RNA ribonucleic acid
• corresponding cDNA complementary DNA
• the hybridization conditions can be established in a constant fashion for example in a hybridization oven. Standard movements of 60 rpm (rounds per minute, revolutions per minute) are produced in such a hybridization oven.
• EST sequence expressed sequence tag
• expression pattern induction pattern
• expression profile which are used synonymously describe, in the present context, the expression differentiated over time and/or the tissue-specific expression of the plant mRNA, the pattern being obtained directly by the generated intensity of the hybridization signal of the RNA obtained from the plant or its corresponding cDNA with the aid of the DNA chip technology.
• the measured “induction values” are obtained by direct numerical processing with the corresponding signals which are obtained by using a synonymous chip, with the hybridization with an untreated/stressed control plant.
• expression state which is obtained by the “gene expression profiling” which has been carried out describes, in the present context, all of the recorded transcriptional activity of cellular genes which is measured with the aid of a DNA chip.
• total RNA describes the representation, which is possible as the result of the disruption method applied, of different plant-endogenous RNA groups which can be present in a plant cell, such as, for example, cytoplasmic rRNA (ribosomal RNA), cytoplasmic tRNA (transfer RNA), cytoplasmic mRNA (messenger RNA) and their respective nuclear precursors, ctRNA (chloroplastidial RNA) and mtRNA (mitochondrial RNA), but it also comprises RNA molecules which can be obtained from exogenous organisms, such as, for example, viruses, or from parasitic bacteria and fungi.
• cytoplasmic rRNA ribosomal RNA
• cytoplasmic tRNA transfer RNA
• cytoplasmic mRNA messenger RNA
• ctRNA chloroplastidial RNA
• mtRNA mitochondrial RNA
• useful plants means, in the present context, crop plants which are employed as plants for obtaining foodstuffs, feed stuffs or for industrial purposes.
• safety refers to a chemical compound which is of non-plant-endogenous origin and which compensates for, or reduces, the phytotoxic properties of a pesticide in relation to useful plants, without substantially reducing the pesticidal activity in relation to harmful organisms such as, for example, weeds, bacteria, viruses and fungi.
• Safeners which, in addition to their function for which they are known per se, also contribute to increasing the tolerance to abiotic stress factors are preferably selected from the group defined hereinbelow, it being possible to select different safeners depending on the abiotic stress factor, and it being possible to use only a single safener or else a plurality of safeners from the same group or from different groups:
• the compounds of the formula (I) are disclosed for example in EP-A-0 333 131 (ZA-89/1960), EP-A-0 269 806 (U.S. Pat. No. 4,891,057), EP-A-0 346 620 (AU-A-89/34951), EP-A-0 174 562, EP-A-0 346 620 (WO-A-91108 202), WO-A-91107 874 or WO-A 95/07 897 (ZA 94/7120) and the literature cited therein or can be prepared by, or in analogy with, the processes described therein.
• EP-A-0 086 750 discloses the compounds of the formula (II) for example in EP-A-0 086 750, EP-A-0 94349 (U.S. Pat. No. 4,902,340), EP-A-0 191736 (U.S. Pat. No. 4,881,966) and EP-A-0 492 366 and the literature cited therein or can be prepared by, or in analogy with, the processes described therein. Some compounds are furthermore described in EP-A-0 582 198 and WO 2002/34048.
• the compounds of group (c) are described in WO-A-97/45016, those of group (d) in WO-A-99/16744, those of group B (e) in EP-A-365484 and those of group (g) in EP-A-1019368.
• Preferred compounds of the formula (I) and/or (II) which are known as safeners are those in which the symbols and indices have the following meanings:
• Preferred compounds of the formula (VI) which are known as safeners are (S3-1), (S3-2), (S3-3), (S3-4) and (S3-5).
• preferred compounds are those of the formulae VIII-1 to VIII-4 of which, in turn, the compound VIII-3 (4-cyclopropylaminocarbonyl-N-(2-methoxybenzoyl)benzenesulfonamide) is very especially preferred for use as agent for increasing the tolerance in plants to abiotic stress factors.
• Especially preferred compounds for use as agents for increasing the tolerance in plants to abiotic stress factors are those which are selected from the group of compounds known as safeners which consists of the compounds of the formulae I-1 (mefenpyr-diethyl), I-9 (isoxadifen-ethyl), II-1 (chloquintocet-mexyl), b-11 (fenclorim), b-14 (dymron), and VIII-3 (4-cyclopropylaminocarbonyl-N-(2-methoxybenzoyl)benzenesulfonamide, with the compounds I-1 and VIII-3 being very especially preferred).
• safeners which consists of the compounds of the formulae I-1 (mefenpyr-diethyl), I-9 (isoxadifen-ethyl), II-1 (chloquintocet-mexyl), b-11 (fenclorim), b-14 (dymron), and VIII-3 (4-cyclopropylaminocarbonyl-
• the genetically modified plants are, as a rule, distinguished by particular advantageous properties, for example by resistances to certain pesticides, especially certain herbicides, resistances to plant diseases or pathogen agents of plant diseases, such as certain insects or microorganisms such as fungi, bacteria or viruses.
• Other particular properties concern for example the harvested material with regard to quantity, quality, storage ability, composition and specific constituents.
• transgenic plants with an increased starch content or a modified starch quality, or those where the harvested material has a different fatty acid composition.
• cereals such as wheat, barley, rye, oats, sorghum and millet
• rice and maize or else crops of sugar beet, cotton, soya, oilseed rape, potato, tomato, pea and other vegetables, especially preferably in crops of maize, wheat, barley,
• transgenic plants can also be treated with substances identified with the aid of DNA microarrays, such as the molecules which are already known as safeners, whose tolerance to abiotic stress factors has already been increased as the result of recombinant methods, so that a synergistic effect of the endogenously encoded tolerance and the extraneously applied tolerance-increasing effect is observed.
• substances identified with the aid of DNA microarrays such as the molecules which are already known as safeners, whose tolerance to abiotic stress factors has already been increased as the result of recombinant methods, so that a synergistic effect of the endogenously encoded tolerance and the extraneously applied tolerance-increasing effect is observed.
• novel plants with modified properties can be generated with the aid of recombinant methods (see, for example, EP-A-0221044, EP-A-0131624). The following have been described in a plurality of cases: for example
• nucleic acid molecules can be introduced into plasmids which permit a mutagenesis or a sequence modification by means of the recombination of DNA sequences.
• base substitutions it is possible for example to carry out base substitutions, to remove part-sequences or to add natural or synthetic sequences.
• adapters or linkers can be added to the fragments.
• the generation of plant cells with a reduced activity of a gene product can be accomplished for example by expressing at least one suitable antisense RNA, a sense RNA for achieving a cosuppression effect or the expression of at least one suitably constructed ribosyme which specifically cleaves transcripts of the abovementioned gene product.
• DNA molecules which comprise all of the coding sequence of a gene product including any flanking sequences which are present or else DNA molecules which only comprise parts of the coding sequence, it being necessary for these parts to be long enough in order to bring about an antisense effect in the cells.
• DNA sequences which have a high degree of homology with the coding sequences of a gene product, but which are not completely identical are also possible.
• the protein synthetized can be located in any compartment of the plant cell.
• sequences are known to a person skilled in the art (see, for example, Braun et al., EMBO J. 11 (1992), 3219-3227; Wolter et al., Proc. Natl. Acad. Sci. USA 85 (1988), 846-850; Sonnewald et al., Plant J. 1 (1991), 95-106).
• the transgenic plant cells can be regenerated to give intact plants, using known techniques.
• the transgenic plants can, in principle, take the form of plants of any plant species, i.e. both monocots and dicots.
• the molecules which have been identified with the aid of the DNA microarrays or which are known as safeners can be employed in transgenic crops which are resistant to herbicides from the group of the sulfonylureas, glufosinate-ammonium or glyphosate-isopropylammonium and analogous active substances and/or which have, as the result of recombinant modification, an endogenous tolerance to abiotic stress factors.
• effects which are specific for the application in the transgenic crop in question are frequently observed in addition to the effects against harmful plants which can be observed in other crops; for example a modified or specifically widened weed spectrum which can be controlled, modification application rates which can be used for the application, preferably good combining ability with the herbicides to which the transgenic crop is resistant, and influencing the growth and yield of the transgenic crop plants.
• the invention therefore also relates to the use of the compounds identified with DNA microarrays, or of compounds which are already known as safeners, for increasing the tolerance to abiotic stress factors in transgenic crop plants, preferably with the purpose of increasing the yield.
• the present invention relates to a method of finding a compound which increases the tolerance to abiotic stress factors in plants, the increase in the transcription or expression of individual or more plant-endogenous genes, such as, for example, genes coding for proteins from the group of the cytochrome oxidases, such as cytochrome oxidase P450, glycosyltransferases, uricases, such as uricase II (E.C.17.3.3), peptidases, different membrane proteins, amidohydrolases and various general stress proteins, being regarded as proof for the induction.
• cytochrome oxidases such as cytochrome oxidase P450, glycosyltransferases, uricases, such as uricase II (E.C.17.3.3)
• uricases such as uricase II (E.C.17.3.3)
• the present invention particularly relates to a method of finding compounds which induce the transcription of the genes which code for plant-endogenous stress tolerance enzymes, which comprises:
• the enzymatic transcription of the resulting cDNA into a cRNA must be considered as the preferred process step since a further amplification of the hybridization sample can thereby be achieved.
• labeling by means of cold nucleotides, especially preferably labeling by means of biotinylated UTP and/or CTP, where the detection is carried out after the hybridization reaction by binding streptavidin-phycoerythrin as fluorophore to the biotinylated cRNA.
• a detection of the specific phycoerythrin fluorescence which serves as the base for the quantitative determination of the expression differentials measured, is carried out after the hybridization step, with the aid of a laser scanner.
• the present invention preferably relates to a process in which the abovementioned procedures a)-h) are maintained, where, in the case of the intended increase in the case of heat stress, the genes for the expression of the cytochrome oxidases, such as cytochrome oxidase P450, glycosyltransferases, uricases, such as uricase II (E.C.17.3.3), peptidases, different membrane proteins, amidohydrolases in the case of heat-stressed and non-heat-stressed plants is compared, preferably of the genes for the expression of “N-carbamyl-L-amino acid amidohydrolase” (Zm.11840.1.A1_at), of “serine carboxypeptidase (Zm.18994.2.A1_a_at), of uricase II (E.C.1.7.3.3) and of glycosyltransferase (Zm.12587.1.S1_s_at), very especially
• the present invention also preferably relates to a process in which the abovementioned process steps a)-h) are maintained, where, in the case of the intended increase in the case of drought stress for example the genes for the expression of the late embryogenesis abundant proteins such as the dehydrins, of the universal stress protein (Zm.818.1.A1_at), non-symbiotic hemoglobin (Zm.485.1.A1_at), the protein which is addressed as “Zm.818.2.A1_a_at” (maize genome array from Affymetrix (Affymetrix Inc., 3380 Central Expressway, Santa Clara, Calif., USA)) and of the protein addressed as “Zm.18682.1.A1_s_at” (maize genome array from Affymetrix (Affymetrix Inc., 3380 Central Expressway, Santa Clara, Calif., USA)) of drought-stressed and non-drought-stressed plants is compared, preferably the genes for the expression of the universal stress protein (
• the present invention furthermore relates to the use of certain DNA microarrays which are used on the basis of genetic information from plants, preferably genetic information from useful plants, especially preferably from useful plants such as, for example, from maize, cereals such as wheat, barley, rye, oats, rice and soya, preferably from maize, wheat, barley, rye, rice and soya, especially preferably from barley, maize, wheat, rice and soya, very especially preferably from maize, wheat and soya, for finding modified gene expression patterns.
• useful plants preferably genetic information from useful plants, especially preferably from useful plants such as, for example, from maize, cereals such as wheat, barley, rye, oats, rice and soya, preferably from maize, wheat, barley, rye, rice and soya, especially preferably from barley, maize, wheat, rice and soya, very especially preferably from maize, wheat and soya, for finding modified gene expression patterns.
• useful plants especially
• the invention furthermore relates to the use of the promoters of the indicator genes described in conjunction with specific reporter genes (for example GUS, GFP, luciferase and the like) for finding substances which have a positive effect on the abiotic stress tolerance in crop plants.
• reporter genes for example GUS, GFP, luciferase and the like
• transgenic test plants are generated which comprise the abovementioned promoter/reporter gene constructs. Active substances which increase the abiotic stress tolerance of plants by the above-described mechanism induce the expression of the reporter gene and can be identified with the aid of a calorimetric, fluorimetric or other suitable assay.
• the invention furthermore relates to the use of the described indicator genes for increasing the abiotic stress tolerance in transgenic crop plants.
• the genes are fused with a suitable promoter which has the desired strength and specificity, and the constructs are transformed into monocotyledonous or dicotyledonous crop plants.
• the resulting transgenic plants are distinguished by an increased tolerance to abiotic stress, for example chill, heat, drought and the like.
• the present invention furthermore also relates to the use of the compounds which have been identified with the aid of the DNA microarray taking into consideration the expression profiles of the genes and/or of compounds which are already known as safeners and which, in the case of abiotic stress conditions such as, for example, abiotic stress factors which act on this plant, such as temperature (chill, frost or heat), water (dryness or drought) or the chemical load (lack or excess of mineral salts, heavy metals, gaseous noxious substances), have a positive effect, i.e.
• abiotic stress conditions such as, for example, abiotic stress factors which act on this plant, such as temperature (chill, frost or heat), water (dryness or drought) or the chemical load (lack or excess of mineral salts, heavy metals, gaseous noxious substances), have a positive effect, i.e.
• an expression-enhancing effect with regard to their inductive effect on single genes or a plurality of genes of the plant-endogenous defense mechanisms, such as, for example, in the case of heat stress on cytochrome oxidases such as cytochrome oxidase P450, on glycosyltransferases, on uricases such as uricase II (E.C.17.3.3), on peptidases, on different membrane proteins, on amidohydrolases and/or various stress proteins, and/or for example in the case of drought stress have a positive effect, i.e.
• non-symbiotic hemoglobin Zm.485.1.A1_at
• Zm.818.2.A1_a_at maize genome array from Affymetrix (Affymetrix Inc., 3380 Central Expressway, Santa Clara, Calif., USA)
• Zm. 18682.1.A1_s_at signature according to maize genome array from Affymetrix (Affymetrix Inc., 3380 Central Expressway, Santa Clara, Calif., USA)
• the invention also relates to the use of substances identified with the aid of the DNA microarray and of the molecules which are already known as safeners for increasing the tolerance to abiotic stress factors in various crop plants such as maize, cereals such as wheat, barley, rye, oats, rice and soya, preferably maize, wheat, barley, rye, rice and soya, especially preferably maize, wheat, rice and soya, very especially preferably maize, wheat and soya.
• the present invention therefore also relates to the use of the compounds which have been identified with the aid of the DNA microarray taking into consideration the expression profiles of the genes and/or of compounds which are already known as safeners which, in plants, directly or indirectly, for example via a signal transduction chain, contribute to increasing the tolerance to abiotic stress factors, such as, for example, temperature (such as chill, frost or heat), water (such as dryness, drought or anoxia), or the chemical load (such as lack or excess of mineral salts, heavy metals, gaseous noxious substances), for increasing the yield, for extending the vegetation period, for making possible an earlier sowing date, for increasing the quality, or for use in plant breeding using otherwise less vital inbred lines.
• abiotic stress factors such as, for example, temperature (such as chill, frost or heat), water (such as dryness, drought or anoxia), or the chemical load (such as lack or excess of mineral salts, heavy metals, gaseous noxious substances), for increasing the yield, for extending the vegetation period, for
• the present invention therefore also relates to a method of increasing the yield in crops of useful plants, for extending the vegetation period, for making possible an earlier sowing date, for increasing the quality, or for use in plant breeding using otherwise less vital inbred lines which comprises treating the useful plants by seed dressing, by foliar sprays or by cell application with one or more compounds which have been identified with the aid of the DNA microarray and/or compounds which are already known as safeners.
• Preferred in this context are those compounds whose use as what are known as safeners is already known in crop protection, such as, for example, from the group of the compounds known as safeners consisting of the compounds of the formulae I-1 (mefenpyr-diethyl), I-9 (isoxadifen-ethyl), II-1 (chloquintocet-mexyl), b-11 (fenclorim), b-14 (dymron), VIII-3 (4-cyclopropylaminocarbonyl-N-(2-methoxybenzoyl)benzenesulfonamide), very especially preferably the compounds I-1 and VIII-3 (4-cyclopropylaminocarbonyl-N-(2-methoxybenzoyl)benzenesulfonamide)).
• the present invention therefore also relates to a method of increasing the tolerance of useful plants in crops of useful plants to abiotic stress factors by the individual or combined application of the compounds identified with the aid of the DNA microarray taking into consideration the expression profiles of the genes and/or of compounds which are already known as safeners.
• the treated seeds were placed into pots with compost (diameter: 10 cm, in each case 10 seeds per pot), and the maize seedlings were raised for 10 days in a controlled-environment chamber under defined light, moisture and temperature conditions [white light, long day (16 hours light, 8 hours dark), 70% atmospheric humidity, 24° C.].
• 2 ⁇ 10 pots were used for the control groups and for the drought stress experiment. While the plants were raised, they were watered from below for 20 minutes every 2 days by raising the water level in a tray. 10 days after the seeds had germinated, the maize plants were exposed to the drought stress.
• control group 1 without dressing with active substance
• test group 2 without dressing with active substance
• test group 2 with dressing with active substance
• the normal irrigation regime was reclaimed.
• the experiment was evaluated as follows. The aerial plant parts were cut off and dried overnight at 50° C. On the next day, the foliar biomass was determined in [g] (dry matter) per pot.
• the seedlings were raised for 10 days in the controlled-environment chamber under defined conditions, likewise as described in example 1.2 ⁇ 10 pots with maize plants were used for the heat stress experiment.
• the control group consisted of undressed plants (solvent), the test group of plants which had been dressed with active substance.
• both plant groups were placed for 2 days into a controlled-environment cabinet at 45° C., white light, long day (16 hours light, 8 hours dark) and 70% atmospheric humidity.
• the plants were irrigated once per day from below by raising the water level in a tray. After the heat stress, it was observed that—especially in the control group—the shoots of many plants had collapsed and that the leaves were lying flat on the ground.
• the experiment was valued quantitatively, taking into consideration the following criteria.
• Abiotic Stress Factor Chill Stress (Greenhouse).
• One control plant contains untreated seed. Approximately 8 days after the seed emerged, the seedlings were in the one-leaf stage and were exposed for 5 days to the following temperature conditions: Maximum: Minimum: Day 1: 16.1° C. 7.2° C. Day 2: 17.8° C. 2.7° C. Day 3: 16.7° C. 0.6° C. Day 4: 16.7° C. 1.1° C. Day 5: 22.8° C. 12.2° C.
• test plots were scored. For this purpose, all plants were assessed individually, and plants with at least 20% chill symptoms based on the total leaf area (burns and/or chloroses) were considered to be damaged.
• control plants of the specific experimental group were kept under the standard conditions described in example 1 (temperature, irrigation).
• RNA probes for the DNA chip hybridization were prepared as described in the protocols (Expression Analysis, Technical Manual) from Affymetrix (Affymetrix Inc., 3380 Central Expressway, Santa Clara, Calif., USA). First, total RNA was isolated from in each case 500 mg of the harvested leaves. In each case 10 ⁇ g of total RNA were used for the cDNA first- and second-strand synthesis. The cDNA was amplified with T7 polymerase and simultaneously labeled with biotin-UTP. In each case 20 ⁇ g of this biotinylated cDNA were employed for the hybridization of the maize genome array from Affymetrix. This DNA microarray contains DNA sequences whose totality represents 13339 genes.
• the DNA microarrays were washed in the Affymetrix Fluidics Station, stained with streptavidin/phycoerythrin (Molecular Probes, P/N S-866) and scanned with the appropriate Agilent Laser Scanner (Agilent Gene Array Scanner).
• the fluorescence data obtained were analyzed using Affymetrix's Microarray Suite 5 software. After the quality assurance had been performed, all DNA chip analyses were stored in a database. To determine relevant expression values (induction factors, repression factors), the absolute expression values of the genes from the respective stress experiments were compared with the values of the respective control experiments (i.e. without abiotic stress and solvent-dressing only), based on the scoring function predetermined by the Affymetrix software.
• the respective sample set no. corresponds to:
• test compound VIII-3 was tested on its own, i.e. without heat stress, the measured expression levels were in the range of the range induced by heat stress, or below or slightly above the range induced by heat stress.
• the respective sample set no. corresponds to:
• the induction patterns derived from table 6 and which are shown directly by the resulting expression values show characteristic inductions in the preence of the compound 4-cyclopropylaminocarbonyl-N-(2-methoxybenzoyl)benzenesulfonamide, the effect on the universal stress protein [Zm.818.1A1_at] and non-symbiotic hemoglobin (HBT) (ZEAMP GLB1) [Zm.485.1A1_at] being most pronounced.

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