Classifications

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  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
  • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
  • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
  • C12N15/8274—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for herbicide resistance
• • 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
  • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
  • A01H6/02—Amaranthaceae or Chenopodiaceae, e.g. beet or spinach
  • A01H6/024—Beta vulgaris [beet]
• • 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
  • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
  • A01H6/04—Amaryllidaceae, e.g. onion
  • A01H6/045—Allium cepa [onion]
• • 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
  • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
  • A01H6/14—Asteraceae or Compositae, e.g. safflower, sunflower, artichoke or lettuce
  • A01H6/1464—Helianthus annuus [sunflower]
• • 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
  • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
  • A01H6/34—Cucurbitaceae, e.g. bitter melon, cucumber or watermelon 
  • A01H6/348—Cucurbita, e.g. squash or pumpkin
• • 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
  • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
  • A01H6/46—Gramineae or Poaceae, e.g. ryegrass, rice, wheat or maize
  • A01H6/4636—Oryza sp. [rice]
• • 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
  • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
  • A01H6/54—Leguminosae or Fabaceae, e.g. soybean, alfalfa or peanut
  • A01H6/542—Glycine max [soybean]
• • 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
  • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
  • A01H6/74—Rosaceae, e.g. strawberry, apple, almonds, pear, rose, blackberries or raspberries
  • A01H6/7409—Fragaria, i.e. strawberries
• • 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
  • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
  • A01H6/82—Solanaceae, e.g. pepper, tobacco, potato, tomato or eggplant
  • A01H6/825—Solanum lycopersicum [tomato]
• • 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
  • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
  • A01H6/82—Solanaceae, e.g. pepper, tobacco, potato, tomato or eggplant
  • A01H6/827—Solanum tuberosum [potato]
• • 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
  • A01N43/54—1,3-Diazines; Hydrogenated 1,3-diazines
• • 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
  • A01N43/84—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 six-membered rings with one nitrogen atom and either one oxygen atom or one sulfur atom in positions 1,4
• • 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
  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/10—Cells modified by introduction of foreign genetic material
  • C12N5/12—Fused cells, e.g. hybridomas
  • C12N5/14—Plant cells
• • 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
  • C12N9/0004—Oxidoreductases (1.)
  • C12N9/001—Oxidoreductases (1.) acting on the CH-CH group of donors (1.3)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y103/00—Oxidoreductases acting on the CH-CH group of donors (1.3)
  • C12Y103/03—Oxidoreductases acting on the CH-CH group of donors (1.3) with oxygen as acceptor (1.3.3)
  • C12Y103/03004—Protoporphyrinogen oxidase (1.3.3.4)

Definitions

• the present invention relates in general to methods for conferring on plants agricultural level tolerance to a herbicide.
• the invention refers to plants having an increased tolerance to PPO-inhibiting herbicides.
• the present invention relates to methods and plants obtained by mutagenesis and cross-breeding and transformation that have an increased tolerance to PPO-inhibiting herbicides.
• Protox Herbicides that inhibit protoporphyrinogen oxidase (hereinafter referred to as Protox or PPO;
• PPO catalyzes the last common step in chlorophyll and heme biosynthesis which is the oxidation of protoporphyrinogen IX to protoporphyrin IX.
• PPO-inhibiting herbicides include many different structural classes of molecules (Duke et al. 1991. Weed Sci. 39: 465; Nandihalli et al. 1992. Pesticide Biochem. Physiol. 43: 193; Matringe et al. 1989. FEBS Lett.
• herbicidal compounds include the diphenylethers ⁇ e.g. lactofen, (+-)- 2-ethoxy-1 -methyl-2-oxoethyl 5- ⁇ 2-chloro-4-(trifluoromethyl)phenoxy ⁇ -2-nitrobenzoate; acifluorfen, 5- ⁇ 2-chloro-4-(trifluoromethyl)phenoxy ⁇ -2-nitrobenzoic acid; its methyl ester; or oxyfluorfen, 2- chloro-1 -(3-ethoxy-4-nitrophenoxy)-4-(trifluorobenzene) ⁇ , oxidiazoles, (e.g.
• oxidiazon 3- ⁇ 2,4- dichloro-5-(1 -methylethoxy)phenyl ⁇ -5-(1 , 1 -dimethylethyl)-1 ,3,4-oxadiazol-2-(3H)-one
• cyclic imides e.g. S-23142, N-(4-chloro-2-fluoro-5-propargyloxyphenyl)-3,4,5,6-tetrahydrophthalimide; chlo- rophthalim, N-(4-chlorophenyl)-3,4,5,6-tetrahydrophthalimide
• phenyl pyrazoles e.g.
• protoporphyrinogen IX in the chloroplast and mitochondria, which is believed to leak into the cytosol where it is oxidized by a peroxidase.
• protoporphyrin IX When exposed to light, protoporphyrin IX causes formation of singlet oxygen in the cytosol and the formation of other reactive oxygen species, which can cause lipid peroxidation and membrane disruption leading to rapid cell death (Lee et al. 1993. Plant Physiol. 102: 881 ).
• US5,767,373 or US5,939,602, and patent family members thereof. discloses nucleotide sequences encoding amino acid sequences having enzymatic activity such that the amino acid sequences are resistant to PPO inhibitor herbicidal chemicals, in particular 3-phenyluracil inhibitor specific PPO mutants.
• the problem is solved by the present invention which refers to a method for controlling undesired vegetation at a plant cultivation site, the method comprising the steps of:
• PPO protoporphyrinogen oxidase
• mut-PPO mutated protoporphy- rinogen oxidase
• the present invention refers to a method for identifying a PPO-inhibiting herbicide by using a wild-type or mut-PPO of the present invention encoded by a nucleic acid which comprises the nucleotide sequence of SEQ ID NO: 1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, 19, 21 , 23, 25, 27, 29, 31 , 33, 35, 37, 39, 41 , 43, or 45, or a variant thereof.
• Said method comprises the steps of:
• test compounds which confer reduced growth to the control cell or plant as compared to the growth of the transgenic cell or plant.
• Another object refers to a method of identifying a nucleotide sequence encoding a mut-PPO which is resistant or tolerant to a PPO-inhibiting herbicide, the method comprising:
• the mut-PPO-encoding nucleic acid selected in step d) provides at least 2-fold as much tolerance to a PPO-inhibiting herbicide as compared to that provided by the control PPO-encoding nucleic acid.
• the resistance or tolerance can be determined by generating a transgenic plant comprising a nu- cleic acid sequence of the library of step a) and comparing said transgenic plant with a control plant.
• Another object refers to a method of identifying a plant or algae containing a nucleic acid encoding a mut-PPO which is resistant or tolerant to a PPO-inhibiting herbicide, the method comprising: a) identifying an effective amount of a PPO-inhibiting herbicide in a culture of plant cells or green algae.
• the mutagenizing agent is ethylmethanesulfonate.
• Another object refers to an isolated nucleic acid encoding a mut-PPO, the nucleic acid comprising the sequence of SEQ ID NO: 1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, 19, 21 , 23, 25, 27, 29, 31 , 33, 35, 37, 39, 41 , 43, or 45, or a variant thereof, as defined hereinafter.
• Another object refers to an isolated mut-PPO polypeptide, the polypeptide comprising the sequence set forth in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, or 46, a variant, derivative, orthologue, paralogue or homologue thereof, as defined hereinafter.
• the nucleic acid being identifiable by a method as defined above.
• the invention refers to a plant cell transformed by and expressing a wild- type or a mut-PPO nucleic acid according to the present invention or a plant which has been mu- tated to obtain a plant expressing, preferably over-expressing a wild-type or a mut-PPO nucleic acid according to the present invention, wherein expression of said nucleic acid in the plant cell results in increased resistance or tolerance to a PPO-inhibiting herbicide as compared to a wild type variety of the plant cell.
• the invention refers to a plant comprising a plant cell according to the present invention, wherein expression of the nucleic acid in the plant results in the plant's increased resistance to PPO-inhibiting herbicide as compared to a wild type variety of the plant.
• the plants of the present invention can be transgenic or non-transgenic.
• the expression of the nucleic acid of the invention in the plant results in the plant's increased resistance to PPO-inhibiting herbicides as compared to a wild type variety of the plant.
• the invention refers to a seed produced by a transgenic plant comprising a plant cell of the present invention, wherein the seed is true breeding for an increased resistance to a PPO-inhibiting herbicide as compared to a wild type variety of the seed.
• the invention refers to a method of producing a transgenic plant cell with an increased resistance to a PPO-inhibiting herbicide as compared to a wild type variety of the plant cell comprising, transforming the plant cell with an expression cassette comprising a wild-type or a mut-PPO nucleic acid.
• the invention refers to a method of producing a transgenic plant comprising, (a) transforming a plant cell with an expression cassette comprising a wild-type or a mut-PPO nucleic acid, and (b) generating a plant with an increased resistance to PPO-inhibiting herbicide from the plant cell.
• the expression cassette further comprises a transcription initiation regulatory region and a translation initiation regulatory region that are functional in the plant.
• the invention relates to using the mut-PPO of the invention as selectable marker.
• the invention provides a method of identifying or selecting a transformed plant cell, plant tissue, plant or part thereof comprising a) providing a transformed plant cell, plant tissue, plant or part thereof, wherein said transformed plant cell, plant tissue, plant or part thereof comprises an isolated nucleic acid encoding a mut-PPO polypeptide of the invention as described hereinafter, wherein the polypeptide is used as a selection marker, and wherein said transformed plant cell, plant tissue, plant or part thereof may optionally comprise a further isolated nucleic acid of interest; b) contacting the transformed plant cell, plant tissue, plant or part thereof with at least one PPO- inhibiting inhibiting compound; c) determining whether the plant cell, plant tissue, plant or part thereof is affected by the inhibitor or inhibiting compound; and d) identifying or selecting the transformed plant cell, plant tissue, plant or part thereof.
• the invention is also embodied in purified mut-PPO proteins that contain the mutations described herein, which are useful in molecular modeling studies to design further improvements to herbicide tolerance.
• Methods of protein purification are well known, and can be readily accomplished using commercially available products or specially designed methods, as set forth for example, in Protein Biotechnology, Walsh and Headon (Wiley, 1994). BRIEF DESCRIPTION OF THE DRAWINGS
• Figure 1 shows an amino acid sequence alignment of Amaranthus tuberculatus (A.tuberculatus), Amaranthus tuberculatus resistant (A.tuberculatus_R), Arabidopsis thaliana long (A.thaliana_2), Spinacia oleracea short (S.oleracea_2), Nicotiana tabacum short (N.tabacum_2), Glycine max (Glycine_max), Arabidopsis thaliana short (A.thaliana_1 ), Nicotiana tabacum long (N.tabacum_1 ), Chlamydomonas reinhardtii long (C.reinhardtiM ), Zea mays (Z.mays), Oryza sativa (0.sativa_1 ), Solanum tuberosum (S.
• Figure 2 shows the selection of Chlamydomonas reinhardtii strains resistant to PPO-inhibiting herbicide 1 ,5-dimethyl-6-thioxo-3-(2,2,7-trifluoro-3-oxo-4-(prop-2-ynyl)-3,4-dihydro-2H- benzo[b][1 ,4]oxazin-6-yl)-1 ,3,5-triazinane-2,4-dione (CAS 1258836-72-4) .
• A Mutagenized cells plated on solid medium without a selecting agent.
• Figure 3 shows growth-characteristics of selected Chlamydomonas reinhardtii strains as seen in Figure 2, resistant to PPO-inhibiting herbicide 1 ,5-dimethyl-6-thioxo-3-(2,2,7-trifluoro-3-oxo-4- (prop-2-ynyl)-3,4-dihydro-2H-benzo[b][1 ,4]oxazin-6-yl)-1 ,3,5-triazinane-2,4-dione (CAS 1258836- 72-4) .
• Figure 4 shows wild type and transgenic T1 soybean plants treated with the indicated spray rate (g ai/ha) of PPO inhibiting herbicides with 1 % MSO.
• A means wild-type soybean plant
• B means soybean plant transformed with a nucleic acid encoding a mut-PPO SEQ ID NO 2, wherein the Leucin at position 397 is substituted by Aspartic acid and the phenylalanin at position 420 is substituted by Valine.
• the present invention refers to a method for controlling undesired vegetation at a plant cultivation site, the method comprising the steps of:
• control of undesired vegetation is to be understood as meaning the killing of weeds and/or otherwise retarding or inhibiting the normal growth of the weeds. Weeds, in the broadest sense, are understood as meaning all those plants which grow in locations where they are undesired.
• the weeds of the present invention include, for example, dicotyledonous and monocotyle- donous weeds.
• Dicotyledonous weeds include, but are not limited to, weeds of the genera: Sinapis, Lepidium, Galium, Stellaria, Matricaria, Anthemis, Galinsoga, Chenopodium, Urtica, Senecio, Ama- ranthus, Portulaca, Xanthium, Convolvulus, Ipomoea, Polygonum, Sesbania, Ambrosia, Cirsium, Carduus, Sonchus, Solanum, Rorippa, Rotala, Lindernia, Lamium, Veronica, Abutilon, Emex, Datura, Viola, Galeopsis, Papaver, Centaurea, Trifolium, Ranunculus, and Taraxacum.
• Monocotyle- donous weeds include, but are not limited to, weeds of of the genera: Echinochloa, Setaria, Pani- cum, Digitaria, Phleum, Poa, Festuca, Eleusine, Brachiaria, Lolium, Bromus, Avena, Cyperus, Sor- ghum, Agropyron, Cynodon, Monochoria, Fimbristyslis, Sagittaria, Eleocharis, Scirpus, Paspalum, Ischaemum, Sphenoclea, Dactyloctenium, Agrostis, Alopecurus, and Apera.
• the weeds of the present invention can include, for example, crop plants that are growing in an undesired location.
• a volunteer maize plant that is in a field that predominantly comprises soybean plants can be considered a weed, if the maize plant is undesired in the field of soybean plants.
• plant is used in its broadest sense as it pertains to organic material and is intended to encompass eukaryotic organisms that are members of the Kingdom Plantae, examples of which include but are not limited to vascular plants, vegetables, grains, flowers, trees, herbs, bushes, grasses, vines, ferns, mosses, fungi and algae, etc, as well as clones, offsets, and parts of plants used for asexual propagation (e.g. cuttings, pipings, shoots, rhizomes, underground stems, clumps, crowns, bulbs, corms, tubers, rhizomes, plants/tissues produced in tissue culture, etc.).
• asexual propagation e.g. cuttings, pipings, shoots, rhizomes, underground stems, clumps, crowns, bulbs, corms, tubers, rhizomes, plants/tissues produced in tissue culture, etc.
• plant further encompasses whole plants, ancestors and progeny of the plants and plant parts, including seeds, shoots, stems, leaves, roots (including tubers), flowers, florets, fruits, pedi- cles, peduncles, stamen, anther, stigma, style, ovary, petal, sepal, carpel, root tip, root cap, root hair, leaf hair, seed hair, pollen grain, microspore, cotyledon, hypocotyl, epicotyl, xylem, phloem, parenchyma, endosperm, a companion cell, a guard cell, and any other known organs, tissues, and cells of a plant, and tissues and organs, wherein each of the aforementioned comprise the gene/nucleic acid of interest.
• plant also encompasses plant cells, suspension cultures, callus tissue, embryos, meristematic regions, gametophytes, sporophytes, pollen and microspores, again wherein each of the aforementioned comprises the gene/nucleic acid of interest.
• Plants that are particularly useful in the methods of the invention include all plants which belong to the superfamily Viridiplantae, in particular monocotyledonous and dicotyledonous plants including fodder or forage legumes, ornamental plants, food crops, trees or shrubs selected from the list comprising Acer spp., Actinidia spp., Abelmoschus spp., Agave sisalana, Agropyron spp., Agrostis stolonifera, Allium spp., Amaranthus spp., Ammophila arenaria, Ananas comosus, Annona spp., Apium graveolens, Arachis spp, Artocarpus spp., Asparagus officinalis, Avena spp.
• Avena sativa e.g. Avena sativa, Avena fatua, Avena byzantina, Avena fatua var. sativa, Avena hybrida
• Averrhoa carambo- la Benincasa hispida
• Bertholletia excelsea
• Beta vulgaris Brassica spp.
• Brassica spp. e.g. Bras- sica napus, Brassica rapa ssp.
• the plant is a crop plant.
• crop plants include inter alia soybean, sunflower, canola, alfalfa, rapeseed, cotton, tomato, potato or tobacco.
• the plant is a monocotyledonous plant, such as sugarcane.
• the plant is a cereal, such as rice, maize, wheat, barley, millet, rye, sorghum or oats.
• the plant has been previously produced by a process comprising re- combinantly preparing a plant by introducing and over-expressing a wild-type or mut-PPO transgene according to the present invention, as described in greater detail hereinfter.
• the plant has been previously produced by a process comprising in situ mutagenizing plant cells, to obtain plant cells which express a mut-PPO.
• the nucleic acids of the invention find use in enhancing the herbicide tolerance of plants that comprise in their genomes a gene encoding a herbicide-tolerant wild-type or mut- PPO protein.
• a gene may be an endogenous gene or a transgene, as described hereinafter.
• the nucleic acids of the present invention can be stacked with any combination of polynucleotide sequences of interest in order to create plants with a desired phenotype.
• nucleic acids of the present invention may be stacked with any other polynucleotides encoding polypeptides having pesticidal and/or insecticidal activity, such as, for example, the Bacillus thuringiensis toxin proteins (described in U.S. Patent Nos. 5,366,892;
• the combinations generated can also include multiple copies of any one of the polynucleotides of interest.
• the term "herbicide” is used herein to mean an active ingredient that kills, controls or otherwise adversely modifies the growth of plants.
• the preferred amount or concentration of the herbicide is an "effective amount” or “effective concentration.”
• effective amount and concentration is intended an amount and concentration, respectively, that is sufficient to kill or inhibit the growth of a similar, wild-type, plant, plant tissue, plant cell, or host cell, but that said amount does not kill or inhibit as severely the growth of the herbicide-resistant plants, plant tissues, plant cells, and host cells of the present invention.
• the effective amount of a herbicide is an amount that is routinely used in agricultural production systems to kill weeds of interest. Such an amount is known to those of ordinary skill in the art.
• Herbicidal activity is exhibited by herbicides useful for the the present invention when they are applied directly to the plant or to the locus of the plant at any stage of growth or before planting or emergence. The effect observed depends upon the plant species to be controlled, the stage of growth of the plant, the application parameters of dilution and spray drop size, the particle size of solid components, the environmental conditions at the time of use, the specific compound employed, the specific adjuvants and carriers employed, the soil type, and the like, as well as the amount of chemical applied.
• herbicide-tolerant or herbbicide-resistant plant it is intended that a plant that is tolerant or resistant to at least one herbicide at a level that would normally kill, or inhibit the growth of, a normal or wild-type plant.
• herbicide-tolerant wildtype or mut-PPO protein or “herbicide -resistant wildtype or mut-PPO protein”
• PPO activity of such a herbicide-tolerant or herbicide-resistant mut-PPO protein may be referred to herein as “herbicide-tolerant” or “herbicide-resistant” PPO activity.
• the PPO-inhibiting herbicides (A) and/or the herbicidal compounds B as described herein which can be employed in the context of the present invention are capable of forming geometrical isomers, for example E/Z isomers, it is possible to use both, the pure isomers and mixtures thereof, in the compositions according to the invention.
• the PPO-inhibting herbicides A and/or the herbicidal compounds B as described herein have one or more centers of chirality and, as a consequence, are present as enantiomers or diastereomers, it is possible to use both, the pure enantiomers and diastereomers and their mixtures, in the compositions according to the invention.
• the PPO-inhibting herbicides A and/or the herbicidal compounds B as described herein have ionizable functional groups, they can also be employed in the form of their agriculturally acceptable salts. Suitable are, in general, the salts of those cations and the acid addition salts of those acids whose cations and anions, respectively, have no adverse effect on the activity of the active compounds.
• Preferred cations are the ions of the alkali metals, preferably of lithium, sodium and potassium, of the alkaline earth metals, preferably of calcium and magnesium, and of the transition metals, preferably of manganese, copper, zinc and iron, further ammonium and substituted ammonium in which one to four hydrogen atoms are replaced by Ci-C 4 -alkyl, hydroxy-Ci-C 4 -alkyl, Ci-C 4 -alkoxy-CrC 4 -alkyl, hydroxy-Ci-C 4 -alkoxy- Ci-C 4 -alkyl, phenyl or benzyl, preferably ammonium, methylammonium, isopropylammonium, dimethylammonium, diisopropylammonium, trimethylammonium, heptylammonium, dodec- ylammonium, tetradecylammonium, tetramethylammonium, tetraethylam
• Anions of useful acid addition salts are primarily chloride, bromide, fluoride, iodide, hy- drogensulfate, methylsulfate, sulfate, dihydrogenphosphate, hydrogenphosphate, nitrate, bi- carbonate, carbonate, hexafluorosilicate, hexafluorophosphate, benzoate and also the anions of Ci-C 4 -alkanoic acids, preferably formate, acetate, propionate and butyrate.
• the PPO-inhibting herbicides A and/or the herbicidal compounds B as described herein hav- ing a carboxyl group can be employed in the form of the acid, in the form of an agriculturally suitable salt as mentioned above or else in the form of an agriculturally acceptable derivative, for example as amides, such as mono- and di-Ci-C6-alkylamides or arylamides, as esters, for example as allyl esters, propargyl esters, Ci-Cio-alkyl esters, alkoxyalkyl esters, tefuryl ((tetra- hydrofuran-2-yl)methyl) esters and also as thioesters, for example as Ci-Cio-alkylthio esters.
• amides such as mono- and di-Ci-C6-alkylamides or arylamides
• esters for example as allyl esters, propargyl esters, Ci-Cio-alkyl est
• Preferred mono- and di-Ci-C6-alkylamides are the methyl and the dimethylamides.
• Preferred arylamides are, for example, the anilides and the 2-chloroanilides.
• Preferred alkyl esters are, for example, the methyl, ethyl, propyl, isopropyl, butyl, isobutyl, pentyl, mexyl (1 -methyl hexyl), meptyl (1 -methylheptyl), heptyl, octyl or isooctyl (2-ethylhexyl) esters.
• Ci-C 4 -alkoxy- Ci-C 4 -alkyl esters are the straight-chain or branched Ci-C 4 -alkoxy ethyl esters, for example the 2-methoxyethyl, 2-ethoxyethyl, 2-butoxyethyl (butotyl), 2-butoxypropyl or 3-butoxypropyl ester.
• An example of a straight-chain or branched Ci-Cio-alkylthio ester is the ethylthio ester.
• PPO inhibiting herbicides which can be used according to the present invention are acifluorfen, acifluorfen-sodium, azafenidin, bencarbazone, benzfendizone, bifenox, butafenacil, carfentrazone, carfentrazone-ethyl, chlomethoxyfen, cinidon-ethyl, fluazolate, flufenpyr, flufenpyr- ethyl, flumiclorac, flumiclorac-pentyl, flumioxazin, fluoroglycofen, fluoroglycofen-ethyl, fluthiacet, fluthiacet-methyl, fomesafen, halosafen, lactofen, oxadiargyl, oxadiazon, oxyfluorfen, pentoxazone, profluazol, pyraclonil, pyraflufen, pyraflufen-eth
• R 30 and R 31 independently of one another are F, CI or CN;
• R 32 is O or S
• R 33 is H, F, CI, CH 3 or OCH 3 ;
• R 34 is CH or N
• R 35 is O or S
• R 36 is H, CN, CH 3 , CF 3 , OCH 3 , OC 2 H 5 , SCH 3 , SC 2 H 5 , (CO)OC 2 H 5 or CH 2 R 38 ,
• R 38 is F, CI, OCH 3 , SCH 3 , SC 2 H 5 , CH 2 F, CH 2 Br or CH 2 OH;
• R 37 is (Ci-C 6 -alkyl)amino, (Ci-C 6 -dialkyl)amino, (NH)OR 39 , OH, OR 40 or SR 40
• R 39 is CH 3 , C 2 H 5 or phenyl
• R 40 is independently of one another Ci-C6-alkyl, C 2 -C6-alkenyl, C 3 -C6- alkynyl, Ci-C6-haloalkyl, Ci-C6-alkoxy-Ci-C6-alkyl, Ci-C6-alkoxy-Ci-C6-al- koxy-Ci -C6-alkyl , C 2 -C6-cyanoal kyl , Ci -C 4 -al koxy-carbonyl-Ci -C 4 -alkyl , Ci -C 4 -alkyl-carbonyl-am ino, Ci -C6-al kylsulfinyl-Ci -C6-al kyl , Ci -C6-al kyl- sulfonyl-Ci -C6-alkyl , Ci -C6-dialkoxy-Ci -C6-al kyl , Ci -
• cycloalkyls indenpently of one another are unsubstituted or substituted by one to five substituents selected from the group consisting of halogen, Ci-C 3 -alkyl and Ci-C 2 -haloalkyl; including their agriculturally acceptable alkali metal salts or ammonium salts.
• Preferred PPO-inhibiting herbicides that can be used according to the present invention are:
• uracils of formula 111.1 (corresponding to uracils of formula III, wherein R 30 is F, R 31 is CI, R 32 is O; R 33 is H; R 34 is CH; R 35 is O and R 37 is OR 40 )
• R 36 is OCH 3 , OC2H5, SCH 3 or SC2H5;
• R 40 is Ci-C6-alkyl, C2-C6-alkenyl, C3-C6-alkynyl, Ci-C6-haloalkyl, Ci-C6-alkoxy-Ci-C6-alkyl, Ci-C6-alkoxy-Ci-C6-alkoxy-Ci-C6-alkyl, Ci-C3-cyanoalkyl, phenyl-Ci-C3-alkyl, pyridyl- Ci-C3-alkyl, C3-C6-cycloalkyl or C3-C6-cycloalkyl-Ci-C 4 -alkyl,
• cycloalkyls are unsubstituted or substituted by one to five substituents selected from the group consisting of halogen, Ci-C3-alkyl and Ci-C2-haloalkyl; and
• uracils of formula III.2 (corresponding to uracils of formula III, wherein R 30 is F; R 31 is CI; R 32 is O; R 33 is H; R 34 is N; R 35 is O and R 37 is OR 40 with R 40 is Ci-C 6 -alkyl)
• PPO-inhibiting herbicides that can be used according to the present invention are:
• acifluorfen acifluorfen-sodium, butafenacil, carfentrazone-ethyl, cinidon-ethyl, flumioxazin, fluthi- acet-methyl, fomesafen, lactofen, oxadiargyl, oxyfluorfen, saflufenacil, sulfentrazone, ethyl [3-[2- chloro-4-fluoro-5-(1 -methyl-6-trifluoromethyl-2,4-dioxo-1 ,2,3,4-tetrahydropyrimidin-3-yl) ⁇ pyridyloxy]acetate (CAS 353292-31 -6; S-3100), 3-[7-fluoro-3-oxo-4-(prop-2-ynyl)-3,4-dihydro-2H- benzo[1 ,4]oxazin-6-yl]-1 ,5-dimethyl-6-thioxo
• uracils of formula III.1.1 (corresponding to uracils of formula III, wherein R 30 is F, R 31 is CI, R 32 is O; R 33 is H; R 34 is CH; R 35 is O, R 36 is OCH 3 and R 37 is OR 40 )
• R 40 is Ci-Ce-alkyl, C 2 -C 6 -alkenyl, C 3 -C 6 -alkynyl, Ci-C 6 -haloalkyl, Ci-C 6 -alkoxy-Ci-C 6 -alkyl, Ci-
• Ci-C3-cyanoalkyl phenyl-Ci-C3-alkyl, pyridyl-Ci-C3- alkyl, C3-C6-cycloalkyl or C3-C6-cycloalkyl-Ci-C 4 -alkyl,
• cycloalkyls are unsubstituted or substituted by one to five substituents selected from the group consisting of halogen, Ci-C3-alkyl andCi-C2-haloalkyl; is preferably CH 3 , CH2CH2OC2H5, CH2CH F2, cyclohexyl, (l -methylcyclopropyl)methyl or CH2(pyridine-4-yl); uracils of formula III.2.1 (corresponding to uracils of formula III, wherein R 30 is F; R 31 is CI; R 32 is O; R 33 is H; R 34 is N; R 35 is O and R 37 is OR 40 with R 40 is CH 3 )
• uracils of formula III.2.2 (corresponding to uracils of formula III, wherein R 30 is F; R 31 is CI; R 32 is O; R 33 is H; R 34 is N; R 35 is O and R 37 is OR 40 with R 40 is C 2 H 5 )
• PPO-inhibiting herbicides are the PPO-inhibiting herbicides.1 to A.14 listed below in table A:
• PPO-inhibiting herbicides described above that are useful to carry out the present invention are often best applied in conjunction with one or more other herbicides to obtain control of a wider variety of undesirable vegetation.
• PPO-inhibiting herbicides may further be used in conjunction with additional herbicides to which the crop plant is naturally tolerant, or to which it is resistant via expression of one or more additional transgenes as mentioned supra.
• the presently claimed compounds can be formulated with the other herbicide or herbicides, tank mixed with the other herbicide or herbicides, or applied sequentially with the other herbicide or herbicides.
• Suitable components for mixtures are, for example, selected from the herbicides of class b1 ) to b15) B) herbicides of class b1 ) to b15):
• ALS inhibitors acetolactate synthase inhibitors
• EBP inhibitors enolpyruvyl shikimate 3-phosphate synthase inhibitors
• DHP inhibitors 7,8-dihydropteroate synthase inhibitors
• VLCFA inhibitors inhibitors of the synthesis of very long chain fatty acids
• herbicides selected from the group consisting of bromobutide, chlorflurenol, chlorflurenol-methyl, cinmethylin, cumyluron, dalapon, dazomet, difenzoquat, difen- zoquat-metilsulfate, dimethipin, DSMA, dymron, endothal and its salts, etobenzanid, flamprop, flamprop-isopropyl, flam prop-methyl, flamprop-M-isopropyl, flamprop-M- methyl, flurenol, flurenol-butyl, flurprimidol, fosamine, fosamine-ammonium, inda- nofan, indaziflam, maleic hydrazide, mefluidide, metam, methiozolin (CAS 403640-
• herbicides B which can be used in combination with the PPO-inhibiting herbicides according to the present invention are: b1 ) from the group of the lipid biosynthesis inhibitors:
• ACC-herbicides such as alloxydim, alloxydim-sodium, butroxydim, clethodim, clodinafop, clodinafop-propargyl, cycloxydim, cyhalofop, cyhalofop-butyl, diclofop, diclofop-methyl, fenoxaprop, fenoxaprop-ethyl, fenoxaprop-P, fenoxaprop-P-ethyl, fluazifop, fluazifop-butyl, fluazifop-P, fluazi- fop-P-butyl, haloxyfop, haloxyfop-methyl, haloxyfop-P, haloxyfop-P-methyl, metamifop, pinoxaden, profoxydim, propaquizafop, quizalofop, quizalofop-ethy
• sulfonylureas such as amidosulfuron, azimsulfuron, bensulfuron, bensulfuron-methyl, chlorimuron, chlorimuron-ethyl, chlorsulfuron, cinosulfuron, cyclosulfamuron, ethametsulfuron, ethametsulfuron- methyl, ethoxysulfuron, flazasulfuron, flucetosulfuron, flupyrsulfuron, flupyrsulfuron-methyl-sodium, foramsulfuron, halosulfuron, halosulfuron-methyl, imazosulfuron, iodosulfuron, iodosulfuron-methyl- sodium, iofensulfuron, iofensulfuron-sodium, mesosulfuron, metazosulfuron, metsulfuron, metsulfuron, metsulfu-
• imidazolinones such as imazamethabenz, imazamethabenz-methyl, imazamox, imazapic, ima- zapyr, imazaquin and imazethapyr, triazolopyrimidine herbicides and sulfonamides such as cloransulam, cloransulam-methyl, diclosulam, flumetsulam, florasulam, metosulam, penoxsulam, pyrimisulfan and pyroxsulam,
• pyrimidinylbenzoates such as bispyribac, bispyribac-sodium, pyribenzoxim, pyriftalid, pyriminobac, pyriminobac-methyl, pyrithiobac, pyrithiobac-sodium, 4-[[[2-[(4,6-dimethoxy-2- pyrimidinyl)oxy]phenyl]methyl]amino]-benzoic acid-1 -methylethyl ester (CAS 420138-41 -6), 4-[[[2- [(4,6-dimethoxy-2-pyrimidinyl)oxy]phenyl]methyl]amino]-benzoic acid propyl ester (CAS 420138- 40-5), N-(4-bromophenyl)-2-[(4,6-dimethoxy-2-pyrimidinyl)oxy]benzenemethanamine (CAS 420138-01 -8),
• sulfonylaminocarbonyl-triazolinone herbicides such as flucarbazone, flucarbazone-sodium, propoxycarbazone, propoxycarbazone-sodium, thiencarbazone and thiencarbazone-methyl; and triafamone;
• compositions comprising at least one imidazolinone herbicide; b3) from the group of the photosynthesis inhibitors:
• inhibitors of the photosystem II e.g. triazine herbicides, including of chlorotriazine, triazinones, triazindiones, methylthiotriazines and pyridazinones such as ametryn, atrazine, chloridazone, cyanazine, desmetryn, dimethametryn,hexazinone, metribuzin, prometon, prometryn, propazine, simazine, simetryn, terbumeton, terbuthylazin, terbutryn and trietazin, aryl urea such as chlorobromuron, chlorotoluron, chloroxuron, dimefuron, diuron, fluometuron, isoproturon, isouron, linuron, metamitron, methabenzthiazuron, metobenzuron, metoxuron, monolinuron, neburon, siduron, te
• a preferred embodiment of the invention relates to those compositions comprising at least one aryl urea herbicide. Among these, likewise a preferred embodiment of the invention relates to those compositions comprising at least one triazine herbicide. Among these, likewise a preferred embodiment of the invention relates to those compositions comprising at least one nitrile herbicide; b4) from the group of the protoporphyrinogen-IX oxidase inhibitors:
• acifluorfen acifluorfen-sodium, azafenidin, bencarbazone, benzfendizone, bifenox, butafenacil, carfentrazone, carfentrazone-ethyl, chlomethoxyfen, cinidon-ethyl, fluazolate, flufenpyr, flufenpyr- ethyl, flumiclorac, flumiclorac-pentyl, flumioxazin, fluoroglycofen, fluoroglycofen-ethyl, fluthiacet, fluthiacet-methyl, fomesafen, halosafen, lactofen, oxadiargyl, oxadiazon, oxyfluorfen, pentoxazone, profluazol, pyraclonil, pyraflufen, pyraflufen-ethyl, saflufenacil, sulfentrazone
• PDS inhibitors beflubutamid, diflufenican, fluridone, flurochloridone, flurtamone, norflurazon, pico- linafen, and 4-(3-trifluoromethylphenoxy)-2-(4-trifluoromethylphenyl)pyrimidine (CAS 180608-33-7), HPPD inhibitors: benzobicyclon, benzofenap, clomazone, isoxaflutole, mesotrione, pyrasulfotole, pyrazolynate, pyrazoxyfen, sulcotrione, tefuryltrione, tembotrione, topramezone and bicyclopyrone, bleacher, unknown target: aclonifen, amitrole and flumeturon; b6) from the group of the EPSP synthase inhibitors:
• glyphosate glyphosate-isopropylammonium, glyposate-potassium and glyphosate-trimesium (sul- fosate); b7) from the group of the glutamine synthase inhibitors:
• bilanaphos (bialaphos), bilanaphos-sodium, glufosinate, glufosinate-P and glufosinate-ammonium; b8) from the group of the DHP synthase inhibitors:
• compounds of group K1 dinitroanilines such as benfluralin, butralin, dinitramine, ethalfluralin, flu- chloralin, oryzalin, pendimethalin, prodiamine and trifluralin, phosphoramidates such as amipro- phos, amiprophos-methyl, and butamiphos, benzoic acid herbicides such as chlorthal, chlorthal- dimethyl, pyridines such as dithiopyr and thiazopyr, benzamides such as propyzamide and tebu- tarn; compounds of group K2: chlorpropham, propham and carbetamide, among these, compounds of group K1 , in particular dinitroanilines are preferred; b10) from the group of the VLCFA inhibitors:
• chloroacetamides such as acetochlor, alachlor, butachlor, dimethachlor, dimethenamid, dimethe- namid-P, metazachlor, metolachlor, metolachlor-S, pethoxamid, pretilachlor, propachlor, propiso- chlor and thenylchlor, oxyacetanilides such as flufenacet and mefenacet, acetanilides such as di- phenamid, naproanilide and napropamide, tetrazolinones such fentrazamide, and other herbicides such as anilofos, cafenstrole, fenoxasulfone, ipfencarbazone, piperophos, pyroxasulfone and isox- azoline compounds of the formulae 11.1 , II.2, II.3, II.4, II.5, II.6, II.7, II.8 and II.9
• 2,4-D and its salts and esters such as clacyfos, 2,4-DB and its salts and esters, aminocyclopyra- chlor and its salts and esters, aminopyralid and its salts such as aminopyralid-tris(2- hydroxypropyl)ammonium and its esters, benazolin, benazolin-ethyl, chloramben and its salts and esters, clomeprop, clopyralid and its salts and esters, dicamba and its salts and esters, dichlorprop and its salts and esters, dichlorprop-P and its salts and esters, fluroxypyr, fluroxypyr-butometyl, fluroxypyr-meptyl, halauxifen and its salts and esters (CAS 943832-60-8); MCPA and its salts and esters, MCPA-thioethyl, MCPB and its salts and esters, mecoprop and its salts and esters,
• methabenzthiazuron metribuzin, paraquat, paraquat-dichloride, phenmedipham, propanil, pyridate, simazine, terbutryn, terbuthylazine and thidiazuron; b4) from the group of the protoporphyrinogen-IX oxidase inhibitors:
• acifluorfen acifluorfen-sodium, azafenidin, bencarbazone, benzfendizone, butafenacil, carfentra- zone-ethyl, cinidon-ethyl, flufenpyr-ethyl, flumiclorac-pentyl, flumioxazin, fluoroglycofen-ethyl, fluthiacet-methyl, fomesafen, lactofen, oxadiargyl, oxadiazon, oxyfluorfen, pentoxazone, pyraflufen- ethyl, saflufenacil, sulfentrazone, ethyl [3-[2-chloro-4-fluoro-5-(1 -methyl-6-trifluoromethyl-2,4-dioxo- 1 ,2,3,4-tetrahydropyrimidin-3-yl)phenoxy]-2-pyridyl
• glyphosate glyphosate-isopropylammonium, glyphosate-potassium and glyphosate-thmesium (sul- fosate); b7) from the group of the glutamine synthase inhibitors:
• glufosinate from the group of the DH P synthase inhibitors: asulam; b9) from the group of the mitosis inhibitors:
• acetochlor alachlor, anilofos, butachlor, cafenstrole, dimethenamid, dimethenamid-P, fentraza- mide, flufenacet, mefenacet, metazachlor, metolachlor, S-metolachlor, naproanilide, napropamide, pretilachlor, fenoxasulfone, ipfencarbazone, pyroxasulfone thenylchlor and isoxazoline-compounds of the formulae 11.1 , II.2, II.3, II.4, II.5, II.6, II.7, II.8 and II.9 as mentioned above; b1 1 ) from the group of the cellulose biosynthesis inhibitors: dichlobenil, flupoxam, isoxaben and 1 - Cyclohexyl-5-pentafluorphenyloxy-1 4 -[1 ,2,4,6]thiatriazin-3-ylamine
• 2,4-D and its salts and esters aminocyclopyrachlor and its salts and esters, aminopyralid and its salts such as aminopyralid-tris(2-hydroxypropyl)ammonium and its esters, clopyralid and its salts and esters, dicamba and its salts and esters, dichlorprop-P and its salts and esters, fluroxypyr- meptyl, halauxifen and its salts and esters (CAS 943832-60-8), MCPA and its salts and esters, MCPB and its salts and esters, mecoprop-P and its salts and esters, picloram and its salts and esters, quinclorac, quinmerac and triclopyr and its salts and esters; b14) from the group of the auxin transport inhibitors: diflufenzopyr and diflufenzopyr-sodium; b15) from the group of the other herbicides: bromobutide, cinmethylin, cumy
• herbicides B that can be used in combination with the PPO-inhibiting herbicides according to the present invention are: b1) from the group of the lipid biosynthesis inhibitors: clodinafop-propargyl, cycloxydim, cyhalofop- butyl, fenoxaprop-P-ethyl, pinoxaden, profoxydim, tepraloxydim, tralkoxydim, 4-(4'-Chloro-4- cyclopropyl-2'-fluoro[1 ,1 '-biphenyl]-3-yl)-5-hydroxy-2,2,6,6-tetramethyl-2H-pyran-3(6H)-one (CAS 1312337-72-6); 4-(2 ⁇ 4'-Dichloro-4-cyclopropyl[1 ,1 , -biphenyl]-3-yl)-5-hydroxy-2,2,6,6-tetramethyl- 2H-pyran-3(6H)-one (
• Safeners are chemical compounds which prevent or reduce damage on useful plants without having a major impact on the herbicidal action of herbicides towards unwanted plants. They can be applied either before sowings (e.g. on seed treatments, shoots or seedlings) or in the pre-emergence application or post-emergence application of the useful plant. Furthermore, the safeners C, the PPO-inhibiting herbicides and/or the herbicides B can be applied simultaneously or in succession. Suitable safeners are e.g.
• Examples of preferred safeners C are benoxacor, cloquintocet, cyometrinil, cyprosulfamide, dichlormid, dicyclonon, dietholate, fenchlorazole, fenclorim, flurazole, fluxofenim, furilazole, isoxadifen, mefenpyr, mephenate, naphthalic anhydride, oxabetrinil, 4-(dichloroacetyl)-1 -oxa-4- azaspiro[4.5]decane (MON4660, CAS 71526-07-3) and 2,2,5-trimethyl-3-(dichloroacetyl)-1 ,3- oxazolidine (R-29148, CAS 52836-31 -4).
• Especially preferred safeners C are benoxacor, cloquintocet, cyprosulfamide, dichlormid, fenchlorazole, fenclorim, flurazole, fluxofenim, furilazole, isoxadifen, mefenpyr, naphthalic anhydride, oxabetrinil, 4-(dichloroacetyl)-1 -oxa-4-azaspiro[4.5]decane (MON4660, CAS 71526-07-3) and 2,2,5- trimethyl-3-(dichloroacetyl)-1 ,3-oxazolidine (R-29148, CAS 52836-31 -4).
• Particularly preferred safeners C are benoxacor, cloquintocet, cyprosulfamide, dichlormid, fenchlorazole, fenclorim, furilazole, isoxadifen, mefenpyr, naphtalic anhydride, 4-(dichloroacetyl)-1 -oxa-4- azaspiro[4.5]decane (MON4660, CAS 71526-07-3), and 2,2,5-trimethyl-3-(dichloroacetyl)-1 ,3- oxazolidine (R-29148, CAS 52836-31 -4).
• Also preferred safeners C are benoxacor, cloquintocet, cyprosulfamide, dichlormid, fenchlorazole, fenclorim, furilazole, isoxadifen, mefenpyr, 4-(dichloroacetyl)-1 -oxa-4-azaspiro[4.5]decane
• safeners C which, as component C, are constituent of the composition according to the invention are the safeners C as defined above; in particular the safeners C.1 - C.12 listed below in table C: Table C
• the PPO-inhibiting herbicides and the active compounds B of groups b1 ) to b15) and the active compounds C are known herbicides and safeners, see, for example, The Compendium of Pesticide Common Names (http://www.alanwood.net/pesticides/); Farm Chemicals Handbook 2000 volume 86, Meister Publishing Company, 2000; B. Hock, C. Fedtke, R. R. Schmidt, Herbizide
• the assignment of the active compounds to the respective mechanisms of action is based on current knowledge. If several mechanisms of action apply to one active compound, this substance was only assigned to one mechanism of action.
• Active compounds B and C having a carboxyl group can be employed in the form of the acid, in the form of an agriculturally suitable salt as mentioned above or else in the form of an agriculturally acceptable derivative in the compositions according to the invention.
• suitable salts include those, where the counterion is an agriculturally acceptable cation.
• suitable salts of dicamba are dicamba-sodium, dicamba-potassium, dicamba-methylammonium, dicamba-dimethylammonium, dicamba-isopropylammonium, dicamba- diglycolamine, dicamba-olamine, dicamba-diolamine, dicamba-trolamine, dicamba-N,N-bis-(3- aminopropyl)methylamine and dicamba-diethylenetriamine.
• suitable ester are dicamba-methyl and dicamba-butotyl.
• Suitable salts of 2,4-D are 2,4-D-ammonium, 2,4-D-dimethylammonium, 2,4-D-diethylammonium, 2,4-D-diethanolammonium (2,4-D-diolamine), 2,4-D-triethanolammonium, 2,4-D- isopropylammonium, 2,4-D-triisopropanolammonium, 2,4-D-heptylammonium, 2,4-D- dodecylammonium, 2,4-D-tetradecylammonium, 2,4-D-triethylammonium, 2,4-D-tris(2- hydroxypropyl)ammonium, 2,4-D-tris(isopropyl)ammonium, 2,4-D-trolamine, 2,4-D-lithium, 2,4-D- sodium.
• esters of 2,4-D are 2,4-D-butotyl, 2,4-D-2-butoxypropyl, 2,4-D-3- butoxypropyl, 2,4-D-butyl, 2,4-D-ethyl, 2,4-D-ethylhexyl, 2,4-D-isobutyl, 2,4-D-isooctyl, 2,4-D- isopropyl, 2,4-D-meptyl, 2,4-D-methyl, 2,4-D-octyl, 2,4-D-pentyl, 2,4-D-propyl, 2,4-D-tefuryl and clacyfos.
• Suitable salts of 2,4-DB are for example 2,4-DB-sodium, 2,4-DB-potassium and 2,4-DB-dimethyl- ammonium.
• Suitable esters of 2,4-DB are for example 2,4-DB-butyl and 2,4-DB-isoctyl.
• Suitable salts of dichlorprop are for example dichlorprop-sodium, dichlorprop-potassium and di- chlorprop-dimethylammonium.
• suitable esters of dichlorprop are dichlorprop-butotyl and dichlorprop-isoctyl.
• Suitable salts and esters of MCPA include MCPA-butotyl, MCPA-butyl, MCPA-dimethylammonium, MCPA-diolamine, MCPA-ethyl, MCPA-thioethyl, MCPA-2-ethylhexyl, MCPA-isobutyl, MCPA- isoctyl, MCPA-isopropyl, MCPA-isopropylammonium, MCPA-methyl, MCPA-olamine, MCPA- potassium, MCPA-sodium and MCPA-trolamine.
• a suitable salt of MCPB is MCPB sodium.
• a suitable ester of MCPB is MCPB-ethyl.
• Suitable salts of clopyralid are clopyralid-potassium, clopyralid-olamine and clopyral id-tris-(2- hydroxypropyl)ammonium.
• Example of suitable esters of clopyralid is clopyralid-methyl.
• Examples of a suitable ester of fluroxypyr are fluroxypyr-meptyl and fluroxypyr-2-butoxy-1 - methylethyl, wherein fluroxypyr-meptyl is preferred.
• Suitable salts of picloram are picloram-dimethylammonium, picloram-potassium, picloram- triisopropanolammonium, picloram-triisopropylammonium and picloram-trolamine.
• a suitable ester of picloram is picloram-isoctyl.
• a suitable salt of triclopyr is thclopyr-triethylammonium.
• Suitable esters of triclopyr are for example triclopyr-ethyl and triclopyr-butotyl.
• Suitable salts and esters of chloramben include chloramben-ammonium, chloramben-diolamine, chloramben-methyl, chloramben-methylammonium and chloramben-sodium.
• Suitable salts and esters of 2,3,6-TBA include 2,3,6-TBA-dimethylammonium, 2,3,6-TBA-lithium, 2,3,6-TBA- potassium and 2,3,6-TBA-sodium.
• Suitable salts and esters of aminopyralid include aminopyralid-potassium and aminopyralid-tris(2- hydroxypropyl)ammonium.
• Suitable salts of glyphosate are for example glyphosate-ammonium, glyphosate-diammonium, glyphoste-dimethylammonium, glyphosate-isopropylammonium, glyphosate-potassium, glypho- sate-sodium, glyphosate-trimesium as well as the ethanolamine and diethanolamine salts, preferably glyphosate-diammonium, glyphosate-isopropylammonium and glyphosate-trimesium (sul- fosate).
• a suitable salt of glufosinate is for example glufosinate-ammonium.
• a suitable salt of glufosinate-P is for example glufosinate-P-ammonium.
• Suitable salts and esters of bromoxynil are for example bromoxynil-butyrate, bromoxynil- heptanoate, bromoxynil-octanoate, bromoxynil-potassium and bromoxynil-sodium.
• Suitable salts and esters of ioxonil are for example ioxonil-octanoate, ioxonil-potassium and ioxo- nil-sodium.
• Suitable salts and esters of mecoprop include mecoprop-butotyl, mecoprop-dimethylammonium, mecoprop-diolamine, mecoprop-ethadyl, mecoprop-2-ethylhexyl, mecoprop-isoctyl, mecoprop- methyl, mecoprop-potassium, mecoprop-sodium and mecoprop-trolamine.
• Suitable salts of mecoprop-P are for example mecoprop-P-butotyl, mecoprop-P- dimethylammonium, mecoprop-P-2-ethylhexyl, mecoprop-P-isobutyl, mecoprop-P-potassium and mecoprop-P-sodium.
• a suitable salt of diflufenzopyr is for example diflufenzopyr-sodium.
• a suitable salt of naptalam is for example naptalam-sodium.
• Suitable salts and esters of aminocyclopyrachlor are for example aminocyclopyrachlor- dimethylammonium, aminocyclopyrachlor-methyl, aminocyclopyrachlor-triisopropanolammonium, aminocyclopyrachlor-sodium and aminocyclopyrachlor-potassium.
• a suitable salt of quinclorac is for example quinclorac-dimethylammonium.
• a suitable salt of quinmerac is for example quinclorac-dimethylammonium.
• a suitable salt of imazamox is for example imazamox-ammonium.
• Suitable salts of imazapic are for example imazapic-ammonium and imazapic-isopropylammonium.
• Suitable salts of imazapyr are for example imazapyr-ammonium and imazapyr- isopropylammonium.
• a suitable salt of imazaquin is for example imazaquin-ammonium.
• Suitable salts of imazethapyr are for example imazethapyr-ammonium and imazethapyr- isopropylammonium.
• a suitable salt of topramezone is for example topramezone-sodium.
• the composition comprises as component B at least one, preferably exactly one herbicide B.
• the composition comprises at least two, preferably exactly two, herbicides B different from each other.
• the composition comprises at least three, preferably exactly three, herbicides B different from each other.
• the composition comprises as com- ponent A at least one, preferably exactly one PPO A, preferably acifluorfen, acifluorfen-sodium, butafenacil, cinidon-ethyl, carfentrazone-ethyl, flumioxazin, fluthiacet-methyl, fomesafen, lactofen, oxadiargyl, oxyfluorfen, saflufenacil, sulfentrazone, ethyl [3-[2-chloro-4-fluoro-5-(1 -methyl-6- trifluoromethyl-2,4-dioxo-1 ,2,3,4-tetrahydropyrimidin-3-yl)phenoxy]-2-pyridyloxy]acetate (CAS 353292-31 -6; S-3100;, 1 ,5-dimethyl-6-thioxo-3-(2,2,7-trifluoro-3-ox
• the composition comprises as component A at least one, preferably exactly preferably exactly one PPO A, preferably acifluorfen, acifluorfen-sodium, butafenacil, cinidon-ethyl, carfentrazone-ethyl, flumioxazin, fluthiacet-methyl, fomesafen, lactofen, oxadiargyl, oxyfluorfen, saflufenacil, sulfentrazone, ethyl [3-[2-chloro-4-fluoro- 5-(1 -methyl-6-trifluoromethyl-2,4-dioxo-1 ,2,3,4-tetrahydropyrimidin-3-yl)phenoxy]-2- pyridyloxy]acetate (CAS 353292-31 -6; S-3100), 1 ,5-dimethyl-6-thioxo-3-(2,2,7-trifluoro-3-oxo
• the composition comprises as component A at least one, preferably exactly preferably exactly one PPO A, preferably acifluorfen, acifluorfen-sodium, butafenacil, cinidon-ethyl, carfentrazone-ethyl, flumioxazin, fluthiacet-methyl, fomesafen, lactofen, oxadiargyl, oxyfluorfen, saflufenacil, sulfentrazone, ethyl [3-[2-chloro-4-fluoro- 5-(1 -methyl-6-trifluoromethyl-2,4-dioxo-1 ,2,3,4-tetrahydropyrimidin-3-yl)phenoxy]-2- pyridyloxy]acetate (CAS 353292-31 -6; S-3100), 1 ,5-dimethyl-6-thioxo-3-(2,2,7-thfluoro-3-oxo
• the composition comprises, in addition to a PPO A, preferably acifluorfen, acifluorfen-sodium, butafenacil, cinidon-ethyl, carfentrazone-ethyl, flumioxazin, fluthiacet-methyl, fomesafen, lactofen, oxadiargyl, oxyfluorfen, saflufenacil, sulfentrazone, ethyl [3-[2-chloro-4-fluoro-5-(1 -methyl-6-trifluoromethyl-2,4-dioxo-1 ,2,3,4- tetrahydropyrimidin-3-yl)phenoxy]-2-pyridyloxy]acetate (CAS 353292-31 -6; S-3100), 1 ,5-dimethyl- 6-thioxo-3-(2,2,7-trifluoro-3-oxo-4-(prop-2-yn
• the composition comprises, in addi- tion to a PPO A, preferably acifluorfen, acifluorfen-sodium, butafenacil, cinidon-ethyl, carfentrazone-ethyl, flumioxazin, fluthiacet-methyl, fomesafen, lactofen, oxadiargyl, oxyfluorfen, saflufenacil, sulfentrazone, ethyl [3-[2-chloro-4-fluoro-5-(1 -methyl-6-trifluoromethyl-2,4-dioxo-1 ,2,3,4- tetrahydropyrimidin-3-yl)phenoxy]-2-pyridyloxy]acetate (CAS 353292-31 -6; S-3100, 1 ,5-dimethyl-6- thioxo-3-(2,2,7-trifluoro-3-oxo-4-(
• the composition comprises, in addition to a a PPO A, preferably acifluorfen, acifluorfen-sodium, butafenacil, cinidon-ethyl, carfentra- zone-ethyl, flumioxazin, fluthiacet-methyl, fomesafen, lactofen, oxadiargyl, oxyfluorfen, saflufenacil, sulfentrazone, ethyl [3-[2-chloro-4-fluoro-5-(1 -methyl-6-trifluoromethyl-2,4-dioxo-1 ,2,3,4- tetrahydropyrimidin-3-yl)phenoxy]-2-pyridyloxy]acetate (CAS 353292-31 -6; S-3100), 1 ,5-dimethyl- 6-thioxo-3-(2,2,7-trifluoro-3-oxo-4-(prop-2
• the composition comprises, in addition to a a PPO A, preferably acifluorfen, acifluorfen-sodium, butafenacil, cinidon-ethyl, carfentra- zone-ethyl, flumioxazin, fluthiacet-methyl, fomesafen, lactofen, oxadiargyl, oxyfluorfen, saflufenacil, sulfentrazone, ethyl [3-[2-chloro-4-fluoro-5-(1 -methyl-6-trifluoromethyl-2,4-dioxo-1 ,2,3,4- tetrahydropyrimidin-3-yl)phenoxy]-2-pyridyloxy]acetate (CAS 353292-31 -6; S-3100), 1 ,5-dimethyl- 6-thioxo-3-(2,2,7-trifluoro-3-oxo-4-(prop-2
• the composition comprises, in addition to a a PPO A, preferably acifluorfen, acifluorfen-sodium, butafenacil, cinidon-ethyl, carfentra- zone-ethyl, flumioxazin, fluthiacet-methyl, fomesafen, lactofen, oxadiargyl, oxyfluorfen, saflufenacil, sulfentrazone, ethyl [3-[2-chloro-4-fluoro-5-(1 -methyl-6-trifluoromethyl-2,4-dioxo-1 ,2,3,4- tetrahydropyrimidin-3-yl)phenoxy]-2-pyridyloxy]acetate (CAS 353292-31 -6; S-3100), 1 ,5-dimethyl- 6-thioxo-3-(2,2,7-trifluoro-3-oxo-4-(prop-2
• the composition comprises, in addition to a a PPO A, preferably acifluorfen, acifluorfen-sodium, butafenacil, cinidon-ethyl, carfentra- zone-ethyl, flumioxazin, fluthiacet-methyl, fomesafen, lactofen, oxadiargyl, oxyfluorfen, saflufenacil, sulfentrazone, ethyl [3-[2-chloro-4-fluoro-5-(1 -methyl-6-trifluoromethyl-2,4-dioxo-1 ,2,3,4- tetrahydropyrimidin-3-yl)phenoxy]-2-pyridyloxy]acetate (CAS 353292-31 -6; S-3100), 1 ,5-dimethyl- 6-thioxo-3-(2,2,7-trifluoro-3-oxo-4-(prop-2
• the composition comprises, in addition to a a PPO A, preferably acifluorfen, acifluorfen-sodium, butafenacil, cinidon-ethyl, carfentra- zone-ethyl, flumioxazin, fluthiacet-methyl, fomesafen, lactofen, oxadiargyl, oxyfluorfen, saflufenacil, sulfentrazone, ethyl [3-[2-chloro-4-fluoro-5-(1 -methyl-6-trifluoromethyl-2,4-dioxo-1 ,2,3,4- tetrahydropyrimidin-3-yl)phenoxy]-2-pyridyloxy]acetate (CAS 353292-31 -6; S-3100), 1 ,5-dimethyl- 6-thioxo-3-(2,2,7-trifluoro-3-oxo-4-(prop-2
• the composition comprises, in addi- tion to a a PPO A, preferably acifluorfen, acifluorfen-sodium, butafenacil, cinidon-ethyl, carfentra- zone-ethyl, flumioxazin, fluthiacet-methyl, fomesafen, lactofen, oxadiargyl, oxyfluorfen, saflufenacil, sulfentrazone, ethyl [3-[2-chloro-4-fluoro-5-(1 -methyl-6-trifluoromethyl-2,4-dioxo-1 ,2,3,4- tetrahydropyrimidin-3-yl)phenoxy]-2-pyridyloxy]acetate (CAS 353292-31 -6; S-3100, 1 ,5-dimethyl-6- thioxo-3-(2,2,7-trifluoro-3-oxo
• the composition comprises, in addition to a PPO A, preferably acifluorfen, acifluorfen-sodium, butafenacil, cinidon-ethyl, carfentra- zone-ethyl, flumioxazin, fluthiacet-methyl, fomesafen, lactofen, oxadiargyl, oxyfluorfen, saflufenacil, sulfentrazone, ethyl [3-[2-chloro-4-fluoro-5-(1 -methyl-6-trifluoromethyl-2,4-dioxo-1 ,2,3,4- tetrahydropyrimidin-3-yl)phenoxy]-2-pyridyloxy]acetate (CAS 353292-31 -6; S-3100, 1 ,5-dimethyl-6- thioxo-3-(2,2,7-trifluoro-3-oxo-4-(prop-2-
• compositions comprising in addition to a a PPO A, preferably acifluorfen, acifluorfen-sodium, butafenacil, cinidon-ethyl, carfentra- zone-ethyl, flumioxazin, fluthiacet-methyl, fomesafen, lactofen, oxadiargyl, oxyfluorfen, saflufenacil, sulfentrazone, ethyl [3-[2-chloro-4-fluoro-5-(1 -methyl-6-trifluoromethyl-2,4-dioxo-1 ,2,3,4- tetrahydropyrimidin-3-yl)phenoxy]-2-pyridyloxy]acetate (CAS 353292-31 -6; S-3100), 1 ,5-dimethyl- 6-thioxo-3-(2,2,7-trifluoro-3-oxo-4-(prop-2-yl
• the composition comprises, in addition to a PPO A, preferably acifluorfen, acifluorfen-sodium, butafenacil, cinidon-ethyl, carfentra- zone-ethyl, flumioxazin, fluthiacet-methyl, fomesafen, lactofen, oxadiargyl, oxyfluorfen, saflufenacil, sulfentrazone, ethyl [3-[2-chloro-4-fluoro-5-(1 -methyl-6-trifluoromethyl-2,4-dioxo-1 ,2,3,4- tetrahydropyrimidin-3-yl)phenoxy]-2-pyridyloxy]acetate (CAS 353292-31 -6; S-3100, 1 ,5-dimethyl-6- thioxo-3-(2,2,7-trifluoro-3-oxo-4-(prop-2-
• triazinane-2,4-dione (CAS 1258836-72-4), especially preferred saflufenacil, 1 ,5-dimethyl-6-thioxo- 3-(2,2,7-trifluoro-3-oxo-4-(prop-2-ynyl)-3,4-dihydro-2H-benzo[b][1 ,4]oxazin-6-yl)-1 ,3,5-triazinane- 2,4-dione (CAS 1258836-72-4), at least one and especially exactly one herbicidally active compound from group b13), in particular selected from the group consisting of 2,4-D and its salts and esters, aminocyclopyrachlor and its salts and esters, aminopyralid and its salts such as aminopyra- lid-tris(2-hydroxypropyl)ammonium and its esters, clopyralid and its salts and esters, dicamba and its salts and esters, fluroxypyr-meptyl, quinclorac
• the composition comprises, in addition to a PPO A, preferably acifluorfen, acifluorfen-sodium, butafenacil, cinidon-ethyl, carfentra- zone-ethyl, flumioxazin, fluthiacet-methyl, fomesafen, lactofen, oxadiargyl, oxyfluorfen, saflufenacil, sulfentrazone, ethyl [3-[2-chloro-4-fluoro-5-(1 -methyl-6-trifluoromethyl-2,4-dioxo-1 ,2,3,4- tetrahydropyrimidin-3-yl)phenoxy]-2-pyridyloxy]acetate (CAS 353292-31 -6; S-3100), 1 ,5-dimethyl- 6-thioxo-3-(2,2,7-trifluoro-3-oxo-4-(prop-2-yl
• the composition comprises, in addition to a PPO A, preferably acifluorfen, acifluorfen-sodium, butafenacil, cinidon-ethyl, carfentra- zone-ethyl, flumioxazin, fluthiacet-methyl, fomesafen, lactofen, oxadiargyl, oxyfluorfen, saflufenacil, sulfentrazone, ethyl [3-[2-chloro-4-fluoro-5-(1 -methyl-6-trifluoromethyl-2,4-dioxo-1 ,2,3,4- tetrahydropyrimidin-3-yl)phenoxy]-2-pyridyloxy]acetate (CAS 353292-31 -6; S-3100), 1 ,5-dimethyl- 6-thioxo-3-(2,2,7-trifluoro-3-oxo-4-(prop-2-yl
• binary compositions includes compositions comprising one or more, for example 1 , 2 or 3, active compounds of the PPO A and either one or more, for example 1 , 2 or 3, herbicides B.
• the weight ratio of the active compounds A:B is generally in the range of from 1 :1000 to 1000:1 , preferably in the range of from 1 :500 to 500:1 , in particular in the range of from 1 :250 to 250:1 and particularly preferably in the range of from 1 :75 to 75:1 .
• herbicides B are the herbicides B as defined above; in particular the herbicides B.1 - B.229 listed below in table B:
• Herbicide B Herbicide B
• compositions 1.1 to 1.229 comprising acifluorfen and the substance(s) as defined in the respective row of table B-1 :
• compositions 2.1. to 2.229 which differ from the corresponding compositions 1.1 to 1.229 only in that they comprise as component A acifluorfen-sodium.
• compositions 3.1. to 3.229 which differ from the corresponding com- positions 1.1 to 1.229 only in that they comprise as component A azafenidin.
• compositions 4.1 . to 4.229 which differ from the corresponding compositions 1.1 to 1.229 only in that they comprise as component A bencarbazone. Also especially preferred are compositions 5.1. to 5.229 which differ from the corresponding compositions 1.1 to 1.229 only in that they comprise as component A benzfendizone.
• compositions 6.1. to 6.229 which differ from the corresponding compositions 1.1 to 1.229 only in that they comprise as component A bifenox.
• compositions 7.1 . to 7.229 which differ from the corresponding compositions 1.1 to 1.227 only in that they comprise as component A butafenacil.
• compositions 8.1. to 8.229 which differ from the corresponding com- positions 1.1 to 1 .229 only in that they comprise as component A carfentrazone.
• compositions 9.1 . to 9.229 which differ from the corresponding compositions 1.1 to 1. 229 only in that they comprise as component A carfentrazone-ethyl. Also especially preferred are compositions 10.1 . to 10.229 which differ from the corresponding compositions 1 .1 to 1.229 only in that they comprise as component A chlomethoxyfen.
• compositions 1 1.1. to 1 1 .229 which differ from the corresponding compositions 1.1 to 1 .229 only in that they comprise as component A cinidon-ethyl.
• compositions 12.1. to 12.229 which differ from the corresponding compositions 1 .1 to 1.229 only in that they comprise as component A fluazolate.
• compositions 13.1. to 13.229 which differ from the corresponding compositions 1 .1 to 1.229 only in that they comprise as component A flufenpyr.
• compositions 14.1. to 14.229 which differ from the corresponding compositions 1.1 to 1 .229 only in that they comprise as component A flufenpyr-ethyl. Also especially preferred are compositions 15.1. to 15.229 which differ from the corresponding compositions 1 .1 to 1.229 only in that they comprise as component A flumiclorac. Also especially preferred are compositions 16.1. to 16.229 which differ from the corresponding compositions 1 .1 to 1.229 only in that they comprise as component A flumiclorac-pentyl.
• compositions 17.1. to 17.229 which differ from the corresponding compositions 1 .1 to 1.229 only in that they comprise as component A flumioxazin.
• compositions 18.1. to 18.229 which differ from the corresponding compositions 1 .1 to 1.229 only in that they comprise as component A fluoroglycofen. Also especially preferred are compositions 19.1. to 19.229 which differ from the corresponding compositions 1 .1 to 1.229 only in that they comprise as component A fluoroglycofen-ethyl.
• compositions 20.1 . to 20.229 which differ from the corresponding compositions 1 .1 to 1.229 only in that they comprise as component A fluthiacet.
• compositions 21.1. to 21 .229 which differ from the corresponding compositions 1 .1 to 1.229 only in that they comprise as component A fluthiacet-methyl.
• compositions 22.1. to 22.229 which differ from the corresponding compositions 1 .1 to 1.229 only in that they comprise as component A fomesafen.
• compositions 23.1. to 23.229 which differ from the corresponding compositions 1 .1 to 1.229 only in that they comprise as component A halosafen. Also especially preferred are compositions 24.1. to 24.229 which differ from the corresponding compositions 1.1 to 1 .229 only in that they comprise as component A lactofen.
• compositions 25.1. to 25.229 which differ from the corresponding compositions 1 .1 to 1.229 only in that they comprise as component A oxadiargyl.
• compositions 26.1. to 26.229 which differ from the corresponding compositions 1 .1 to 1.229 only in that they comprise as component A oxadiazon.
• compositions 27.1. to 27.229 which differ from the corresponding compositions 1 .1 to 1.229 only in that they comprise as component A oxyfluorfen.
• compositions 28.1. to 28.229 which differ from the corresponding compositions 1 .1 to 1.229 only in that they comprise as component A pentoxazone.
• compositions 29.1. to 29.229 which differ from the corresponding compositions 1 .1 to 1.229 only in that they comprise as component A profluazol.
• compositions 30.1 . to 30.229 which differ from the corresponding compositions 1 .1 to 1.229 only in that they comprise as component A pyraclonil.
• compositions 31.1. to 31 .229 which differ from the corresponding compositions 1 .1 to 1 .229 only in that they comprise as component A pyraflufen.
• compositions 32.1. to 32.229 which differ from the corresponding compositions 1 .1 to 1.229 only in that they comprise as component A pyraflufen-ethyl.
• compositions 33.1 . to 33.229 which differ from the corresponding compositions 1 .1 to 1.229 only in that they comprise as component A saflufenacil.
• compositions 34.1. to 34.229 which differ from the corresponding compositions 1 .1 to 1.229 only in that they comprise as component A sulfentrazone.
• compositions 35.1. to 35.229 which differ from the corresponding compositions 1 .1 to 1.229 only in that they comprise as component A thidiazimin.
• compositions 36.1. to 36.229 which differ from the corresponding compositions 1 .1 to 1.229 only in that they comprise as component A tiafenacil.
• compositions 37.1. to 37.229 which differ from the corresponding compositions 1.1 to 1.229 only in that they comprise as component A ethyl [3-[2-chloro-4-fluoro-5- (1 -methyl-6-trifluoromethyl-2,4-dioxo-1 ,2,3,4-tetrahydropyrimidin-3-yl)phenoxy]-2- pyridyloxy]acetate (CAS 353292-31 -6; S-3100).
• compositions 38.1 . to 38.229 which differ from the corresponding compositions 1 .1 to 1.229 only in that they comprise as component A 1 ,5-dimethyl-6-thioxo-3- (2,27-trifluoro-3-oxo-4-(prop-2-ynyl)-3,4-dihydro-2H-benzo[b][1 ,4]oxazin-6-yl)-1 ,3,5-triazinane-2,4- dione (CAS 1258836-72-4)
• compositions 39.1. to 39.229 which differ from the corresponding compositions 1 .1 to 1.229 only in that they comprise as component A
• compositions 40.1. to 40.229 which differ from the corresponding compositions 1 .1 to 1.229 only in that they comprise as component A N-tetrahydrofurfuryl-3-(2,6- dichloro-4-trifluoromethylphenoxy)-5-methyl-1 H-pyrazole-1 -carboxamide (CAS 915396-43-9).
• compositions 41.1. to 41 .229 which differ from the corresponding compositions 1 .1 to 1.229 only in that they comprise as component A N-ethyl-3-(2-chloro-6-fluoro-4-trifluoromethylphenoxy)-5-methyl-1 H-pyrazole-1 -carboxamide (CAS 452099-05-7).
• compositions 42.1. to 42.229 which differ from the corresponding compositions 1 .1 to 1.229 only in that they comprise as component A N-tetrahydrofurfuryl-3-(2- chloro-6-fluoro-4-trifluoromethylphenoxy)-5-methyl-1 H-pyrazole-1 -carboxamide (CAS 452100-03- 7).
• compositions 43.1. to 43.229 which differ from the corresponding compositions 1 .1 to 1.229 only in that they comprise as component A
• compositions 44.1. to 44.229 which differ from the corresponding compositions 1 .1 to 1.229 only in that they comprise as component A methyl (E)-4-[2-chloro-5-[4- chloro-5-(difluoromethoxy)-1 /-/-methyl-pyrazol-3-yl]-4-fluoro-phenoxy]-3-methoxy-but-2-enoate (CAS 948893-00-3).
• compositions 45.1. to 45.229 which differ from the corresponding compositions 1 .1 to 1.229 only in that they comprise as component A
• compositions 46.1 . to 46.229 which differ from the corresponding compositions 1 .1 to 1.229 only in that they comprise as component A
• compositions 47.1. to 47.229 which differ from the corresponding compositions 1 .1 to 1.229 only in that they comprise as component A
• compositions 48.1. to 48.229 which differ from the corresponding compositions 1.1 to 1 .229 only in that they additionally comprise benoxacor as safener C.
• compositions 49.1. to 49.229 which differ from the corresponding compositions 1.1 to 1 .229 only in that they additionally comprise cloquintocet as safener C.
• compositions 50.1 . to 50.229 which differ from the corresponding compositions 1.1 to 1 .229 only in that they additionally comprise cyprosulfamide as safener C.
• compositions 51.1. to 51 .229 which differ from the corresponding compositions 1 .1 to 1.229 only in that they additionally comprise dichlormid as safener C.
• compositions 52.1. to 52.229 which differ from the corresponding compositions 1.1 to 1 .229 only in that they additionally comprise fenchlorazole as safener C.
• compositions 53.1 . to 53.229 which differ from the corresponding compositions 1.1 to 1 .229 only in that they additionally comprise fenclorim as safener C.
• compositions 54.1. to 54.229 which differ from the corresponding compositions 1 .1 to 1 .229 only in that they additionally comprise furilazole as safener C.
• compositions 55.1. to 55.229 which differ from the corresponding compositions 1.1 to 1 .229 only in that they additionally comprise isoxadifen as safener C.
• compositions 56.1. to 56.229 which differ from the corresponding compositions 1.1 to 1 .229 only in that they additionally comprise mefenpyr as safener C.
• compositions 57.1. to 57.229 which differ from the corresponding compositions 1.1 to 1 .229 only in that they additionally comprise 4-(dichloroacetyl)-1 -oxa-4- azaspiro[4.5]decane (MON4660, CAS 71526-07-3) as safener C.
• compositions 58.1. to 58.229 which differ from the corresponding compositions 1 .1 to 1.229 only in that they additionally comprise 2,2,5-trimethyl-3-(dichloroacetyl)- 1 ,3-oxazolidine (R-29148, CAS 52836-31 -4) as safener C.
• the compounds of the invention in combination with herbicides that are selective for the crop being treated and which complement the spectrum of weeds controlled by these compounds at the application rate employed. It is further generally preferred to apply the compounds of the invention and other complementary herbicides at the same time, either as a combination formulation or as a tank mix.
• polynucleotide molecules and polypeptides of the invention encompass polynucleotide molecules and polypeptides comprising a nucleotide or an amino acid sequence that is sufficiently identical to nucleotide sequences set forth in SEQ ID Nos: 1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, 19, 21 , 23, 25, 27, 29, 31 , 33, 35, 37, 39, 41 , 43, or 45, or to the amino acid sequences set forth in SEQ ID Nos: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, or 46.
• sufficiently identical is used herein to refer to a first amino acid or nucleotide sequence that contains a sufficient or minimum number of identical or equivalent (e.g., with a similar side chain) amino acid residues or nucleotides to a second amino acid or nucleotide sequence such that the first and second amino acid or nucleotide sequences have a common structural domain and/or common functional activity.
• sequence identity refers to the extent to which two optimally aligned DNA or amino acid sequences are invariant throughout a window of alignment of components, e.g., nucleotides or amino acids.
• An “identity fraction” for aligned segments of a test sequence and a reference sequence is the number of identical components that are shared by the two aligned sequences divided by the total number of components in reference sequence segment, i.e., the entire reference sequence or a smaller defined part of the reference sequence. "Percent identity” is the identity fraction times 100.
• Optimal alignment of sequences for aligning a comparison window are well known to those skilled in the art and may be conducted by tools such as the local homology algorithm of Smith and Waterman, the homology alignment algorithm of Needleman and Wunsch, the search for similarity method of Pearson and Lipman, and preferably by computerized implementa- tions of these algorithms such as GAP, BESTFIT, FASTA, and TFASTA available as part of the GCG. Wisconsin Package. (Accelrys Inc. Burlington, Mass.)
• an “isolated polynucleotide” including DNA, RNA, or a combination of these, single or double stranded, in the sense or antisense orientation or a combination of both, dsRNA or otherwise, we mean a polynucleotide which is at least partially separated from the polynucleotide sequences with which it is associated or linked in its native state.
• the isolated polynucleotide is at least 60% free, preferably at least 75% free, and most preferably at least 90% free from other compo- nents with which they are naturally associated.
• an isolated polynucleotide can be an exogenous polynucleotide present in, for example, a transgenic organism which does not naturally comprise the polynucleotide.
• polynu- cleotide(s) refers to nucleotides, either ribonucleotides or deox- yribonucleotides or a combination of both, in a polymeric unbranched form of any length.
• mut-PPO nucleic acid refers to a PPO nucleic acid having a sequence that is mutated from a wild-type PPO nucleic acid and that confers increased PPO-inhibiting herbicide tolerance to a plant in which it is expressed.
• mutated protoporphyrinogen oxidase refers to the replacement of an amino acid of the wild-type primary sequences SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, or 46, or a variant, a derivative, a homologue, an orthologue, or paralogue thereof, with another amino acid.
• mutated amino acid will be used below to designate the amino acid which is replaced by another amino acid, thereby designating the site of the mutation in the primary sequence of the protein.
• the PPO nucleotide sequence comprises the sequence of SEQ ID NO: 1 , 25, 37 or 39 or a variant or derivative thereof.
• the PPO nucleotide sequences encompasse homologues, paralogues and and orthologues of SEQ ID NO: 1 , 25, 37 or 39 as defined hereinafter.
• variants with respect to a sequence (e.g., a polypeptide or nucleic acid sequence such as - for example - a transcription regulating nucleotide sequence of the invention) is intended to mean substantially similar sequences.
• variants include those sequences that, because of the degeneracy of the genetic code, encode the identical amino acid sequence of the native protein.
• Naturally occurring allelic variants such as these can be identified with the use of well-known molecular biology techniques, as, for example, with polymerase chain reaction (PCR) and hybridization techniques.
• Variant nucleotide sequences also include synthetically derived nucleotide sequences, such as those generated, for example, by using site-directed mutagenesis and for open reading frames, encode the native protein, as well as those that encode a polypeptide having amino acid substitutions relative to the na- tive protein.
• nucleotide sequence variants of the invention will have at least 30, 40, 50, 60, to 70%, e.g., preferably 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, to 79%, generally at least 80%, e.g., 81 %-84%, at least 85%, e.g., 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, to 98% and 99% nucleotide "sequence identity" to the nucleotide sequence of SEQ ID NO: SEQ ID NO: 1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, 19, 21 , 23, 25, 27, 29, 31 , 33, 35, 37, 39, 41 , 43, or 45.
• the query sequence is at least 45 nucleotides in length, and the GAP analysis aligns the two sequences over a region of at least 45 nucleotides.
• the query sequence is at least 150 nucleotides in length, and the GAP analysis aligns the two sequences over a region of at least 150 nucleotides. More preferably, the query sequence is at least 300 nucleotides in length and the GAP analysis aligns the two sequences over a region of at least 300 nucleotides. Even more preferably, the GAP analysis aligns the two sequences over their entire length.
• substantially purified polypeptide or “purified” a polypeptide is meant that has been separated from one or more lipids, nucleic acids, other polypeptides, or other contaminating molecules with which it is associated in its native state. It is preferred that the substantially purified polypeptide is at least 60% free, more preferably at least 75% free, and more preferably at least 90% free from other components with which it is naturally associated. As the skilled addressee will appreciate, the purified polypeptide can be a recombinantly produced polypeptide.
• polypeptide and “protein” are generally used interchangeably and refer to a single polypeptide chain which may or may not be modified by addition of non-amino acid groups.
• polypeptide chains may associate with other polypeptides or proteins or other molecules such as co- factors.
• proteins and “polypeptides” as used herein also include variants, mutants, modifications, analogous and/or derivatives of the polypeptides of the invention as described herein.
• the query sequence is at least 25 amino acids in length, and the GAP analysis aligns the two sequences over a region of at least 25 amino acids.
• the query sequence is at least 50 amino acids in length, and the GAP analysis aligns the two sequences over a region of at least 50 amino acids. More preferably, the query sequence is at least 100 amino acids in length and the GAP analysis aligns the two sequences over a region of at least 100 amino acids. Even more preferably, the query sequence is at least 250 amino acids in length and the GAP analysis aligns the two sequences over a region of at least 250 amino acids. Even more preferably, the GAP analysis aligns the two sequences over their entire length.
• the PPO polypeptide of the invention comprises an amino acid sequence which is at least 40%, more preferably at least 45%, more preferably at least 50%, more preferably at least 55%, more preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91 %, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.1 %, more preferably at least 99.2%, more preferably at least 99.3%, more preferably at least 99.
• variant polypeptide is intended a polypeptide derived from the protein of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, or 46 by deletion (so-called truncation) or addition of one or more amino acids to the N-terminal and/or C-terminal end of the native protein; deletion or addition of one or more amino acids at one or more sites in the native protein; or substitution of one or more amino acids at one or more sites in the native protein.
• variants may result from, for example, genetic polymorphism or from human manipulation. Methods for such manipulations are generally known in the art.
• Derivatives of a protein encompass peptides, oligopeptides, polypeptides, proteins and enzymes having amino acid substitutions, deletions and/or insertions relative to the unmodified protein in question and having similar biological and functional activity as the unmodified protein from which they are derived.
• "Homologues” of a protein encompass peptides, oligopeptides, polypeptides, proteins and enzymes having amino acid substitutions, deletions and/or insertions relative to the unmodified protein in question and having similar biological and functional activity as the unmodified protein from which they are derived.
• a deletion refers to removal of one or more amino acids from a protein.
• Insertions refers to one or more amino acid residues being introduced into a predetermined site in a protein. Insertions may comprise N-terminal and/or C-terminal fusions as well as intra- sequence insertions of single or multiple amino acids. Generally, insertions within the amino acid sequence will be smaller than N- or C-terminal fusions, of the order of about 1 to 10 residues.
• N- or C-terminal fusion proteins or peptides include the binding domain or activation domain of a transcriptional activator as used in the yeast two-hybrid system, phage coat proteins, (histidine)-6-tag, glutathione S-transferase-tag, protein A, maltose-binding protein, dihydrofolate reductase, Tag « 100 epitope, c-myc epitope, FLAG ® -epitope, lacZ, CMP (calmodulin-binding peptide), HA epitope, protein C epitope and VSV epitope.
• a transcriptional activator as used in the yeast two-hybrid system
• phage coat proteins phage coat proteins
• glutathione S-transferase-tag glutathione S-transferase-tag
• protein A maltose-binding protein
• dihydrofolate reductase dihydrofolate reductase
• a substitution refers to replacement of amino acids of the protein with other amino acids having similar properties (such as similar hydrophobicity, hydrophilicity, antigenicity, propensity to form or break a-helical structures or ⁇ -sheet structures).
• Amino acid substitutions are typically of single residues, but may be clustered depending upon functional constraints placed upon the polypeptide and may range from 1 to 10 amino acids; insertions will usually be of the order of about 1 to 10 amino acid residues.
• the amino acid substitutions are preferably conservative amino acid substitutions. Conservative substitution tables are well known in the art (see for example Creighton (1984) Proteins. W.H. Freeman and Company (Eds).
• Amino acid substitutions, deletions and/or insertions may readily be made using peptide synthetic techniques well known in the art, such as solid phase peptide synthesis and the like, or by recombinant DNA manipulation. Methods for the manipulation of DNA sequences to produce substitution, insertion or deletion variants of a protein are well known in the art. For example, techniques for making substitution mutations at predetermined sites in DNA are well known to those skilled in the art and include M 13 mutagenesis, T7-Gen in vitro mutagenesis (USB, Cleveland, OH), Quick- Change Site Directed mutagenesis (Stratagene, San Diego, CA), PCR-mediated site-directed mutagenesis or other site-directed mutagenesis protocols.
• “Derivatives” further include peptides, oligopeptides, polypeptides which may, compared to the amino acid sequence of the naturally-occurring form of the protein, such as the protein of interest, comprise substitutions of amino acids with non-naturally occurring amino acid residues, or additions of non-naturally occurring amino acid residues.
• “Derivatives” of a protein also encompass peptides, oligopeptides, polypeptides which comprise naturally occurring altered (glycosylated, acylated, prenylated, phosphorylated, myristoylated, sulphated etc.) or non-naturally altered amino acid residues compared to the amino acid sequence of a naturally-occurring form of the polypeptide.
• a derivative may also comprise one or more non-amino acid substituents or additions compared to the amino acid sequence from which it is derived, for example a reporter molecule or oth- er ligand, covalently or non-covalently bound to the amino acid sequence, such as a reporter molecule which is bound to facilitate its detection, and non-naturally occurring amino acid residues relative to the amino acid sequence of a naturally-occurring protein.
• reporter molecule or oth- er ligand covalently or non-covalently bound to the amino acid sequence, such as a reporter molecule which is bound to facilitate its detection, and non-naturally occurring amino acid residues relative to the amino acid sequence of a naturally-occurring protein.
• derivatives also include fusions of the naturally-occurring form of the protein with tagging peptides such as FLAG, HIS6 or thioredoxin (for a review of tagging peptides, see Terpe, Appl. Microbiol. Biotechnol
• orthologues and “paralogues” encompass evolutionary concepts used to describe the ancestral relationships of genes. Paralogues are genes within the same species that have originated through duplication of an ancestral gene; orthologues are genes from different organisms that have origi- nated through speciation, and are also derived from a common ancestral gene. A non-limiting list of examples of such orthologues are shown in Table 1.
• paralogues and orthologues may share distinct domains harboring suitable amino acid residues at given sites, such as binding pockets for particular substrates or binding motifs for interaction with other proteins.
• domain refers to a set of amino acids conserved at specific positions along an alignment of sequences of evolutionahly related proteins. While amino acids at other positions can vary between homologues, amino acids that are highly conserved at specific positions indicate amino acids that are likely essential in the structure, stability or function of a protein. Identified by their high degree of conservation in aligned sequences of a family of protein homologues, they can be used as identifiers to determine if any polypeptide in question belongs to a previously identified polypeptide family.
• motif or "consensus sequence” refers to a short conserved region in the sequence of evolutionahly related proteins.
• Motifs are frequently highly conserved parts of domains, but may also include only part of the domain, or be located outside of conserved domain (if all of the amino acids of the motif fall outside of a defined domain).
• GAP uses the algorithm of Needleman and Wunsch ((1970) J Mol Biol 48: 443-453) to find the global (i.e. spanning the complete sequences) alignment of two sequences that maximizes the number of matches and minimizes the number of gaps.
• the BLAST algorithm (Altschul et al. (1990) J Mol Biol 215: 403-10) calculates percent sequence identity and performs a statistical analysis of the similarity between the two sequences.
• the software for performing BLAST analysis is publicly available through the National Centre for Biotechnology Information (NCBI).
• Homologues may readily be identified using, for example, the ClustalW multiple sequence alignment algorithm (version 1 .83), with the default pairwise alignment parameters, and a scoring method in percentage. Global percentages of similarity and identity may also be determined using one of the methods available in the MatGAT software package (Campanella et al., BMC Bioinformatics. 2003 Jul 10;4:29. MatGAT: an application that generates similarity/identity matrices using protein or DNA sequences.). Minor manual editing may be performed to optimise alignment between conserved motifs, as would be apparent to a person skilled in the art. Furthermore, instead of using full-length sequences for the identification of homologues, specific domains may also be used.
• sequence identity values may be determined over the entire nucleic acid or amino acid sequence or over selected domains or conserved motif(s), using the programs mentioned above using the default parameters.
• the Smith- Waterman algorithm is particularly useful (Smith TF, Waterman MS (1981 ) J. Mol. Biol 147(1 );195- 7).
• the inventors of the present invention have found that by substituting one or more of the key amino acid residues the herbicide tolerance or resistance could be remarkably increased as compared to the activity of the wild type PPO enzymes with SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, or 46.
• Preferred substitutions of mut-PPO are those that increase the herbicide tolerance of the plant, but leave the biological activitiy of the oxidase activity substantially unaffected.
• the key amino acid residues of a PPO enzyme is substituted by any other amino acid.
• the key amino acid residues of a PPO enzyme is substituted by a conserved amino acid as depicted in Table 2. It will be understood by the person skilled in the art that amino acids located in a close proximity to the positions of amino acids mentioned below may also be substituted.
• a variant, derivative, orthologue, paralogue or homologue thereof comprises a mut-PPO, wherein an amino acid ⁇ 3, ⁇ 2 or ⁇ 1 amino acid positions from a key amino acid is sub- stituted by any other amino acid.
• sequence pattern is not limited by the exact distances between two adjacent amino acid residues of said pattern.
• Each of the distances between two neighbours in the above patterns may, for ex- ample, vary independently of each other by up to ⁇ 10, ⁇ 5, ⁇ 3, ⁇ 2 or ⁇ 1 amino acid positions without substantially affecting the desired activity.
• the inventors of the present invention have identified specific combinations of mutations, which combination refers to a substi- tution of the Phenylalanine residue at position 420 in SEQ ID NO:2 or 4, combined with a second substitution of the Leucin at position 397 in SEQ ID NO:2 or 4.
• the variant or derivative of the mut-PPO of SEQ ID NO: 2 or SEQ ID NO: 4 is selected from the combined amino acid substitutions of the following Table 3a.
• Table 3a SEQ ID NO: 2 or SEQ ID NO:4 (combined amino acid substitutions obtained by site directed mutagenesis.)
• the variant or derivative of the mut-PPO of SEQ ID NO: 2 or SEQ ID NO: 4 is selected from the combined amino acid substitutions of the following Table 3b.
• Table 3b SEQ ID NO: 2 or SEQ ID NO:4 (combined amino acid substitutions)
• the amino acid sequence differs from an amino acid sequence of a PPO of SEQ ID NO: 2 or SEQ ID NO: 4 at one or more of the following positions: 128, 204, 208, 397, 400, 420, 457. Examples of differences at these amino acid positions include, but are not limited to, one or more of the following:
• amino acid at position 128 is other than Arginine
• amino acid at position 204 is other than Phenylalanine
• the amino acid at position 208 is other than Threonine
• amino acid at position 397 is other than Leucine
• amino acid at position 400 is other than Leucine
• amino acid at position 420 is other than Phenylalanine
• the amino acid at position 457 is other than Phenylalanine.
• the mut-PPO enzyme of SEQ ID NO: 2 or SEQ ID NO: 4 comprises one or more of the following: the amino acid at position 128 is Leu, Ala, Val, or lie;
• amino acid at position 204 is Ala, Leu, lie, or Val; the amino acid at position 208 is Ser;
• amino acid at position 397 is Gly, Ala, Ser, Thr, Cys, Val, lie, Met, Pro, Phe, Tyr, Trp, His, Lys, Arg, Asn, Asp, Glu, or Gin;
• amino acid at position 400 is Ala, lie, Val, or Met;
• amino acid at position 420 is Val, Met, Ala, lie, or Leu;
• amino acid at position 457 is Met, Ala, Leu, lie, Val;
• the mut-PPO comprises a sequence of SEQ ID NO: 2, a variant, derivative, orthologue, paralogue or homologue thereof, in which:
• amino acid at position 397 is Gly, Ala, Ser, Thr, Cys, Val, lie, Met, Pro, Tyr, Trp, Asp, Glu, Asn, Gin, His, Lys, or Arg
• amino acid at position 420 is Met, Ala, Leu, lie, or Val.
• the mut-PPO comprises a sequence of SEQ ID NO: 2, a variant, derivative, orthologue, paralogue or homologue thereof, in which:
• amino acid at position 397 is Gly
• amino acid at position 420 is Met
• the mut-PPO comprises a sequence of SEQ ID NO: 2, a variant, derivative, orthologue, paralogue or homologue thereof, in which:
• amino acid at position 397 is Gly
• amino acid at position 420 is Ala
• the mut-PPO comprises a sequence of SEQ ID NO: 2, a variant, derivative, orthologue, paralogue or homologue thereof, in which:
• the amino acid at position 397 is Gly, and the amino acid at position 420 is Leu.
• the mut-PPO comprises a sequence of SEQ ID NO: 2, a variant, derivative, orthologue, paralogue or homologue thereof, in which:
• the amino acid at position 397 is Gly, and the amino acid at position 420 is lie.
• the mut-PPO comprises a sequence of SEQ ID NO: 2, a variant, derivative, orthologue, paralogue or homologue thereof, in which:
• amino acid at position 397 is Gly
• amino acid at position 420 is Val
• the mut-PPO comprises a sequence of SEQ ID NO: 2, a variant, derivative, orthologue, paralogue or homologue thereof, in which:
• amino acid at position 397 is Ala
• amino acid at position 420 is Met
• the mut-PPO comprises a sequence of SEQ ID NO: 2, a variant, derivative, orthologue, paralogue or homologue thereof, in which:
• the amino acid at position 397 is Ala
• the amino acid at position 420 is Ala
• the mut-PPO comprises a sequence of SEQ ID NO: 2, a variant, derivative, orthologue, paralogue or homologue thereof, in which:
• the amino acid at position 397 is Ala
• the amino acid at position 420 is Leu.
• the mut-PPO comprises a sequence of SEQ ID NO: 2, a variant, derivative, orthologue, paralogue or homologue thereof, in which:
• the amino acid at position 397 is Ala, and the amino acid at position 420 is lie.
• the mut-PPO comprises a sequence of SEQ ID NO: 2, a variant, derivative, orthologue, paralogue or homologue thereof, in which:
• the amino acid at position 397 is Ala
• the amino acid at position 420 is Val.
• the mut-PPO comprises a sequence of SEQ ID NO: 2, a variant, derivative, orthologue, paralogue or homologue thereof, in which:
• amino acid at position 397 is Ser
• amino acid at position 420 is Met
• the mut-PPO comprises a sequence of SEQ ID NO: 2, a variant, derivative, orthologue, paralogue or homologue thereof, in which:
• amino acid at position 397 is Ser
• amino acid at position 420 is Ala
• the mut-PPO comprises a sequence of SEQ ID NO: 2, a variant, derivative, orthologue, paralogue or homologue thereof, in which:
• amino acid at position 397 is Ser
• amino acid at position 420 is Leu.
• the mut-PPO comprises a sequence of SEQ ID NO: 2, a variant, derivative, orthologue, paralogue or homologue thereof, in which:
• amino acid at position 397 is Ser
• amino acid at position 420 is lie.
• the mut-PPO comprises a sequence of SEQ ID NO: 2, a variant, derivative, orthologue, paralogue or homologue thereof, in which:
• the amino acid at position 397 is Ser
• the amino acid at position 420 is Val.
• the mut-PPO comprises a sequence of SEQ ID NO: 2, a variant, derivative, orthologue, paralogue or homologue thereof, in which:
• amino acid at position 397 is Thr
• amino acid at position 420 is Met
• the mut-PPO comprises a sequence of SEQ ID NO: 2, a variant, derivative, orthologue, paralogue or homologue thereof, in which:
• the amino acid at position 397 is Thr, and the amino acid at position 420 is Ala.
• the mut-PPO comprises a sequence of SEQ ID NO: 2, a variant, derivative, orthologue, paralogue or homologue thereof, in which:
• the amino acid at position 397 is Thr, and the amino acid at position 420 is Leu.
• the mut-PPO comprises a sequence of SEQ ID NO: 2, a variant, derivative, orthologue, paralogue or homologue thereof, in which:
• the amino acid at position 397 is Thr, and the amino acid at position 420 is lie.
• the mut-PPO comprises a sequence of SEQ ID NO: 2, a variant, derivative, orthologue, paralogue or homologue thereof, in which:
• amino acid at position 397 is Thr
• amino acid at position 420 is Val
• the mut-PPO comprises a sequence of SEQ ID NO: 2, a variant, derivative, orthologue, paralogue or homologue thereof, in which:
• amino acid at position 397 is Cys
• amino acid at position 420 is Met
• the mut-PPO comprises a sequence of SEQ ID NO: 2, a variant, derivative, orthologue, paralogue or homologue thereof, in which:
• amino acid at position 397 is Cys
• amino acid at position 420 is Ala
• the mut-PPO comprises a sequence of SEQ ID NO: 2, a variant, derivative, orthologue, paralogue or homologue thereof, in which:
• the amino acid at position 397 is Cys
• the amino acid at position 420 is Leu.
• the mut-PPO comprises a sequence of SEQ ID NO: 2, a variant, derivative, orthologue, paralogue or homologue thereof, in which:
• amino acid at position 397 is Cys
• amino acid at position 420 is lie.
• the mut-PPO comprises a sequence of SEQ ID NO: 2, a variant, derivative, orthologue, paralogue or homologue thereof, in which:
• amino acid at position 397 is Cys
• amino acid at position 420 is Val
• the mut-PPO comprises a sequence of SEQ ID NO: 2, a variant, derivative, orthologue, paralogue or homologue thereof, in which:
• amino acid at position 397 is Val
• amino acid at position 420 is Met
• the mut-PPO comprises a sequence of SEQ ID NO: 2, a variant, derivative, orthologue, paralogue or homologue thereof, in which:
• amino acid at position 397 is Val
• amino acid at position 420 is Ala
• the mut-PPO comprises a sequence of SEQ ID NO: 2, a variant, derivative, orthologue, paralogue or homologue thereof, in which:
• the amino acid at position 397 is Val
• the amino acid at position 420 is Leu.
• the mut-PPO comprises a sequence of SEQ ID NO: 2, a variant, derivative, orthologue, paralogue or homologue thereof, in which:
• the amino acid at position 397 is Val
• the amino acid at position 420 is lie.
• the mut-PPO comprises a sequence of SEQ ID NO: 2, a variant, derivative, orthologue, paralogue or homologue thereof, in which:
• amino acid at position 397 is Val
• amino acid at position 420 is Val
• the mut-PPO comprises a sequence of SEQ ID NO: 2, a variant, derivative, orthologue, paralogue or homologue thereof, in which:
• the amino acid at position 397 is lie, and the amino acid at position 420 is Met.
• the mut-PPO comprises a sequence of SEQ ID NO: 2, a variant, derivative, orthologue, paralogue or homologue thereof, in which:
• amino acid at position 397 is lie, and the amino acid at position 420 is Ala.
• the mut-PPO comprises a sequence of SEQ ID NO: 2, a variant, derivative, orthologue, paralogue or homologue thereof, in which:
• amino acid at position 397 is lie, and the amino acid at position 420 is Leu.
• the mut-PPO comprises a sequence of SEQ ID NO: 2, a variant, derivative, orthologue, paralogue or homologue thereof, in which:
• the amino acid at position 397 is lie, and the amino acid at position 420 is lie.
• the mut-PPO comprises a sequence of SEQ ID NO: 2, a variant, derivative, orthologue, paralogue or homologue thereof, in which:
• amino acid at position 397 is lie, and the amino acid at position 420 is Val.
• the mut-PPO comprises a sequence of SEQ ID NO: 2, a variant, derivative, orthologue, paralogue or homologue thereof, in which:
• the amino acid at position 397 is Met
• the amino acid at position 420 is Met
• the mut-PPO comprises a sequence of SEQ ID NO: 2, a variant, derivative, orthologue, paralogue or homologue thereof, in which:
• amino acid at position 397 is Met
• amino acid at position 420 is Ala
• the mut-PPO comprises a sequence of SEQ ID NO: 2, a variant, derivative, orthologue, paralogue or homologue thereof, in which:
• amino acid at position 397 is Met
• amino acid at position 420 is Leu.
• the mut-PPO comprises a sequence of SEQ ID NO: 2, a variant, derivative, orthologue, paralogue or homologue thereof, in which:
• the amino acid at position 397 is Met, and the amino acid at position 420 is lie.
• the mut-PPO comprises a sequence of SEQ ID NO: 2, a variant, derivative, orthologue, paralogue or homologue thereof, in which:
• amino acid at position 397 is Met
• amino acid at position 420 is Val
• the mut-PPO comprises a sequence of SEQ ID NO: 2, a variant, derivative, orthologue, paralogue or homologue thereof, in which:
• amino acid at position 397 is Pro
• amino acid at position 420 is Met
• the mut-PPO comprises a sequence of SEQ ID NO: 2, a variant, derivative, orthologue, paralogue or homologue thereof, in which:
• amino acid at position 397 is Pro
• amino acid at position 420 is Ala
• the mut-PPO comprises a sequence of SEQ ID NO: 2, a variant, derivative, orthologue, paralogue or homologue thereof, in which:
• amino acid at position 397 is Pro
• amino acid at position 420 is Leu.
• the mut-PPO comprises a sequence of SEQ ID NO: 2, a variant, derivative, orthologue, paralogue or homologue thereof, in which:
• the amino acid at position 397 is Pro, and the amino acid at position 420 is lie.
• the mut-PPO comprises a sequence of SEQ ID NO: 2, a variant, derivative, orthologue, paralogue or homologue thereof, in which:
• amino acid at position 397 is Pro
• amino acid at position 420 is Val
• the mut-PPO comprises a sequence of SEQ ID NO: 2, a variant, derivative, orthologue, paralogue or homologue thereof, in which:
• amino acid at position 397 is Tyr
• amino acid at position 420 is Met
• the mut-PPO comprises a sequence of SEQ ID NO: 2, a variant, derivative, orthologue, paralogue or homologue thereof, in which:
• amino acid at position 397 is Tyr
• amino acid at position 420 is Ala
• the mut-PPO comprises a sequence of SEQ ID NO: 2, a variant, derivative, orthologue, paralogue or homologue thereof, in which:
• amino acid at position 397 is Tyr
• amino acid at position 420 is Leu.
• the mut-PPO comprises a sequence of SEQ ID NO: 2, a variant, derivative, orthologue, paralogue or homologue thereof, in which:
• the amino acid at position 397 is Tyr, and the amino acid at position 420 is lie.
• the mut-PPO comprises a sequence of SEQ ID NO: 2, a variant, derivative, orthologue, paralogue or homologue thereof, in which:
• the amino acid at position 397 is Tyr, and the amino acid at position 420 is Val.
• the mut-PPO comprises a sequence of SEQ ID NO: 2, a variant, derivative, orthologue, paralogue or homologue thereof, in which:
• the amino acid at position 397 is Trp, and the amino acid at position 420 is Met.
• the mut-PPO comprises a sequence of SEQ ID NO: 2, a variant, derivative, orthologue, paralogue or homologue thereof, in which:
• the amino acid at position 397 is Trp
• the amino acid at position 420 is Ala.
• the mut-PPO comprises a sequence of SEQ ID NO: 2, a variant, derivative, orthologue, paralogue or homologue thereof, in which:

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