Herbicide-detoxifying enzymes

Abstract

The present invention refers to novel herbicide-detoxyfying enzymes capable of demethylating thereby inactivating herbicidal compounds, in particular of the diaminotriazine class.

Description

[0001] HERBICIDE-DETOXYFYING ENZYMES

[0002] REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

[0003] The Sequence Listing, which is a part of the present disclosure, is submitted concurrently with the specification as an XML file. The name of the file containing the Sequence Listing is “231201_SeqListing.xml”, which was created on 16 January 2025 and is 264,400 bytes in size. The entire contents of the Sequence Listing are incorporated herein by reference.

[0004] FIELD OF THE INVENTION

[0005] The present invention relates in general to methods for conferring on plants agricultural level tolerance to herbicides. Particularly, the invention refers to novel demethylases capable of inactivating herbicides, more specifically to herbicides which inhibit cellulose biosynthesis, thus, interfere with cell wall biosynthesis.

[0006] BACKGROUND OF THE INVENTION

[0007] Three main strategies are available for making plants tolerant to herbicides, i.e. (1) detoxifying the herbicide with an enzyme which transforms the herbicide, or its active metabolite, into non-toxic products, such as, for example, the enzymes for tolerance to bromoxynil or to basta (EP242236, EP337899); (2) mutating the target enzyme into a functional enzyme which is less sensitive to the herbicide, or to its active metabolite, such as, for example, the enzymes for tolerance to glyphosate (EP293356, Padgette S. R. et al., J. Biol. Chem., 266, 33, 1991); or (3) overexpressing the sensitive enzyme so as to produce quantities of the target enzyme in the plant which are sufficient in relation to the herbicide, in view of the kinetic constants of this enzyme, so as to have enough of the functional enzyme available despite the presence of its inhibitor.

[0008] The present invention provides new enzymes, in particular demethylases and their potential use to increase herbicide tolerance in plants by their capability to demethylate, and, thus, detoxify the herbicide, in particular cellulose biosynthesis inhibitors named azines. The inventors of the present invention have surprisingly found that contacting of the demethylase of the present invention with herbicidal compounds leads to inactivation of said herbicidal compounds by removing a methyl group. The expression of such demethylases in crop plants may find use in generating novel herbicide-tolerant mode of action.

[0009] The problem of the present invention can be seen as to the provision of novel traits by identifying target polypeptides, the expression of which makes plants tolerant to herbicides.

[0010] The problem is solved by the subject-matter of the present invention.

[0011] SUMMARY OF THE INVENTION

[0012] Accordingly, in one aspect, the present invention! provides an isolated, recombinant and / or chemically synthesized nucleic acid encoding a demethylase, the nucleic acid comprising the sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129 or 131, or a variant thereof, as defined hereinafter. A preferred embodiment refers to an isolated and / or recombinantly produced and / or synthetic nucleic acid molecule comprising a nucleic acid molecule encoding a demethylase polypeptide selected from the group consisting of:

[0013] (a) a nucleic acid molecule encoding a demethylase polypeptide comprising the sequence 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, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102,

[0014] 104. 106. 108. 110. 112. 114. 116. 118. 120. 122. 124. 126. 128. 130, or 132, or a variant, paralogue, orthologue or homolog thereof;

[0015] (b) a nucleic acid molecule comprising the sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71 , 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117,

[0016] 119. 121. 123. 125. 127. 129 or 131, or a variant, paralogue, orthologue or homolog thereof;

[0017] (c) a nucleic acid molecule, which, as a result of the degeneracy of the genetic code, can be derived from a demethylase polypeptide sequence 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, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120,

[0018] 122. 124. 126. 128. 130, or 132, or a variant, paralogue, orthologue or homolog thereof, and confers increased herbicide tolerance or resistance, as compared to a corresponding, e.g. nontransformed, wild type plant cell, a plant or a part thereof;

[0019] (d) a nucleic acid molecule having 30% or more identity, preferably 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99,5%, or more with the nucleic acid molecule sequence of a polynucleotide comprising the nucleic acid molecule of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113,

[0020] 115. 117. 119. 121. 123. 125. 127. 129 or 131, or a variant, paralogue, orthologue or homolog thereof, and confers increased herbicide tolerance or resistance, as compared to a corresponding, e.g. non-transformed, wild type plant cell, a plant or a part thereof;

[0021] (e) a nucleic acid molecule encoding a demethylase polypeptide having 30% or more identity, preferably at least 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99,5% or more, with the amino acid sequence of the demethylase polypeptide sequence 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, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100,

[0022] 102. 104. 106. 108. 110. 112. 114. 116. 118. 120. 122. 124. 126. 128. 130, or 132, and confers increased herbicide tolerance or resistance, as compared to a corresponding, e.g. nontransformed, wild type plant cell, a plant or a part thereof;

[0023] (f) nucleic acid molecule which hybridizes with a nucleic acid molecule of (a), (b), (c), (d) or (e) under stringent hybridization conditions and confers increased herbicide tolerance or resistance, as compared to a corresponding, e.g. non-transformed, wild type plant cell, a plant or a part thereof; wherein the encoded demethylase is capable of demethylating a herbicide.

[0024] Another object refers to an expression cassette, a vector and a recombinant microorganism comprising the nucleic acid molecule of the present invention. Another object refers to an isolated, recombinant and / or chemically synthesized demethylase 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, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, or 132, a variant, derivative, orthologue, paralogue or homologue thereof, as defined hereinafter.

[0025] Another object refers to a plant or plant part comprising a polynucleotide encoding a demethylase polypeptide of the present invention, the expression of said polynucleotide confers to the plant or plant part tolerance to herbicides.

[0026] In some aspects, the present invention provides a seed capable of germination into a plant comprising in at least some of its cells a polynucleotide operably linked to a promoter operable in plant cells, the promoter capable of expressing a demethylase polypeptide encoded by the polynucleotide, the expression of the demethylase polypeptide conferring to the plant tolerance to herbicides.

[0027] In one aspect, the present invention provides a plant cell capable of regenerating a plant comprising in at least some of its cells a polynucleotide operably linked to a promoter operable in plant cells, the promoter capable of expressing a demethylase polypeptide encoded by the polynucleotide, the expression of the demethylase polypeptide conferring to the plant tolerance to herbicides, wherein the plant cell comprises the polynucleotide operably linked to a promoter.

[0028] In another aspect, the present invention provides a plant cell comprising a polynucleotide operably linked to a promoter operable in a cell, the promoter capable of expressing a demethylase polypeptide encoded by the polynucleotide, the expression of the demethylase polypeptide conferring to the plant tolerance to herbicides.

[0029] In other aspects, the present invention provides a plant product prepared from a plant or plant part comprising in at least some of its cells a polynucleotide operably linked to a promoter operable in plant cells, the promoter capable of expressing a demethylase polypeptide encoded by the polynucleotide, the expression of the demethylase polypeptide conferring to the plant tolerance to herbicides.

[0030] In some aspects, the present invention provides a progeny or descendant plant derived from a plant comprising in at least some of its cells a polynucleotide operably linked to a promoter operable in plant cells, the promoter capable of expressing a demethylase polypeptide encoded by the polynucleotide, wherein the progeny or descendant plant comprises in at least some of its cells the recombinant polynucleotide operably linked to the promoter, the expression of the demethylase polypeptide conferring to the progeny or descendant plant tolerance to the herbicides.

[0031] In other aspects, the present invention provides a method for demethylating a herbicide, the method comprising contacting said herbicide with the demethylase of the present invention.

[0032] Preferably, the demethylase comprises sequence having at least 80%, 90%, 95%, 98%, 99% identity to the sequence 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, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, or 132, or a variant, paralogue, orthologue or homologue thereof. In some aspects, the present invention provides the use of the demethylase of the present invention for demethylating a herbicidal composition.

[0033] In other aspects, the present invention provides a method of producing a plant having tolerance to herbicides, the method comprising regenerating a plant from a plant cell transformed with a polynucleotide operably linked to a promoter operable in plant cells, the promoter capable of expressing a demethylase polypeptide encoded by the polynucleotide, the expression of the demethylase polypeptide conferring to the plant tolerance to herbicides.

[0034] In one aspect, the present invention provides a method of producing a progeny plant having tolerance to herbicides, the method comprising: crossing a first herbicide- tolerant plant with a second plant to produce a herbicide- tolerant progeny plant, wherein the first plant and the progeny plant comprise in at least some of their cells a polynucleotide operably linked to a promoter operable in plant cells, the promoter capable of expressing a demethylase polypeptide encoded by the polynucleotide, the expression of the demethylase polypeptide conferring to the plant tolerance to herbicides.

[0035] In addition, the present invention refers to a method for identifying a herbicide by using a demethylase of the present invention encoded by a nucleic acid which comprises the nucleotide sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129 or 131, or a variant, homologue, paralogue or orthologue thereof.

[0036] Said method comprises the steps of: a) generating a transgenic cell or plant comprising a nucleic acid encoding a demethylase of the present invention, wherein the demethylase of the present invention is expressed; b) applying a herbicide to the transgenic cell or plant of a) and to a control cell or plant of the same variety; c) determining the growth or the viability of the transgenic cell or plant and the control cell or plant after application of said test compound, and d) selecting test compounds which confer reduced growth to the control cell or plant as compared to the growth of the transgenic cell or plant.

[0037] Another object refers to a method of identifying a nucleotide sequence encoding a demethylase which is capable of demethylating a herbicide, the method comprising: a) generating a library of demethylase-encoding nucleic acids, b) screening a population of the resulting demethylase-encoding nucleic acids by expressing each of said nucleic acids in a cell or plant and treating said cell or plant with an herbicide, c) comparing the herbicide-tolerance levels provided by said population of demethylase encoding nucleic acids with the herbicide-tolerance level provided by a control demethylase-encoding nucleic acid, d) selecting at least one demethylase-encoding nucleic acid that provides a significantly increased level of tolerance to a herbicide as compared to that provided by the control demethylase-encoding nucleic acid.

[0038] In a preferred embodiment, the demethylase-encoding nucleic acid selected in step d) provides at least 2-fold as much tolerance to a herbicide as compared to that provided by the control demethylase-encoding nucleic acid.

[0039] The resistance or tolerance can be determined by generating a transgenic plant comprising a nucleic acid sequence of the library of step a) and comparing said transgenic plant with a control plant.

[0040] In another embodiment, the invention refers to a plant cell transformed by and expressing a nucleic acid encoding a demethylase according to the present invention.

[0041] In a preferred embodiment, the expression of said nucleic acid in the plant cell results in increased resistance or tolerance to a herbicide as compared to a wildtype variety of the plant cell

[0042] In another embodiment, 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 herbicide as compared to a wildtype variety of the plant.

[0043] Preferably, the expression of the nucleic acid of the invention in the plant results in the plant's increased resistance to herbicides as compared to a wildtype variety of the plant.

[0044] In another embodiment, 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 herbicide as compared to a wildtype variety of the seed.

[0045] In another embodiment, the invention refers to a method of producing a transgenic plant cell with an increased resistance to a herbicide as compared to a wildtype variety of the plant cell comprising, transforming the plant cell with an expression cassette comprising a polynucleotide operably linked to a promoter operable in plant cells, the promoter capable of expressing a demethylase polypeptide encoded by the polynucleotide.

[0046] In another embodiment, the invention refers to a method of producing a transgenic plant comprising, (a) transforming a plant cell with an expression cassette comprising a polynucleotide operably linked to a promoter operable in plant cells, the promoter capable of expressing a demethylase polypeptide encoded by the polynucleotide, and (b) generating a plant with an increased resistance to herbicide from the plant cell.

[0047] Preferably, the expression cassette further comprises a transcription initiation regulatory region and a translation initiation regulatory region that are functional in the plant.

[0048] DESCRIPTION OF THE DRAWINGS

[0049] Figures 1 A ■ 1 K: show a multiple alignment identifying conserved regions among the demethylases according to the present invention.

[0050] Figure 2. shows a schematic overview of the enzymatic reaction catalyzed by the demethylase of the present invention, i.e. the demethylation of N4-(5-chloro-2,3-difluoro-6-methoxy-phenyl)-6-(1-fluoro-1-methyl-ethyl)- 1 ,3, 5-tri azi ne-2, 4-d i am ine .

[0051] Figure 3. shows that the demethylase of the present invention (e.g. SEQ ID NO: 2) completely demethylated N4-(5-chloro-2,3-difluoro-6-methoxy-phenyl)-6-(1-fluoro-1-methyl-ethyl)-1 ,3,5-triazine-2,4-diamine in the crude cell lysate assay; values on the Y-axis are given in percentage of demethylation.

[0052] Figure 4: shows native demethylase of SEQ ID NO: 2 is superior over known deaminase (TriA variants comprising mutated variants of SEQ ID NO: 133 as disclosed in WO2016 / 116870, WO2018 / 011750, or WO2019 / 142099) in the Arabidopsis germination assay performed in the presence of N4-(5-chloro-2,3-difluoro- 6-methoxy-phenyl)-6-(1 -fluoro-1 -methyl-ethyl)-1 ,3,5-triazine-2,4-diamine. (A) TriA-variant 1 (>0.49uM azine); (B) TriA-variant 2 (0.195uM azine); (C) TriA-variant 3 (6.25uM azine); (D)-(G) demethylase SEQ ID NO: 2; (H) no azine

[0053] Figure 5 shows the result of a soybean hairy root assay and demonstrates that the demethyase of the present invention (e.g. SEQ ID NO: 2) provides tolerance to N4-(5-chloro-2,3-difluoro-6-methoxy-phenyl)-6-(1 -fluoro-1 - methyl-ethyl)-1 ,3,5-triazine-2,4-diamine when transformed into soy seedlings via Agrobacterium rhizogenes mediated transformation. Roots were collected after two weeks and fresh weight (FW) was measured.

[0054] (1) untransformed Control; 3nM Azine; FW=2.47g

[0055] (2) untransformed Control, no Azine; FW=47.6g

[0056] (3) transformed with cytosol-localized demethylase; 3nM Azine FW=41.1g

[0057] (4) transformed with plastid-localized demethylase; 3nM Azine FW=27.77g

[0058] (5) transformed with mitochondria-localized demethylase;3nM Azine FW=26.42g

[0059] (6) transformed with apoplast-localized demethylase; 3nM Azine FW=31.62g

[0060] DETAILED DESCRIPTION

[0061] The articles "a" and "an" are used herein to refer to one or more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one or more elements.

[0062] As used herein, the word "comprising," or variations such as "comprises" or "comprising," will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[0063] The term “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.). The term “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, pedicles, 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. The term “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.

[0064] 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. (e.g. Avena sativa, Avena fatua, Avena byzantina, Avena fatua var. sativa, Avena hybrida), Averrhoa carambola, Bambusa sp., Benincasa hispida, Bertholletia excelsea, Beta vulgaris, Brassica spp. (e.g. Brassica napus, Brassica rapa ssp. [canola, oilseed rape, turnip rape]), Cadaba farinosa, Camellia sinensis, Canna indica, Cannabis sativa, Capsicum spp., Carex elata, Carica papaya, Carissa macrocarpa, Carya spp., Carthamus tinctorius, Castanea spp., Ceiba pentandra, Cichorium endivia, Cinnamomum spp., Citrullus lanatus, Citrus spp., Cocos spp., Coffea spp., Colocasia esculenta, Cola spp., Corchorus sp., Coriandrum sativum, Corylus spp., Crataegus spp., Crocus sativus, Cucurbita spp., Cucumis spp., Cynara spp., Daucus carota, Desmodium spp., Dimocarpus longan, Dioscorea spp., Diospyros spp., Echinochloa spp., Elaeis (e.g. Elaeis guineensis, Elaeis oleifera), Eleusine coracana, Eragrostis tef, Erianthus sp., Eriobotrya japonica, Eucalyptus sp., Eugenia uniflora, Fagopyrum spp., Fagus spp., Festuca arundinacea, Ficus carica, Fortunella spp., Fragaria spp., Ginkgo biloba, Glycine spp. (e.g. Glycine max, Soja hispida or Soja max), Gossypium hirsutum, Helianthus spp. (e.g. Helianthus annuus), Hemerocallis fulva, Hibiscus spp., Hordeum spp. (e.g. Hordeum vulgare), Ipomoea batatas, Juglans spp., Lactuca sativa, Lathyrus spp., Lens culinaris, Linum usitatissimum, Litchi chinensis, Lotus spp., Luffa acutangula, Lupinus spp., Luzula sylvatica, Lycopersicon spp. (e.g. Lycopersicon esculentum, Lycopersicon lycopersicum, Lycopersicon pyriforme), Macrotyloma spp., Malus spp., Malpighia emarginata, Mammea americana, Mangifera indica, Manihot spp., Manilkara zapota, Medicago sativa, Melilotus spp., Mentha spp., Miscanthus sinensis, Momordica spp., Morus nigra, Musa spp., Nicotiana spp., Olea spp., Opuntia spp., Ornithopus spp., Oryza spp. (e.g. Oryza sativa, Oryza latifolia), Panicum miliaceum, Panicum virgatum, Passiflora edulis, Pastinaca sativa, Pennisetum sp., Persea spp., Petroselinum crispum, Phalaris arundinacea, Phaseolus spp., Phleum pratense, Phoenix spp., Phragmites australis, Physalis spp., Pinus spp., Pistacia vera, Pisum spp., Poa spp., Populus spp., Prosopis spp., Prunus spp., Psidium spp., Punica granatum, Pyrus communis, Quercus spp., Raphanus sativus, Rheum rhabarbarum, Ribes spp., Ricinus communis, Rubus spp., Saccharum spp., Salix sp., Sambucus spp., Secale cereale, Sesamum spp., Sinapis sp., Solanum spp. (e.g. Solanum tuberosum, Solanum integrifolium or Solanum lycopersicum), Sorghum bicolor, Spinacia spp., Syzygium spp., Tagetes spp., Tamarindus indica, Theobroma cacao, Trifolium spp., Tripsacum dactyloides, Triticosecale rimpaui, Triticum spp. (e.g. Triticum aestivum, Triticum durum, Triticum turgidum, Triticum hybernum, Triticum macha, Triticum sativum, Triticum monococcum or Triticum vulgare), Tropaeolum minus, Tropaeolum majus, Vaccinium spp., Vicia spp., Vigna spp., Viola odorata, Vitis spp., Zea mays, Zizania palustris, Ziziphus spp., amaranth, artichoke, asparagus, broccoli, Brussels sprouts, cabbage, canola, carrot, cauliflower, celery, collard greens, flax, kale, lentil, oilseed rape, okra, onion, potato, rice, soybean, strawberry, sugar beet, sugar cane, sunflower, tomato, squash, tea and algae, amongst others. According to a preferred embodiment of the present invention, the plant is a crop plant. Examples of crop plants include inter alia soybean, sunflower, canola, alfalfa, rapeseed, cotton, tomato, potato or tobacco. Further preferebly, the plant is a monocotyledonous plant, such as sugarcane. Further preferably, the plant is a cereal, such as rice, maize, wheat, barley, millet, rye, sorghum or oats.

[0065] Generally, 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." By "effective amount" and "effective 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 herbicideresistant plants, plant tissues, plant cells, and host cells of the present invention. Typically, 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. These and other factors can be adjusted as is known in the art to promote non-selective or selective herbicidal action. Generally, the herbicide treatments can be applied PPI (Pre Plant Incorporated), PPSA (Post plant surface applied), PRE- or POST-emergent. Postemergent treatment typically occurs to relatively immature undesirable vegetation to achieve the maximum control of weeds.

[0066] By a "herbicide-tolerant" or "herbicide-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 wildtype plant. Levels of herbicide that normally inhibit growth of a non-tolerant plant are known and readily determined by those skilled in the art. Examples include the amounts recommended by manufacturers for application. The maximum rate is an example of an amount of herbicide that would normally inhibit growth of a non-tolerant plant. For the present invention, the terms "herbicide-tolerant" and "herbicide-resistant" are used interchangeably and are intended to have an equivalent meaning and an equivalent scope. Similarly, the terms "herbicide-tolerance" and "herbicide-resistance" are used interchangeably and are intended to have an equivalent meaning and an equivalent scope. Similarly, the terms "tolerant" and "resistant" are used interchangeably and are intended to have an equivalent meaning and an equivalent scope. As used herein, in regard to an herbicidal composition useful in various embodiments hereof, terms such as herbicides, and the like, refer to those agronomically acceptable herbicide active ingredients (A. I.) recognized in the art. Similarly, terms such as fungicide, nematicide, pesticide, and the like, refer to other agronomically acceptable active ingredients recognized in the art.

[0067] When used in reference to a particular enzyme or polypeptide, terms such as herbicide-tolerant and herbicidetolerance refer to the ability of such enzyme or polypeptide to perform its physiological activity in the presence of an amount of an herbicide A. I. that would normally inactivate or inhibit the activity of a sensitive version of said enzyme or polypeptide.

[0068] On the other hand, when used specifically in regard to the demethylase of the present invention, terms such as herbicide-tolerant and herbicide-tolerance refer specifically to the ability to metabolize, in particular demethylate, and thereby inactivate herbicides which inhibit cellulose biosynthesis, so-called cellulose biosynthesis inhibitors (CBI) By "herbicide-tolerant demethylase protein" or "herbicide -resistant demethylase protein", it is intended that such a demethylase displays higher metabolizing activity, relative to the metabolizing activity of an alternative demethylase, when in the presence of at least one herbicide that is known to interfere with cellulose biosynthesis and at a concentration or level of the herbicide that is known to inhibit cellulose biosynthesis. The metabolizing activity is dependent on the protein’s inherent catalytic activity and the amount of catalytically active protein present in the plant tissue. For protein to be catalytically active, it needs to be correctly folded. Incorrectly folded protein or partially unfolded protein is susceptible to degradation by cytosolic proteases which lowers the amount of catalytically active protein in the plant tissue if expressed therein. Therefore, it is desirable that the demethylase is resistant to degradation by proteases.

[0069] As used herein, "recombinant," when referring to nucleic acid or polypeptide, indicates that such material has been altered as a result of human application of a recombinant technique, such as by polynucleotide restriction and ligation, by polynucleotide overlap-extension, or by genomic insertion or transformation. A gene sequence open reading frame is recombinant if that nucleotide sequence has been removed from it natural text and cloned into any type of artificial nucleic acid vector. The term recombinant also can refer to an organism having a recombinant material, e.g., a plant that comprises a recombinant nucleic acid can be considered a recombinant plant. The term "transgenic plant" refers to a plant that comprises a heterologous polynucleotide. Preferably, the heterologous polynucleotide is stably integrated within the genome such that the polynucleotide is passed on to successive generations. The heterologous polynucleotide may be integrated into the genome alone or as part of a recombinant expression cassette. "Transgenic" is used herein to refer to any cell, cell line, callus, tissue, plant part or plant, the genotype of which has been so altered by the presence of heterologous nucleic acid including those transgenic organisms or cells initially so altered, as well as those created by crosses or asexual propagation from the initial transgenic organism or cell. In some embodiments, a "recombinant" organism is a "transgenic" organism. The term "transgenic" as used herein is not intended to encompass the alteration of the genome (chromosomal or extra-chromosomal) by conventional plant breeding methods (e.g., crosses) or by naturally occurring events such as, e.g., self-fertilization, random cross-fertilization, non-recombinant viral infection, non-recombinant bacterial transformation, non- recombinant transposition, or spontaneous mutation.

[0070] As used herein, "mutagenized" refers to an organism or DNA thereof having alteration(s) in the biomolecular sequence of its native genetic material as compared to the sequence of the genetic material of a corresponding wildtype organism or DNA, wherein the alteration(s) in genetic material were induced and / or selected by human action. Examples of human action that can be used to produce a mutagenized organism or DNA include, but are not limited to treatment with a chemical mutagen such as EMS and subsequent selection with herbicide(s); or by treatment of plant cells with x-rays and subsequent selection with herbicide(s). Any method known in the art can be used to induce mutations. Methods of inducing mutations can induce mutations in random positions in the genetic material or can induce mutations in specific locations in the genetic material (i.e., can be directed mutagenesis techniques), such as by use of a genoplasty technique.

[0071] As used herein, a "genetically modified organism" (GMO) is an organism whose genetic characteristics contain alteration(s) that were produced by human effort causing transfection that results in transformation of a target organism with genetic material from another or "source" organism, or with synthetic or modified-native genetic material, or an organism that is a descendant thereof that retains the inserted genetic material. The source organism can be of a different type of organism (e.g., a GMO plant can contain bacterial genetic material) or from the same type of organism (e.g., a GMO plant can contain genetic material from another plant). As used herein in regard to plants and other organisms, "recombinant," "transgenic," and "GMO" are considered synonyms and indicate the presence of genetic material from a different source; in contrast, "mutagenized" is used to refer to a plant or other organism, or the DNA thereof, in which no such transgenic material is present, but in which the native genetic material has become mutated so as to differ from a corresponding wild-type organism or DNA.

[0072] As used herein, "wildtype" or "corresponding wildtype plant" means the typical form of an organism or its genetic material, as it normally occurs, as distinguished from, e.g., mutagenized and / or recombinant forms. Similarly, by “control cell” or "similar, wildtype, plant, plant tissue, plant cell or host cell" is intended a plant, plant tissue, plant cell, or host cell, respectively, that lacks the herbicide-resistance characteristics and / or particular polynucleotide of the invention that are disclosed herein. The use of the term "wildtype" is not, therefore, intended to imply that a plant, plant tissue, plant cell, or other host cell lacks recombinant DNA in its genome, and / or does not possess herbicide-resistant characteristics that are different from those disclosed herein.

[0073] As used herein, "descendant" refers to any generation plant. In some embodiments, a descendant is a first, second, third, fourth, fifth, sixth, seventh, eight, ninth, or tenth generation plant. As used herein, "progeny" refers to a first generation plant.

[0074] The term "seed" comprises seeds of all types, such as, for example, true seeds, caryopses, achenes, fruits, tubers, seedlings and similar forms. In the context of Brassica and Sinapis species, "seed" refers to true seed(s) unless otherwise specified. For example, the seed can be seed of transgenic plants or plants obtained by traditional breeding methods. Examples of traditional breeding methods can include cross-breeding, selfing, back-crossing, embryo rescue, in-crossing, out-crossing, inbreeding, selection, asexual propagation, and other traditional techniques as are known in the art.

[0075] Although exemplified with reference to specific plants or plant varieties and their hybrids, in various embodiments, the presently described methods using herbicides can be employed with a variety of commercially valuable plants. Herbicide-tolerant plant lines described as useful herein can be employed in weed control methods either directly or indirectly, i. e. either as crops for herbicide treatment or as herbicide-tolerance trait donor lines for development, to produce other varietal and / or hybrid crops containing such trait or traits. All such resulting variety or hybrids crops, containing the ancestral herbicide-tolerance trait or traits can be referred to herein as progeny or descendant of the ancestral, herbicide-tolerant line(s). Such resulting plants can be said to retain the "herbicide tolerance characteristic(s)" of the ancestral plant, i.e. meaning that they possess and express the ancestral genetic molecular components responsible for the trait.

[0076] In one aspect, the present invention refers to an isolated and / or recombinantly produced and / or synthetic nucleic acid molecule comprising a nucleic acid molecule encoding a demethylase selected from the group consisting of:

[0077] (a) a nucleic acid molecule encoding a demethylase comprising the sequence 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, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106,

[0078] 108. 110. 112. 114. 116. 118. 120. 122. 124. 126. 128. 130, or 132, or a variant, paralogue, orthologue or homolog thereof;

[0079] (b) a nucleic acid molecule comprising the sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71 , 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117,

[0080] 119. 121. 123. 125. 127. 129, or 131, or a variant, paralogue, orthologue or homolog thereof;

[0081] (c) a nucleic acid molecule, which, as a result of the degeneracy of the genetic code, can be derived from a demethylase sequence ef-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, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124,

[0082] 126. 128. 130, or 132, or a variant, paralogue, orthologue or homolog thereof,;

[0083] (d) a nucleic acid molecule having 30% or more identity, preferably 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99,5%, or more with the nucleic acid molecule sequence of a polynucleotide comprising the nucleic acid molecule of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113,

[0084] 115. 117. 119. 121. 123. 125. 127. 129, or 131, or a variant, paralogue, orthologue or homolog thereof,; (e) a nucleic acid molecule encoding a demethylase having 30% or more identity, preferably at least 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99,5% or more, with the amino acid sequence of the demethylase sequence ef-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, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, or 132,;

[0085] (f) nucleic acid molecule which hybridizes with a nucleic acid molecule of (a), (b), (c), (d) or (e) under stringent hybridization conditions, wherein the encoded demethylase is capable of demethylating a herbicide.

[0086] In a preferred embodiment, said herbicide belongs to the class of cellulose biosynthesis inhibitors, preferably azines, as described in further detail hereinafter.

[0087] In other embodiments, the demethylase polypeptide according to the present invention is a functional variant having, over the full-length of the variant, at least about 80%, illustratively, at least about 80%, 90%, 95%, 98%, 99% or more amino acid sequence identity to 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, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, or 132.

[0088] In another embodiment, the demethylase polypeptide for use according to the present invention is a functional fragment of a polypeptide having the amino acid 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, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, or 132.

[0089] It is recognized that the demethylase nucleic acid molecules and demethylase polypeptides of the invention encompass polynucleotide molecules and polypeptides comprising a nucleotide or an amino acid sequence that is sufficiently identical to nucleotide sequence set forth in SEQ ID Nos: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81,

[0090] 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129 or

[0091] 131, or to the amino acid sequence 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, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88,

[0092] 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, or 132. The term "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.

[0093] Generally, "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 implementations of these algorithms such as GAP, BESTFIT, FASTA, and TFASTA available as part of the GCG. Wisconsin Package. (Accelrys Inc. Burlington, Mass.)

[0094] Polynucleotides and Oligonucleotides

[0095] By an "isolated polynucleotide", or “nucleic acid molecule” 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. Preferably, the isolated polynucleotide is at least 60% free, preferably at least 75% free, and most preferably at least 90% free from other components with which they are naturally associated. As the skilled addressee would be aware, an isolated polynucleotide can be an exogenous polynucleotide present in, for example, a transgenic organism which does not naturally comprise the polynucleotide. Furthermore, the terms "polynucleotide(s)", "nucleic acid sequence(s)", "nucleotide sequence(s)", “nucleic acid(s)”, “nucleic acid molecule” are used interchangeably herein and refer to nucleotides, either ribonucleotides or deoxyribonucleotides or a combination of both, in a polymeric unbranched form of any length.

[0096] The term “mutated demethylase nucleic acid” refers to a demethylase nucleic acid having a sequence that is mutated from a wild-type demethylase nucleic acid. Furthermore, the term “mutated demethylase” refers to the replacement of an amino acid of the wild-type primary sequence 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, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, or 132, or a variant, a derivative, a homologue, an orthologue, or paralogue thereof, with another amino acid. The expression "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.

[0097] In a preferred embodiment, the demethylase nucleotide sequence encoding a demethylase comprises the sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129 or 131, or a variant or derivative thereof

[0098] Furthermore, it will be understood by the person skilled in the art that the demethylase nucleotide sequences encompasse homologues, paralogues and and orthologues of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129 or 131, as defined hereinafter.

[0099] The term "variant" 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. For nucleotide sequences comprising an open reading frame, 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 comprising the sequence 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, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, or 132, as well as those that encode a polypeptide having amino acid substitutions relative to the native protein, e.g. the demethylase according to the present invention as disclosed herein.

[0100] Generally, 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: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129 or 131,. The % identity of a polynucleotide is determined by GAP (Needleman and Wunsch, 1970) analysis (GCG program) with a gap creation penalty=5, and a gap extension penalty=0.3. Unless stated otherwise, 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. Preferably, 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.

[0101] Polypeptides

[0102] By "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. The terms "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. It would be understood that such polypeptide chains may associate with other polypeptides or proteins or other molecules such as co-factors. The terms "proteins" and "polypeptides" as used herein also include variants, mutants, modifications, analogous and / or derivatives of the polypeptides of the invention as described herein.

[0103] The % identity of a polypeptide is determined by GAP (Needleman and Wunsch, 1970) analysis (GCG program) with a gap creation penalty=5, and a gap extension penalty=0.3. 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. More preferably, 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.

[0104] With regard to a defined polypeptide, it will be appreciated that % identity figures higher than those provided above will encompass preferred embodiments. Thus, where applicable, in light of the minimum % identity figures, it is preferred that the demethylase 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 99.4%, more preferably at least 99.5%, more preferably at least 99.6%, more preferably at least 99.7%, more preferably at least 99.8%, and even more preferably at least 99.9% identical to 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, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, or 132.

[0105] By "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, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, or 132, 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. Such variants may result from, for example, genetic polymorphism or from human manipulation. Methods for such manipulations are generally known in the art.

[0106] “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. Thus, functional variants and fragments of the demethylase polypeptides, and nucleic acid molecules encoding them, also are within the scope of the present invention, and unless specifically described otherwise, irrespective of the origin of said polypeptide and irrespective of whether it occurs naturally. Various assays for functionality of a demethylase polypeptide can be employed. For example, a functional variant or fragment of the demethylase polypeptide can be assayed to determine its ability to confer herbicides detoxification. By way of illustration, a herbicides detoxification rate can be defined as a catalytic rate sufficient to provide a determinable increase in tolerance to herbicides in a plant or plant part comprising a recombinant polynucleotide encoding the variant or fragment of the demethylase polypeptide, wherein the plant or plant part expresses the variant or fragment at up to about 0.5%, illustratively, about 0.05 to about 0.5%, about 0.1 to about 0.4%, and about 0.2 to about 0.3%, of the total cellular protein relative to a similarly treated control plant that does not express the variant or fragment.

[0107] In a preferred embodiment, the demethylase polypeptide is a functional variant or fragment of a demethylase having the amino acid 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, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, or 132, wherein the functional variant or fragment has at least about 80% amino acid sequence identity to 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, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, or 132.

[0108] In other embodiments, the functional variant or fragment further has a herbicide detoxification rate defined as a catalytic rate sufficient to provide a determinable increase in tolerance to herbicides in a plant or plant part comprising a recombinant polynucleotide encoding the variant or fragment, wherein the plant or plant part expresses the variant or fragment at up to about 0.5% of the total cellular protein to a similarly treated control plant that does not express the variant or fragment.

[0109] “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.

[0110] In addition, one of ordinary skill in the art will further appreciate that changes can be introduced by mutation into the nucleotide sequences of the invention thereby leading to changes in the amino acid sequence of the encoded proteins without altering the biological activity of the proteins.

[0111] Thus, for example, an isolated polynucleotide molecule encoding a demethylase polypeptide having an amino acid sequence that differs from that 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, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, or 132 can be created by introducing one or more nucleotide substitutions, additions, or deletions into the corresponding nucleotide sequence such as shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, or 131, such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein. Mutations can be introduced by standard techniques, such as site-directed mutagenesis and PCR- mediated mutagenesis. Such variant nucleotide sequences are also encompassed by the present invention. For example, preferably, conservative amino acid substitutions may be made at one or more predicted preferably nonessential amino acid residues. A "nonessential" amino acid residue is a residue that can be altered from the wild-type sequence of a protein without altering the biological activity, whereas an "essential" amino acid residue is required for biological activity. A deletion refers to removal of one or more amino acids from a protein.

[0112] An insertion 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. Examples of 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.

[0113] 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 p-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. A conservative amino acid substitution is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Such substitutions would not be made for conserved amino acid residues, or for amino acid residues residing within a conserved motif. Conservative substitution tables are well known in the art (see for example Creighton (1984) Proteins. W.H. Freeman and Company (Eds).

[0114] 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 M13 mutagenesis, T7-Gen in vitro mutagenesis (USB, Cleveland, OH), QuickChange Site Directed mutagenesis (Stratagene, San Diego, CA), PCR-mediated site-directed mutagenesis or other site-directed mutagenesis protocols.

[0115] “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 other 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. Furthermore, “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. 60, 523-533, 2003).

[0116] “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 originated through specification, and are also derived from a common ancestral gene.

[0117] It is well-known in the art that 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.

[0118] The term "domain" refers to a set of amino acids conserved at specific positions along an alignment of sequences of evolutionarily 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.

[0119] The term “motif’ or “consensus sequence” refers to a short conserved region in the sequence of evolutionarily 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).

[0120] Specialist databases exist for the identification of domains, for example, SMART (Schultz et al. (1998) Proc. Natl. Acad. Sci. USA 95, 5857-5864; Letunic et al. (2002) Nucleic Acids Res 30, 242-244), InterPro (Mulder et al., (2003) Nucl. Acids. Res. 31, 315-318), Prosite (Bucher and Bairoch (1994), A generalized profile syntax for biomolecular sequences motifs and its function in automatic sequence interpretation. (In) ISMB-94; Proceedings 2nd International Conference on Intelligent Systems for Molecular Biology. Altman R., Brutlag D., Karp P., Lathrop R., Searls D., Eds., pp53-61, AAAI Press, Menlo Park; Hulo et al., Nucl. Acids. Res. 32:D134-D137, (2004)), or Pfam (Bateman et al., Nucleic Acids Research 30(1): 276-280 (2002)). A set of tools for in silico analysis of protein sequences is available on the ExPASy proteomics server (Swiss Institute of Bioinformatics (Gasteiger et al., ExPASy: the proteomics server for in-depth protein knowledge and analysis, Nucleic Acids Res. 31 :3784-3788(2003)). Domains or motifs may also be identified using routine techniques, such as by sequence alignment.

[0121] Methods for the alignment of sequences for comparison are well known in the art, such methods include GAP, BESTFIT, BLAST, FASTA and TFASTA. 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 (See Figure 1). 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. The 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. For local alignments, the Smith-Waterman algorithm is particularly useful (Smith TF, Waterman MS (1981) J. Mol. Biol 147(1 ); 195-7).

[0122] The proteins of the invention may be altered in various ways including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known in the art. For example, amino acid sequence variants can be prepared by mutations in the DNA. Methods for mutagenesis and nucleotide sequence alterations are well known in the art. See, for example, Kunkel (1985) PNAS, 82:488-492; Kunkel et al. (1987) Methods in Enzymol. 154:367-382; U.S. Patent No. 4,873,192; Walker and Gaastra, eds. (1983) Techniques in Molecular Biology (MacMillan Publishing Company, New York) and the references cited therein. Guidance as to appropriate amino acid substitutions that do not affect biological activity of the protein of interest may be found in the model of Dayhoff et al. (1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, D. C), herein incorporated by reference. Conservative substitutions, such as exchanging one amino acid with another having similar properties, may be preferable.

[0123] Alternatively, variant nucleotide sequences can be made by introducing mutations randomly along all or part of a coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened to identify mutants that encode proteins that retain activity. For example, following mutagenesis, the encoded protein can be expressed recombinantly, and the activity of the protein can be determined using standard assay techniques.

[0124] It will be within the knowledge of the skilled artisan to identify conserved regions and motifs shared between the homologues, orthologues and paralogues encoded by SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129 or 131,. Having identified such conserved regions that may represent suitable binding motifs, amino acids can be chosen to be subsituted by any other amino acid, for example by conserved amino acids, preferably by the amino acid substitutions described SUPRA using SEQ ID NO:2 as reference.

[0125] The present invention further provides a method for demethylating a herbicide, which method comprises contacting said herbicide with the demethylase of the present invention. In a preferred embodiment at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, most preferably 100% of the herbicide is demethylated after being contacted with the demethylase of the present invention. It will be understood by the person skilled in the art that the demethylase of the present invention may act as a hydroxylase where the leaving group is just methanol, or may act as a methyl tranferase where the methyl group is transferred from the herbicide of interest to another methyl acceptor.

[0126] In a further preferred embodiment, said demethylation leads to an inactivation of the respective herbicide.

[0127] In a further preferred embodiment said contacting occurs at a plant cultivation site where crop plants, as described SUPRA, are grown, which crop plants or plant parts are capable of expressing the demethylase of the present invention, as further described hereinafter.

[0128] Preferably, said demethylase comprises a sequence having at least 80%, 90%, 95%, 98%, 99% identity to the sequence 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, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, or 132, or a variant, paralogue, orthologue or homolog thereof.

[0129] In another aspect, the present invention relates to the use of a nucleic acid molecule encoding a demethylase of the present invention for demethylating a herbicide.

[0130] Herbicides

[0131] As decribed above, the present invention provides nucleic acids, polypeptides, which re capable of demethylating compounds / herbicides interfering or inhibiting cell wall (cellulose) biosynthesis.

[0132] Examples of herbicides which are demethylated by the demethylases according to the present invention, are compounds known to the skilled artisan as azines. Examples of Azines which are metabolized by the demethylase polypeptides of the present invention are described in detail in the following patent applications depicted in the following Table 2.

[0133] Table 2

[0134]

[0135] Examples of preferred herbicides which are particularly useful for the present invention are diaminotriazine compounds as described in WO2022 / 161801 , having the Formula (I).. wherein

[0136] R1is F;

[0137] R2is selected from the group consisting of H, halogen, CR2A; wherein R2Ais H or halogen;

[0138] R3is H, F;

[0139] R4is selected from the group consisting of Cl, Br, I, CR4A; wherein R4Ais H or halogen;

[0140] R5is selected from the group consisting of H, halogen, CN, Ci-Ce-alkyl, (Ci-C6-alkoxy)-Ci-Ce-alkyl, C3-C6- cycloalkyl, (C3-C6-cycloalkyl)-Ci -Chalky I, Ci-Ce-alkoxy, C2-Ce-alkenyloxy, C2-Ce-alkynyloxy, C3-C6- cycloalkoxy, (C3-C6-cycloalkyl)-Ci-C4-alkoxy, where the aliphatic and cycloaliphatic parts of the radicals are unsubstituted, partly or completely halogenated;

[0141] R6is selected from the group consisting of H, halogen, CN, Ci-Ce-alkyl, Ci-Ce-haloalkyl, Ci-Ce-alkoxy and Ci-Ce-haloalkoxy;

[0142] R7is selected from the group consisting of halogen, CN, Ci-Ce-alkyl, C2-Ce-alkenyl, Cs-Ce-alkynyl, C3-C6- cycloalkyl, (C3-Ce-cycloalkyl)-Ci-C4-alkyl, Cs-Ce-cycloalkenyl and Ci-Ce-alkoxy-Ci-Ce-alkyl, where the aliphatic and cycloaliphatic parts of the radicals are unsubstituted, partly or completely halogenated;

[0143] R6and R7together with the carbon atom to which they are attached form a moiety selected from the group consisting of carbonyl, Cs-Ce-cycloalky I, Cs-Ce-cycloalkenyl, three- to six-membered saturated or partially unsaturated heterocyclyl, and the moiety >C=CRxRy, where Rxand Ry are hydrogen, Ci-C4-alkyl, C1-C4- haloalky I, Cs-Ce-cycloalkyl or CRxRy form a 3- to 6-membered cycloalkyl;

[0144] R8is selected from the group consisting of Ci-Ce-alkyl, C2-Ce-alkenyl, C2-Ce-alkynyl, (Ci-Ce-alkoxy)-Ci-Ce- alkyl, (Ci-Ce-alkoxy)-C2-Ce-alkenyl, (Ci-Ce-alkoxy)-C2-Ce-alkynyl, (Ci-Ce-cycloalkyl)-C2-Ce-alkynyl, (C3- Ce-cycloalkyl)-Ci-C4-alkyl, (C3-Ce-cycloalkoxy)-Ci-C4-alkyl, where the aforementioned radicals are unsubstituted, partly or completely halogenated and where the cycloaliphatic parts of the last 6 mentioned radicals may carry 1, 2, 3, 4, 5 or 6 methyl groups, including their agriculturally acceptable salts

[0145] Particular groups of the embodiment relate to the diaminotriazine compound of formula (I), wherein: R1is F.

[0146] Also preferred are diaminotriazine compounds of formula (I), wherein

[0147] R2is selected from the group consisting of H, halogen, CH3, Ci -haloalkyl; in particular consisting of H, halogen, CH3; more particularly consisting of H, F, Cl, CH3.

[0148] Further particular groups of embodiments relate to the diaminotriazine compounds of formula (I), wherein R3is H or F; preferably H. Further particular groups of embodiments relate to the diaminotriazine compounds of formula (I), wherein R4is selected from the group consisting of halogen, CH3, Ci -haloalkyl; in particular consisting of halogen, CH3; more particularly consisting of Br, Cl, CH3.

[0149] Also preferred are diaminotriazine compounds of formula (I), wherein

[0150] R5is selected from the group consisting of H, halogen, CN, Ci-Ce-alkyl, Ci-Ce-haloalkyl, Ci-Ce-alkoxy and Ci-Ce-haloalkoxy, in particular from the group consisting of hydrogen, fluorine, Ci-C4-alkyl, such as methyl, ethyl, n-propyl, 2-propyl, n-butyl, 2-butyl, isobutyl or tert.-butyl, Ci-C4-haloalkyl, such as difluoromethyl, trifluoromethyl, 2,2,2-trifluoroethyl, 1,1 -difluoroethyl, 1 , 1 ,2,2-tetrafluoroethyl or pentafluoroethyl, Ci-C4-alkoxy, such as methoxy or ethoxy and Ci-C4-haloalkoxy, such as difluoromethoxy or trifluoromethoxy.

[0151] Further particular groups embodiments relate to the diaminotriazine compounds of formula (I), wherein R6is selected from the group consisting of H, halogen, CN, Ci-Ce-alkyl, Ci-Ce-haloalkyl, Ci-Ce-alkoxy and Ci-Ce-haloalkoxy, in particular from the group consisting of hydrogen, fluorine and Ci-C4-alkyl, more particularly from hydrogen, fluorine and methyl, especially from fluorine and methyl.

[0152] In groups (1) of embodiments, R7is as defined above. Preferably

[0153] R7is selected from the group consisting of halogen, Ci-Ce-alkyl, Ci-Ce-haloalkyl, C2-Ce-alkenyl, C3-C6- alkynyl, Cs-Ce-cycloalkyl, Cs-Ce-cycloalkyl-Ci-Ce-alkyl, Cs-Ce-cycloalkenyl, Ci -Ce-alkoxy-Ci-Ce-alkyL

[0154] Further particular groups of embodiments relate to the diaminotriazine compounds of formula (I), wherein R6and R7together with the carbon atom to which they are attached form a moiety selected from the group consisting of carbonyl, Cs-Ce-cycloalkan, Cs-Ce-cycloalkenyl, three- to six-membered saturated or partially unsaturated heterocycly I, where the carbocycle and the heterocycle are unsubstituted, partly or completely halogenated or carry from 1 to 6 Ci -Ce-alkyl groups, and the moiety >C=CRxRy, where Rxand Ry are hydrogen, Ci-C4-alkyl, Ci -C4-haloalkyl, Cs-Ce-cycloalkyl or CRxRy form a 3- to 6-membered cycloalkyl. Preferably, R6and R7together with the carbon atom to which they are attached form Cs-Ce-cycloalkan

[0155] Other examples of preferred herbicides which are particularly useful for the present invention are diaminotriazine compounds are described in WO 2015 / 155271, having the Formula (I).. ach other, selected from the group consisting of hydrogen, halogen, CN, NO2, Ci-Ce-alkyl, Ci-Ce-haloalkyl, OH, Ci-Ce-alkoxy, Ci-Ce-alkylthio, (Ci-Ce- alkyl)sulfinyl, (Ci-Ce-alkyl)sulfonyl, amino, (Ci-C6-alkyl)amino, di(Ci-Ce-alkyl)amino, (Ci-Ce- alkyl)carbonyl and (Ci-Ce-alkoxy)carbonyl; Rbis selected from the group consisting of hydrogen, halogen, Ci-Ce-alkyl, Ci -Ce-haloalkyl, CN and Ci-Ce-alkoxy,

[0156] Rdis selected from the group consisting of halogen, Ci -Ce-alky I, C 1 -Ce-haloalkyl, CN and Ci- Ce-alkoxy, provided that either Rbor Rdis Ci-Ce-alkoxy;

[0157] R1is selected from the group consisting of H, OH, S(O)2NH2, CN, Ci-Ce-alkyl, C2-Ce-alkenyl, C2-Ce-alkynyl, (C3-Ce-cycloalkyl)-Ci-C4-alkyl, Ci-Ce-alkoxy, (Ci-Ce-alkoxy)-Ci-Ce-alkyl, (Ci- Ce-alkyl)-carbonyl, (Ci-Ce-alkoxy)carbonyl, (Ci-Ce-alkyl)sulfonyl, (Ci-Ce- alkylamino)carbonyl, di(Ci-Ce-alkyl)aminocarbonyl, (Ci-Ce-alkylamino)sulfonyl, di(Ci-Ce- alkyl)aminosulfonyl and (Ci -Ce-alkoxy)sulfonyl, where the aliphatic and cycloaliphatic parts of the 14 aforementioned radicals are unsubstituted, partly or completely halogenated, phenyl, phenyl-Ci -Ce-alkyl, phenylsulfonyl, phenylaminosulfonyl, phenylcarbonyl and phenoxycarbonyl, wherein phenyl in the last 6 mentioned radicals are unsubstituted or substituted by 1, 2, 3, 4 or 5 identical or different substituents selected from the group consisting of halogen, CN, NO2, Ci-Ce-alkyl, Ci-Ce-haloalkyl, Ci-Ce-alkoxy and Ci-Ce-haloalkoxy;

[0158] R2is selected from the group consisting of H, OH, S(O)2NH2, CN, Ci-Ce-alkyl, C2-Ce-alkenyl, C2-Ce-alkynyl, (C3-Ce-cycloalkyl)-Ci-C4-alkyl, Ci-Ce-alkoxy, (Ci-Ce-alkoxy)-Ci -Ce-alkyl, (C1- Ce-alkyl)carbonyl, (Ci-Ce-alkoxy)carbonyl, (Ci-Ce-alkyl)sulfonyl, (Ci-Ce-alkylamino)carbonyl, di(Ci-Ce-alkyl)aminocarbonyl, (Ci-Ce-alkylamino)sulfonyl, di(Ci-Ce-alkyl)aminosulfonyl and (Ci-Ce-alkoxy)sulfonyl, where the aliphatic and cycloaliphatic parts of the 14 aforementioned radicals are unsubstituted, partly or completely halogenated, phenyl, phenylsulfonyl, phenylaminosulfonyl, phenyl-Ci-Ce alkyl, phenoxy, phenylcarbonyl and phenoxycarbonyl, wherein phenyl in the last 6 mentioned radicals is unsubstituted or substituted by 1, 2, 3, 4 or 5 identical or different substituents selected from the group consisting of halogen, CN, NO2, Ci-Ce-alkyl, Ci-Ce-haloalkyl, Ci-Ce-alkoxy and Ci-Ce-haloalkoxy;

[0159] R3is selected from the group consisting of H, halogen, OH, CN, Ci-Ce-alkyl, Ci -Ce-haloalkyl, Ci-Ce-alkoxy and Ci-Ce-haloalkoxy;

[0160] R4is selected from the group consisting of H, halogen, CN, Ci -Ce-alkyl, Ci -Ce-haloalkyl, Ci-Ce- alkoxy and Ci-Ce-haloalkoxy;

[0161] R5is selected from the group consisting of halogen, CN, C 1 -Ce-alkyl, C 1 -Ce-haloalkyl, Ci-Ce- alkoxy, C2-Ce-alkenyl, Cs-Ce-alkynyl, Cs-Ce-cycloalkyl, Cs-Ce-cycloalkyl-Ci -Ce-alkyl, C3-C6- cycloalkenyl and Ci-Ce-alkoxy-Ci-Ce-alkyl; or

[0162] R4and R5together with the carbon atom to which they are attached may form a moiety selected from the group consisting of carbonyl, Cs-Ce-cycloalkan-l ,1-diyl, ipso-Cs-Ce-cycloalkendiyl, three- to six-membered saturated or partially unsaturated ipso-heterocyclodiy I, where the carbocycle and the heterocycle are unsubstituted, partly or completely halogenated or carry from 1 to 6 Ci-Ce-alkyl groups, and the moiety >C=CRxRy, where Rxand Ry are hydrogen, Ci -Czi-alkyl or Ci-C4-haloalkyl; including their agriculturally acceptable salts;

[0163] Particularly preferred is the compound of formula.

[0164] 6’(1-fluoro-1»methyl~ethyl)-N4-(2>3.5 rilluoro-6-methoxy-phenyl)-1.3(5~triazine-2>4-diam!ne

[0165] Another object refers to a method of identifying a nucleotide sequence encoding a demethylase demethylating, thereby inactivating a herbicide, the method comprising: a) generating a library of demethylase-encoding nucleic acids, b) screening a population of the resulting demethylase-encoding nucleic acids by expressing each of said nucleic acids in a cell or plant and treating said cell or plant with a herbicide, c) comparing the herbicide-tolerance levels provided by said population of demethylase encoding nucleic acids with the herbicide-tolerance level provided by a control demethylase-encoding nucleic acid, d) selecting at least one demethylase-encoding nucleic acid that provides a significantly increased level of tolerance to a herbicide as compared to that provided by the control demethylase-encoding nucleic acid.

[0166] Herbicide-tolerance levels may also be determined by measuring the detoxification rate in a cell, tissue, or plant. Detoxification rate is the rate of herbicide degradation, preferably demethylation within a certain timeframe in a respective tissue. The degradation and product formation can be determined analytically for instance by liquid chromatographie (LC) coupled to a high resolution (HR) mass spectromter (MS). Product can be determined by comparison to authentic standards and / or by structure elucidation.

[0167] In a preferred embodiment, the demethylase-encoding nucleic acid selected in step d) provides at least 2-fold as much resistance or tolerance of a cell or plant to a herbicide as compared to that provided by the control demethylase-encoding nucleic acid.

[0168] In a further preferred embodiment, the demethylase-encoding nucleic acid selected in step d) provides at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 500-fold, as much resistance or tolerance of a cell or plant to a herbicide as compared to that provided by the control demethylase-encoding nucleic acid.

[0169] The resistance or tolerance can be determined by generating a transgenic plant or host cell, preferably a plant cell, comprising a nucleic acid sequence of the library of step a) and comparing said transgenic plant with a control plant or host cell, preferably a plant cell.

[0170] Many methods well known to the skilled artisan are available for obtaining suitable candidate nucleic acids for identifying a nucleotide sequence encoding a demethylase from a variety of different potential source organisms including microbes, plants, fungi, algae, mixed cultures etc. as well as environmental sources of DNA such as soil. These methods include inter alia the preparation of cDNA or genomic DNA libraries, the use of suitably degenerate oligonucleotide primers, the use of probes based upon known sequences or complementation assays (for example, for growth upon tyrosine) as well as the use of mutagenesis and shuffling in order to provide recombined or shuffled demethylase-encoding sequences.

[0171] Nucleic acids comprising candidate and control demethylase encoding sequences can be expressed in yeast, in a bacterial host strain, in an alga or in a higher plant such as tobacco or Arabidopsis and the relative levels of inherent tolerance of the organism expressing the demethylase encoding sequences screened according to a visible indicator phenotype of the transformed strain or plant in the presence of different concentrations of the selected herbicide. Dose responses and relative shifts in dose responses associated with these indicator phenotypes (formation of brown color, growth inhibition, herbicidal effect etc) are conveniently expressed in terms, for example, of GR50 (concentration for 50% reduction of growth) or MIC (minimum inhibitory concentration) values where increases in values correspond to increases in inherent tolerance of the expressed demethylase. For example, in a relatively rapid assay system based upon transformation of a bacterium such as E. coli, each demethylase encoding sequence may be expressed, for example, as a DNA sequence under expression control of a controllable promoter such as the lacZ promoter and taking suitable account, for example by the use of synthetic DNA, of such issues as codon usage in order to obtain as comparable a level of expression as possible of different demethylase sequences.

[0172] In another embodiment, candidate nucleic acids are transformed into plant material to generate a transgenic plant, regenerated into morphologically normal fertile plants which are then measured for differential tolerance to selected herbicides, preferably cellulose biosynthesis inhibitors named azines, as described in the Example section hereinafter. Many suitable methods for transformation using suitable selection markers such as kanamycin, binary vectors such as from Agrobacterium and plant regeneration as, for example, from tobacco leaf discs are well known in the art. Optionally, a control population of plants is likewise transformed with a nucleic acid expressing the control demethylase. The average, and distribution, of herbicide tolerance levels of a range of primary plant transformation events or their progeny to herbicides described supra are evaluated in the normal manner based upon plant damage, meristematic bleaching symptoms etc. at a range of different concentrations of herbicides. These data can be expressed in terms of, for example, GR50 values derived from dose / response curves having "dose" plotted on the x-axis and "percentage kill", "herbicidal effect", "numbers of emerging green plants" etc. plotted on the y-axis where increased GR50 values correspond to increased levels of inherent tolerance of the expressed demethylase. Herbicides can suitably be applied pre-emergence or postemergence.

[0173] In other aspects, the present invention encompasses a progeny or a descendant of a herbicide-tolerant plant of the present invention as well as seeds derived from the herbicide-tolerant plants of the invention and cells derived from the herbicide-tolerant plants of the invention.

[0174] In some embodiments, the present invention provides a progeny or descendant plant derived from a plant comprising in at least some of its cells a polynucleotide operably linked to a promoter operable in plant cells, the promoter capable of expressing a demethylase polypeptide encoded by the polynucleotide, wherein the progeny or descendant plant comprises in at least some of its cells the recombinant polynucleotide operably linked to the promoter, the expression of the demethylase polypeptide conferring to the progeny or descendant plant tolerance to the herbicides.

[0175] In one embodiment, seeds of the present invention preferably comprise the herbicide-tolerance characteristics of the herbicide-tolerant plant. In other embodiments, a seed is capable of germination into a plant comprising in at least some of its cells a polynucleotide operably linked to a promoter operable in plant cells, the promoter capable of expressing a demethylase polypeptide encoded by the polynucleotide, the expression of the demethylase polypeptide conferring to the progeny or descendant plant tolerance to the herbicides.

[0176] In some embodiments, plant cells of the present invention are capable of regenerating a plant or plant part. In other embodiments, plant cells are not capable of regenerating a plant or plant part. Examples of cells not capable of regenerating a plant include, but are not limited to, endosperm, seed coat (testa & pericarp), and root cap.

[0177] In another embodiment, the present invention provides a plant cell of or capable of regenerating a plant comprising in at least some of its cells a polynucleotide operably linked to a promoter operable in plant cells, the promoter capable of expressing a demethylase polypeptide encoded by the polynucleotide, the expression of the demethylase polypeptide conferring to the plant tolerance to the herbicides, wherein the plant cell comprises the recombinant polynucleotide operably linked to a promoter.

[0178] In other embodiments, the present invention provides a plant cell comprising a polynucleotide operably linked to a promoter operable in plant cells, the promoter capable of expressing a demethylase polypeptide encoded by the polynucleotide, the expression of the demethylase polypeptide conferring to the cell tolerance to the herbicides.

[0179] In another embodiment, the invention refers to a plant cell transformed by a nucleic acid encoding a demethylase polypeptide according to the present invention, wherein expression of the nucleic acid in the plant cell results in increased resistance or tolerance to a herbicide as compared to a wild type variety of the plant cell. Preferably, the demethylase polypeptide encoding nucleic acid comprises a polynucleotide sequence selected from the group consisting of: a) a polynucleotide as shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129 or 131, or a variant or derivative thereof; b) a polynucleotide encoding a polypeptide as shown 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, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, or 132, or a variant or derivative thereof; c) a polynucleotide comprising at least 60 consecutive nucleotides of any of a) or b); and d) a polynucleotide complementary to the polynucleotide of any of a) through c).

[0180] In some aspects, the present invention provides a plant product prepared from the herbicide-tolerant plants hereof. In some embodiments, examples of plant products include, without limitation, grain, oil, and meal. In one embodiment, a plant product is plant grain (e.g., grain suitable for use as feed or for processing), plant oil (e.g., oil suitable for use as food or biodiesel), or plant meal (e.g., meal suitable for use as feed).

[0181] In one embodiment, a plant product prepared from a plant or plant part is provided, wherein the plant or plant part comprises in at least some of its cells a polynucleotide operably linked to a promoter operable in plant cells, the promoter capable of expressing a demethylase polypeptide encoded by the polynucleotide, the expression of the demethylase polypeptide conferring to the plant or plant part tolerance to the herbicides.

[0182] In another embodiment, the invention refers to a method of producing a transgenic plant cell with an increased resistance to a herbicide as compared to a wild type variety of the plant cell comprising, transforming the plant cell with an expression cassette comprising a polynucleotide operably linked to a promoter operable in plant cells, the promoter capable of expressing a demethylase polypeptide encoded by the polynucleotide.

[0183] In another embodiment, the invention refers to a method of producing a transgenic plant comprising, (a) transforming a plant cell with an expression cassette comprising a polynucleotide operably linked to a promoter operable in plant cells, the promoter capable of expressing a demethylase polypeptide encoded by the polynucleotide, and (b) regenerating a plant with an increased resistance to herbicide from the plant cell.

[0184] In some aspects, the present invention provides a method for producing a herbicide-tolerant plant. In one embodiment, the method comprises: regenerating a plant from a plant cell transformed with a polynucleotide operably linked to a promoter operable in plant cells, the promoter capable of expressing a demethylase polypeptide encoded by the polynucleotide, the expression of the demethylase polypeptide conferring to the plant tolerance to the herbicides.

[0185] The term “expression / expressing” or “gene expression” means the transcription of a specific gene or specific genes or specific genetic construct. The term “expression” or “gene expression” in particular means the transcription of a gene or genes or genetic construct into structural RNA (rRNA, tRNA) or mRNA with or without subsequent translation of the latter into a protein. The process includes transcription of DNA and processing of the resulting mRNA product.

[0186] To obtain the desired effect, i.e. plants that are tolerant or resistant to the herbicide, preferable cellulose biosynthesis inhibitors named azines, it will be understood that the at least one nucleic acid is “over-expressed” by methods and means known to the person skilled in the art.

[0187] The term “increased expression” or “overexpression” as used herein means any form of expression that is additional to the wild-type expression level. Methods for increasing expression of genes or gene products are well documented in the art and include, for example, overexpression driven by appropriate promoters, the use of transcription enhancers or translation enhancers. Isolated nucleic acids which serve as promoter or enhancer elements may be introduced in an appropriate position (typically upstream) of a non-heterologous form of a polynucleotide so as to upregulate expression of a nucleic acid encoding the polypeptide of interest. For example, endogenous promoters may be altered in vivo by mutation, deletion, and / or substitution (see, Kmiec, US 5,565,350; Zarling et al., WO9322443), or isolated promoters may be introduced into a plant cell in the proper orientation and distance from a gene of the present invention so as to control the expression of the gene. If polypeptide expression is desired, it is generally desirable to include a polyadenylation region at the 3'-end of a polynucleotide coding region. The polyadenylation region can be derived from the natural gene, from a variety of other plant genes, or from T-DNA. The 3' end sequence to be added may be derived from, for example, the nopaline synthase or octopine synthase genes, or alternatively from another plant gene, or less preferably from any other eukaryotic gene. An intron sequence may also be added to the 5' untranslated region (UTR) or the coding sequence of the partial coding sequence to increase the amount of the mature message that accumulates in the cytosol. Inclusion of a spliceable intron in the transcription unit in both plant and animal expression constructs has been shown to increase gene expression at both the mRNA and protein levels up to 1000-fold (Buchman and Berg (1988) Mol. Cell biol. 8: 4395-4405; Callis et al. (1987) Genes Dev 1 :1183-1200). Such intron enhancement of gene expression is typically greatest when placed near the 5' end of the transcription unit. Use of the maize introns Adh1-S intron 1, 2, and 6, the Bronze-1 intron are known in the art. For general information see: The Maize Handbook, Chapter 116, Freeling and Walbot, Eds., Springer, N.Y. (1994).

[0188] Where appropriate, nucleic acid sequences may be optimized for increased expression in a transformed plant. For example, coding sequences that comprise plant-preferred codons for improved expression in a plant can be provided. See, for example, Campbell and Gowri (1990) Plant Physiol., 92: 1-11 for a discussion of hostpreferred codon usage. Methods also are known in the art for preparing plant-preferred genes. See, for example, U.S. Patent Nos. 5,380,831, and 5,436,391, and Murray et al. (1989) Nucleic Acids Res. 17:477-498, herein incorporated by reference.

[0189] Consequently, demethylase nucleic acids of the invention are provided in expression cassettes for expression in the plant of interest. The cassette will include regulatory sequences operably linked to a demethylase nucleic acid sequence of the invention. The term "regulatory element" as used herein refers to a polynucleotide that is capable of regulating the transcription of an operably linked polynucleotide. It includes, but not limited to, promoters, enhancers, introns, 5' UTRs, and 3' UTRs. By "operably linked" is intended a functional linkage between a promoter and a second sequence, wherein the promoter sequence initiates and mediates transcription of the DNA sequence corresponding to the second sequence. Generally, operably linked means that the nucleic acid sequences being linked are contiguous and, where necessary to join two protein coding regions, contiguous and in the same reading frame. The cassette may additionally contain at least one additional gene to be cotransformed into the organism. Alternatively, the additional gene(s) can be provided on multiple expression cassettes.

[0190] Such an expression cassette is provided with a plurality of restriction sites for insertion of the wildtype / demethylase nucleic acid sequence to be under the transcriptional regulation of the regulatory regions. The expression cassette may additionally contain selectable marker genes. The expression cassette of the present invention will include in the 5'-3' direction of transcription, a transcriptional and translational initiation region (i.e., a promoter), a wildtype / demethylase encoding nucleic acid sequence of the invention, and a transcriptional and translational termination region (i.e., termination region) functional in plants. The promoter may be native or analogous, or foreign or heterologous, to the plant host and / or to the wildtype / demethylase nucleic acid sequence of the invention. Additionally, the promoter may be the natural sequence or alternatively a synthetic sequence. Where the promoter is "foreign" or "heterologous" to the plant host, it is intended that the promoter is not found in the native plant into which the promoter is introduced. Where the promoter is "foreign" or "heterologous" to the wildtype / demethylase nucleic acid sequence of the invention, it is intended that the promoter is not the native or naturally occurring promoter for the operably linked wildtype / mutateddemethylase nucleic acid sequence of the invention. As used herein, a chimeric gene comprises a coding sequence operably linked to a transcription initiation region that is heterologous to the coding sequence. While it may be preferable to express the wildtype / demethylase nucleic acids of the invention using heterologous promoters, the native promoter sequences may be used. Such constructs would change expression levels of the wildtype / demethylase protein in the plant or plant cell. Thus, the phenotype of the plant or plant cell is altered.

[0191] The termination region may be native with the transcriptional initiation region, may be native with the operably linked wildtype / demethylase sequence of interest, may be native with the plant host, or may be derived from another source (i.e., foreign or heterologous to the promoter, the wildtype / demethylase nucleic acid sequence of interest, the plant host, or any combination thereof). Convenient termination regions are available from the Ti- plasmid of A. tumefaciens, such as the octopine synthase and nopaline synthase termination regions. See also Guerineau et al. (1991) Mol. Gen. Genet. 262: 141-144; Proudfoot (1991) Cell 64:671-674; Sanfacon et al. (1991) Genes Dev. 5: 141-149; Mogen et al. (1990) Plant Cell 2: 1261-1272; Munroe et al. (1990) Gene 91 : 151-158; Ballas t ai. (1989) Nucleic Acids Res. 17:7891-7903; and Joshi et al. (1987) Nucleic Acid Res. 15:9627-9639. Where appropriate, the gene(s) may be optimized for increased expression in the transformed plant. That is, the genes can be synthesized using plant-preferred codons for improved expression. See, for example, Campbell and Gowri (1990) Plant Physiol. 92: 1-11 for a discussion of host-preferred codon usage. Methods are available in the art for synthesizing plant-preferred genes. See, for example, U.S. Patent Nos. 5,380,831, and 5,436,391, and Murray et al. (1989) Nucleic Acids Res. 17:477-498, herein incorporated by reference. Thus, the present invention provides an expression cassette comprising a demethylase nucleic acid nucleic acid molecule according to the present invention and a promoter operable in plant cells.

[0192] While the polynucleotides of the invention may find use as selectable marker genes for plant transformation, the expression cassettes of the invention can include another selectable marker gene for the selection of transformed cells. Selectable marker genes, including those of the present invention, are utilized for the selection of transformed cells or tissues. Marker genes include, but are not limited to, genes encoding antibiotic resistance, such as those encoding neomycin phosphotransferase II (NEO) and hygromycin phosphotransferase (HPT), as well as genes conferring resistance to herbicidal compounds, such as glufosinate ammonium, bromoxynil, imidazolinones, and 2,4-dichlorophenoxyacetate (2,4-D). See generally, Yarranton (1992) Curr. Opin. Biotech. 3 :506-511 ; Christophers on et al (1992) Proc. Natl. Acad. ScL USA 89:6314-6318; Yao et al. (1992) Cell 71:63-72; Reznikoff (1992) Mol Microbiol 6:2419-2422; Barkley et al (1980) in The Operon, pp. 177-220; Hu et al (1987) Cell 48:555-566; Brown et al (1987) Cell 49:603-612; Figge et al (1988) Cell 52:713-722; Deuschle et al (1989) Proc. Natl Acad. AcL USA 86:5400-5404; Fuerst et al (1989) Proc. Natl Acad. ScL USA 86:2549-2553; Deuschle et al (1990) Science 248:480-483; Gossen (1993) Ph.D. Thesis, University of Heidelberg; Reines et al (1993) Proc. Natl Acad. ScL USA 90: 1917-1921; Labow et al (1990) Mol Cell Biol 10:3343-3356; Zambretti et al (1992) Proc. Natl Acad. ScL USA 89:3952-3956; Bairn et al (1991) Proc. Natl Acad. ScL USA 88:5072-5076; Wyborski et al (1991) Nucleic Acids Res. 19:4647-4653; Hillenand- Wissman (1989) Topics Mol Struc. Biol 10: 143- 162; Degenkolb et al (1991) Antimicrob. Agents Chemother. 35: 1591-1595; Kleinschnidt et al (1988) Biochemistry 27: 1094-1104; Bonin (1993) Ph.D. Thesis, University of Heidelberg; Gossen et al (1992) Proc. Natl Acad. ScL USA 89:5547- 5551; Oliva et al (1992) Antimicrob. Agents Chemother. 36:913-919; Hlavka et al (1985) Handbook of Experimental Pharmacology, Vol. 78 (Springer- Verlag, Berlin); Gill et al (1988) Nature 334:721-724. Such disclosures are herein incorporated by reference. The above list of selectable marker genes is not meant to be limiting. Any selectable marker gene can be used in the present invention.

[0193] Further, additional sequence modifications are known to enhance gene expression in a cellular host. These include elimination of sequences encoding spurious polyadenylation signals, exon-intron splice site signals, transposon-like repeats, and other such well -characterized sequences that may be deleterious to gene expression. The G-C content of the sequence may be adjusted to levels average for a given cellular host, as calculated by reference to known genes expressed in the host cell. Also, if desired, sequences can be readily modified to avoid predicted hairpin secondary mRNA structures. Nucleotide sequences for enhancing gene expression can also be used in the plant expression vectors. These include, for example, introns of the maize Adh gene Adh 1 -S intron 1 , 2, and 6 (Callis et al. Genes and Development 1 : 1183-1200, 1987), and leader sequences, (W-sequence) from the Tobacco Mosaic virus (TMV), Maize Chlorotic Mottle Virus and Alfalfa Mosaic Virus (Gallie et al. Nucleic Acid Res. 15:8693-8711 , 1987 and Skuzeski et al. Plant Mol. Biol. 15:65-79, 1990). The first intron from the shrunken-1 locus of maize has been shown to increase expression of genes in chimeric gene constructs. U.S. Pat. Nos. 5,424,412 and 5,593,874 disclose the use of specific introns in gene expression constructs, and Gallie et al. (Plant Physiol. 106:929-939, 1994) also have shown that introns are useful for regulating gene expression on a tissue specific basis. To further enhance or to optimize gene expression, the plant expression vectors of the invention also may contain DNA sequences containing matrix attachment regions (MARs). Plant cells transformed with such modified expression systems, then, may exhibit overexpression or constitutive expression of a nucleotide sequence of the invention.

[0194] The invention further provides an isolated recombinant expression vector comprising the expression cassette containing a wildtype / demethylase nucleic acid nucleic acid as described above, wherein expression of the vector in a host cell results in increased tolerance to a herbicide as compared to a wild type variety of the host cell. As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a “plasmid,” which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “expression vectors.” In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, “plasmid” and “vector” can be used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses, and adeno-associated viruses), which serve equivalent functions.

[0195] The recombinant expression vectors of the invention comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, which is operably linked to the nucleic acid sequence to be expressed. Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cells and those that direct expression of the nucleotide sequence only in certain host cells or under certain conditions. It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of polypeptide desired, etc. The expression vectors of the invention can be introduced into host cells to thereby produce polypeptides or peptides, including fusion polypeptides or peptides, encoded by nucleic acids as described herein (e.g., demethylase polypeptides, fusion polypeptides, etc.) Expression vectors may additionally contain 5' leader sequences in the expression construct. Such leader sequences can act to enhance translation. Translation leaders are known in the art and include: picornavirus leaders, for example, EMCV leader (Encephalomyo carditis 5' noncoding region) (Elroy-Stein et al. (1989) PNAS, 86:6126- 6130); potyvirus leaders, for example, TEV leader (Tobacco Etch Virus) (Gallie et al. (1995) Gene 165(2):233-238), MDMV leader (Maize Dwarf Mosaic Virus) (Virology 154:9-20), and human immunoglobulin heavy-chain binding protein (BiP) (Macejak et al. (1991) Nature 353:90-94); untranslated leader from the coat protein mRNA of alfalfa mosaic virus (AMV RNA 4) (Jobling et al. (1987) Nature 325:622-625); tobacco mosaic virus leader (TMV) (Gallie et al. (1989) in Molecular Biology of RNA, ed. Cech (Liss, New York), pp. 237-256); and maize chlorotic mottle virus leader (MCMV) (Lommel et al. (1991) Virology 81 :382- 385). See also, Della-Cioppa et al. (1987) Plant Physiol. 84:965-968.

[0196] Other methods known to enhance translation also can be utilized, for example, introns, and the like. In preparing an expression vector, the various nucleic acid fragments may be manipulated, so as to provide for the nucleic acid sequences in the proper orientation and, as appropriate, in the proper reading frame. Toward this end, adapters or linkers may be employed to join the nucleic acid fragments or other manipulations may be involved to provide for convenient restriction sites, removal of superfluous nucleic acid, removal of restriction sites, or the like. For this purpose, in vitro mutagenesis, primer repair, restriction, annealing, resubstitutions, e.g., transitions and transversions, may be involved. A number of promoters can be used in the practice of the invention. The promoters can be selected based on the desired outcome. The nucleic acids can be combined with constitutive, tissue-preferred, or other promoters for expression in plants.

[0197] Constitutive promoters include, for example, the core promoter of the Rsyn7 promoter and other constitutive promoters disclosed in WO 99 / 43838 and U.S. Patent No. 6,072,050; the core CaMV 35S promoter (Odell et al. (1985) Nature 313:810-812); rice actin (McElroy et al. (1990) Plant Cell 2: 163-171); ubiquitin (Christensen et al. (1989) Plant Mol. Biol. 12:619-632 and Christensen et al. (1992) Plant Mol. Biol. 18:675-689); pEMU (Last et al. (1991) Theor. Appl. Genet. 81 :581- 588); MAS (Velten et al. (1984) EMBO J. 3:2723- 2730); ALS promoter (U.S. Patent No. 5,659,026), and the like. Other constitutive promoters include, for example, U.S. Patent Nos. 5,608,149; 5,608, 144; 5,604,121 ; 5,569,597; 5,466,785; 5,399,680; 5,268,463; 5,608,142; and 6,177,611.

[0198] Tissue-preferred promoters can be utilized to target enhanced expression within a particular plant tissue. Such tissue-preferred promoters include, but are not limited to, leaf- preferred promoters, root-preferred promoters, seed- preferred promoters, and stem-preferred promoters. Some examples of tissue-preferred promoters are described by, e.g., Yamamoto et al. (1997) Plant J. 12(2):255-265; Kawamata et al. (1997) Plant Cell Physiol. 38(7)792-803; Hansen et al. (1997) Mol. Gen Genet. 254(3):337-343; Russell et al. (1997) Transgenic Res. 6(2): 157-168; Rinehart et al. (1996) Plant Physiol. 1 12(3): 1331-1341; Van Camp et al. (1996) Plant Physiol. 112(2):525-535; Canevascini et al. (1996) Plant Physiol. 1 12(2):513- 524; Yamamoto et al. (1994) Plant Cell Physiol. 35(5)773-778; Lam (1994) Results Probl. Cell Differ. 20:181- 196; Orozco ef al. (1993) Plant Mol Biol. 23(6): 1 129-1138; Matsuoka et al. (1993) Voc Natl. Acad. ScL USA 90(20):9586-9590; and Guevara-Garcia et al. (1993) Plant J 4(3):495-505. Promoters can be modified, if necessary, for weak expression.

[0199] In some embodiments, the nucleic acids of interest can be targeted to the chloroplast for expression. In this manner, where the nucleic acid of interest is not directly inserted into the chloroplast, the expression vector will additionally contain a chloroplast- targeting sequence comprising a nucleotide sequence that encodes a chloroplast transit peptide to direct the gene product of interest to the chloroplasts. Such transit peptides are known in the art. With respect to chloroplast-targeting sequences, "operably linked" means that the nucleic acid sequence encoding a transit peptide (i.e., the chloroplast-targeting sequence) is linked to the desired coding sequence of the invention such that the two sequences are contiguous and in the same reading frame. See, for example, Von Heijne et al. (1991) Plant Mol. Biol. Rep. 9: 104-126; Clark et al. (1989) J Biol. Chem. 264:17544- 17550; Della-Cioppa et al. (1987) Plant Physiol. 84:965-968; Romer et al. (1993) Biochem. Biophys. Res. Commun. 196: 1414-1421; and Shah et al. (1986) Science 233:478-481. For example, a chloroplast transit peptide known in the art can be fused to the amino acid sequence of a demethylase polypeptide of the invention by operably linking a chloroplast-targeting sequence to the 5'-end of a nucleotide sequence encoding the demethylase polypeptide.

[0200] Chloroplast targeting sequences are known in the art and include the chloroplast small subunit of ribu lose-l,5- bisphosphate carboxylase (Rubisco) (de Castro Silva Filho et al. (1996) Plant Mol. Biol. 30:769-780; Schnell et al. (1991) J Biol. Chem. 266(5):3335-3342); EPSPS (Archer et al. (1990) J Bioenerg. Biomemb. 22(6)789-810); tryptophan synthase (Zhao et al. (1995) J Biol. Chem. 270(11):6081 -6087); plastocyanin (Lawrence et al. (1997) J Biol. Chem. 272(33):20357-20363); chorismate synthase (Schmidt et al. (1993) J Biol. Chem. 268(36):27447- 27457); and the light harvesting chlorophyll a / b binding protein (LHBP) (Lamppa et al. (1988) J Biol. Chem. 263: 14996-14999). See also Von Heijne et al. (1991) Plant Mol. Biol. Rep. 9: 104- 126; Clark et al. (1989) J Biol. Chem. 264: 17544-17550; Della-Cioppa et al. (1987) Plant Physiol. 84:965-968; Romer et al. (1993) Biochem Biophys. Res. Commun. 196: 1414-1421; and Shah et al. (1986) Science 233:478-481. Methods for transformation of chloroplasts are known in the art. See, for example, Svab et al. (1990) Proc. Natl. Acad. ScL USA 87:8526-8530; Svab and Maliga (1993) Proc. Natl. Acad. Sci. USA 90:913-917; Svab and Maliga (1993) EMBO J. 12:601-606. The method relies on particle gun delivery of DNA containing a selectable marker and targeting of the DNA to the plastid genome through homologous recombination. Additionally, plastid transformation can be accomplished by transactivation of a silent plastid-borne transgene by tissue-preferred expression of a nuclear-encoded and plastid-directed RNA polymerase. Such a system has been reported in McBride et al. (1994) Proc. Natl. Acad. Sci. USA 91 :7301- 7305.

[0201] The nucleic acids of interest to be targeted to the chloroplast may be optimized for expression in the chloroplast to account for differences in codon usage between the plant nucleus and this organelle. In this manner, the nucleic acids of interest may be synthesized using chloroplast-preferred codons. See, for example, U.S. Patent No. 5,380,831, herein incorporated by reference.

[0202] Numerous plant transformation vectors and methods for transforming plants are available. See, for example, An, G. et al. (1986) Plant PysioL, 81 :301-305; Fry, J., et al. ( 1987) Plant Cell Rep. 6:321-325; Block, M. (1988) Theor. Appl. Genet .16: 161 -1 1 A; Hinchee, et al. (1990) Stadler. Genet. Symp.2032\2.203-2\2; Cousins, et al. (1991) Aust. J. Plant Physiol. 18:481-494; Chee, P. P. and Slightom, J. L. (1992) Gene.l 1 8:255-260; Christou, et al. (1992) Trends. Biotechnol. 10:239-246; Halluin, et al. (1992) Bio / Technol. 10:309-314; Dhir, et al. (1992) Plant Physiol. 99:81-88; Casas et al. (1993) Proc. Nat. Acad Sd. USA 90: 1 1212-1 1216; Christou, P. (1993) In Vitro Cell. Dev. Biol.-Plant; 29P.119-124; Davies, et al. (1993) Plant Cell Rep. 12: 180-183; Dong, J. A. and Mchughen, A. (1993) Plant ScL 91 : 139-148; Franklin, C. I. and Trieu, T. N. (1993) Plant. Physiol. 102: 167; Golovkin, et al. (1993) Plant ScL 90:41-52; Guo Chin ScL Bull. 38:2072-2078; Asano, et al. (1994) Plant Cell Rep. 13; Ayeres N. M. and Park, W. D. (1994) Crit. Rev. Plant. Sci. 13:219-239; Barcelo, et al. (1994) Plant. J. 5:583-592; Becker, et al. (1994) Plant. J. 5:299-307; Borkowska et al. (1994) Acta. Physiol Plant. 16:225-230; Christou, P. (1994) Agro. Food. Ind. Hi Tech. 5: 17-27; Eapen et al. (1994) Plant Cell Rep. 13:582-586;

[0203] Hartman, et al. (1994) Bio-Technology 12: 919923; Ritala, et al. (1994) Plant. Mol. Biol. 24:317-325; and Wan, Y. C. and Lemaux, P. G. (1994) Plant Physiol. 104:3748.

[0204] In some embodiments, the methods of the invention involve introducing a polynucleotide construct into a plant. By "introducing" is intended presenting to the plant the polynucleotide construct in such a manner that the construct gains access to the interior of a cell of the plant. The methods of the invention do not depend on a particular method for introducing a polynucleotide construct to a plant, only that the polynucleotide construct gains access to the interior of at least one cell of the plant. Methods for introducing polynucleotide constructs into plants are known in the art including, but not limited to, stable transformation methods, transient transformation methods, and virus-mediated methods. The term “introduction” or “transformation” as referred to herein further means the transfer of an exogenous polynucleotide into a host cell, irrespective of the method used for transfer. Plant tissue capable of subsequent clonal propagation, whether by organogenesis or embryogenesis, may be transformed with a genetic construct of the present invention and a whole plant regenerated there from. The particular tissue chosen will vary depending on the clonal propagation systems available for, and best suited to, the particular species being transformed. Exemplary tissue targets include leaf disks, pollen, embryos, cotyledons, hypocotyls, megagametophytes, callus tissue, existing meristematic tissue (e.g., apical meristem, axillary buds, and root meristems), and induced meristem tissue (e.g., cotyledon meristem and hypocotyl meristem). The polynucleotide may be transiently or stably introduced into a host cell and may be maintained non-integrated, for example, as a plasmid. Alternatively, it may be integrated into the host genome. The resulting transformed plant cell may then be used to regenerate a transformed plant in a manner known to persons skilled in the art. By "stable transformation" is intended that the polynucleotide construct introduced into a plant integrates into the genome of the plant and is capable of being inherited by descendent thereof. By "transient transformation" is intended that a polynucleotide construct introduced into a plant does not integrate into the genome of the plant.

[0205] For the transformation of plants and plant cells, the nucleotide sequences of the invention are inserted using standard techniques into any vector known in the art that is suitable for expression of the nucleotide sequences in a plant or plant cell. The selection of the vector depends on the preferred transformation technique and the target plant species to be transformed. In an embodiment of the invention, the encoding nucleotide sequence is operably linked to a plant promoter, e.g. a promoter known in the art for high-level expression in a plant cell, and this construct is then introduced into a plant cell that is susceptible to herbicides; and a transformed plant is regenerated. In some embodiments, the transformed plant is tolerant to exposure to a level of herbicides that would kill or significantly injure a plant regenerated from an untransformed cell. This method can be applied to any plant species or crops.

[0206] Methodologies for constructing plant expression vectors and introducing foreign nucleic acids into plants are generally known in the art. For example, foreign DNA can be introduced into plants, using tumor-inducing (Ti) plasmid vectors. Other methods utilized for foreign DNA delivery involve the use of PEG mediated protoplast transformation, electroporation, microinjection whiskers, and biolistics or microprojectile bombardment for direct DNA uptake. Such methods are known in the art. (U.S. Pat. No. 5,405,765 to Vasil et al.; Bilang et a (1991) Gene 100: 247-250; Scheid et al.al, (1991) MoL Gen. Genet., 228: 104- 1 12; Guerche et al., (1987) Plant Science 52: 1 1 1 -1 16; Neuhause et al., (1987) Theor. Appl Genet. 75: 30-36; Klein et al., (1987) Nature 327: 70-73; Howell et al., (1980) Science 208: 1265; Horsch et al., (1985) Science 227: 1229-1231 ; DeBlock et al., (1989) Plant Physiology 91 : 694-701 ; Methods for Plant Molecular Biology (Weissbach and Weissbach, eds.) Academic Press, Inc. (1988) and Methods in Plant Molecular Biology (Schuler and Zielinski, eds.) Academic Press, Inc. (1989).

[0207] Other suitable methods of introducing nucleotide sequences into plant cells include microinjection as described by, e.g., Crossway et al. (1986) Biotechniques 4:320-334, electroporation as described by e.g., Riggs et al. (1986) Proc. Natl. Acad. ScL USA 83:5602- 5606, Agrobacterium-mediated transformation as described by e.g., Townsend et al., U.S. Patent No. 5,563,055, Zhao et al., U.S. Patent No. 5,981,840, direct gene transfer as described by, e.g., Paszkowski et al. (1984) EMBO J. 3:2717-2722, and ballistic particle acceleration as described by, e.g., U.S. Patent Nos. 4,945,050; 5,879,918; 5,886,244; and 5,932,782; Tomes et al. (1995) "Direct DNA Transfer into Intact Plant Cells via Microprojectile Bombardment," in Plant Cell, Tissue, and Organ Culture: Fundamental Methods, ed. Gamborg and Phillips (Springer- Verlag, Berlin); McCabe et al. (1988) Biotechnology 6:923-926); and Led transformation (WO 00 / 28058). Also see, Weissinger et al., (1988) Ann. Rev. Genet. 22:421-477; Sanford et al, (1987) Particulate Science and Technology 5:27-37 (onion); Christou et al, (1988) Plant Physiol. 87:671-674 (soybean); McCabe et al., (1988) Bio / Technology 6:923-926 (soybean); Finer and McMullen (1991) In Vitro Cell Dev. Biol. 27P: 175-182 (soybean); Singh et al, (1998) Theor. Appl. Genet. 96:319-324 (soybean); Datta et al., (1990) Biotechnology 8:736-740 (rice); Klein et al., (1988) PNAS, 85:4305-4309 (maize); Klein et al., (1988) Biotechnology 6:559-563 (maize); U.S. Patent Nos. 5,240,855; 5,322,783; and 5,324,646; Tomes et al., (1995) "Direct DNA Transfer into Intact Plant Cells via Microprojectile Bombardment," in Plant Cell, Tissue, and Organ Culture: Fundamental Methods, ed. Gamborg (Springer- Verlag, Berlin) (maize); Klein et al., (1988) Plant Physiol. 91 :440-444 (maize); Fromm et al., (1990) Biotechnology 8:833-839 (maize); Hooykaas-Van Slogteren et al., (1984) Nature (London) 31 1 :763-764; Bowen et al, U.S. Patent No. 5,736,369 (cereals); Bytebier et al, (1987) PNAS 84:5345- 5349 (Liliaceae); De Wet et al., (1985) in The Experimental Manipulation of Ovule Tissues, ed. Chapman et al, (Longman, New York), pp. 197-209 (pollen); Kaeppler et al., (1990) Plant Cell Reports 9:415-418 and Kaeppler et al., (1992) Theor. Apph Genet. 84:560-566 (whisker-mediated transformation); D'Halluin et al., (1992) Plant Cell 4: 1495- 1505 (electroporation); Li et al., (1993) Plant Cell Reports 12:250- 255 and Christou and Ford (1995) Annals of Botany 75:407-413 (rice); Osjoda et al, (1996) Nature Biotechnology 14:745-750 (maize via Agrobacterium tumefaciens); each of which is herein incorporated by reference.

[0208] Transgenic plants, including transgenic crop plants, are preferably produced via Agrobacterium-mediated transformation. An advantageous transformation method is the transformation in planta. To this end, it is possible, for example, to allow the agrobacteria to act on plant seeds or to inoculate the plant meristem with agrobacteria. It has proved particularly expedient in accordance with the invention to allow a suspension of transformed agrobacteria to act on the intact plant or at least on the flower primordia. The plant is subsequently grown on until the seeds of the treated plant are obtained (Clough and Bent, Plant J. (1998) 16, 735-743). Methods for Agrobacterium-mediated transformation of rice include well known methods for rice transformation, such as those described in any of the following: European patent application EP 1198985 A1, Aldemita and Hodges (Planta 199: 612-617, 1996); Chan et al. (Plant Mol Biol 22 (3): 491-506, 1993), Hiei et al. (Plant J 6 (2): 271-282, 1994), which disclosures are incorporated by reference herein as if fully set forth. In the case of corn transformation, the preferred method is as described in either Ishida et al. (Nat. Biotechnol 14(6): 745-50, 1996) or Frame et al. (Plant Physiol 129(1): 13-22, 2002), which disclosures are incorporated by reference herein as if fully set forth. Said methods are further described by way of example in B. Jenes et al., Techniques for Gene Transfer, in: Transgenic Plants, Vol. 1, Engineering and Utilization, eds. S.D. Kung and R. Wu, Academic Press (1993) 128-143 and in Potrykus Annu. Rev. Plant Physiol. Plant Molec. Biol. 42 (1991) 205-225). The nucleic acids or the construct to be expressed is preferably cloned into a vector, which is suitable for transforming Agrobacterium tumefaciens, for example pBin 19 (Bevan et al., Nucl. Acids Res. 12 (1984) 8711). Agrobacteria transformed by such a vector can then be used in known manner for the transformation of plants, such as plants used as a model, like Arabidopsis (Arabidopsis thaliana is within the scope of the present invention not considered as a crop plant), or crop plants such as, by way of example, tobacco plants, for example by immersing bruised leaves or chopped leaves in an agrobacterial solution and then culturing them in suitable media. The transformation of plants by means of Agrobacterium tumefaciens is described, for example, by Hofgen and Willmitzer in Nucl. Acid Res. (1988) 16, 9877 or is known inter alia from F.F. White, Vectors for Gene Transfer in Higher Plants; in Transgenic Plants, Vol. 1, Engineering and Utilization, eds. S.D. Kung and R. Wu, Academic Press, 1993, pp. 15-38.

[0209] One transformation method known to those of skill in the art is the dipping of a flowering plant into an Agrobacteria solution, wherein the Agrobacteria contains the demethylase nucleic acid, followed by breeding of the transformed gametes. Agrobacterium mediated plant transformation can be performed using for example the GV3101(pMP90) (Koncz and Schell, 1986, Mol. Gen. Genet. 204:383-396) or LBA4404 (Clontech) Agrobacterium tumefaciens strain. Transformation can be performed by standard transformation and regeneration techniques (Deblaere et al., 1994, Nucl. Acids. Res. 13:4777-4788; Gelvin, Stanton B. and Schilperoort, Robert A, Plant Molecular Biology Manual, 2nd Ed. - Dordrecht : Kluwer Academic Publ., 1995. - in Sect., Ringbuc Zentrale Signatur: BT11-P ISBN 0-7923-2731-4; Glick, Bernard R. and Thompson, John E., Methods in Plant Molecular Biology and Biotechnology, Boca Raton : CRC Press, 1993 360 S., ISBN 0-8493- 5164-2). For example, rapeseed can be transformed via cotyledon or hypocotyl transformation (Moloney et al., 1989, Plant Cell Report 8:238-242; De Block et al., 1989, Plant Physiol. 91 :694-701). Use of antibiotics for Agrobacterium and plant selection depends on the binary vector and the Agrobacterium strain used for transformation. Rapeseed selection is normally performed using kanamycin as selectable plant marker. Agrobacterium mediated gene transfer to flax can be performed using, for example, a technique described by Mlynarova et al., 1994, Plant Cell Report 13:282-285. Additionally, transformation of soybean can be performed using for example a technique described in European Patent No. 0424 047, U.S. Patent No. 5,322,783, European Patent No. 0397 687, U.S. Patent No. 5,376,543, or U.S. Patent No. 5,169,770. Transformation of maize can be achieved by particle bombardment, polyethylene glycol mediated DNA uptake, or via the silicon carbide fiber technique. (See, for example, Freeling and Walbot “The maize handbook” Springer Verlag: New York (1993) ISBN 3-540-97826-7). A specific example of maize transformation is found in U.S. Patent No. 5,990,387, and a specific example of wheat transformation can be found in PCT Application No. WO 93 / 07256.

[0210] In some embodiments, polynucleotides of the present invention may be introduced into plants by contacting plants with a virus or viral nucleic acids. Generally, such methods involve incorporating a polynucleotide construct of the invention within a viral DNA or RNA molecule. It is recognized that the polypeptides of the invention may be initially synthesized as part of a viral polyprotein, which later may be processed by proteolysis in vivo or in vitro to produce the desired recombinant polypeptide. Further, it is recognized that promoters of the invention also encompass promoters utilized for transcription by viral RNA polymerases. Methods for introducing polynucleotide constructs into plants and expressing a protein encoded therein, involving viral DNA or RNA molecules, are known in the art. See, for example, U.S. Patent Nos. 5,889,191, 5,889,190, 5,866,785, 5,589,367 and 5,316,931; herein incorporated by reference. The cells that have been transformed may be grown into plants in accordance with conventional ways. See, for example, McCormick et a (1986) Plant Cell Reports 5:81-84. These plants may then be grown, and either pollinated with the same transformed strain or different strains, and the resulting hybrid having constitutive expression of the desired phenotypic characteristic identified. Two or more generations may be grown to ensure that expression of the desired phenotypic characteristic is stably maintained and inherited and then seeds harvested to ensure expression of the desired phenotypic characteristic has been achieved.

[0211] The present invention may be used for transformation of any plant species, including, but not limited to, monocots and dicots. Examples of plant species of interest include, but are not limited to, corn or maize (Zea mays), Brassica sp. (e.g., B. napus, B. rapa, B. juncea), particularly those Brassica species useful as sources of seed oil, alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), millet e.g., pearl millet (Pennisetum glaucum), proso millet (Panicum miliaceum), foxtail millet (Setaria italica), finger millet (Eleusine coracana)), sunflower (Helianthus annu ), safflower (Carthamus tinctorius), wheat (Triticum aestivum, T. Turgidum ssp. durum), soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solarium tuberosum), peanuts (Arachis hypogaea), cotton (Gossypium barbadense, Gossypium hirsutum), sweet potato (Ipomoea batatus), cassava (Manihot esculenta), coffee (Coffea spp.), coconut (Cocos nucifera), pineapple (Ananas comosus), citrus trees (Citrus spp.), cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musa spp.), avocado (Persea americana), fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica), olive (Olea europaea), papaya (Carica papaya), cashew (Anacardium occidentale), macadamia (Macadamia integrifolia), almond (Prunus amygdalus), sugar beets (Beta vulgaris), sugarcane (Saccharum spp.), oats, barley, vegetables, ornamentals, and conifers. Preferably, plants of the present invention are crop plants (for example, sunflower, Brassica sp., cotton, sugar, beet, soybean, peanut, alfalfa, safflower, tobacco, corn, rice, wheat, rye, barley triticale, sorghum, millet, etc.).

[0212] In addition to the transformation of somatic cells, which then have to be regenerated into intact plants, it is also possible to transform the cells of plant meristems and in particular those cells which develop into gametes. In this case, the transformed gametes follow the natural plant development, giving rise to transgenic plants. Thus, for example, seeds of Arabidopsis are treated with agrobacteria and seeds are obtained from the developing plants of which a certain proportion is transformed and thus transgenic [Feldman, KA and Marks MD (1987). Mol Gen Genet 208:274-289; Feldmann K (1992). In: C Koncz, N-H Chua and J Shell, eds, Methods in Arabidopsis Research. Word Scientific, Singapore, pp. 274-289], Alternative methods are based on the repeated removal of the inflorescences and incubation of the excision site in the center of the rosette with transformed agrobacteria, whereby transformed seeds can likewise be obtained at a later point in time (Chang (1994). Plant J. 5: 551-558; Katavic (1994). Mol Gen Genet, 245: 363-370). However, an especially effective method is the vacuum infiltration method with its modifications such as the “floral dip” method. In the case of vacuum infiltration of Arabidopsis, intact plants under reduced pressure are treated with an agrobacterial suspension [Bechthold, N (1993). C R Acad Sci Paris Life Sci, 316: 1194-1199], while in the case of the ’’floral dip” method the developing floral tissue is incubated briefly with a surfactant-treated agrobacterial suspension [Clough, SJ and Bent AF (1998) The Plant J. 16, 735-743], A certain proportion of transgenic seeds are harvested in both cases, and these seeds can be distinguished from non-transgenic seeds by growing under the above-described selective conditions. In addition the stable transformation of plastids is of advantages because plastids are inherited maternally is most crops reducing or eliminating the risk of transgene flow through pollen. The transformation of the chloroplast genome is generally achieved by a process which has been schematically displayed in Klaus et al., 2004 [Nature Biotechnology 22 (2), 225-229], Briefly the sequences to be transformed are cloned together with a selectable marker gene between flanking sequences homologous to the chloroplast genome. These homologous flanking sequences direct site specific integration into the plastome. Plastidal transformation has been described for many different plant species and an overview is given in Bock (2001) Transgenic plastids in basic research and plant biotechnology. J Mol Biol. 2001 Sep 21; 312 (3):425-38 or Maliga, P (2003) Progress towards commercialization of plastid transformation technology. Trends Biotechnol. 21, 20-28. Further biotechnological progress has recently been reported in form of marker free plastid transformants, which can be produced by a transient co-integrated maker gene (Klaus et al., 2004, Nature Biotechnology 22(2), 225-229). The genetically modified plant cells can be regenerated via all methods with which the skilled worker is familiar. Suitable methods can be found in the abovementioned publications by S.D. Kung and R. Wu, Potrykus or Hofgen and Willmitzer.

[0213] Generally after transformation, plant cells or cell groupings are selected for the presence of one or more markers which are encoded by plant-expressible genes co-transferred with the gene of interest, following which the transformed material is regenerated into a whole plant. To select transformed plants, the plant material obtained in the transformation is, as a rule, subjected to selective conditions so that transformed plants can be distinguished from untransformed plants. For example, the seeds obtained in the above-described manner can be planted and, after an initial growing period, subjected to a suitable selection by spraying. A further possibility consists in growing the seeds, if appropriate after sterilization, on agar plates using a suitable selection agent so that only the transformed seeds can grow into plants. Alternatively, the transformed plants are screened for the presence of a selectable marker such as the ones described above.

[0214] Following DNA transfer and regeneration, putatively transformed plants may also be evaluated, for instance using Southern analysis or whole genome sequencing, for the presence of the gene of interest, copy number and / or genomic organisation. Alternatively or additionally, expression levels of the newly introduced DNA may be monitored using Northern and / or Western analysis, both techniques being well known to persons having ordinary skill in the art.

[0215] The generated transformed plants may be propagated by a variety of means, such as by clonal propagation or classical breeding techniques. For example, a first generation (or T1) transformed plant may be selfed and homozygous second-generation (or T2) transformants selected, and the T2 plants may then further be propagated through classical breeding techniques. The generated transformed organisms may take a variety of forms. For example, they may be chimeras of transformed cells and non-transformed cells; clonal transformants (e.g., all cells transformed to contain the expression cassette); grafts of transformed and untransformed tissues (e.g., in plants, a transformed rootstock grafted to an untransformed scion).

[0216] Preferably, the expression of the nucleic acid in the plant results in the plant's increased resistance to herbicide as compared to a wild type variety of the plant.

[0217] In another embodiment, 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 herbicide as compared to a wild type variety of the plant.

[0218] The plants described herein can be either transgenic crop plants or non-transgenic plants.

[0219] In addition to the general definition, give SUPRA, "transgenic", “transgene” or "recombinant" means with regard to, for example, a nucleic acid sequence, an expression cassette, gene construct or a vector comprising the nucleic acid sequence or an organism transformed with the nucleic acid sequences, expression cassettes or vectors according to the invention, all those constructions brought about by recombinant methods in which either

[0220] (a) the nucleic acid sequences encoding proteins useful in the methods of the invention, or

[0221] (b) genetic control sequence(s) which is operably linked with the nucleic acid sequence according to the invention, for example a promoter, or

[0222] (c) a) and b) are not located in their natural genetic environment or have been modified by recombinant methods, it being possible for the modification to take the form of, for example, a substitution, addition, deletion, inversion or insertion of one or more nucleotide residues in order to allow for the expression of the demethylase of the present invention. The natural genetic environment is understood as meaning the natural genomic or chromosomal locus in the original plant or the presence in a genomic library. In the case of a genomic library, the natural genetic environment of the nucleic acid sequence is preferably retained, at least in part. The environment flanks the nucleic acid sequence at least on one side and has a sequence length of at least 50 bp, preferably at least 500 bp, especially preferably at least 1000 bp, most preferably at least 5000 bp. A naturally occurring expression cassette - for example the naturally occurring combination of the natural promoter of the nucleic acid sequences with the corresponding nucleic acid sequence encoding a polypeptide useful in the methods of the present invention, as defined above - becomes a transgenic expression cassette when this expression cassette is modified by non-natural, synthetic ("artificial") methods such as, for example, mutagenic treatment. Suitable methods are described, for example, in US 5,565,350 or WO 00 / 15815.

[0223] A transgenic plant for the purposes of the invention is thus understood as meaning, as above, that the nucleic acids of the invention are not at their natural locus in the genome of said plant, it being possible for the nucleic acids to be expressed homologously or heterologously. However, as mentioned, transgenic also means that, while the nucleic acids according to the invention or used in the inventive method are at their natural position in the genome of a plant, the sequence has been modified with regard to the natural sequence, and / or that the regulatory sequences of the natural sequences have been modified. Transgenic is preferably understood as meaning the expression of the nucleic acids according to the invention at an unnatural locus in the genome, i.e. homologous or, preferably, heterologous expression of the nucleic acids takes place. Preferred transgenic plants are mentioned herein. Furthermore, the term “transgenic” refers to any plant, plant cell, callus, plant tissue, or plant part, that contains all or part of at least one recombinant polynucleotide. In many cases, all or part of the recombinant polynucleotide is stably integrated into a chromosome or stable extra-chromosomal element, so that it is passed on to successive generations. For the purposes of the invention, the term “recombinant polynucleotide” refers to a polynucleotide that has been altered, rearranged, or modified by genetic engineering. Examples include any cloned polynucleotide, or polynucleotides, that are linked or joined to heterologous sequences. The term “recombinant’ does not refer to alterations of polynucleotides that result from naturally occurring events, such as spontaneous mutations, or from non-spontaneous mutagenesis followed by selective breeding.

[0224] “Alleles” or “allelic variants” are alternative forms of a given gene, located at the same chromosomal position. Allelic variants encompass Single Nucleotide Polymorphisms (SNPs), as well as Small I nsertion / Deletion Polymorphisms (INDELs). The size of INDELs is usually less than 100 bp. SNPs and INDELs form the largest set of sequence variants in naturally occurring polymorphic strains of most organisms

[0225] The term “variety” refers to a group of plants within a species defined by the sharing of a common set of characteristics or traits accepted by those skilled in the art as sufficient to distinguish one cultivar or variety from another cultivar or variety. There is no implication in either term that all plants of any given cultivar or variety will be genetically identical at either the whole gene or molecular level or that any given plant will be homozygous at all loci. A cultivar or variety is considered “true breeding” for a particular trait if, when the true-breeding cultivar or variety is self-pollinated, all of the progeny contain the trait. The terms “breeding line” or “line” refer to a group of plants within a cultivar defined by the sharing of a common set of characteristics or traits accepted by those skilled in the art as sufficient to distinguish one breeding line or line from another breeding line or line. There is no implication in either term that all plants of any given breeding line or line will be genetically identical at either the whole gene or molecular level or that any given plant will be homozygous at all loci. A breeding line or line is considered “true breeding” for a particular trait if, when the true-breeding line or breeding line is self-pollinated, all of the progeny contain the trait. In the present invention, the trait arises from a mutation in a demethylase gene of the plant or seed.

[0226] The herbicide-resistant plants of the invention that comprise polynucleotides encoding demethylase polypeptides also find use in methods for increasing the herbicide-resistance of a plant through conventional plant breeding involving sexual reproduction. The methods comprise crossing a first plant that is a herbicideresistant plant of the invention to a second plant that may or may not be resistant to the same herbicide or herbicides as the first plant or may be resistant to different herbicide or herbicides than the first plant. The second plant can be any plant that is capable of producing viable progeny plants (i.e., seeds) when crossed with the first plant. Typically, but not necessarily, the first and second plants are of the same species. The methods can optionally involve selecting for progeny plants that comprise the demethylase polypeptides of the first plant and the herbicide resistance characteristics of the second plant. The progeny plants produced by this method of the present invention have increased resistance to a herbicide when compared to either the first or second plant or both. When the first and second plants are resistant to different herbicides, the progeny plants will have the combined herbicide tolerance characteristics of the first and second plants. The methods of the invention can further involve one or more generations of backcrossing the progeny plants of the first cross to a plant of the same line or genotype as either the first or second plant. Alternatively, the progeny of the first cross or any subsequent cross can be crossed to a third plant that is of a different line or genotype than either the first or second plant.

[0227] The present invention also provides plants, plant organs, plant tissues, plant cells, seeds, and non-human host cells that are transformed with the at least one polynucleotide molecule, expression cassette, or transformation vector of the invention. Such transformed plants, plant organs, plant tissues, plant cells, seeds, and non-human host cells have enhanced tolerance or resistance to at least one herbicide, at levels of the herbicide that kill or inhibit the growth of an untransformed plant, plant tissue, plant cell, or non-human host cell, respectively. Preferably, the transformed plants, plant tissues, plant cells, and seeds of the invention are Arabidopsis thaliana and crop plants.

[0228] In another embodiment, 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 herbicide as compared to a wild type variety of the seed.

[0229] In other aspects, herbicide-tolerant plants of the present invention can be employed as herbicide-tolerance trait donor lines for development, as by traditional plant breeding, to produce other varietal and / or hybrid crops containing such trait or traits. All such resulting variety or hybrids crops, containing the ancestral herbicidetolerance trait or traits can be referred to herein as progeny or descendant of the ancestral, herbicide-tolerant line(s).

[0230] In other embodiments, the present invention provides a method for producing a herbicide-tolerant plant. The method comprises: crossing a first herbicide-tolerant plant with a second plant to produce a herbicide-tolerant progeny plant, wherein the first plant and the progeny plant comprise in at least some of their cells a polynucleotide operably linked to a promoter operable in plant cells, the recombinant polynucleotide being effective in the cells of the first plant to express a demethylase polypeptide encoded by the polynucleotide, the expression of the demethylase polypeptide conferring to the plant tolerance to herbicides.

[0231] Traditional plant breeding might be employed whereby the herbicide-tolerant trait is introduced in the progeny plant resulting therefrom. In one embodiment, the present invention provides a method for producing a herbicide-tolerant progeny plant, the method comprising: crossing a parent plant with a herbicide-tolerant plant to introduce the herbicide-tolerance characteristics of the herbicide-tolerant plant into the germplasm of the progeny plant, wherein the progeny plant has increased tolerance to the herbicides relative to the parent plant. In other embodiments, the method further comprises the step of introgressing the herbicide-tolerance characteristics through traditional plant breeding techniques to obtain a descendent plant having the herbicidetolerance characteristics.

[0232] In another aspect, the present invention provides a method for preparing a descendent seed. The method comprises planting a seed of or capable of producing a plant of the present invention. In one embodiment, the method further comprises growing a descendent plant from the seed; and harvesting a descendant seed from the descendent plant. In other embodiments, the method further comprises applying a herbicide or a herbicidal composition to the descendent plant.

[0233] In another embodiment, the invention refers to harvestable parts of the transgenic plant according to the present invention. Preferably, the harvestable parts comprise the demethylase nucleic acid or demethylase protein of the present invention. The harvestable parts may be seeds, roots, leaves and / or flowers comprising the demethylase nucleic acid or demethylase protein or parts thereof. Preferred parts of soy plants are soy beans comprising the demethylase nucleic acid or demethylase protein.

[0234] In another embodiment, the invention refers to products derived from a transgenic plant according to the present invention, parts thereof or harvestable parts thereof. A preferred plant product is fodder, seed meal, oil, or seed- treatment-coated seeds. Preferably, the meal and / or oil comprise the demethylase nucleic acids or demethylase proteins.

[0235] In another embodiment, the invention refers to a method for the production of a product, which method comprises a) growing the plants of the invention or obtainable by the methods of invention and b) producing said product from or by the plants of the invention and / or parts, e.g. seeds, of these plants.

[0236] In a further embodiment the method comprises the steps a) growing the plants of the invention, b) removing the harvestable parts as defined above from the plants and c) producing said product from or by the harvestable parts of the invention.

[0237] The product may be produced at the site where the plant has been grown, the plants and / or parts thereof may be removed from the site where the plants have been grown to produce the product. Typically, the plant is grown, the desired harvestable parts are removed from the plant, if feasible in repeated cycles, and the product made from the harvestable parts of the plant. The step of growing the plant may be performed only once each time the methods of the invention is performed, while allowing repeated times the steps of product production e.g. by repeated removal of harvestable parts of the plants of the invention and if necessary further processing of these parts to arrive at the product. It is also possible that the step of growing the plants of the invention is repeated and plants or harvestable parts are stored until the production of the product is then performed once for the accumulated plants or plant parts. Also, the steps of growing the plants and producing the product may be performed with an overlap in time, even simultaneously to a large extend or sequentially. Generally the plants are grown for some time before the product is produced.

[0238] In one embodiment the products produced by said methods of the invention are plant products such as, but not limited to, a foodstuff, feedstuff, a food supplement, feed supplement, fiber, cosmetic and / or pharmaceutical. Foodstuffs are regarded as compositions used for nutrition and / or for supplementing nutrition. Animal feedstuffs and animal feed supplements, in particular, are regarded as foodstuffs.

[0239] In another embodiment the inventive methods for the production are used to make agricultural products such as, but not limited to, plant extracts, proteins, amino acids, carbohydrates, fats, oils, polymers, vitamins, and the like.

[0240] It is possible that a plant product consists of one or more agricultural products to a large extent.

[0241] In another embodiment, the present invention refers to a method for identifying a herbicide by using a demethylase encoded by a nucleic acid which comprises the nucleotide sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61 , 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129 or 131, or a variant or derivative thereof.

[0242] Said method comprises the steps of: a) generating a transgenic cell or plant comprising a nucleic acid encoding a demethylase, wherein the demethylase is expressed; b) applying a herbicide to the transgenic cell or plant of a) and to a control cell or plant of the same variety; c) determining the growth or the viability of the transgenic cell or plant and the control cell or plant after application of said herbicide, and d) selecting “ herbicides” which confer reduced growth to the control cell or plant as compared to the growth of the transgenic cell or plant.

[0243] As described above, the present invention teaches compositions and methods for increasing the tolerance of a crop plant or seed as compared to a wild-type variety of the plant or seed.

[0244] In these methods, the herbicide can be applied by any method known in the art including, but not limited to, seed treatment, soil treatment, and foliar treatment. Prior to application, the herbicide can be converted into the customary formulations, for example solutions, emulsions, suspensions, dusts, powders, pastes and granules. The use form depends on the particular intended purpose; in each case, it should ensure a fine and even distribution of the compound according to the invention.

[0245] By providing plants having increased tolerance to herbicide, a wide variety of formulations can be employed for protecting plants from weeds, so as to enhance plant growth and reduce competition for nutrients. A herbicide can be used by itself for pre-emergence, post-emergence, pre-planting, and at-planting control of weeds in areas surrounding the crop plants described herein, or a herbicide formulation can be used that contains other additives. The herbicide can also be used as a seed treatment. Additives found in a herbicide formulation include other herbicides, detergents, adjuvants, spreading agents, sticking agents, stabilizing agents, or the like. The herbicide formulation can be a wet or dry preparation and can include, but is not limited to, flowable powders, emulsifiable concentrates, and liquid concentrates. The herbicide and herbicide formulations can be applied in accordance with conventional methods, for example, by spraying, irrigation, dusting, or the like.

[0246] Suitable formulations are described in detail in PCT / EP2009 / 063387 and PCT / EP2009 / 063386, which are incorporated herein by reference.

[0247] As disclosed herein, the demethylase 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 demethylase protein. Such a gene may be an endogenous gene or a transgene, as described above. Additionally, in certain embodiments, 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. For example, the 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; 5,747,450; 5,737,514; 5,723,756; 5,593,881; and Geiser et al (1986) Gene 48: 109), 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), Glyphosate acetyl transferase (GAT), cytochrome P450 monooxygenase, phosphinothricin acetyltransferase (PAT), Acetohydroxyacid synthase (AHAS; EC 4.1.3.18, also known as acetolactate synthase or ALS), hydroxyphenyl pyruvate dioxygenase (HPPD), Phytoene desaturase (PD), Protoporphyrinogen oxidase (PPG) and dicamba degrading enzymes as disclosed in WO 02 / 068607, or phenoxyaceticacid- and phenoxypropionicacid-derivative degrading enzymes as disclosed in WO 2008141154 or WO 2005107437. The combinations generated can also include multiple copies of any one of the polynucleotides of interest.

[0248] It should also be understood that the foregoing relates to preferred embodiments of the present invention and that numerous changes may be made therein without departing from the scope of the invention. The invention is further illustrated by the following examples, which are not to be construed in any way as imposing limitations upon the scope thereof. On the contrary, it is to be clearly understood that resort may be had to various other embodiments, modifications, and equivalents thereof, which, after reading the description herein, may suggest themselves to those skilled in the art without departing from the spirit of the present invention and / or the scope of the appended claims.

[0249] EXAMPLES

[0250] EXAMPLE 1 : Genomic DNA extraction and creation of fosmid library

[0251] In brief, genomic bacterial DNA is purified and subsequently randomly sheared and end-repaired, and ligated into the fosmid vector e.g. CopyControl™. Colonies are plated on EPI300 cells according to the manufacturer’s instructions, and screened for desired activity.

[0252] EXAMPLE 2: Identifying Azine-detoxifying enzymes.

[0253] Fosmid samples were pooled to facilitate sufficient throughput on the LCMS (1,152 samples). To overcome the dilution effect, cells are concentrated by spinning, resuspended in small volume of fresh media with high concentration of azine (with induction), grow 24 hrs. Supernatants are analyzed using the LC / MS and looking for demethylated demethylated diaminotriazine N4-(5-chloro-2,3-difluoro-6-methoxy-phenyl)-6-(1-fluoro-1-methyl- ethyl)-1 ,3,5-triazine-2,4-diamine was tracked by LCMS.

[0254] EXAMPLE 3: Arabidopsis Germination Assay

[0255] Bacterial strain expressing protein of interest, (comprising e.g. SEQ ID NO: 2 plus - as control - TriA variants comprising mutated variants of SEQ ID NO: 133 as disclosed in WO2016 / 116870, WO2018 / 011750, or WO2019 / 142099) is incubated in the presence of 0 - 500nM herbicide (e.g. diaminotriazine N4-(5-chloro-2,3- difluoro-6-methoxy-phenyl)-6-(1 -fluoro-1 -methyl-ethyl)-1 ,3,5-triazine-2,4-diamine) overnight (e.g 24 hrs at 30°C) and the culture spun, 40microliter of the supernatant sterile filtered and applied to agar medium containing the Arabidopsis seeds in the assay. Cultures a kept 48 hrs at 4°C and subsequently grown 7 days in a growth chamber. Ability to germinate and grow is scored. The results are shown in Figure 4.

[0256] EXAMPLE 4: Protein Purification.

[0257] Demethylase and variants thereof are produced in E. coli BL21(DE3) Gold (Agilent Technologies, Germany). Therefore E. coli was transformed with appropriate pET24d N-HIS tag expression vector and chaperone plasmid pGro7 (chaperones groEL and groES). Bacterial strains are grown at 30°C in 100 mL LB for 20 h and protein expression induced with 0.1mM IPTG at 25°C for 20hs. Cells are harvested by centrifugation at 3000 rpm at 4°C for 20 min, resuspended in Bug Buster protein extraction reagent (Novagen, Germany) according to manufactures instructions. Lysates are clarified by centrifugation. Samples of bovine serum albumin (5, 10, and 20 g) are loaded onto each gel analyzed by densitometry to provide an internal standard. Protein determinations are verified using Coomassie protein assay dye, according to manufactures instruction (Thermo Scientific;

[0258] USA). The HIS-tagged enzymes are purified by metal ion affinity chromatography using Ni-IDA 1000 kit (Macherey-Nagel, Germany) following manufactures instructions. Protein purity was accessed by SDS-PAGE using NuPAGE Novex 4-12% Bis-Tris pre-cast gels (Life Technologies; USA) stained with Coomassie Brilliant Blue (Serva, Germany). Protein concentrations are estimated by measuring absorbance at 280 nm using Lambda Bio+ (Perkin Elmer, USA).

[0259] EXAMPLE 5: Enzyme kinetics. E. coli clones expressing demethylase genes, generated as described in EXAMPLE 4, were used in resting cell suspensions which were incubated with various azines (e.g. diaminotriazine N4-(5-chloro-2,3-difluoro-6- methoxy-phenyl)-6-(1-fluoro-1-methyl-ethyl)-1,3,5-triazine-2,4-diamine), melamine and atrazine. Culture filtrate was analyzed by UPLC-HR-MS. Substrates are obtained either from Sigma-Aldrich or synthesized internally. Synthetic standards and enzyme reaction products are analysed by UPLC-HR-MS (Thermo / Dionex UPLC UltiMate3000 coupled to a QExactive high resolution mass spectrometer). A Waters Acquity HSS T3 column (2.1mm; 100mm; 1.8 uM) was used with a mobile phase water / acetonitrile (0.1% formic acid) with a flow rat e of 0.6 ul min1. Enzymes are used solved 25 mM sodium phosphate buffer (pH 7.2) with substrate concentration in the range of 1 nM to 10 nM at 30°C. Over time, the original azine peak disappeared whereas the formed OH- Metabolite (reaction product) increased. The product was identified by determination of the exact formula and by analysis of the accurate MS-MS fragments. Moreover for some of the formed products authentic standards are co-eluted. Degradation in %was calculated against the cells harboring the empty vector as control. The results are shown in Figure 1 .

[0260] EXAMPLE 6: Soybean Hairy Root Assay via Agrobacterium rhizogenes mediated transformation In brief, explants are prepared from 5 days old soybean seedlings, and are subsequently inoculated with Agrobacterium rhizogenes K599 containing the construct of interest (i.e. nucleic acid encoding the demethylase of the present invention). Construct of interest may further contain various transit peptides targeting the demethylase of the present invention to different compartments of the plant cell (e.g. cytosol, plastids, mitochondria, apoplast)

[0261] After 5 days of co-culivation, a selected arsenal is further cultivated 14-20 days to allow for developing hairy roots. Herbicide of interest (e.g. diaminotriazine N4-(5-chloro-2,3-difluoro-6-methoxy-phenyl)-6-(1-fluoro-1- methy l-ethy l)-1 ,3,5-tri azi ne-2,4-d iam ine)) is applied to the plate and root growth of transformed seedlings is compared to untransformed seedlings. The result is shown in Figure 5.

[0262] EXAMPLE 7: Generation of herbicide tolerant model plants.

[0263] Generation of azine-tolerant Arabidopsis plants expressing demethylase sequences. For transformation of Arabidopsis thaliana, demethylase sequences based on SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129 or 131, encoding 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, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, or 132, are cloned with standard cloning techniques as described in Sambrook et al. (Molecular cloning (2001) Cold Spring Harbor Laboratory Press) in a binary vector containing resistance marker gene cassette (AHAS) and mutated amidohydrolase sequence (marked as GOI) in between ubiquitin promoter (PcUbi) and nopaline synthase terminator (NOS) sequence. Binary plasmids are introduced to Agrobacterium tumefaciens for plant transformation. Arabidopsis thaliana are transformed with mutated amidohydrolase sequences by floral dip method as decribed by McElver and Singh (WO 2008 / 124495). Transgenic Arabidopsis plants are subjected to TaqMan analysis for analysis of the number of integration loci.

[0264] EXAMPLE 8: Test for herbicide tolerant model plants. For selection of azine resistant Arabidopsis thaliana plants, expressing the demethylase of the present invention, are used. Selected Arabidopsis thaliana lines are assayed for improved resistance to azines like 6-cyclopentyl-N4-(2,3,4,5,6-pentafluorophenyl)-1,3,5-triazine-2,4- diamine or diaminotriazine N4-(5-chloro-2,3-difluoro-6-methoxy-phenyl)-6-(1-fluoro-1-methyl-ethyl)-1 ,3,5- triazine-2,4-diamine) in 48-well plates. Therefore, T2-seeds are surface sterilized by stirring for 5 min in ethanol + water (70+30 by volume), rinsing one time with ethanol + water (70+30 by volume) and two times with sterile, deionized water. The seeds are resuspended in 0.1% agar dissolved in water (w / v). Four to five seeds per well are plated on solid nutrient medium consisting of half-strength Murashige Skoog nutrient solution, pH 5.8 (Murashige and Skoog (1962) Physiologia 40 Plantarum 15: 473-497). Compounds are dissolved in dimethylsulfoxid (DMSO) and added to the medium prior to solidification (final DMSO concentration 0.1%). Multi well plates are incubated in a growth chamber at 22°C, 75% relative humidity and 110 pmol Phot * m-2 * s-1 with 14 : 10 h light : dark photoperiod. Growth inhibition is evaluated seven to ten days after seeding in comparison to wild type plants. Tolerance factors are calculated based on IC50 values of growth inhibition of transformed versus non-transformed Arabidopsis plants. Additionally, transgenic T2 or T3 Arabidopsis plants are tested for improved tolerance to cellulose biosynthesis-inhibiting herbicides in greenhouse studies.

[0265] EXAMPLE 9: Generation and test of herbicide tolerance crops (soybean and corn). Binary vectors are generated as described in EXAMPLE 10. Soybean cv Jake are transformed as previously described by Siminszky et al., Phytochem Rev. 5:445-458 (2006). After regeneration, transformants are transplanted to soil in small pots, placed in growth chambers (16 hr day / 8 hr night; 25°C day / 23°C night; 65% relative humidity; 130- 150 microE m-2 s-1) and subsequently tested for the presence of the T-DNA via Taqman analysis. After a few weeks, healthy, transgenic positive, single copy events are transplanted to larger pots and allowed to grow in the growth chamber. Plants are subsequently transferred to the greenhouse for T 1 seed production and harvest.

[0266] An optimal shoot for cutting is about 3-4 inches tall, with at least two nodes present. Each cutting is taken from the original transformant (mother plant) and dipped into rooting hormone powder (indole-3-butyric acid, I BA). The cutting is then placed in oasis wedges inside a bio-dome. The mother plant is taken to maturity in the greenhouse and harvested for seed. Wild type cuttings are also taken simultaneously to serve as negative controls. The cuttings are kept in the bio-dome for 5-7 days. 7-10 days after transfer to oasis wedges, the roots are treated via nutrient solution with the herbicide. Typical phytotox symptoms, like club shaped root, are evaluated 3-4 days after treatment. Less or no injury of transgenic plants compared to wildtype plants are interpreted as herbicide tolerance.

[0267] For the pre-emergence treatment, the culture containers used can be plastic flowerpots containing loamy sand with approximately 3.0% of humus as the substrate. The seeds of the test plants are sown separately for each species / event. The used active ingredients, which are suspended or emulsified in water, are applied directly after sowing by means of finely distributing nozzles. The containers are irrigated gently to promote germination and growth and subsequently covered with transparent plastic hoods until the plants had rooted. This cover causes uniform germination of the test plants, unless this had been impaired by the active ingredients.

[0268] Immature corn embryos are transformed according to the procedure outlined in Peng et al. (W02006 / 136596). Plants are tested for the presence of the T-DNA by Taqman analysis with the target being the nos terminator which is present in all constructs. Healthy looking plants are sent to the greenhouse for hardening and subsequent spray testing. The plants are individually transplanted into MetroMix 360 soil in 4” pots. Once in the greenhouse (day / night cycle of 27°C / 21 °C with 14 hour day length supported by 600W high pressure sodium lights), they are allowed to grow for 14 days. Transgenic corn plants are cultivated to T1 seeds for herbicide tolerance testing. 14 days after transfer, the roots are treated via nutrient solution with the herbicide. Typical phytotox symptoms, like club shaped root, are evaluated 3-4 days after treatment. Less or no injury of transgenic plants compared to wildtype plants are interpreted as herbicide tolerance.

[0269] For the pre-emergence treatment, the culture containers used are plastic flowerpots containing loamy sand with approximately 3.0% of humus as the substrate. The seeds of the test plants are sown separately for each species / event. The used active ingredients, which are suspended or emulsified in water, are applied directly after sowing by means of finely distributing nozzles. The containers are irrigated gently to promote germination and growth and subsequently covered with transparent plastic hoods until the plants have rooted. This cover causes uniform germination of the test plants, unless this is impaired by the active ingredients.

[0270] For post emergence treatment, the test plants are first grown to a height of 3 to 15 cm, depending on the plant habit, and only then treated with the herbicides. For this purpose, the test plants are either sown directly, grown in the same containers or they are first grown separately and transplanted into the test containers a few days prior to treatment. Herbicide injury evaluations are taken at 2 and 3 weeks after treatment. Plant injury is rated on a scale of 0% to 100%, 0% being no injury and 100% being complete death. 100 means no emergence of the plants, or complete destruction of at least the aerial moieties, and 0 means no damage, or normal course of growth. A moderate herbicidal activity is given at values of at least 60, a good herbicidal activity is given at values of at least 70, and a very good herbicidal activity is given at values of at least 85.

[0271] EXAMPLE 10: Binary Vector Construction. Cloning methods e.g. use of restriction endonucleases to cut double stranded DNA at specific sites, agarose gel electrophoreses, purification of DNA fragments, transfer of nucleic acids onto nitrocellulose and nylon membranes, joining of DNA-fragments, transformation of E.coli cells and culture of bacteria are performed as described in Sambrook et al. (1989) (Cold Spring Harbor Laboratory Press: ISBN 0-87965-309-6). Polymerase chain reaction is performed using Phusion™ High-Fidelity DNA Polymerase (NEB, Frankfurt, Germany) according to the manufactures instructions. In general, primers used in PCR are designed such, that at least 20 nucleotides of the 3’ end of the primer anneal perfectly with the template to amplify. Restriction sites are added by attaching the corresponding nucleotides of the recognition sites to the 5’ end of the primer. Fusion PCR, for example described by K. Heckman and L. R. Pease, Nature Protocols (2207) 2, 924—932 was used as an alternative method to join two fragments of interest, e.g. a promoter to a gene or a gene to a terminator. Gene Synthesis, as for example described by Czar et al. (Trends in Biotechnology, 2009, 27(2): 63-72), was performed by Life Technologies using their Geneart® service.

[0272] Genes are assessed for codon usage and the presence of restriction sites that might impede cloning efforts. Where necessary genes are codon optimized using standard protocols for maximum expression in the crop plant (for example see Puigbo et al. 2007 and Gasper et al. 2012) as well as removal of undesired restriction sites. Genes are either synthesized by GeneArt (Regensburg) or PCR-amplified using Phusion™ High-Fidelity DNA Polymerase (NEB, Frankfurt, Germany) according to the manufactures instructions from cDNA. In both cases a Ncol and / or Asci restriction site at the 5’terminus, and a Sbfl restriction site at the 3’ terminus are introduced to enable cloning of these genes between functional elements such as promoters and terminators using these restriction sites. Promoter-terminator modules or promoter-intron-terminator modules are created by nucleotide synthesis. While joining terminator sequences to promoter sequences or promoter-intron sequences either via synthesis, recognition sequences for the restriction endonucleases are added to either side of the modules, and the recognition sites for the restriction endonucleases Ncol, Asci and Sbfl are introduced between promoter and terminator or between introns and terminator. To obtain the final expression modules, PCR- amplified genes are cloned between promoter and terminator or intron and terminator via Asci and / or Sbfl restriction sites. Completed binary vectors are used for transformation into E. coll as described in Sambrook et al. 1989.

[0273] Alternatively, gene synthesis, as for example described by Czar et al. (Trends in Biotechnology, 2009, 27(2): 63- 72), can be performed by Life Technologies using their Geneart® service. Standard methods like cloning, restriction, molecular analysis, transformation of E.coli cells and culture of bacteria can be performed as described in Sambrook et al. (1989) (Cold Spring Harbor Laboratory Press: ISBN 0-87965-309-6). Polymerase chain reaction can be performed using Phusion™ High-Fidelity DNA Polymerase (NEB, Frankfurt, Germany) according to the manufactures instructions. Fusion PCR can be done as described by K. Heckman and L. R. Pease, Nature Protocols (2207) 2, 924-932. In both cases a Ncol and / or Asci restriction site at the 5’terminus, and a Pad restriction site at the 3’ terminus can be introduced to enable cloning of these genes between functional elements. Promoter-terminator modules or promoter-intron-terminator modules are created by complete synthesis by GeneArt (Regensburg) or by joining the corresponding expression elements using fusion PCR and cloning the PCR-product into the TOPO-vector pCR2.1 (Invitrogen) according to the manufactures instructions. While joining terminator sequences to promoter sequences or promoter-intron sequences either via synthesis of whole cassettes or using fusion PCR, recognition sequences for the restriction endonucleases can be added to either side of the modules, and the recognition sites for the restriction endonucleases Ncol, Asci and Pad can be introduced between promoter and terminator or between introns and terminator. To obtain the final expression modules, PCR-amplified genes can be cloned between promoter and terminator or intron and terminator via Ncol and / or Pac I restriction sites. Genes of interest can be codon optimized using standard protocols for maximum expression in the crop plant (for example see Puigbo et al. 2007 and Gasper et al. 2012) as well as removal of undesired restriction sites and synthesized by GeneArt (Regensburg, Germany).

[0274] EXAMPLE 11

[0275] Herbicide demethylation by different Demethylases

[0276] Demethylase Sequence Identification: Protein sequence of SEQ ID NO: 2 was used to perform a BLAST search (BLASTp) of an internal microbial protein database using BLASTp default parameters. The retrieved protein sequences were filtered for >80% length and >50% sequence identity with respect to Protein SEQ ID NO: 2. Demethylase sequences with >95% identity were clustered. From the 123 non-redundant Demethylase sequences obtained, 65 additional sequences were selected for further testing (giving 66 in total including SEQ ID NO: 2) .

[0277] Protein expression, extraction, and quantification: Demethylase sequences were expressed in E. coli BL21 (DE3) Gold (Agilent Technologies, Germany) to produce the proteins. E. coli was transformed with the pET28a N-HIS tag expression vector containing the Demethylase sequences. Bacterial strains were grown at 30°C in 100 mL LB for 20 h and protein expression induced with 0.1mM IPTG at 25°C for 20h. Cells were harvested by centrifugation at 3000 rpm at 4°C for 20 min, resuspended in Bug Buster protein extraction reagent (Novagen, Germany) according to manufacturer’s instructions. Lysates were clarified by centrifugation at 3000 rpm at 4°C for 10 min to obtain a crude protein extract. To verify expression of the tested Demethylases in E. coli and to quantify them in order to normalize activity measurements across the different Demethylases mass spectrometry proteomics using data independent acquisition was used [DIA] (Doerr, A. DIA mass spectrometry. Nat Methods 12, 35 (2015), https: / / doi.org / 10.1038 / nmeth.3234). Sample preparation for DIA mass spectrometry: Clarified protein lysates were reduced, alkylated, and digested with Trypsin / Lys-C Mix overnight according to manufacturer’s instructions (Promega, USA). Trypsin-digested lysates were further purified through desalting and buffer exchange using PD-10 desalting columns packed with Sephadex G-25 resin according to manufacturer’s instructions (Cytiva.USA). Elution of trypsin-digested lysates was performed with 100 pL HEPES buffer. DIA proteomics for target demethylase identification and quantification from lysates was performed in a nano-flow UPLC-HR mass spectrometry setup (Thermo Scientific Vanquish Neo nano UHPLC System coupled to a Thermo Scientific Orbitrap Exploris 240 mass spectrometer). A Thermo Scientific pPAC Neo high-throughput HPLC column (5.5 cm) was used with a mobile phase water / acetonitrile (0.1% formic acid) with a varying flow rate of 2.5 - 1 .25 pL / min. Data analysis for target demethylase identification and quantification was performed with the universal automated software suite for DIA proteomics data analysis DIA- NN using default parameters (Demichev V. et al. Nature Methods volume 17, pages41-44 (2020), https: / / doi.org / 10.1038 / s41592-019-0638-x). Total protein concentration of the clarified lysates is typically determined by measuring absorbance at 280nm with a microvolume spectrophotometer (NanoDrop, Thermo Scientific, USA) as a reference for absolute demethylase quantification. Activity measurements: To determine the catalytic constant (Kcat, turnover number in catalyzing reactions, unit: min-1), clarified E. coli lysate deriving from crude protein extracts after Bug Buster protein extraction (Novagen, Germany) and subsequent desalting (Sephadex G-25 columns, Cytiva, USA) for the various demethylases were used. Extracts were incubated with 500 pM of diaminotriazine N4-(5-chloro-2,3-difluoro-6-methoxy-phenyl)-6- (1 -fluoro-1 -methyl-ethyl)-1 ,3,5-triazine-2,4-diamine) for 5 min at 28°C before the reaction was stopped with 5x volume of 10 mM formic acid. The concentrations of the herbicide and the formed OH-metabolite (reaction product) were analyzed by mass spectrometry. Substrates are obtained either from Sigma-Aldrich or synthesized internally. Synthetic standards and enzyme reaction products are analyzed by UPLC-HR mass spectrometry setup (Thermo Scientific Vanquish UHPLC System coupled to a Thermo Scientific Orbitrap Exploris 240 mass spectrometer). A Thermo Scientific Accucore Vanquish C18+ UHPLC column (50mm; 1 .5 pM) was used with a mobile phase water / acetonitrile (0.1% formic acid) with a flow rate of 350 pL min-1 . Availably of synthetic standards for the herbicide and the OH-metabolite allowed for precise metabolite confirmation via MS-MS fragmentation and accurate quantification using peak areas referenced to standards in each assay. Protein lysate from E. coli expressing an empty vector and a reaction buffer blank were used as negative controls. Results are shown in Table 3.

[0278] As shown in Table 3 no activity was detected in the buffer control, indicating that the herbicide was chemically stable and did not demethylate spontaneously under these assay conditions. The lack of detectable activity in the empty vector control indicates that the crude E. coli extract does not contain endogenous enzymes with demethylation activity on the herbicide. Therefore, the presence of the OH-metabolite in the assays with the different expressed demethylases is caused by their enzymatic conversion of the herbicide. All demethylases tested showed detectable activity. Of the 66 demethylases tested, 15 showed a HIGH, 29 a MEDIUM and 22 a LOW activity level (Table 3). The level of metabolic conversion of the herbicide to the OH-metabolite is a good basis to develop a metabolism-based herbicide tolerance trait for crops.

[0279] Table 3: Catalytic constant (Kcat, turnover number in catalyzing reactions) activity levels for the tested demethylases. kcat (min-1) activity level - >1 = HIGH, >0.01 = MEDIUM, >0.0001 = LOW

[0280] EXAMPLE 12

[0281] Herbicide tolerance of transgenic TO soybean plants expressing demethylases

[0282] Eight of the 66 demethylases were selected, and their ability to provide herbicide tolerance in planta was tested in soybean (cultivar Thorne). Constructs were generated for all eight demethylases and their expression is driven by the strong Ubi 10 promoter from Arabidopsis. For each construct transgenic events (TO-plants) were generated as described in EXAMPLE 9

[0283] Soybean was transformed as previously described by Siminszky et al., Phytochem Rev. 5:445-458 (2006). Before transfer to the greenhouse, all selected transgenic lines were analyzed by PCR, and only single-copy lines tested further. Transformants were transplanted to soil in small pots, placed in growth chambers (16 hr day / 8 hr night; 25°C day / 23°C night; 65% relative humidity; 130-150 15 microE m-2 s-1). After 1-2 weeks, healthy transgenic events were sprayed over the top with the herbicide diaminotriazine N4-(5-chloro-2,3- difluoro-6-methoxy-phenyl)-6-(1-fluoro-1-methyl-ethyl)-1,3,5-triazine-2,4-diamine) at the rate of 50g ai / ha. Herbicide injury was scored 7 days after treatment, and rated on a scale of 0% to 100%, with 0% being no injury and 100% being complete death. Wild-type (untransformed plants of cv. Thorne) were included in all herbicide treatment experiments to ensure the herbicide treatment was effective.

[0284] Results of the experiment are shown in Table 4 below. For each demethylase, between 20 and 52 TO plants were scored and the average injury per demethylase as well as the minimum and maximum injury scores are reported. The results for herbicide injury for plants expressing the eight demethylases were spread over a certain range as all TO plants for a given construct are independent events and differ in the level of expression of the demethylase resulting in different level of herbicide injury.

[0285] As expected, wild-type plants showed very high levels of injury ranging from 90 to 100% (91% on average). Expression of all demethylases from the different accession significantly reduces the level of herbicide injury of the TO-plants. For four demethylases, SEQ ID NO: 37 / 38, SEQ ID NO: 59 / 60, SEQ ID NO: 45 / 46, SEQ ID NO: 1 / 2 there were some TO plants which showed a very low level of injury, less than 10%. Such demethylases would be good candidates for a commercial herbicide tolerance trait.

[0286] Expression of demethylase from accession SEQ ID NO:119 / 120 did not result in a significant reduction of the average level of herbicide injury. However, some TO plants were generated with injury level of 80% which is better than the lowest score of wild-type and suggests a significant herbicidal tolerance. Since this demethylase is the only one of the 8 that had a “MEDIUM” level of activity and the other 7 had “HIGH” activity (Table 3 and 4), it is likely assumed that a higher expression level of this demethylase would be required to achieve a similar herbicide tolerance as the demethylases with “HIGH” activity levels. Table 4

Claims

Claims:1 . An isolated and / or recombinantly produced and / or synthetic nucleic acid molecule comprising a nucleic acid molecule encoding a demethylase selected from the group consisting of:1 . (a) a nucleic acid molecule encoding a demethylase comprising the sequence 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, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104,106.

108.

110.

112.

114.

116.

118.

120.

122.

124.

126.

128. 130, or 132, or a variant, paralogue, orthologue or homolog thereof;2. (b) a nucleic acid molecule comprising the sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15,17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67,69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113,115.

117.

119.

121.

123.

125.

127. 129, or 131, or a variant, paralogue, orthologue or homolog thereof;3. (c) a nucleic acid molecule, which, as a result of the degeneracy of the genetic code, can be derived from a demethylase sequence 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, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122,124.

126.

128. 130, or 132, or a variant, paralogue, orthologue or homolog thereof,;4. (d) a nucleic acid molecule having 30% or more identity, preferably 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99,5%, or more with the nucleic acid molecule sequence of a polynucleotide comprising the nucleic acid molecule of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61 , 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109,111.

113.

115.

117.

119.

121.

123.

125.

127. 129, or 131, or a variant, paralogue, orthologue or homolog thereof,(e) a nucleic acid molecule encoding a demethylase having 30% or more identity, preferably at least 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99,5% or more, with the amino acid sequence of the demethylase sequence 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, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68,70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114,116.

118.

120.

122.

124.

126.

128. 130, or 132,;(f) nucleic acid molecule which hybridizes with a nucleic acid molecule of (a), (b), (c), (d) or (e) under stringent hybridization conditions, wherein the encoded demethylase is capable of demethylating a herbicide.

2. The nucleic acid molecule of claim 1 , wherein said herbicide belongs to the class of cellulose biosynthesis inhibitors, preferably azines.

3. A recombinant expression cassette comprising the nucleic acid molecule as defined in claim 1 .

4. The expression cassette of claim 3, wherein the nucleic acid molecule encoding the demethylase is functionally linked to a heterologous promoter.

5. A vector comprising the nucleic acid molecule as defined in claim 1 or the expression cassette as defined in claim 3.

6. A recombinant microorganism comprising the recombinant expression cassette of claim 3 or 4, or the recombinant vector of claim 5.

7. An isolated, recombinantly produced and / or chemically synthesized demethylase encoded by the nucleic acid molecule as claimed in claim 1 or a polypeptide having at least 80%, 90%, 95%, 98%, 99% identity to the sequence 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, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, or 132, or a variant, paralogue, orthologue or homolog thereof8. A method for demethylating a herbicide, the method comprising contacting said herbicide with a demethylase.

9. The method of claim 8, wherein said herbicide belongs to the class of cellulose biosynthesis inhibitors, preferably azines, more preferably diaminotriazines.

10. The method of claim 8 or 9, wherein said demethylation leads to the inactivation of said herbicide.11 . The method of claim 8 to 10, wherein said contacting occurs at a plant cultivation site comprising plants or plant parts expressing said demethylase.

12. The method of any of claims 8 to 11 , wherein said demethylase comprises a sequence having at least 80%, 90%, 95%, 98%, 99% identity to the sequence 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, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, or 132, or a variant, paralogue, orthologue or homolog thereof.

13. Use of a nucleic acid molecule encoding a demethylase as defined in claim 1 or a demethylase as defined in claim 7 for demethylating a herbicide.

14. A transgenic plant cell, plant or plant part comprising a nucleic acid molecule encoding a demethylase as defined in claim 1 or an expression cassette as defined in claim 3 or a vector as defined in claim 5, or a demethylase as defined in claim 7.

15. A method of producing a plant having tolerance to herbicides, the method comprising regenerating a plant from a plant cell transformed with a recombinant polynucleotide operably linked to a promoter operable in plant cells, the promoter capable of expressing a demethylase encoded by the polynucleotide as defined in claim 1, the expression of the demethylase conferring to the plant tolerance to herbicides.

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