Herbicide resistant plants

Description

HERBICIDE-RESISTANT PLANTS AREA OF THE INVENTION

[0001] The present invention relates to modified protoporphyrinogen oxidase (PPO) enzymes having one or more mutations at specific amino acid positions that increase the enzyme's resistance to PPO-inhibiting compounds such as herbicides. The invention further relates to plants or parts thereof comprising the modified PPO enzymes, methods for preparing the plants, and methods for controlling unwanted vegetation in the vicinity of said plants. BACKGROUND

[0002] The present invention relates to the production of modified PPO enzymes, and plants containing said enzymes, which are resistant to herbicides.

[0003] Protoporphyrinogen oxidase (PPO) enzymes (EC 1.3.3.4) are well-conserved eukaryotic enzymes that convert protoporphyrinogen IX to protoporphyrin IX. PPO is a flavoprotein located in the inner mitochondrial membrane that catalyzes the removal of six hydrogens (four from methylene bridges and two from ring nitrogens) to form protoporphyrin-IX. This involves a three-step, six-electron flavin adenine dinucleotide-dependent oxidation that consumes molecular oxygen. It is essential for mitochondrial heme functions.

[0004] Herbicides of particular interest in agriculture include those that inhibit PPO enzymes, referred to as PPO-inhibiting herbicides. Given that heme function is essential for plant survival, it has been identified as a herbicide target. Recently, compounds Petition 870250095065, dated 10 / 17 / 2025, page 16 / 375 2 / 346 Effective herbicides that inhibit PPO enzymes have been developed. Upon contact with plants, herbicides cause cell death and eventually plant death. Industrially, herbicides are used in agriculture to remove unwanted vegetation such as weeds from cultivated crops. However, non-specific herbicides that target essential pathways, such as those targeting heme synthesis, will affect both crops and unwanted vegetation, making them difficult to use without destroying the valuable crop. Therefore, in order to effectively use these herbicides, it would be desirable to protect crop plants so that they have resistance to herbicide compounds. Plants that have herbicide resistance can then be contacted with the herbicide and will not be affected, while non-resistant unwanted vegetation is affected and controlled.

[0005] Agricultural crop production frequently utilizes transgenic traits created using biotechnology methods. A heterologous gene, also known as a transgene, can be introduced into a plant to produce a transgenic trait. The expression of the transgene in the plant confers a trait, such as herbicide tolerance, to the plant. Herbicide-tolerant traits allow a plant to grow, while non-tolerant weed species are eradicated by contact with the herbicide. Examples of transgenic herbicide-tolerant traits include glyphosate tolerance, glufosinate tolerance, and dicamba tolerance. With the increase in weed species resistant to commonly used herbicides, new herbicides and new herbicide-tolerant traits Petition 870250095065, dated 10 / 17 / 2025, page 17 / 375 3 / 346 companions are needed in the area. Given that several herbicides targeting PPO have been developed, there is a need to develop traits conferring tolerance to PPO-inhibiting herbicides, which will be particularly useful in crops.

[0006] The present invention aims to solve one or more of the problems mentioned above in the art. INVENTION STATEMENTS

[0007] According to a first aspect of the present invention, a modified Protoporphyrinogen Oxidase (PPO) enzyme or functional fragment thereof is provided, wherein the modified Protoporphyrinogen Oxidase or functional fragment thereof comprises at least one mutation in one or more amino acid residues selected from residues 362, 365 and / or 479 of SEQ ID NO:1 or residues corresponding thereto.

[0008] In a second aspect of the present invention, a modified Protoporphyrinogen Oxidase (PPO) enzyme or functional fragment thereof is provided, wherein the modified Protoporphyrinogen Oxidase or functional fragment thereof comprises a mutation in amino acid residues 305 and 426 of SEQ ID NO:1, or residues corresponding thereto, wherein the mutation in amino acid residue 305, or in a residue corresponding thereto, is a substitution for leucine (305L) and wherein the mutation in amino acid residue 426, or in a residue corresponding thereto, is a substitution for valine (426V). In certain embodiments, the modified PPO enzyme comprises the substitutions S305L and Y426V with reference to SEQ ID NO:1 or a residue corresponding thereto. In certain embodiments, the Petition 870250095065, dated 10 / 17 / 2025, p. 18 / 375 4 / 346 The modified PPO enzyme comprises the T305L and Y426V substitutions with reference to SEQ ID NO: 2 or a residue corresponding thereto. In certain embodiments, the modified PPO enzyme comprises or consists of an amino acid sequence according to SEQ ID NO: 125. In certain embodiments, the modified PPO enzyme comprises or consists of an amino acid sequence according to SEQ ID NO: 139.

[0009] According to a third aspect of the present invention, a nucleic acid is provided comprising a polynucleotide encoding a modified PPO enzyme or a functional fragment thereof according to the first or second aspect.

[0010] According to a fourth aspect of the present invention, a recombinant vector comprising a nucleic acid according to the third aspect is provided.

[0011] According to a fifth aspect of the present invention, a plant or part thereof is provided comprising a modified PPO enzyme or a functional fragment thereof according to the first or second aspect, a nucleic acid according to the third aspect or a recombinant vector according to the fourth aspect.

[0012] According to a sixth aspect of the present invention, a method of producing a plant or part thereof is provided according to the fifth aspect, the method comprising: modifying the plant or part thereof to comprise a PPO enzyme according to the first or second aspect.

[0013] According to a seventh aspect of the present invention, a method is provided for controlling unwanted vegetation in the vicinity of a plant or part thereof according to Petition 870250095065, dated 10 / 17 / 2025, p. 19 / 375 5 / 346 the fifth aspect or at a locus for plant growth according to the fifth aspect, the method comprising applying an effective amount of at least one compound that inhibits a PPO enzyme to the unwanted vegetation, to the plant or part thereof and / or to the locus and / or, optionally, planting a seed at the locus where the seed is capable of producing a plant according to the fifth aspect.

[0014] The invention will now be further described under the following headed sections, the features under any given section may apply to any embodiment or aspect in any combination. Detailed Description of Modified PPO Enzymes

[0015] The invention relates primarily to modified Protoporphyrinogen Oxidase (PPO) enzymes that have increased resistance to compounds such as herbicides, compared with wild-type or control PPO enzymes.

[0016] As used herein, reference to a PPO enzyme or modified PPO enzyme and its amino acid sequences also refers to, and is intended to encompass, the isolated polynucleotides encoding such an enzyme. The terms modified polynucleotide, mutated polynucleotide, mutated polypeptide, or modified polypeptide refer to a polynucleotide or polypeptide that has been altered through human intervention. Such a mutated polynucleotide, modified polynucleotide, mutated polypeptide, or modified polypeptide has a sequence that differs from the sequence of the corresponding non-mutated or non-modified polynucleotide or polypeptide (e.g., a Petition 870250095065, dated 10 / 17 / 2025, page 20 / 375 6 / 346 wild-type or naturally occurring polynucleotide or polypeptide) by at least one nucleotide or amino acid. In certain examples, the mutated or modified polynucleotide comprises an alteration resulting from a Cas guide polynucleotide / endonuclease system as disclosed here.

[0017] Protoporphyrinogen Oxidase (PPO) refers to a critical enzyme in the biosynthesis of protoporphyrin IX. PPO catalyzes the last common step in chlorophyll and heme biosynthesis, which is the oxidation of protoporphyrinogen IX to protoporphyrin IX. (Matringe et al. 1989. Biochem. 1. 260: 231). Inhibition of protoporphyrinogen oxidase is a mechanism of action for several commercial herbicides, including the nitrophenyl ethers acifluorfen and fomesafen and the pyrimidinediones butafenacil and saflufenacil. Visible symptoms of treatment are chlorosis and desiccation. The damage is caused by an accumulation of protoporphyrin IX in plant cells due to protox inhibition within the tetrapyrrole biosynthesis pathway. In plants, it is encoded by two homologous genes located in the nucleus, PPOX1 and PPOX2. PPOX1 is targeted exclusively to plastids, providing proto-protein for heme and chlorophyll synthesis, while PPOX2 has been found in plastid envelopes and mitochondria in spinach.However, the PPOX2 from N. tabacum proved to be merely a mitochondrial protein.

[0018] Appropriately, the PPO enzyme was modified to increase the enzyme activity, appropriately within a plant or part thereof. Appropriately, the plant may have been modified to comprise a modified PPO enzyme having increased activity compared to the Petition 870250095065, dated 10 / 17 / 2025, p. 21 / 375 7 / 346 unmodified PPO enzyme, suitably of wild type, suitably within the plant or a part thereof.

[0019] Appropriately, the activity of the modified PPO enzyme is increased to a level that provides the plant with increased resistance to a compound that inhibits PPO enzymatic activity compared to an unmodified enzyme or control enzyme.

[0020] Appropriately, the activity of the modified PPO enzyme is increased to a level that provides the plant with increased resistance to a compound that inhibits PPO enzymatic activity compared to an unmodified enzyme or control enzyme.

[0021] Appropriately, the activity of the modified PPO enzyme is increased to a level at least 5%, 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%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150% higher than the activity of the same enzyme in an unmodified or control plant when exposed to a compound that inhibits the enzymatic activity of PPO.

[0022] Appropriately, the increase in enzyme activity can be measured by an enzyme assay, which measures the consumption of a substrate or the production of a product over time, appropriately in an in vitro environment. Such assays may be spectrophotometric, fluorometric, calorimetric, chemiluminescent, light scattering, or microscale thermophoresis.

[0023] Appropriately, the modified PPO enzyme comprises one or more modifications, appropriately one or more mutations. Petition 870250095065, dated 10 / 17 / 2025, p. 22 / 375 8 / 346 Properly, one or more mutations are a selection of deletions, insertions, substitutions, etc. By mutation, we mean any substitution, deletion, or insertion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids.

[0024] The modified PPO enzyme may be a modified PPO1 enzyme.

[0025] The modified PPO enzyme may include at least one amino acid modification at any of the amino acid positions 362, 365 and / or 479 of SEQ ID NO:1 or residues corresponding thereto.

[0026] Suitably, the modified PPO enzyme may comprise an amino acid sequence having at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity or 100% identity with an amino acid sequence of SEQ ID NO: 1, 2, 4 - 151, 153 -302 or 305 - 336, respectively, or a functional fragment thereof.Suitably, the modified PPO enzyme may comprise an amino acid sequence having at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with an amino acid sequence of SEQ ID NO: 1, 2, 4 - 151, 153 - 302, or 305 - 336, respectively, or a functional fragment thereof. Suitably, the modified PPO enzyme may comprise an amino acid sequence having at least 30%, at least 35%, or at least 40% identity with an amino acid sequence of SEQ ID NO: 1, 2, 4 - 151, 153 - 302, or 305 - 336, respectively. Petition 870250095065, dated 10 / 17 / 2025, page 23 / 375 9 / 346 at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity, or 100% identity with an amino acid sequence of SEQ ID NO: 37- 39, 58, 59, 97 - 99, 118, 119, 125 - 137, 139 - 151, 188 - 190, 209, 210, 248 - 250, 269, 270, 277 288 or 291 - 302, respectively, or a functional fragment thereof. Appropriately, the modified PPO enzyme may comprise an amino acid sequence having at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with an amino acid sequence of SEQ ID NO: 37-39, 58, 59, 97-99, 118, 119, 125-137, 139-151, 188-190, 209, 210, 248-250, 269, 270, 277-288 or 291-302, respectively, or a functional fragment thereof.

[0027] Suitably, the modified PPO enzyme may comprise an amino acid sequence having at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity or 100% identity with an amino acid sequence of SEQ ID NO: 58, 59, 126, 127, 130, 131, 132, 134, 136, 137, 209, 210, 277, 278, 281, 282, 283, 285, 287, 288, 118, 119, 140, 141, 144, 145, 146, 148, 150, 151, 269, 270, 291, 292, 295, 296, Petition 870250095065, dated 10 / 17 / 2025, page 24 / 375 10 / 346 297, 299, 301, and 302, respectively, or a functional fragment thereof. Appropriately, the modified PPO enzyme may comprise an amino acid sequence having at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity with an amino acid sequence of SEQ ID NO: 58, 59, 126, 127, 130, 131, 132, 134, 136, 137, 209, 210, 277, 278, 281, 282, 283, 285, 287, 288, 118, 119, 140, 141, 144, 145, 146, 148, 150, 151, 269, 270, 291, 292, 295, 296, 297, 299, 301, and 302, respectively, or a functional fragment thereof.

[0028] Suitably, the modified PPO enzyme may comprise an amino acid sequence having at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity or 100% identity with an amino acid sequence of SEQ ID NO: 125, 139, 276, 290, 126, 131, 134, 140, 145, 148, 277, 282, 285, 291, 296 or 299, respectively, or a functional fragment thereof.Appropriately, the modified PPO enzyme may comprise an amino acid sequence having at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity with an amino acid sequence of SEQ ID NO: 125, 139, 276, 290, 126, 131, 134, 140, 145, 148, 277, 282. Petition 870250095065, dated 10 / 17 / 2025, p. 25 / 375 11 / 346 285, 291, 296, and 299, respectively, or a functional fragment thereof.

[0029] Appropriately, the modified PPO enzyme may comprise an amino acid sequence having at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity or 100% identity with an amino acid sequence of SEQ ID NO: 125, 126, 127, 131, 132, 134, 276, 277, 278, 282, 283, 285, 139, 140, 141, 145, 146, 148, 290, 291, 292, 296, 297, or 299, respectively, or a functional fragment thereof.Suitablely, the modified PPO enzyme may comprise an amino acid sequence having at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with an amino acid sequence of SEQ ID NO: 125, 126, 127, 131, 132, 134, 276, 277, 278, 282, 283, 285, 139, 140, 141, 145, 146, 148, 290, 291, 292, 296, 297, or 299, respectively, or a functional fragment thereof. Appropriately, the modified PPO enzyme may comprise an amino acid sequence having at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity. Petition 870250095065, dated 10 / 17 / 2025, p. 26 / 375 12 / 346 or 100% identity with an amino acid sequence of SEQ ID NO: 125, 131, 134, 139, 145, 148, 276, 282, 285, 290, 296 or 299, respectively, or a functional fragment thereof. Suitablely, the modified PPO enzyme may comprise an amino acid sequence having at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with an amino acid sequence of SEQ ID NO: 125, 131, 134, 139, 145, 148, 276, 282, 285, 290, 296, or 299, respectively, or a functional fragment thereof.

[0030] Suitably, the modified PPO enzyme may comprise an amino acid sequence having at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity or 100% identity with an amino acid sequence of SEQ ID NO: 125, 139, 276 or 290, respectively, or a functional fragment thereof. Appropriately, the modified PPO enzyme may comprise an amino acid sequence having at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with an amino acid sequence of SEQ ID NO: 125, 139, 276, or 290, respectively, or a functional fragment thereof. Petition 870250095065, dated 10 / 17 / 2025, p. 27 / 375 13 / 346

[0031] Suitably, the modified PPO enzyme may comprise an amino acid sequence having at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity or 100% identity with an amino acid sequence of SEQ ID NO: 131, 145, 282 or 296, respectively, or a functional fragment thereof. Appropriately, the modified PPO enzyme may comprise an amino acid sequence having at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with an amino acid sequence of SEQ ID NO: 131, 145, 282, or 296, respectively, or a functional fragment thereof.

[0032] Suitably, the modified PPO enzyme may comprise an amino acid sequence having at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity or 100% identity with an amino acid sequence of SEQ ID NO: 126, 140, 277 or 291, respectively, or a functional fragment thereof. Suitably, the modified PPO enzyme may comprise an amino acid sequence having at least Petition 870250095065, dated 10 / 17 / 2025, page 28 / 375 14 / 346 less than 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity with an amino acid sequence of SEQ ID NO: 126, 140, 277 or 291, respectively, or a functional fragment thereof.

[0033] Suitably, the modified PPO enzyme may comprise an amino acid sequence having at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity or 100% identity with an amino acid sequence of SEQ ID NO: 134, 148, 285 or 299, respectively, or a functional fragment thereof. Appropriately, the modified PPO enzyme may comprise an amino acid sequence having at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with an amino acid sequence of SEQ ID NO: 134, 148, 285, or 299, respectively, or a functional fragment thereof.

[0034] Appropriately, the modified PPO enzyme may comprise an amino acid sequence having at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, by Petition 870250095065, dated 10 / 17 / 2025, page 29 / 375 15 / 346 less than 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity or 100% identity with an amino acid sequence of SEQ ID NO: 132, 283, 146 or 297, respectively, or a functional fragment thereof. Suitably, the modified PPO enzyme may comprise an amino acid sequence having at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity with an amino acid sequence of SEQ ID NO: 132, 283, 146 or 297, respectively, or a functional fragment thereof.

[0035] Suitably, the modified PPO enzyme may comprise an amino acid sequence having at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity or 100% identity with an amino acid sequence of SEQ ID NO: 127, 278, 141 or 292, respectively, or a functional fragment thereof. Suitablely, the modified PPO enzyme may comprise an amino acid sequence having at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with an amino acid sequence of SEQ ID NO: 127, 278, 141, or 292, respectively, or a functional fragment thereof. Petition 870250095065, dated 10 / 17 / 2025, p. 30 / 375 16 / 346

[0036] In one embodiment, the modified PPO enzyme may be derived from Arabidopsis thaliana. In another embodiment, the modified PPO enzyme may be derived from Setaria italica.

[0037] A modified PPO enzyme of the invention may comprise an amino acid sequence according to SEQ ID NO:1 (PPO from Arabidopsis thaliana) wherein positions 362, 365 and / or 479 or residues corresponding thereto have been modified.

[0038] A modified PPO enzyme of the invention may comprise an amino acid sequence according to SEQ ID NO: 2 (PPO from Setaria italica) wherein positions 360, 363 and / or 477 or residues corresponding thereto have been modified. Positions 360, 363 and 477 correspond respectively to positions 362, 365 and 479 of SEQ ID NO: 1.

[0039] A modified PPO enzyme of the invention may comprise an amino acid sequence according to SEQ ID NO:1 (PPO from Arabidopsis thaliana) wherein positions 305 and / or 426 or residues corresponding thereto have been modified suitably to be leucine and valine, respectively.

[0040] A modified PPO enzyme of the invention may comprise an amino acid sequence according to SEQ ID NO:2 (PPO from Setaria italica) wherein positions 303 and / or 424 or residues corresponding thereto have been modified suitably to be leucine and valine, respectively.

[0041] For example, a modified PPO enzyme of the invention may comprise an amino acid sequence according to SEQ ID NO:1 (PPO from Arabidopsis thaliana) wherein position 362 has been modified.

[0042] For example, a modified PPO enzyme of the invention may comprise an amino acid sequence according to Petition 870250095065, dated 10 / 17 / 2025, p. 31 / 375 17 / 346 a SEQ ID NO:1 (PPO of Arabidopsis thaliana) in which position 365 was modified.

[0043] For example, a modified PPO enzyme of the invention may comprise an amino acid sequence according to SEQ ID NO:1 (PPO from Arabidopsis thaliana) wherein position 479 has been modified.

[0044] For example, a modified PPO enzyme of the invention may comprise an amino acid sequence according to SEQ ID NO:2 (PPO from Setaria italica) wherein position 360 has been modified.

[0045] For example, a modified PPO enzyme of the invention may comprise an amino acid sequence according to SEQ ID NO:2 (PPO from Setaria italica) wherein position 363 has been modified.

[0046] For example, a modified PPO enzyme of the invention may comprise an amino acid sequence according to SEQ ID NO:2 (PPO from Setaria italica) wherein position 477 has been modified.

[0047] Suitably, the modified PPO enzyme may comprise an amino acid sequence having at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 98%, at least 99% identity with an amino acid sequence of SEQ ID NO: 1, wherein at least one of the positions 362, 365 and 479 thereof, or positions corresponding thereto, has been modified.

[0048] Appropriately, the modified PPO enzyme may comprise an amino acid sequence having at least Petition 870250095065, dated 10 / 17 / 2025, page 32 / 375 18 / 346 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 98%, at least 99% identity with an amino acid sequence of SEQ ID NO: 2, in which at least one of the positions 360, 363 and 477 thereof, or positions corresponding to them, has been modified.

[0049] Appropriately, a modified PPO of the invention may comprise one or more amino acid sequence motifs as presented in Table 27.

[0050] Appropriately, position 362 of SEQ ID NO: 1 or positions corresponding to it may be replaced by a cysteine. In some examples, position 362 of SEQ ID NO: 1 or positions corresponding to it may be replaced by a phenylalanine. Appropriately, position 360 of SEQ ID NO: 2 or positions corresponding to it may be replaced by a cysteine. In some examples, position 360 of SEQ ID NO: 2 or positions corresponding to it may be replaced by a phenylalanine.

[0051] Appropriately, position 365 of SEQ ID NO: 1 or positions corresponding to it may be replaced by a methionine. In some examples, position 365 of SEQ ID NO: 1 or positions corresponding to it may be replaced by a leucine. Appropriately, position 363 of SEQ ID NO: 2 or positions corresponding to it may be replaced by a methionine. In some examples, position 363 of SEQ ID NO: 2 or positions corresponding to it may be replaced by a leucine. Petition 870250095065, dated 10 / 17 / 2025, p. 33 / 375 19 / 346

[0052] Appropriately, position 479 of SEQ ID NO: 1 or positions corresponding to it may be replaced by methionine. In some examples, position 479 of SEQ ID NO: 1 or positions corresponding to it may be replaced by asparagine. Appropriately, position 477 of SEQ ID NO: 2 or positions corresponding to it may be replaced by methionine. In some examples, position 477 of SEQ ID NO: 2 or positions corresponding to it may be replaced by asparagine.

[0053] In some examples, the modified PPOs of the invention may include one or more additional modifications in addition to those detailed above. For example, the modification of any of the amino acid residues 305, 361, 404, 426, 431 and / or 461 of SEQ ID NO: 1 or residues corresponding thereto.

[0054] Appropriately, residue 305 of SEQ ID NO: 1 or a residue corresponding thereto may be replaced by leucine.

[0055] Suitably, residue 361 of SEQ ID NO: 1 or a corresponding residue may be replaced by threonine. Suitably, residue 361 of SEQ ID NO: 1 or a corresponding residue may be replaced by cysteine. Suitably, residue 361 of SEQ ID NO: 1 or a corresponding residue may be replaced by aspartic acid. Suitably, residue 361 of SEQ ID NO: 1 or a corresponding residue may be replaced by glutamine.

[0056] Appropriately, residue 404 of SEQ ID NO: 1 or a residue corresponding thereto may be replaced by alanine. Petition 870250095065, dated 10 / 17 / 2025, p. 34 / 375 20 / 346

[0057] Suitably, residue 426 of SEQ ID NO: 1 or a residue corresponding to it may be replaced by leucine. Suitably, residue 426 of SEQ ID NO: 1 or a residue corresponding to it may be replaced by cysteine. Suitably, residue 426 of SEQ ID NO: 1 or a residue corresponding to it may be replaced by threonine. Suitably, residue 426 of SEQ ID NO: 1 or a residue corresponding to it may be replaced by valine. Suitably, residue 426 of SEQ ID NO: 1 or a residue corresponding to it may be replaced by methionine.

[0058] Suitably, residue 431 of SEQ ID NO: 1 or a residue corresponding to it may be replaced by phenylalanine. Suitably, residue 431 of SEQ ID NO: 1 or a residue corresponding to it may be replaced by alanine. Suitably, residue 431 of SEQ ID NO: 1 or a residue corresponding to it may be replaced by arginine.

[0059] Appropriately, residue 461 of SEQ ID NO: 1 or a residue corresponding to it may be replaced by glutamine.

[0060] For example, the modification of any of the amino acid residues 303, 359, 402, 424, 429 and / or 459 of SEQ ID NO: 2 or residues corresponding to them.

[0061] Appropriately, residue 303 of SEQ ID NO: 2 or a residue corresponding to it may be replaced by leucine.

[0062] Suitably, residue 359 of SEQ ID NO: 2 or a residue corresponding to it may be replaced by threonine. Suitably, residue 359 of SEQ ID NO: 2 or a Petition 870250095065, dated 10 / 17 / 2025, p. 35 / 375 Residue 21 / 346 corresponding to it may be replaced by cysteine. Suitablely, residue 359 of SEQ ID NO: 2 or a residue corresponding to it may be replaced by aspartic acid. Suitablely, residue 359 of SEQ ID NO: 2 or a residue corresponding to it may be replaced by glutamine.

[0063] Appropriately, residue 402 of SEQ ID NO: 2 or a residue corresponding to it can be replaced by alanine.

[0064] Suitably, residue 424 of SEQ ID NO: 2 or a residue corresponding to it may be replaced by leucine. Suitably, residue 424 of SEQ ID NO: 2 or a residue corresponding to it may be replaced by cysteine. Suitably, residue 424 of SEQ ID NO: 2 or a residue corresponding to it may be replaced by threonine. Suitably, residue 424 of SEQ ID NO: 2 or a residue corresponding to it may be replaced by valine. Suitably, residue 424 of SEQ ID NO: 2 or a residue corresponding to it may be replaced by methionine.

[0065] Suitably, residue 429 of SEQ ID NO: 2 or a residue corresponding to it may be replaced by phenylalanine. Suitably, residue 429 of SEQ ID NO: 2 or a residue corresponding to it may be replaced by asparagine.

[0066] Appropriately, residue 459 of SEQ ID NO: 2 or a residue corresponding to it may be replaced by glutamine.

[0067] In some examples, the modified PPO enzyme may include modifications to the positions of amino acid residues. Petition 870250095065, dated 10 / 17 / 2025, p. 36 / 375 22 / 346 365, 426 and 479 of SEQ ID NO: 1 or residues corresponding to them.

[0068] In some examples, the modified PPO enzyme may include modifications at amino acid residue positions 305, 404, 426, 431, and 479 of SEQ ID NO: 1 or residues corresponding to them.

[0069] In some examples, the modified PPO enzyme may include modifications at amino acid residue positions 305 and 365 of SEQ ID NO: 1 or residues corresponding to them.

[0070] In some examples, the modified PPO enzyme may include modifications at amino acid residue positions 305, 362, and 404 of SEQ ID NO: 1 or residues corresponding to them.

[0071] In some examples, the modified PPO enzyme may include modifications at amino acid residue positions 305, 361, 365, 431, and 479 of SEQ ID NO: 1 or residues corresponding to them.

[0072] In some examples, the modified PPO enzyme may include modifications at amino acid residue positions 305, 426, and 479 of SEQ ID NO: 1 or residues corresponding to them.

[0073] In some examples, the modified PPO enzyme may include modifications at the amino acid residue positions 426, 461, and 479 of SEQ ID NO: 1 or residues corresponding to them.

[0074] In some examples, the modified PPO enzyme may include modifications at amino acid residue positions 305, 361, and 365 of SEQ ID NO: 1 or residues corresponding to them. Petition 870250095065, dated 10 / 17 / 2025, p. 37 / 375 23 / 346

[0075] In some examples, the modified PPO enzyme may include modifications at amino acid residue positions 305, 361, 362, 426, and 479 of SEQ ID NO: 1 or residues corresponding to them.

[0076] In some examples, the modified PPO enzyme may include modifications at amino acid residue positions 361 and 365 of SEQ ID NO: 1 or residues corresponding to them.

[0077] In some examples, the modified PPO enzyme may include modifications at amino acid residue positions 365, 404, and 479 of SEQ ID NO: 1 or residues corresponding to them.

[0078] In some examples, the modified PPO enzyme may include modifications at amino acid residue positions 305, 404, and 479 of SEQ ID NO: 1 or residues corresponding to them.

[0079] In some examples, the modified PPO enzyme may include modifications at amino acid residue positions 305 and 426 of SEQ ID NO: 1 or residues corresponding to them.

[0080] In some examples, the modified PPO enzyme may include modifications at amino acid residue positions 363, 424, and 477 of SEQ ID NO: 2 or residues corresponding to them.

[0081] In some examples, the modified PPO enzyme may include modifications at amino acid residue positions 303, 402, 424, 429, and 477 of SEQ ID NO: 2 or residues corresponding to them.

[0082] In some examples, the modified PPO enzyme may include modifications to the positions of amino acid residues. Petition 870250095065, dated 10 / 17 / 2025, p. 38 / 375 24 / 346 303 and 363 of SEQ ID NO: 2 or residues corresponding to them.

[0083] In some examples, the modified PPO enzyme may include modifications at amino acid residue positions 303, 360, and 402 of SEQ ID NO: 2 or residues corresponding to them.

[0084] In some examples, the modified PPO enzyme may include modifications at the amino acid residue positions 303, 359, 363, 429, and 477 of SEQ ID NO: 2 or residues corresponding to them.

[0085] In some examples, the modified PPO enzyme may include modifications at amino acid residue positions 303, 424, and 477 of SEQ ID NO: 2 or residues corresponding to them.

[0086] In some examples, the modified PPO enzyme may include modifications at the amino acid residue positions 424, 459, and 477 of SEQ ID NO: 2 or residues corresponding to them.

[0087] In some examples, the modified PPO enzyme may include modifications at amino acid residue positions 303, 359, and 363 of SEQ ID NO: 2 or residues corresponding to them.

[0088] In some examples, the modified PPO enzyme may include modifications at amino acid residue positions 303, 359, 360, 424, and 477 of SEQ ID NO: 2 or residues corresponding to them.

[0089] In some examples, the modified PPO enzyme may include modifications at amino acid residue positions 359 and 363 of SEQ ID NO: 2 or residues corresponding to them. Petition 870250095065, dated 10 / 17 / 2025, p. 39 / 375 25 / 346

[0090] In some examples, the modified PPO enzyme may include modifications at amino acid residue positions 363, 402, and 477 of SEQ ID NO: 2 or residues corresponding to them.

[0091] In some examples, the modified PPO enzyme may include modifications at the amino acid residue positions 303, 402, and 477 of SEQ ID NO: 2 or residues corresponding to them.

[0092] In some examples, the modified PPO enzyme may include modifications at amino acid residue positions 303 and 424 of SEQ ID NO: 2 or residues corresponding to them.

[0093] Appropriately, the modified PPO may have an amino acid sequence having at least 30% sequence identity with SEQ ID NO: 37 (where the amino acid sequence includes the Y362F substitution compared to SEQ ID NO:1). Appropriately, the modified PPO may have an amino acid sequence having at least 50% sequence identity with SEQ ID NO: 37. Appropriately, the modified PPO may have an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 37. Appropriately, the modified PPO may comprise an amino acid sequence according to SEQ ID NO: 37. Appropriately, the modified PPO may consist essentially of an amino acid sequence according to SEQ ID NO: 37.

[0094] Appropriately, the modified PPO may have an amino acid sequence having at least 30% sequence identity with SEQ ID NO: 38 (where the sequence of Petition 870250095065, dated 10 / 17 / 2025, p. 40 / 375 26 / 346 amino acids includes the V365L substitution compared to SEQ ID NO:1). Suitably, the modified PPO may have an amino acid sequence having at least 50% sequence identity with SEQ ID NO: 38. Suitably, the modified PPO may have an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 38. Suitably, the modified PPO may comprise an amino acid sequence according to SEQ ID NO: 38. Suitably, the modified PPO may consist essentially of an amino acid sequence according to SEQ ID NO: 38.

[0095] Appropriately, the modified PPO may have an amino acid sequence having at least 30% sequence identity with SEQ ID NO: 39 (where the amino acid sequence includes the V365M substitution compared to SEQ ID NO:1). Appropriately, the modified PPO may have an amino acid sequence having at least 50% sequence identity with SEQ ID NO: 39. Appropriately, the modified PPO may have an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 39. Appropriately, the modified PPO may comprise an amino acid sequence according to SEQ ID NO: 39. Appropriately, the modified PPO may consist essentially of an amino acid sequence according to SEQ ID NO: 39.

[0096] Appropriately, the modified PPO may have an amino acid sequence having at least 30% sequence identity with SEQ ID NO: 58 (where the sequence of Petition 870250095065, dated 10 / 17 / 2025, p. 41 / 375 27 / 346 amino acids includes the L479M substitution compared to SEQ ID NO:1). Appropriately, the modified PPO may have an amino acid sequence having at least 50% sequence identity with SEQ ID NO: 58. Appropriately, the modified PPO may have an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 58. Appropriately, the modified PPO may comprise an amino acid sequence according to SEQ ID NO: 58. Appropriately, the modified PPO may consist essentially of an amino acid sequence according to SEQ ID NO: 58.

[0097] Appropriately, the modified PPO may have an amino acid sequence having at least 30% sequence identity with SEQ ID NO: 59 (where the amino acid sequence includes the L479N substitution compared to SEQ ID NO:1). Appropriately, the modified PPO may have an amino acid sequence having at least 50% sequence identity with SEQ ID NO: 59. Appropriately, the modified PPO may have an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 59. Appropriately, the modified PPO may comprise an amino acid sequence according to SEQ ID NO: 59. Appropriately, the modified PPO may consist essentially of an amino acid sequence according to SEQ ID NO: 59.

[0098] Appropriately, the modified PPO may have an amino acid sequence having at least 30% sequence identity with SEQ ID NO: 97 (where the sequence of Petition 870250095065, dated 10 / 17 / 2025, p. 42 / 375 28 / 346 amino acids includes the Y360F substitution compared to SEQ ID NO:2). Suitably, the modified PPO may have an amino acid sequence having at least 50% sequence identity with SEQ ID NO: 97. Suitably, the modified PPO may have an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 97. Suitably, the modified PPO may comprise an amino acid sequence according to SEQ ID NO: 97. Suitably, the modified PPO may consist essentially of an amino acid sequence according to SEQ ID NO: 97.

[0099] Appropriately, the modified PPO may have an amino acid sequence having at least 30% sequence identity with SEQ ID NO: 98 (where the amino acid sequence includes the V363L substitution compared to SEQ ID NO:2). Appropriately, the modified PPO may have an amino acid sequence having at least 50% sequence identity with SEQ ID NO: 98. Appropriately, the modified PPO may have an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 98. Appropriately, the modified PPO may comprise an amino acid sequence according to SEQ ID NO: 98. Appropriately, the modified PPO may consist essentially of an amino acid sequence according to SEQ ID NO: 98.

[0100] Appropriately, the modified PPO may have an amino acid sequence having at least 30% sequence identity with SEQ ID NO: 99 (where the sequence of Petition 870250095065, dated 10 / 17 / 2025, p. 43 / 375 29 / 346 amino acids includes the V363M substitution compared to SEQ ID NO:2). Appropriately, the modified PPO may have an amino acid sequence having at least 50% sequence identity with SEQ ID NO: 99. Appropriately, the modified PPO may have an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 99. Appropriately, the modified PPO may comprise an amino acid sequence according to SEQ ID NO: 99. Appropriately, the modified PPO may consist essentially of an amino acid sequence according to SEQ ID NO: 99.

[0101] Appropriately, the modified PPO may have an amino acid sequence having at least 30% sequence identity with SEQ ID NO: 118 (where the amino acid sequence includes the V477M substitution compared to SEQ ID NO:2). Appropriately, the modified PPO may have an amino acid sequence having at least 50% sequence identity with SEQ ID NO: 118. Appropriately, the modified PPO may have an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 118. Appropriately, the modified PPO may comprise an amino acid sequence according to SEQ ID NO: 118. Appropriately, the modified PPO may consist essentially of an amino acid sequence according to SEQ ID NO: 118.

[0102] Appropriately, the modified PPO may have an amino acid sequence having at least 30% sequence identity with SEQ ID NO: 119 (where the sequence of Petition 870250095065, dated 10 / 17 / 2025, p. 44 / 375 30 / 346 amino acids includes the V477N substitution compared to SEQ ID NO:2). Suitably, the modified PPO may have an amino acid sequence having at least 50% sequence identity with SEQ ID NO: 119. Suitably, the modified PPO may have an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 119. Suitably, the modified PPO may comprise an amino acid sequence according to SEQ ID NO: 119. Suitably, the modified PPO may consist essentially of an amino acid sequence according to SEQ ID NO: 119.

[0103] Appropriately, the modified PPO may have an amino acid sequence having at least 30% sequence identity with SEQ ID NO: 125 (where the amino acid sequence includes S305L and Y426V substitutions compared to SEQ ID NO:1). Appropriately, the modified PPO may have an amino acid sequence having at least 50% sequence identity with SEQ ID NO: 125. Appropriately, the modified PPO may have an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 125. Appropriately, the modified PPO may comprise an amino acid sequence according to SEQ ID NO: 125. Appropriately, the modified PPO may consist essentially of an amino acid sequence according to SEQ ID NO: 125.

[0104] Appropriately, the modified PPO may have an amino acid sequence having at least 30% sequence identity with SEQ ID NO: 126 (where the sequence of Petition 870250095065, dated 10 / 17 / 2025, p. 45 / 375 31 / 346 amino acids includes the V365L, Y426V, and L479N substitutions compared to SEQ ID NO:1). Suitably, the modified PPO may have an amino acid sequence having at least 50% sequence identity with SEQ ID NO: 126. Suitably, the modified PPO may have an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 126. Suitably, the modified PPO may comprise an amino acid sequence according to SEQ ID NO: 126. Suitably, the modified PPO may consist essentially of an amino acid sequence according to SEQ ID NO: 126.

[0105] Appropriately, the modified PPO may have an amino acid sequence having at least 30% sequence identity with SEQ ID NO: 127 (where the amino acid sequence includes S305L, G404A, Y426V, T431R and L479N substitutions compared to SEQ ID NO:1). Appropriately, the modified PPO may have an amino acid sequence having at least 50% sequence identity with SEQ ID NO: 127. Appropriately, the modified PPO may have an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 127. Appropriately, the modified PPO may comprise an amino acid sequence according to SEQ ID NO: 127. Appropriately, the modified PPO may consist essentially of an amino acid sequence according to SEQ ID NO: 127.

[0106] Appropriately, the modified PPO may have an amino acid sequence having at least 30% sequence identity with SEQ ID NO: 128 (where the sequence of Petition 870250095065, dated 10 / 17 / 2025, p. 46 / 375 32 / 346 amino acids includes the S305L and V365M substitutions compared to SEQ ID NO:1). Suitably, the modified PPO may have an amino acid sequence having at least 50% sequence identity with SEQ ID NO: 128. Suitably, the modified PPO may have an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 128. Suitably, the modified PPO may comprise an amino acid sequence according to SEQ ID NO: 128. Suitably, the modified PPO may consist essentially of an amino acid sequence according to SEQ ID NO: 128.

[0107] Appropriately, the modified PPO may have an amino acid sequence having at least 30% sequence identity with SEQ ID NO: 129 (where the amino acid sequence includes S305L, Y362F and G404A substitutions compared to SEQ ID NO:1). The modified PPO may suitably have an amino acid sequence having at least 50% sequence identity with SEQ ID NO: 129. The modified PPO may have an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 129. The modified PPO may suitably comprise an amino acid sequence according to SEQ ID NO: 129. The modified PPO may suitably consist essentially of an amino acid sequence according to SEQ ID NO: 129.

[0108] Appropriately, the modified PPO may have an amino acid sequence having at least 30% sequence identity with SEQ ID NO: 130 (where the sequence of Petition 870250095065, dated 10 / 17 / 2025, p. 47 / 375 33 / 346 amino acids includes the substitutions S305L, Y361T, V365L, T431R and L479M compared to SEQ ID NO:1). Appropriately, the modified PPO may have an amino acid sequence having at least 50% sequence identity with SEQ ID NO: 130. Appropriately, the modified PPO may have an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 130. Appropriately, the modified PPO may comprise an amino acid sequence according to SEQ ID NO: 130. Appropriately, the modified PPO may consist essentially of an amino acid sequence according to SEQ ID NO: 130.

[0109] Appropriately, the modified PPO may have an amino acid sequence having at least 30% sequence identity with SEQ ID NO: 131 (where the amino acid sequence includes the S305L, Y426V and L479M substitutions compared to SEQ ID NO:1). Appropriately, the modified PPO may have an amino acid sequence having at least 50% sequence identity with SEQ ID NO: 131. Appropriately, the modified PPO may have an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 131. Appropriately, the modified PPO may comprise an amino acid sequence according to SEQ ID NO: 131. Appropriately, the modified PPO may consist essentially of an amino acid sequence according to SEQ ID NO: 131.

[0110] Appropriately, the modified PPO may have an amino acid sequence having at least 30% sequence identity with SEQ ID NO: 132 (where the sequence of Petition 870250095065, dated 10 / 17 / 2025, p. 48 / 375 34 / 346 amino acids includes the Y426M, T461Q, and L479M substitutions compared to SEQ ID NO:1). Suitably, the modified PPO may have an amino acid sequence having at least 50% sequence identity with SEQ ID NO: 132. Suitably, the modified PPO may have an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 132. Suitably, the modified PPO may comprise an amino acid sequence according to SEQ ID NO: 132. Suitably, the modified PPO may consist essentially of an amino acid sequence according to SEQ ID NO: 132.

[0111] Appropriately, the modified PPO may have an amino acid sequence having at least 30% sequence identity with SEQ ID NO: 133 (where the amino acid sequence includes the S305L, Y361D and V365M substitutions compared to SEQ ID NO:1). Appropriately, the modified PPO may have an amino acid sequence having at least 50% sequence identity with SEQ ID NO: 133. Appropriately, the modified PPO may have an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 133. Appropriately, the modified PPO may comprise an amino acid sequence according to SEQ ID NO: 133. Appropriately, the modified PPO may consist essentially of an amino acid sequence according to SEQ ID NO: 133.

[0112] Appropriately, the modified PPO may have an amino acid sequence having at least 30% sequence identity with SEQ ID NO: 134 (where the sequence of Petition 870250095065, dated 10 / 17 / 2025, p. 49 / 375 35 / 346 amino acids includes the substitutions S305L, Y361C, Y362F, Y426M and L479M compared to SEQ ID NO:1). Appropriately, the modified PPO may have an amino acid sequence having at least 50% sequence identity with SEQ ID NO: 134. Appropriately, the modified PPO may have an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 134. Appropriately, the modified PPO may comprise an amino acid sequence according to SEQ ID NO: 134. Appropriately, the modified PPO may consist essentially of an amino acid sequence according to SEQ ID NO: 134.

[0113] Appropriately, the modified PPO may have an amino acid sequence having at least 30% sequence identity with SEQ ID NO: 135 (where the amino acid sequence includes Y361D and V365L substitutions compared to SEQ ID NO:1). Appropriately, the modified PPO may have an amino acid sequence having at least 50% sequence identity with SEQ ID NO: 135. Appropriately, the modified PPO may have an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 135. Appropriately, the modified PPO may comprise an amino acid sequence according to SEQ ID NO: 135. Appropriately, the modified PPO may consist essentially of an amino acid sequence according to SEQ ID NO: 135.

[0114] Appropriately, the modified PPO may have an amino acid sequence having at least 30% sequence identity with SEQ ID NO: 136 (where the sequence of Petition 870250095065, dated 10 / 17 / 2025, p. 50 / 375 36 / 346 amino acids includes the V365L, G404A, and L479N substitutions compared to SEQ ID NO:1). Suitably, the modified PPO may have an amino acid sequence having at least 50% sequence identity with SEQ ID NO: 136. Suitably, the modified PPO may have an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 136. Suitably, the modified PPO may comprise an amino acid sequence according to SEQ ID NO: 136. Suitably, the modified PPO may consist essentially of an amino acid sequence according to SEQ ID NO: 136.

[0115] Appropriately, the modified PPO may have an amino acid sequence having at least 30% sequence identity with SEQ ID NO: 137 (where the amino acid sequence includes S305L, G404A and L479N substitutions compared to SEQ ID NO:1). Appropriately, the modified PPO may have an amino acid sequence having at least 50% sequence identity with SEQ ID NO: 137. Appropriately, the modified PPO may have an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 137. Appropriately, the modified PPO may comprise an amino acid sequence according to SEQ ID NO: 137. Appropriately, the modified PPO may consist essentially of an amino acid sequence according to SEQ ID NO: 137.

[0116] Appropriately, the modified PPO may have an amino acid sequence having at least 30% sequence identity with SEQ ID NO: 138 (where the sequence of Petition 870250095065, dated 10 / 17 / 2025, p. 51 / 375 37 / 346 amino acids includes T303L and Y424M substitutions compared to SEQ ID NO:2). Suitably, the modified PPO may have an amino acid sequence having at least 50% sequence identity with SEQ ID NO: 138. Suitably, the modified PPO may have an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 138. Suitably, the modified PPO may comprise an amino acid sequence according to SEQ ID NO: 138. Suitably, the modified PPO may consist essentially of an amino acid sequence according to SEQ ID NO: 138.

[0117] Appropriately, the modified PPO may have an amino acid sequence having at least 30% sequence identity with SEQ ID NO: 139 (where the amino acid sequence includes T303L and Y424V substitutions compared to SEQ ID NO:2). Appropriately, the modified PPO may have an amino acid sequence having at least 50% sequence identity with SEQ ID NO: 139. Appropriately, the modified PPO may have an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 139. Appropriately, the modified PPO may comprise an amino acid sequence according to SEQ ID NO: 139. Appropriately, the modified PPO may consist essentially of an amino acid sequence according to SEQ ID NO: 139.

[0118] Appropriately, the modified PPO may have an amino acid sequence having at least 30% sequence identity with SEQ ID NO: 140 (where the sequence of Petition 870250095065, dated 10 / 17 / 2025, p. 52 / 375 38 / 346 amino acids includes the V363L, Y424V, and L477N substitutions compared to SEQ ID NO:2). Suitably, the modified PPO may have an amino acid sequence having at least 50% sequence identity with SEQ ID NO: 140. Suitably, the modified PPO may have an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 140. Suitably, the modified PPO may comprise an amino acid sequence according to SEQ ID NO: 140. Suitably, the modified PPO may consist essentially of an amino acid sequence according to SEQ ID NO: 140.

[0119] Appropriately, the modified PPO may have an amino acid sequence having at least 30% sequence identity with SEQ ID NO: 141 (where the amino acid sequence includes the substitutions T303L, G402A, Y424V, T429R and L477N compared to SEQ ID NO:2). Appropriately, the modified PPO may have an amino acid sequence having at least 50% sequence identity with SEQ ID NO: 141. Appropriately, the modified PPO may have an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 141. Appropriately, the modified PPO may comprise an amino acid sequence according to SEQ ID NO: 141. Appropriately, the modified PPO may consist essentially of an amino acid sequence according to SEQ ID NO: 141.

[0120] Appropriately, the modified PPO may have an amino acid sequence having at least 30% sequence identity with SEQ ID NO: 142 (where the sequence of Petition 870250095065, dated 10 / 17 / 2025, p. 53 / 375 39 / 346 amino acids includes T303L and V363M substitutions compared to SEQ ID NO:2). Suitably, the modified PPO may have an amino acid sequence having at least 50% sequence identity with SEQ ID NO: 142. Suitably, the modified PPO may have an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 142. Suitably, the modified PPO may comprise an amino acid sequence according to SEQ ID NO: 142. Suitably, the modified PPO may consist essentially of an amino acid sequence according to SEQ ID NO: 142.

[0121] Appropriately, the modified PPO may have an amino acid sequence having at least 30% sequence identity with SEQ ID NO: 143 (where the amino acid sequence includes the T303L, Y360F and G402A substitutions compared to SEQ ID NO:2). Appropriately, the modified PPO may have an amino acid sequence having at least 50% sequence identity with SEQ ID NO: 143. Appropriately, the modified PPO may have an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 143. Appropriately, the modified PPO may comprise an amino acid sequence according to SEQ ID NO: 143. Appropriately, the modified PPO may consist essentially of an amino acid sequence according to SEQ ID NO: 143.

[0122] Appropriately, the modified PPO may have an amino acid sequence having at least 30% sequence identity with SEQ ID NO: 144 (where the sequence of Petition 870250095065, dated 10 / 17 / 2025, p. 54 / 375 40 / 346 amino acids includes the substitutions T303L, Y359T, V363L, T429R and L477M compared to SEQ ID NO:2). Appropriately, the modified PPO may have an amino acid sequence having at least 50% sequence identity with SEQ ID NO: 144. Appropriately, the modified PPO may have an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 144. Appropriately, the modified PPO may comprise an amino acid sequence according to SEQ ID NO: 144. Appropriately, the modified PPO may consist essentially of an amino acid sequence according to SEQ ID NO: 144.

[0123] Appropriately, the modified PPO may have an amino acid sequence having at least 30% sequence identity with SEQ ID NO: 145 (where the amino acid sequence includes the T303L, Y424V and L477M substitutions compared to SEQ ID NO:2). Appropriately, the modified PPO may have an amino acid sequence having at least 50% sequence identity with SEQ ID NO: 145. Appropriately, the modified PPO may have an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 145. Appropriately, the modified PPO may comprise an amino acid sequence according to SEQ ID NO: 145. Appropriately, the modified PPO may consist essentially of an amino acid sequence according to SEQ ID NO: 145.

[0124] Appropriately, the modified PPO may have an amino acid sequence having at least 30% sequence identity with SEQ ID NO: 146 (where the sequence of Petition 870250095065, dated 10 / 17 / 2025, p. 55 / 375 41 / 346 amino acids includes the Y424M, V459Q, and L477M substitutions compared to SEQ ID NO:2). Suitably, the modified PPO may have an amino acid sequence having at least 50% sequence identity with SEQ ID NO: 146. Suitably, the modified PPO may have an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 146. Suitably, the modified PPO may comprise an amino acid sequence according to SEQ ID NO: 146. Suitably, the modified PPO may consist essentially of an amino acid sequence according to SEQ ID NO: 146.

[0125] Appropriately, the modified PPO may have an amino acid sequence having at least 30% sequence identity with SEQ ID NO: 147 (where the amino acid sequence includes the T303L, Y359D and V363M substitutions compared to SEQ ID NO:2). Appropriately, the modified PPO may have an amino acid sequence having at least 50% sequence identity with SEQ ID NO: 147. Appropriately, the modified PPO may have an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 147. Appropriately, the modified PPO may comprise an amino acid sequence according to SEQ ID NO: 147. Appropriately, the modified PPO may consist essentially of an amino acid sequence according to SEQ ID NO: 147.

[0126] Appropriately, the modified PPO may have an amino acid sequence having at least 30% sequence identity with SEQ ID NO: 148 (where the sequence of Petition 870250095065, dated 10 / 17 / 2025, p. 56 / 375 42 / 346 amino acids includes the substitutions T303L, Y359C, Y360F, Y424M and L477M compared to SEQ ID N0:2). Appropriately, the modified PPO may have an amino acid sequence having at least 50% sequence identity with SEQ ID NO: 148. Appropriately, the modified PPO may have an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 148. Appropriately, the modified PPO may comprise an amino acid sequence according to SEQ ID NO: 148. Appropriately, the modified PPO may consist essentially of an amino acid sequence according to SEQ ID NO: 148.

[0127] Appropriately, the modified PPO may have an amino acid sequence having at least 30% sequence identity with SEQ ID NO: 149 (where the amino acid sequence includes Y359D and V363L substitutions compared to SEQ ID NO:2). Appropriately, the modified PPO may have an amino acid sequence having at least 50% sequence identity with SEQ ID NO: 149. Appropriately, the modified PPO may have an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 149. Appropriately, the modified PPO may comprise an amino acid sequence according to SEQ ID NO: 149. Appropriately, the modified PPO may consist essentially of an amino acid sequence according to SEQ ID NO: 149.

[0128] Appropriately, the modified PPO may have an amino acid sequence having at least 30% sequence identity with SEQ ID NO: 150 (where the sequence of Petition 870250095065, dated 10 / 17 / 2025, p. 57 / 375 43 / 346 amino acids includes the V363L, G402A, and L477N substitutions compared to SEQ ID NO:2). Suitably, the modified PPO may have an amino acid sequence having at least 50% sequence identity with SEQ ID NO: 150. Suitably, the modified PPO may have an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 150. Suitably, the modified PPO may comprise an amino acid sequence according to SEQ ID NO: 150. Suitably, the modified PPO may consist essentially of an amino acid sequence according to SEQ ID NO: 150.

[0129] Appropriately, the modified PPO may have an amino acid sequence having at least 30% sequence identity with SEQ ID NO: 151 (where the amino acid sequence includes T303L, G402A and L477N substitutions compared to SEQ ID NO:2). Appropriately, the modified PPO may have an amino acid sequence having at least 50% sequence identity with SEQ ID NO: 151. Appropriately, the modified PPO may have an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 151. Appropriately, the modified PPO may comprise an amino acid sequence according to SEQ ID NO: 151. Appropriately, the modified PPO may consist essentially of an amino acid sequence according to SEQ ID NO: 151.

[0130] In any aspect of the invention as described herein, a modified PPO enzyme having resistance to a compound that inhibits the enzymatic activity of PPO, whether partially or Petition 870250095065, dated 10 / 17 / 2025, p. 58 / 375 44 / 346 complete, can include an amino acid that has at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, by less than 98% or at least 99% identity with any of the SEQ IDs NOS 1 or 2 or any modified PPO disclosed here, which may also be referred to as reference sequences.

[0131] In any aspect of the invention, a modified PPO enzyme that has resistance to a compound that inhibits the enzymatic activity of PPO may be a homologue of any of the SEQ ID NOs: 1, 2, 4 - 151, 153 - 302 or 305 - 336. As used herein, homologue refers to a protein that is functionally equivalent, i.e., has the same enzymatic activity as an enzyme having an amino acid sequence according to the SEQ ID NOs 1, 2, 4 - 151, 153 - 302 or 305 - 336 (i.e., acts as a PPO enzyme as defined herein), but may have a limited number of amino acid substitutions, deletions, insertions or additions in the amino acid sequence. Homologous enzymes need not exhibit the same Petition 870250095065, dated 10 / 17 / 2025, p. 59 / 375 45 / 346 level of enzymatic activity. Homologs may have lower sequence identities, for example, at least 20%, at least 25%, at least 30%, at least 35% or at least 40% or more sequence identity with a PPO enzyme identified herein, but are capable of performing the same enzymatic reaction. The invention, therefore, includes any isoforms of PPO enzymes and their mutations as defined herein.

[0132] Identity or percentage identity refers to the degree of sequence variation between two given nucleic acid or amino acid sequences. For sequence comparison, typically one sequence acts as a reference sequence against which the test sequences are compared. When using a sequence comparison algorithm, the test and reference sequences are entered into a computer, subsequence coordinates are assigned if necessary, and sequence algorithm program parameters are assigned. The sequence comparison algorithm then calculates the percentage of sequence identity for the test sequence(s) relative to the reference sequence, based on the assigned program parameters. Optimal sequence alignment for comparison can be conducted, for example, by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2: 482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J.Mol. Biol. 48: 443 (1970), by searching for the Pearson & Lipman similarity method, Proc. Natl. Acad. Sci. USA 85: 2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics. Petition 870250095065, dated 10 / 17 / 2025, page 60 / 375 46 / 346 Computer Group, 575 Science Dr., Madison, WI) or by visual inspection. An example of an algorithm that is suitable for determining sequence identity percentage and sequence similarity is the BLAST algorithm, which is described in Altschul et al., J. Mol. Biol. 215: 403-410 (1990). The software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (on the internet at ncbi.nlm.nih.gov / ). This algorithm involves first identifying high-score sequence pairs (HSPs) by identifying short words of length W in the query sequence that match or satisfy some positive value threshold score T when aligned with a word of the same length in a database sequence. T is referred to as a neighborhood word threshold score (Altschul et al., J. Mol. Biol. 215: 403-410 (1990)).These initial neighborhood word matches act as seeds to initiate searches to find longer HSPs containing the same. The word matches are then extended in both directions along each sequence as much as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a matching residue pair; always > 0) and N (penalty score for non-matching residues; always < 0). For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. The extension of word matches in each direction is stopped when the cumulative alignment score falls by an amount X of its value. Petition 870250095065, dated 10 / 17 / 2025, page 61 / 375 47 / 346 maximum reached, the cumulative score goes to zero or below due to the accumulation of one or more alignments of negative scoring residues, or the end of any sequence is reached. The W, T, and X parameters of the BLAST algorithm determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, an expectation (E) of 10, a cutoff of 100, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word length (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89: 10915 (1989)). In addition to calculating the percentage of sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Natl. Acad. Sci. USA 90: 5873-5787 (1993)).One measure of similarity provided by the BLAST algorithm is the lowest sum probability (P(N)), which provides an indication of the probability that a match between two nucleotide or amino acid sequences would occur by chance. For example, a test nucleic acid sequence is considered similar to a reference sequence if the lowest sum probability in a comparison of the test nucleic acid sequence with the reference nucleic acid sequence is less than about 0.1. In one embodiment, less than about 0.01, and in another embodiment, less than about 0.001.

[0133] Unless otherwise indicated, the identity percentage as used here refers to the value obtained Petition 870250095065, dated 10 / 17 / 2025, p. 62 / 375 48 / 346 using the Needleman and Wunsch algorithm ((1970) J. Mol. Biol. 48: 443-453) implemented in the EMBOSS Needle alignment tool using standard EBLOSUM62 matrix files for protein with standard parameters (Gap Opening = 10, Gap Extension = 0.5, Terminal Gap Penalty = False, Terminal Gap Opening = 10, Terminal Gap Extension = 0.5) or DNafull for nucleic acids with standard parameters (Gap Opening = 10, Gap Extension = 0.5, Terminal Gap Penalty = False, Terminal Gap Opening = 10, Terminal Gap Extension = 0.5); or any equivalent program thereof. EMBOSS Needle is available, p. e.g., from EMBL-EBI as on the following website: ebi.ac.uk / Tools / psa / emboss_needle / and as described in the following publication: The EMBL-EBI search and sequence analysis tools APIs in 2019. Madeira et al. Nucleic Acids Research, June 2019, 47 (Wl): W636-W641.The term equivalent program as used here refers to any sequence comparison program that, for any two sequences in question, generates an alignment having identical matches of nucleotide or amino acid residues and an identical percentage of sequence identity compared to the corresponding alignment generated by EMBOSS Needle.

[0134] A nucleic acid-encoded PPO enzyme or PPO enzyme of the invention may be a functional fragment of a PPO enzyme as described herein. A functional fragment refers to a protein fragment that retains protein function. As such, a functional fragment of a PPO enzyme is a fragment, portion, or part of a PPO protein that is Petition 870250095065, dated 10 / 17 / 2025, p. 63 / 375 49 / 346 capable of catalyzing the 6-electron oxidation of protoporphyrinogen-IX to form protoporphyrin-IX.

[0135] The mutations defined here may be located at a position corresponding to a listed amino acid position in another PPO enzyme. It is possible to compare PPO polypeptides by comparing sequences and locating conserved regions that correspond to the listed amino acid positions. The term equivalent amino acids or corresponding amino acids refers to amino acids in a sequence of interest that correspond to those amino acids in an identified reference sequence; typically, here the reference sequence is SEQ ID NO:1 or 2 for PPO enzymes. An equivalent or corresponding amino acid region can be determined by aligning the amino acid sequences of proteins from different species using an alignment program such as BLAST® or ClustalW. Note that the corresponding positions in a sequence of interest must be determined by comparison with a similar-to-similar reference sequence.If it is desired to determine the corresponding positions in a PPO enzyme lacking a targeting peptide, then the reference PPO sequence must also lack a targeting peptide. Appropriately, in such embodiments, the reference sequence used here may be SEQ ID NO: 1, 2 (with transit peptides), 153 or 154 (without transit peptides). Any amino acid positions listed here in relation to a PPO enzyme sequence comprising a targeting peptide still apply to a PPO enzyme sequence from the same organism without a targeting peptide. Petition 870250095065, dated 10 / 17 / 2025, page 64 / 375 50 / 346

[0136] An amino acid position corresponding to a given SEQ ID NO is determined using Geneious as a global alignment with free-end gaps having the following parameters: Blossum cost matrix 62, gap opening penalty 12, gap extension penalty 3, refinement iterations 2; or an equivalent program of the same.

[0137] Unless otherwise indicated, an amino acid position corresponding to a given SEQ ID NO is determined using the standard EMBOSS Needle parameters: BLOSUM62; GAP OPENING 10, GAP EXTENSION 0.5; END GAP OPENING 10 and END GAP EXTENSION 0.5 or an equivalent program thereof. See Madeira et al. (2022) Nucleic Acids Research, July 1, 2022, 50 (Wl): W276-W279; PMID: 35412617 PMCID: PMC9252731. The term equivalent program as used here refers to any sequence comparison program that, for any two sequences in question, generates an alignment having identical matches of nucleotide or amino acid residues and an identical percentage of sequence identity compared to the matching alignment generated by EMBOSS Needle.

[0138] Mutations can include deletions or substitutions or combinations thereof. For example, mutations can be conservative or non-conservative amino acid substitutions.

[0139] Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains and thus typically involve the substitution of one amino acid in a polypeptide by another. Petition 870250095065, dated 10 / 17 / 2025, p. 65 / 375 51 / 346 amino acids within the same defined amino acid class or a similar defined amino acid class. For example, an amino acid with an aliphatic side chain can be replaced by another aliphatic amino acid, e.g., alanine, valine, leucine, and isoleucine; an amino acid with a hydroxyl side chain can be replaced by another amino acid with a hydroxyl side chain, e.g., serine and threonine; an amino acid having aromatic side chains can be replaced by another amino acid having an aromatic side chain, e.g., phenylalanine, tyrosine, tryptophan, and histidine; an amino acid with a basic side chain can be replaced by another amino acid with a basic side chain, e.g., lysine and arginine; an amino acid with an acidic side chain can be replaced by another amino acid with an acidic side chain, e.g.Aspartic acid or glutamic acid; and a hydrophobic or hydrophilic amino acid can be replaced by another hydrophobic or hydrophilic amino acid, respectively. Exemplary conservative substitutions are provided below: Residue Possible Conservative Substitutions A, L, V, I Other aliphatics (A, L, V, I) Other nonpolar elements (A, L, V, I) G, M) G, M Other nonpolar elements (A, L, V, I) G, M) D, E Other acids (D, E) K, R Other basics (K, R) N, Q, S, T Other polar Η, Y, W, F Other aromatics (Η, Y, W, F) Petition 870250095065, dated 10 / 17 / 2025, p. 66 / 375 52 / 346 Residue Possible Conservative Substitutions C None None

[0140] Nonconservative substitution refers to the replacement of an amino acid in a polypeptide by an amino acid with significantly different side chain properties. Nonconservative substitutions may use amino acids between, rather than within, defined groups and may affect (a) the structure of the peptide backbone in the area of ​​the substitution (e.g., proline for glycine) (b) the charge or hydrophobicity or (c) the bulk of the side chain. As an example, an exemplary nonconservative substitution might be an acidic amino acid replaced by a basic or aliphatic amino acid; an aromatic amino acid replaced by a small amino acid; and a hydrophilic amino acid replaced by a hydrophobic amino acid.

[0141] Deletion refers to the modification of a polypeptide by removing one or more amino acids compared to a wild-type or control polypeptide. Deletions may comprise the removal of 1 or more amino acids, 2 or more amino acids, or 3 or more amino acids from the polypeptide, while maintaining enzymatic activity. Deletions may comprise a continuous segment or may be discontinuous.

[0142] Suitably, the modified PPO enzyme may further comprise a transit peptide, suitably a mitochondrial and / or chloroplast transit peptide, suitably at the N- or C-terminus thereof. Suitably, at the N-terminus thereof. In one embodiment, the Petition 870250095065, dated 10 / 17 / 2025, page 67 / 375 53 / 346 Transit peptide is a mitochondrial transit peptide. In one embodiment, the transit peptide is a chloroplast transit peptide. In another embodiment, the transit peptide comprises a chloroplast transit peptide and a mitochondrial transit peptide.

[0143] Suitablely, such mitochondrial or chloroplast transit peptides are present in wild-type PPO enzymes. Suitablely, therefore, the modified PPO enzyme may comprise an endogenous, native mitochondrial or chloroplast transit peptide. Alternatively, the endogenous mitochondrial or chloroplast transit peptide may be replaced by a heterologous mitochondrial or chloroplast transit peptide derived from a different PPO enzyme or enzyme.

[0144] Suitably therefore, in some embodiments, the modified PPO enzyme can be further modified by the addition of a sequence. Suitably by the addition of a sequence to its N or C terminus. Suitably, the sequence is a heterologous transit peptide. Suitably, the heterologous transit peptide is added to the C terminus of the PPO enzyme. Suitably, the heterologous transit peptide is added to the N terminus of the PPO enzyme.

[0145] In one embodiment, therefore, the modified PPO enzyme comprises a heterologous transit peptide, suitably a heterologous mitochondrial or chloroplast transit peptide, suitably at the N-terminus thereof. In one embodiment, the transit peptide is a heterologous mitochondrial transit peptide. In one embodiment, the transit peptide is a heterologous chloroplast transit peptide. Petition 870250095065, dated 10 / 17 / 2025, page 68 / 375 54 / 346 In one embodiment, the transit peptide is a heterologous dual-targeting chloroplast / mitochondrial transit peptide. Suitably, the heterologous transit peptide is added to the modified PPO enzyme, suitably such that the modified PPO enzyme is produced as a fusion protein with the heterologous transit peptide.

[0146] Properly, a heterologous mitochondrial or chloroplast transit peptide is a plant mitochondrial or chloroplast transit peptide. Properly, a heterologous mitochondrial or chloroplast transit peptide may be derived from a heterologous PPO enzyme, properly from a wild-type heterologous PPO enzyme. Properly, a heterologous mitochondrial or chloroplast transit peptide may be derived from a heterologous plant PPO enzyme. Non-limiting examples of transit peptides include those presented in WO2017198859, US10745712, and US10563220, each of which is incorporated by reference in its entirety.

[0147] Suitable transit peptides can be selected from: MELSLLRPTTQSLLPSFSKPNLRLNVYKPLRLRC (SEQ ID NO: 303); or MVAAAMATAPSAGVPPLRGTRGPARFRIRGVSVRC (SEQ ID NO: 304).

[0148] For example, the modified PPO enzyme may comprise a sequence conforming to any of the SEQ ID NOs: 153 to 302 or 321 to 336. Appropriately, the modified PPO enzyme may comprise a sequence having at least 30% sequence identity with a sequence conforming to any of the SEQ ID NOs: 153 to 302 or 321 to 336. For example, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 70%, at least 75%, at least 80%, at least Petition 870250095065, dated 10 / 17 / 2025, p. 69 / 375 55 / 346 less 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 98% or at least 99%. Suitably, such a modified PPO enzyme comprising a sequence according to any of the SEQ ID NOS: 153 to 302 or 321 to 336 may further comprise a transit peptide at the C or N terminus thereof, suitably which may be added thereto. Suitably which may be a mitochondrial transit peptide and / or a chloroplastic transit peptide. Suitably, in any case, which may be a heterologous transit peptide.

[0149] Appropriately, the modified PPO enzyme may comprise both a mitochondrial transit peptide and a chloroplastic transit peptide. For example, a chloroplastic and mitochondrial transit peptide as described herein.

[0150] It will be understood that, when reference is made to specific amino acid positions with reference to a sequence including a transit peptide, these positions will differ by the number of amino acids comprising a transit peptide in a modified PPO enzyme that does not include a transit peptide. Any references herein to sequences including a transit peptide apply equally to the corresponding sequence provided herein without a transit peptide under SEQ IDs NOS 153-302 or 321336.

[0151] For example, positions 362, 365 and 479 of SEQ ID NO: 1 (wild-type Arabidopsis thaliana PPO including naturally occurring transit peptide) would be denoted as positions 328, 331 and 445 of the Petition 870250095065, dated 10 / 17 / 2025, page 70 / 375 56 / 346 SEQ ID NO: 153 (Wild-type Arabidopsis thaliana PPO without transit peptide).

[0152] For example, positions 360, 363 and 477 of SEQ ID NO: 2 (wild-type Setaria Italica PPO including naturally occurring transit peptide) would be denoted respectively as positions 325, 328 and 442 of SEQ ID NO: 154 (wild-type Setaria Italica PPO without transit peptide).

[0153] In some instances, the modified PPO enzyme may comprise one or more additional elements, regions, or domains that act to enhance or assist in the purification, expression, and / or stability of the modified PPO enzyme. For example, the modified PPO enzymes provided herein may include one or more purification tags, detection tags, and / or cleavage sites.

[0154] A detection tag may be an amino acid sequence that can be bound by a specific binding partner or emit a signal to allow the modified PPO enzyme to be detected or visualized. Examples of detection tags include V5 tag, Xpress tag, myc tag, 6XHis, GST, BioEase tag, capTEV tag, fluorescent tags (such as GFP), Lumio tag, HA tag, and FLAG tag.

[0155] A purification tag can be an amino acid sequence that can be used to help purify modified PPO enzymes when produced by recombinant methods. For example, purification tags can allow the modified PPO enzyme to be attached to a resin or spherule via the purification tag. Examples of purification tags include 6XHis, GST, Petition 870250095065, dated 10 / 17 / 2025, p. 71 / 375 57 / 346 BioEase label, capTEV label. For example, the modified PPO enzymes provided here may include a 6XHis label (i.e., a label including 6 histidine residues). Appropriately, the modified PPO enzymes provided here may include a 6XHis label according to SEQ ID NO: 375.

[0156] A cleavage site refers to an amino acid sequence that can be cleaved or cut by a specific cleavage enzyme such as a protease. Cleavage sites can allow for the simplified removal of tags that may be included. Cleavage can be carried out after purification and before the transfer of a recombinant protein to a host. For example, a cleavage site can be placed adjacent to a tag allowing the tag to be cleavaged using a protease enzyme. Examples of cleavage sites include the Tobacco Etch Virus (TEV) site, enterokinase (EK) cleavage site, HRV3C site, and Factor Xa site. Appropriately, the modified PPO enzymes provided herein may include a TEV site. For example, a VTE site according to SEQ ID NO: 376. In some instances, the modified PPO enzymes provided here include a His tag and VTE site as defined in SEQ ID NO: 377 or 378. Combined Strength and / or Additional Traces of Interest

[0157] The plants or parts thereof of the invention may be further modified to comprise an additional trait of interest. In one embodiment, the additional trait of interest increases resistance to a different compound that inhibits a different plant metabolic process. Suitably, the plants or parts thereof of the invention Petition 870250095065, dated 10 / 17 / 2025, page 72 / 375 58 / 346 can be further modified to comprise increased resistance to a compound that inhibits a plant metabolic process other than those involving a PPO enzyme. Appropriately, this can be considered as resistance stacking. Appropriately, resistance to a compound that inhibits PPO enzymes can be stacked with resistance to another compound that inhibits a different enzyme or metabolic pathway in the plants of the invention. Appropriately, therefore, the plant or part thereof exhibits a second compound-resistant trait.

[0158] Suitably, the plants or parts thereof of the invention may be further modified to comprise increased resistance to a compound that inhibits a plant enzyme other than PPO. Suitably, the plants or parts thereof of the invention may be further modified to comprise increased resistance to a compound that inhibits an essential plant metabolic process. By inhibition of a plant metabolic process, this may mean inhibiting one or more enzymes of a plant metabolic process. Suitably, the plants or parts thereof of the invention may be further modified to comprise increased resistance to a compound that is not a compound targeting PPO enzymes, but that targets a different essential plant enzyme or metabolic process.

[0159] Suitably, the plant or part thereof may comprise an additional modified enzyme, suitably modified to increase its resistance to the compound that inhibits a plant enzyme other than PPO. Suitably, such compounds are herbicides. Suitably, Petition 870250095065, dated 10 / 17 / 2025, page 73 / 375 59 / 346 the plant or part thereof may comprise increased resistance to a herbicide that inhibits PPO enzymatic activity and increased resistance to another herbicide, suitably inhibiting a different plant metabolic process. Suitably therefore, the plant or part thereof exhibits a second herbicide-resistant trait.

[0160] The modified PPO enzymes and variants thereof provided herein may therefore be stacked with one or more additional modified enzymes that confer a desirable trait such as, for example, resistance to insects, diseases or herbicides or other desirable agronomic traits of interest including, but not limited to, traits associated with high oil content; traits associated with increased protein content, increased digestibility; balanced amino acid content; improved drought resistance, modified maturity and / or flowering time or high energy content. Such traits may refer to plant tissue properties from both seeds and non-seeds or to food or feed prepared from plants or seeds having such traits.

[0161] As used herein, stacking genes or traits comprises combining desired genes or traits in a transgenic plant line.Stacking can include the introduction of transgenic traits of interest, genome-edited traits of interest, or native traits of interest. The additional polynucleotide can be introduced by a variety of approaches, as described here in relation to polynucleotides encoding modified PPO enzymes, including by transgenic means, by breeding, targeted integration, through genome editing systems, or... Petition 870250095065, dated 10 / 17 / 2025, page 74 / 375 60 / 346 by genome editing. As one approach, plant breeders stack transgenic traits by making crosses between relatives that each have a desired trait and then identifying offspring that have both of these desired traits (called breeding stacks). Another way to stack genes is by transferring two or more genes into the cell nucleus of a plant at the same time during transformation. In embodiments, the two or more genes can be transferred via distinct expression cassettes or via a common expression cassette. Another way to stack genes is to retransform a transgenic plant comprising a desired trait with another gene of interest conferring another desired trait to thereby provide a transgenic plant of the offspring comprising the combination of traits.Such methods may include, for example, random integration techniques or targeted integration through a gene editing system such as CRISPR or meganucleases. For example, gene stacking can be used to combine two different insect resistance traits, two different herbicide resistance traits, two different agronomic performance traits, an insect resistance trait with a disease resistance trait, a herbicide resistance trait (such as, for example, Bt1) or an agronomic performance trait, etc. The use of a selectable marker in addition to a gene of interest would also be considered gene stacking. In embodiments, the progeny or offspring plant having the desired combination of traits is identified through the use of genetic markers or molecular markers including but not limited to. Petition 870250095065, dated 10 / 17 / 2025, page 75 / 375 61 / 346 SNPs, QTLs, primers or probes targeting genes or transgenes associated with desired traits, promoters, microRNAs, siRNAs, mRNAs, dsRNAs, transcriptional profiles, and methylation patterns.

[0162] In some embodiments, a polynucleotide or vector described herein may include an additional coding sequence for one or more polypeptides or double-stranded RNA (dsRNA) molecules of interest for agronomic traits that primarily benefit a seed company, grain producer, or processor. A polypeptide of interest may be any polypeptide encoded by a nucleotide sequence of interest, such as an enzyme. Non-limiting examples of polypeptides of interest that are suitable for production in plants include those resulting in agronomically important traits such as herbicide resistance (also sometimes referred to as herbicide tolerance), virus resistance, bacterial pathogen resistance, insect resistance, nematode resistance, or fungal resistance, such as modified enzymes conferring these traits. See, e.g., U.S. Patents No. 035,569,823; 5,304,730; 5,495,071; 6,329,504; and 6,337,431.A polypeptide can also be one that increases plant vigor or yield (including traits that allow a plant to grow at different temperatures, soil conditions, and levels of sunlight and precipitation) or one that allows the identification of a plant exhibiting a trait of interest (e.g., a selectable marker, seed coat color, relative maturity group, etc.). Several polypeptides of interest, as well as methods for introducing these polypeptides into a plant, are... Petition 870250095065, dated 10 / 17 / 2025, p. 76 / 375 62 / 346 described, for example, in U.S. Patents Nos. 4,761,373; 4,769,061; 4,810,648; 4,940,835; 4,975,374; 5,013,659; 5,162,602; 5,276,268; 5,304,730; 5,495,071; 5,554,798; 5,561,236; 5,569,823; 5,767,366; 5,879,903; 5,928,937; 6,084,155; 6,329,504 and 6,337,431; as well as in U.S. Patent Publication No. 2001 / 0016956.

[0163] Suitably, the additional polypeptide of interest to be stacked with the modified PPO enzyme provided herein may comprise any known polypeptide of interest or an enzyme modified in the art that has been modified to increase resistance to a known compound that inhibits a plant metabolic process. Suitably, such compounds are herbicides. Suitably, therefore, the additional polypeptide of interest or modified enzyme may provide additional herbicide resistance trait, suitably by providing increased herbicide resistance. Suitably, the plant or part thereof may comprise a modified PPO enzyme that provides increased resistance to a PPO-inhibiting herbicide and an additional enzyme that provides increased resistance to another herbicide, suitably inhibiting a different plant metabolic process.

[0164] In some embodiments, the polynucleotides of interest stacked with the modified PPO enzymes provided herein conferring resistance / tolerance to a growth point or meristem-inhibiting herbicide, such as an imidazoline or a sulfonylurea, may be suitable in some embodiments. Exemplary polynucleotides in this category encode mutant ALS and AHAS enzymes as described, p. (e.g., in U.S. Patents) Petition 870250095065, dated 10 / 17 / 2025, p. 77 / 375 63 / 346 U.S. Patent Nos. 5,767,366 and 5,928,937. U.S. Patent Nos. 4,761,373 and 5,013,659 are directed to plants resistant to various imidazoline or sulfonamide herbicides. U.S. Patent No. 4,975,374 relates to plant cells and plants containing a nucleic acid encoding a modified glutamine synthetase (GS) enzyme resistant to inhibition by herbicides known to inhibit GS, e.g., phosphinothricin and methionine sulfoximine. U.S. Patent No. 5,162,602 discloses plants resistant to inhibition by cyclohexanedione and aryloxyphenoxypropanoic acid herbicides. The resistance is conferred by a modified acetyl coenzyme A carboxylase (ACCase) enzyme. Additional herbicide-tolerant traits that confer tolerance to ACCase inhibitors can be found in WO2014144951, WO2017138986, WO2018205995, WO2021088601, WO2011028836 and WO2011028833.

[0165] In some embodiments, the polypeptide of interest stacked with the modified PPO enzymes provided herein conferring glyphosate resistance is also suitable for disclosure. See, e.g., U.S. Patent No. 4,940,835 and U.S. Patent No. 4,769,061. U.S. Patent No. 5,554,798 discloses more transgenic plants resistant to glyphosate, resistance conferred by a modified 5-enolpyruvyl-3-phosphoshikimate (EPSP) synthase gene. Polynucleotides encoding resistance to phosphono compounds such as ammonium glufosinate or phosphinothricin and pyridinoxy- or phenoxypropionic acids and cyclohexones are also suitable. See European Patent Application No. 0 242 246. See also U.S. Patents Nos. 5,879, 903, 5,276, 268 and 5,561,236. Petition 870250095065, dated 10 / 17 / 2025, page 78 / 375 64 / 346

[0166] Additional herbicide-tolerant traits that can be stacked with the modified PPOs include PPO-tolerant traits including, for example, one or more PPO traits presented in US20190062777, US10370677, US11124803, WO2017217793, WO2020251313, US10392630, US10378023, WO2016099153, WO2019117579, WO2019117578 and US10100329, each of which is incorporated herein by reference in its entirety. HPPD-tolerant traits include: WO2009144079, US8642748, EP2453012, WO2013026740, US9078446, US10793872, US10508089, US10400249, US10597674, WO2018119364, WO2018119361, US11180770, US20200157086, US20210147866, US11279944, US202000331866, WO2019227036, WO2019227028, WO2022115296, WO2011068567 and those disclosed in Maeda et al. (2019) Science, vol 365, issue 6451, pp 393-396, each of which is incorporated herein by reference in its entirety.ACCase-tolerant traits include: US20120284812, US20120284853, US20160108423, US20160244780, US20160264990, US20170275645, US20210153448, US10696975B2, US10370678, CN109082416, US10694694, US20170265469, US20170231225, each of which is incorporated herein by reference. Dicamba-tolerant traits include, for example, RE45048 or US7884262. Several traits conferring tolerance to AOPP herbicides, phenoxy acid herbicides, and / or pyridinyloxy acid herbicides include, for example, US10174337, US8278505, WO05107437, WO11022469, US10023874, and US2019241903 (and other traits therein), each of which is incorporated herein by reference. Additional herbicide-tolerant traits of interest for stacking include glucosyl transferase polypeptides as presented in 2018213022 or. Petition 870250095065, dated 10 / 17 / 2025, page 79 / 375 65 / 346 Solanesyl Diphosphate Synthase polypeptides as presented in WO2020236790 or a BIO3-BIO1 and / or BioA enzyme as described in European patent application EP23154964.3, each of which is incorporated herein by reference in its entirety.

[0167] Other suitable polynucleotides include those conferring resistance to herbicides that inhibit photosynthesis, such as a triazine and a benzonitrile (nitrilase). See U.S. Patent No. 4,810,648. Additional suitable polynucleotides encoding herbicide resistance include those encoding resistance to 2,2-dichloropropionic acid, sethoxydim, haloxyfop, imidazolinone herbicides, sulfonylurea herbicides, triazolopyrimidine herbicides, s-triazine herbicides, and bromoxynil.Also suitable are polynucleotides encoding modified enzymes that confer resistance to a protox enzyme or that provide enhanced resistance to plant diseases; enhanced tolerance to adverse environmental conditions (abiotic stress), including but not limited to drought, excessive cold, excessive heat or excessive soil salinity or extreme acidity or alkalinity; and alterations in plant architecture or development, including changes in developmental timing. See, e.g., U.S. Patent Publication No. 2001 / 0016956 and U.S. Patent No. 6,084,155.

[0168] Disease resistance proteins such as enzymes that increase resistance to various plant diseases including rust include, but are not limited to, one or more of the various resistance genes presented in: WO2019103918; WO202100878; WO2021022022; WO2021260673; Petition 870250095065, dated 10 / 17 / 2025, page 80 / 375 66 / 346 WO2022173659; WO2022159341; WO2021154632A1, WO2021022026, WO2021022101, US20220135997; US10842097; or WO2022140257; each of which is incorporated by reference in its entirety. In other embodiments, the modified PPO enzymes described herein or variant or active fragments thereof are stacked with a native trait that confers disease resistance. For example, the various ranges, loci, or resistance genes as presented in WO2009079729, US9091681, WO2010009404, WO2017222827, WO2021000878, WO2021022026, WO2021022101, WO2021154632, WO2022173659 (each of which is incorporated by reference in its entirety) can be used to introduce a trait of interest.Disease resistance proteins and / or native traits that increase resistance to various plant diseases, including NCLB, include, for example, US8921646, US2021000059, US10858668, US20200199610, WO2022 / 013268, WO2022 / 013268, US9040772, US10897862, EP3839073, each of which is incorporated herein by reference.

[0169] Additional suitable polynucleotides include those encoding insecticidal polypeptides such as insecticidal enzymes. These polypeptides can be produced in sufficient quantities to control, for example, insect pests (i.e., insect-controlling quantities). It is recognized that the amount of an insecticidal polypeptide produced in a plant needed to control insects or other pests can vary depending on the cultivar, pest type, environmental factors, and the like. Useful polynucleotides for additional insect or pest resistance include, for example, those encoding toxins identified in Bacillus organisms. Polynucleotides comprising Petition 870250095065, dated 10 / 17 / 2025, page 81 / 375 67 / 346 nucleotide sequences encoding Cry proteins from Bacillus thuringiensis (Bt) of various subspecies were cloned, and recombinant clones were found to be toxic to larvae of lepidopteran, dipteran, and / or coleopteran insects. Examples of such insecticidal Bt proteins include Cry proteins such as CrylAa, CrylAb, CrylAc, CrylB, CrylC, CrylD, CrylEa, CrylFa, Cry3A, Cry9A, Cry9B, Cry9C, and similar proteins, as well as vegetative insecticidal proteins such as Vipl, Vip2, Vip3, and similar proteins. A complete list of Bt-derived proteins can be found online at the Bacillus thuringiensis Toxin Nomenclature Database maintained by the University of Sussex (see also Crickmore et al. (1998) Microbiol. Mol. Biol. Rev. 62: SOTSIS).

[0170] In embodiments, an additional polypeptide is an insecticidal polypeptide, such as an enzyme, derived from a source other than Bt, including, without limitation, an alpha-amylase, a peroxidase, a cholesterol oxidase, a patatin, a protease, a protease inhibitor, a urease, an alpha-amylase inhibitor, a pore-forming protein, a chitinase, a lectin, an antibody or manipulated antibody fragment, an insecticidal protein from Bacillus cereus, an insecticidal protein from Xenorhabdus spp. (such as X. nematophila or X. bovienii), an insecticidal protein from Photorhabdus spp. (such as P. luminescens or P. asymobiotica), an insecticidal protein from Brevibacillus spp. (such as B. laterosporous), an insecticidal protein from Lysinibacillus spp. (such as L. sphearicus), an insecticidal protein from Chromobacterium spp. (such as C. subtsugae or C. piscinae), an insecticidal protein from Yersinia spp. (such Petition 870250095065, dated 10 / 17 / 2025, page 82 / 375 68 / 346 as Y. entomophaga), an insecticidal protein from Paenibacillus spp. (such as P. propylaea), an insecticidal protein from Clostridium spp. (such as C. bifermentans), Pseudomonas spp. (such as P. fluorescens) and a lignin.

[0171] In certain embodiments, the additional polypeptide or enzyme is a resistance protein such as an enzyme conferring enhanced resistance to pathogens, such as enhanced resistance to any of the following pathogens: soybean cyst nematode, bacterial pustule, root nodule nematode, frog-eye leaf spot, Phytophthora, brown stem rot, nematode, Asian soybean rust, sooty mold, Golovinomyces cichoracearum, Erysiphe cichoracearum, Blumeria graminis, Podosphaera xanthii, Sphaerotheca fuliginea, Pythium ultimum, Uncinula necator, Mycosphaerella pinodes, Magnaporthe grisea, Bipolaris oryzae, Magnaporthe grisea, Rhizoctonia solani, Phytophthora sojae, Schizaphis graminum, Bemisia tabaci, Rhopalosiphum maidis, Deroceras reticulatum, Diatraea saccharalis, Schizaphis graminum, Myzus persicae, Sclerotinia sclerotiorum, Macrophomina phaseolina, Fusarium virguliforme.Exemplary polynucleotides encoding proteins that confer enhanced resistance to pathogens that can be stacked with the modified PPO enzymes of the invention include polynucleotides encoding proteins such as enzymes that confer enhanced resistance to ASR as described in U.S. Patent Publication No. 20200354739 and PCT Publications Nos. WO2019103918, WO2021154632A1, WO2021022022, WO2021022026, WO2021022101, WO2021260673 and WO2021263249, each of which is incorporated by reference in its entirety. Petition 870250095065, dated 10 / 17 / 2025, page 83 / 375 69 / 346

[0172] Polypeptides suitable for production in plants additionally include those that improve or otherwise facilitate the conversion of harvested plants or plant parts into a commercially useful product, including, for example, increased or altered carbohydrate content or distribution, improved fermentation properties, increased oil content, increased protein content, improved digestibility, and increased nutraceutical content, e.g., increased phytosterol content, increased tocopherol content, increased stanol content, or increased vitamin content. Polypeptides of interest also include, for example, those resulting in or contributing to a reduced content of an undesirable component in a harvested crop, e.g., phytic acid or sugar-degrading enzymes.By resulting in or contributing to, it is intended that the polypeptide of interest may contribute directly or indirectly to the existence of a trait of interest (e.g., increased cellulose degradation by the use of a heterologous cellulase enzyme). Any such polypeptides may be stacked with the modified PPO enzymes of the invention.

[0173] In some embodiments, the polypeptide contributes to the improved digestibility of food or feed. Xylanases are hemicellulolytic enzymes that improve the degradation of plant cell walls, leading to better utilization of plant nutrients by an animal. This leads to improved growth rate and feed conversion. Similarly, the viscosity of feeds containing xylan can be reduced. Heterologous production of xylanases Petition 870250095065, dated 10 / 17 / 2025, p. 84 / 375 70 / 346 in plant cells can also facilitate lignocellulosic conversion into fermentable sugars in industrial processing. Numerous xylanases from fungal and bacterial microorganisms have been identified and characterized (see, e.g., U.S. Patent No. 5,437,992; Coughlin et al. (1993) Proceedings of the Second TRICEL Symposium on Trichoderma reesei Cellulases and Other Hydrolases Espoo; Souminen and Reinikainen, eds. (1993) Foundation for Biotechnical and Industrial Fermentation Research 8: 125-135; U.S. Patent Publication No. 2005 / 0208178; and PCT Publication No. WO 03 / 16654). In particular, three specific xylanases (XYL-I, XYL-II, and XYL-III) have been identified in T. reesei (Tenkanen et al. (1992) Enzyme Microb. Technol. 14: 566; Torronen et al. (1992) Bio / Technology 10: 1461; and Xu et al. (1998) Appl. Microbiol. Biotechnol. 49: 718). Any such polypeptides can be stacked with the modified PPO enzymes of the invention.

[0174] In other embodiments, a useful polypeptide for dissemination may be a polysaccharide-degrading enzyme. Plants producing such an enzyme in this dissemination may be useful for generating, for example, fermentation feedstocks for bioprocessing. In some embodiments, enzymes useful for a fermentation process include alpha-amylases, proteases, pullulanases, isoamylases, cellulases, hemicellulases, xylanases, cyclodextrin glucotransferases, lipases, phytases, laccases, oxidases, esterases, cutinases, granular starch hydrolysis enzyme, and other glucoamylases. Polysaccharide-degrading enzymes include: starch-degrading enzymes such as α-amylases (EC 3.2.1.1), glucuronidases (EC Petition 870250095065, dated 10 / 17 / 2025, page 85 / 375 71 / 346 3.2.1.131), exo-1,4-aD-glucanases such as amyloglucosidases and glucoamylases (EC 3.2.1.3), β-amylases (EC 3.2.1.2), α-glucosidases (EC 3.2.1.20) and other exoamylases; enzimas de desramificação do starch, tais como: a) isoamylase (EC 3.2.1.68), pululanase (EC 3.2.1.41) e similares, b) celulases tais como exo-1,4-3-celobiohydrolase (EC 3.2.1.91), exo-1,3-β-D-glucanase (EC 3.2.1.39), βglucosidase (EC 3.2.1.21), c) L-arabinases, tais como endo1,5-aL-arabinase (EC 3.2.1.99), α-arabinosidases (EC 3.2.1.55) and congeners; d) galactanases such as endo-1,4β-D-galactanase (EC 3.2.1.89), endo-1,3^-D-galactanase (EC 3.2.1.90), α-galactosidase (EC 3.2.1.22), β-galactosidase (EC 3.2.1.23) and similar; e) mannanases, such as endo-1,4β-D-mannanase (EC 3.2.1.78), β-mannosidase (EC 3.2.1.25), amannosidase (EC 3.2.1.24) and the like, f) xylanases, such as endo-1,4^-xylanase (EC 3.2.1.8), β-D-xylosidase (EC 3.2.1.37), 1,3^-D-xylanase, and the like; and g) other enzymes such as α-L-fucosidase (EC 3.2.1.51), alpha-Lramnosidase (EC 3.2.1.40), levanase (EC 3.2.1.65), inulanase (EC 3.2.1.7) and the like. In one embodiment, the α-amylase is the synthetic α-amylase, Amy797E, described in U.S. Patent No. 8,093,453, which is incorporated herein by reference in its entirety. Any such polypeptides may be stacked with the modified PPO enzymes of the invention.

[0175] Additional enzymes that may be used with the disclosure include proteases, such as fungal and bacterial proteases.Fungal proteases include, but are not limited to, those obtained from Aspergillus, Trichoderma, Mucor, and Rhizopus, such as A. niger, A. awamori, A. oryzae, and M. miehei. In some embodiments, the... Petition 870250095065, dated 10 / 17 / 2025, p. 86 / 375 72 / 346 polypeptides of this disclosure may be cellobiohydrolase (CBH) enzymes (EC 3.2.1.91). In one embodiment, the cellobiohydrolase enzyme may be CBH1 or CBH2. Any such polypeptides may be stacked with the modified PPO enzymes of the invention.

[0176] Other useful enzymes with disclosure include, but are not limited to, hemicellulases such as mannases and arabinofuranosidases (EC 3.2.1.55); ligninases; lipases (e.g., EC 3.1.1.3), glucose oxidases, pectinases, xylanases, transglucosidases, alpha-1,6-glucosidases (e.g., EC 3.2.1.20); esterases such as ferulic acid esterase (EC 3.1.1.73) and acetyl xylan esterases (EC 3.1.1.72); and cutinases (e.g., EC 3.1.1.74). Any such polypeptides can be stacked with the modified PPO enzymes of the invention.

[0177] In other embodiments, the modified PPO enzymes described herein may be stacked with polynucleotides encoding polypeptides that increase protein content and / or alter seed composition and / or fatty acid content. Such sequences include, but are not limited to, sequences disclosed in PCT Application No. PCT / CN2022 / 075977 and PCT Application No. PCT / CN2022 / 075982, both filed on 2 / 11 / 2022, WO2021 / 044027; US2020 / 0131524; and US2021 / 0403933, each of which is incorporated by reference in its entirety.

[0178] In one embodiment, the modified PPO enzymes described herein are stacked with a modified BIO3-BIO1 and / or BioA enzyme and / or a modified biotin synthesis pathway. Appropriately therefore, the plant or part thereof described herein comprises BIO3-BIO1 and / or BioA enzyme. Petition 870250095065, dated 10 / 17 / 2025, p. 87 / 375 73 / 346 modified. In one embodiment, therefore, the additional modified enzyme is a modified BIO3-BIO1 and / or BioA enzyme.

[0179] Suitably therefore, the plant or part thereof may be further modified to comprise a BIO3-BIO1 and / or BioA enzyme that provides the plant or part thereof with increased resistance to a compound that inhibits a BIO3-BIO1 and / or BioA enzyme and / or the biotin synthesis pathway relative to an unmodified plant. Suitably therefore, the plant or part thereof may be modified to comprise a BIO3-BIO1 and / or BioA enzyme having one or more modifications that provide the plant or part thereof with a resistance trait to BIO3-BIO1 and / or BioA, suitably by providing increased resistance to a compound that inhibits biotin synthesis and therefore the BIO3-BIO1 and / or BioA enzyme relative to an unmodified plant. Suitably, the plant or part thereof may comprise a recombinant polynucleotide encoding a BIO3-BIO1 and / or BioA enzyme having one or more modifications.Suitablely, the modification(s) provide the plant with increased resistance to the compound that inhibits the BIO3-BIO1 and / or BioA enzyme compared to an unmodified plant. Suitablely, the modification(s) provide the plant with increased resistance to a herbicide that inhibits the BIO3-BIO1 and / or BioA enzymes and the biotin synthesis pathway. Suitablely, the plant or part thereof may be further modified to comprise a BIO3-BIO1 and / or BioA enzyme as described in European patent application EP23154964.3, which is incorporated herein by reference. Plants or Parts of Plants Petition 870250095065, dated 10 / 17 / 2025, p. 88 / 375 74 / 346

[0180] The plants of the present invention include both non-transgenic and transgenic plants.

[0181] By non-transgenic plant is meant a plant lacking recombinant DNA in its genome, but containing a mutant nucleic acid molecule in the plant cell genome that has been mutated through human intervention using mutagenic techniques, such as chemical mutagenesis, gene editing or by those methods provided herein. Non-transgenic plants may encompass those plants having mutant or modified sequences as a result of natural processes, such as plants including spontaneous PPO enzymes that provide the desired resistance to compounds that inhibit PPO enzymatic activity or by the use of gene editing techniques. In specific embodiments, the non-transgenic plant comprises a modified PPO enzyme that has been altered through gene editing to comprise at least one or more of the modifications disclosed herein. Such gene editing modifications will increase the plant's resistance to the herbicide of interest.

[0182] By transgenic plant is meant a plant comprising recombinant DNA in its genome. As used herein, recombinant, when referring to nucleic acid or polypeptide, indicates that such material was designed and created in the laboratory using one or more of the following techniques of biotechnology, protein design or protein manipulation, such as molecular biology, protein biochemistry, bacterial transformation, plant transformation, site-directed mutagenesis, directed evolution using random mutagenesis, genome editing, gene editing, gene cloning, DNA ligation, DNA synthesis, synthesis of Petition 870250095065, dated 10 / 17 / 2025, page 89 / 375 75 / 346 proteins and DNA scrambling. Including, for example, being altered as a result of the human application of a recombinant technique, such as by restriction and ligation of polynucleotides, by polynucleotide overlap-extension, or by genomic insertion or transformation. An open reading frame of gene sequence is recombinant if that nucleotide sequence has been removed from its natural context and cloned into any type of artificial nucleic acid vector. The term recombinant may also refer to an organism having recombinant material, e.g., a plant comprising recombinant nucleic acid may be considered a recombinant plant. Such a transgenic plant may be produced by introducing recombinant DNA into the plant genome. When such recombinant DNA is incorporated into the genome of the transgenic plant, the plant's offspring may also comprise recombinant DNA.A descendant plant that includes at least a portion of recombinant DNA from at least one transgenic parent plant is also a transgenic plant.

[0183] As used herein, heterologous in reference to a polypeptide or polynucleotide sequence is a sequence that originates, for example, from a cell or organism of a foreign species. Alternatively, if the sequence originates from the same species, it is derived from a cell or organism having a different genetic background; or, if from the same genetic background, it is substantially modified from its native form in composition and / or genomic locus by deliberate human intervention. As such, sequences Petition 870250095065, dated 10 / 17 / 2025, page 90 / 375 76 / 346 heterologous groups are in a configuration not found in nature.

[0184] The term spontaneous mutant refers to mutants or variants that arise from the parental strain without the intentional use of mutagens, i.e., they are considered non-genetically modified (non-GMO). Spontaneous mutants with respect to plants may also be known as sports, breaks or chimeras.

[0185] Suitablely, the plant or part thereof of the invention is transgenic. In other embodiments, the plant or part thereof of the invention is not transgenic and comprises a gene edit that increases the tolerance of the plant or part thereof to a herbicide of interest.

[0186] In one embodiment, therefore, the plant or part thereof comprises a recombinant polynucleotide encoding a modified PPO enzyme. In some embodiments, the plant or part thereof comprises a polynucleotide encoding a modified or mutated PPO enzyme. Suitably, the polynucleotide encoding the modified PPO enzyme may comprise one or more modifications. Suitably, therefore, the polynucleotide may be operable to express a modified PPO enzyme having one or more modifications or mutations. Suitably, in that the expression of said polynucleotide provides or confers upon the plant or part thereof increased resistance to a compound that inhibits the enzymatic activity of PPO. Suitably, one or more modifications in the modified PPO enzyme provide increased resistance to a compound that inhibits the enzymatic activity of PPO. Suitable PPO modifications are defined herein and may include any of the SEQ ID NO: 1, 2, 4-151, 153-302 and Petition 870250095065, dated 10 / 17 / 2025, page 91 / 375 77 / 346 305-336 or active variants thereof as described elsewhere herein. For example, such PPO modifications are defined herein and may include any of the SEQ ID NO: 37-39, 58, 59, 97-99, 118, 119, 125-137, 139-151, 188-190, 209, 210, 248-250, 269, 270, 277-288 or 291-302 or active variants thereof as described elsewhere herein. In specific embodiments, the modified or mutated PPO enzyme is encoded by a recombinant polynucleotide stably integrated into the plant genome. Alternatively, the modified or mutated PPO enzyme is encoded by a polynucleotide, specifically a gene, comprising a non-transgenic modification, such as an edit, within the plant genome.

[0187] In one embodiment, the plant or part thereof may be modified to comprise a modified PPO enzyme wherein the modified PPO enzyme is overexpressed and wherein the modified PPO enzyme comprises one or more mutations provided herein that provide the plant or part thereof with increased resistance to a compound that inhibits PPO enzymatic activity relative to an unmodified or control PPO enzyme or plant. Suitable modified PPOs may include any of the SEQ ID NO: 1, 2, 4-151, 153-302 and 305-336. For example, modified PPOs may include any of the following SEQ ID numbers: 37-39, 58, 59, 97-99, 118, 119, 125-137, 139-151, 188-190, 209, 210, 248-250, 269, 270, 277-288 or 291-302, or active variants thereof as described elsewhere herein.

[0188] In one embodiment, the plant was transformed with the aforementioned recombinant polynucleotide. Suitable transformation methods are described hereafter. Petition 870250095065, dated 10 / 17 / 2025, page 92 / 375 78 / 346

[0189] The transformed plant parts, transformed plant cells or transformed plant protoplasts as described herein can be regenerated to produce a modified plant as described herein.

[0190] When adequate numbers of transformed cells or protoplasts containing a recombinant modified PPO enzyme are obtained, the cells can be cultured and then regenerated into whole plants. Regeneration refers to the process of growing a plant from a plant cell (e.g., protoplasts or plant explants). Such regeneration techniques rely on the manipulation of certain phytohormones in a tissue culture growth medium, typically based on a biocidal and / or herbicidal marker that has been introduced along with the desired nucleotide sequences. The choice of methodology for the regeneration step is not critical. See, for example, Ammirato et al., Handbook of Plant Cell Culture — Crop Species. Macmillan Publ. Co. (1984); Shimamoto et al., Nature 338: 274-276 (1989); Fromm, UCLA Symposium on Molecular Strategies for Crop Improvement, April 1622, 1990. Keystone, Colo. (1990); Vasil et al., Bio / Technology 8: 429-434 (1990); Vasil et al., Bio / Technology 10: 667-674 (1992); Hayashimoto, Plant Physiol. 93: 857-863 (1990); and Datta et al., Bio-technology 8: 736-740 (1990). Such regeneration techniques are generally described in Klee et al., Ann. Rev. Plant Phys. 38: 467-486 (1987).

[0191] The present invention can be used with any plant species and its offspring, including, but not limited to, monocotyledons and dicotyledons. Petition 870250095065, dated 10 / 17 / 2025, p. 93 / 375 79 / 346

[0192] Examples of plant species of interest include, but are not limited to, maize or more (Zea mays), Brassica sp. (e.g., B. napus, B. rapa, B. juncea), including 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 annuus), safflower (Carthamus tinctorius), wheat (Triticum aestivum, T. turgidum ssp. durum), soybean (Glycine max), tobacco (Nicotiana taba cum), 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 (Primus amygdalus), sugar beet (Beta vulgaris), sugarcane (Saccharum spp.), oats (Avena sativa), barley (Hordeum vulgare), legumes and vegetables, ornamental plants and conifers.

[0193] In one embodiment, the plants of the present invention are crop plants (e.g., sunflower, Brassica sp., cotton, sugar, beet, soybean, peanut, alfalfa, safflower, tobacco, Petition 870250095065, dated 10 / 17 / 2025, p. 94 / 375 80 / 346 corn, rice, wheat, rye, barley, triticale, sorghum, millet, etc.).

[0194] In another embodiment, the plants of the present invention may also include various types of cover crops. Exemplary cover crops include, but are not limited to, Brassica sp. (e.g., B. carinata, B. napus, B. rapa, B. hirta, B. juncea, B. nigra), radish (Raphanus sativus), Camelina sp. (e.g., C. sativa), watercress (Thlaspi arvense), clover (Trifolium sp., e.g., T. encarnatum, T. pratense, T. repens, T. subterranean), field peas (Pisum sativum), Vicia sp. (e.g., V. villosa, V. lutea, V. nigricans, V. sativa), rye (Secale cereale), barley (Hordeum vulgare), winter wheat (Triticum aestivum), oats (Avena sativa), annual ryegrass (Lolium multiflorum), buckwheat (Fagopyrum esculentum), Sinapsis alba, alfalfa (Medicago sativa).

[0195] As used herein, the terms offspring and offspring plant refer to a plant generated from vegetative or sexual reproduction of one or more parent plants. An offspring plant may be obtained by cloning or self-fertilization of a single parent plant or by crossing two parent plants.

[0196] As used herein unless clearly indicated otherwise, the term plant is intended to mean a plant at any stage of development, as well as any part or parts of a plant that may be attached to or separate from an intact whole plant. Such parts of a plant include, but are not limited to, plant organs, tissues and cells, including plant calluses, plant clumps, plant protoplasts and plant cultures. Petition 870250095065, dated 10 / 17 / 2025, page 95 / 375 81 / 346 plant cell tissues from which plants can be regenerated. Examples of particular plant parts include a stem, a leaf, a root, an inflorescence, a flower, a floret, a fruit, a pedicel, a peduncle, a stamen, an anther, a stigma, a style, an ovary, a petal, a sepal, a carpel, a root tip, a root cap, a root hair, a leaf hair, a seed hair, a pollen grain, a microspore, an embryo, an ovule, a cotyledon, a hypocotyl, an epicotyl, xylem, phloem, parenchyma, endosperm, a companion cell, a guard cell, and any other known organs, tissues, and cells of a plant. Furthermore, a seed is recognized as a plant part.

[0197] A plant cell is a structural and physiological unit of a plant, comprising a protoplast and a cell wall. The plant cell may be in the form of a single isolated cell or a cultured cell or as part of a higher organizational unit such as, for example, plant tissue, a plant organ or an entire plant. A plant part is a distinct and visibly structured and differentiated part of a plant such as a root, stem, leaf, flower bud or embryo.

[0198] The plants, offspring thereof, or parts thereof of the invention express at least one of a modified PPO enzyme as disclosed herein. The expression of the enzymes, polynucleotides encoding said enzymes, or expression vectors of the invention provides a plant that is at least partially resistant to compounds that inhibit the enzymatic activity of PPO, such as those herbicides described herein. For example, the plants, offspring of Petition 870250095065, dated 10 / 17 / 2025, page 96 / 375 82 / 346 The same or parts thereof of the invention have increased resistance to a herbicide that inhibits the enzymatic activity of PPO. The increase in resistance can be determined by comparison with a wild-type or control plant as described herein. For example, a plant that has not been modified to include or express the modified PPO enzymes, polynucleotide encoding the same, or expression vectors of the invention. Methods of producing modified plants or increasing the resistance of a plant.

[0199] Methods of conferring enhanced resistance are provided herein to compounds that inhibit PPO enzymes (i.e., PPO enzymatic activity) to a plant or part thereof by modifying the plant to comprise a modified PPO enzyme that confers said resistance. Appropriately, such methods may include (i) a method of producing a modified plant or part thereof having enhanced resistance to a compound that inhibits PPO enzymatic activity and (ii) a method of increasing the resistance of a plant or part thereof to a compound that inhibits PPO enzymatic activity.

[0200] Appropriately, either method comprises a step of modifying the plant or part thereof to comprise a modified PPO enzyme that provides increased resistance.

[0201] Properly modifying the plant may involve providing the plant or part thereof with a modified PPO enzyme having one or more modifications, wherein the modification or modifications provide increased resistance. Petition 870250095065, dated 10 / 17 / 2025, page 97 / 375 83 / 346

[0202] Appropriately, such steps may involve providing the plant or part thereof with a recombinant polynucleotide encoding a modified PPO enzyme as described herein.

[0203] Appropriately, providing may comprise introducing the recombinant polynucleotide encoding a modified PPO enzyme as provided herein into the plant or part thereof, or introducing the modified PPO protein into the plant or part thereof. Appropriately, the modified PPO enzyme may be introduced into a plant or part thereof by introducing a polynucleotide of the invention encoding a modified PPO enzyme. Thus, the plants of the invention may be referred to as modified or transgenic plants.

[0204] Appropriately, introducing the modified PPO enzyme into a plant or part thereof can be carried out by transforming the plant or part thereof with a recombinant polynucleotide encoding a modified PPO enzyme.

[0205] Suitably, the recombinant polynucleotide may additionally encode a transit peptide, such as a mitochondrial or chloroplast transit peptide. Suitably as a fusion with the modified PPO enzyme. Suitably therefore, the recombinant polynucleotide may comprise a chimeric polynucleotide encoding a modified PPO as described above and a transit peptide operationally linked thereto, suitably a mitochondrial or chloroplast transit peptide operationally linked thereto. Suitable transit peptides are described elsewhere herein. Petition 870250095065, dated 10 / 17 / 2025, page 98 / 375 84 / 346

[0206] Suitably, the recombinant polynucleotide may be part of an expression construct or be comprised in an expression vector. Suitably, the expression construct or vector may comprise one or more expression elements such as a promoter, as described elsewhere herein. Suitably, therefore, the methods may comprise providing, introducing or transforming the plant or part thereof with an expression construct or expression vector comprising a polynucleotide encoding a modified PPO enzyme, suitably which may be a recombinant polynucleotide.

[0207] Suitably, the methods may further comprise a step of inducing the expression of the recombinant polynucleotide to produce the modified PPO enzyme provided herein in the plant or part thereof. Alternatively, the expression of the polynucleotide may comprise contacting the plant or part thereof with an inducer. Suitably in such embodiments, the polynucleotide encoding the modified PPO enzyme may be under the control of an inducible promoter, suitably therefore the expression construct or vector may comprise an inducible promoter operationally linked to the polynucleotide encoding the modified PPO enzyme. Suitable inducible promoters and inducers are well known in the art.

[0208] Alternatively, a plant can be modified by in situ editing of endogenous genetic material in order to provide a gene that expresses a modified PPO enzyme that provides increased resistance to compounds that inhibit PPO enzymatic activity. Petition 870250095065, dated 10 / 17 / 2025, page 99 / 375 85 / 346

[0209] Suitablely in such embodiments, a plant may be provided with the components of a gene editing system to modify an endogenous plant gene sequence encoding PPO enzyme at one or more positions to produce a modified gene sequence encoding a PPO enzyme that provides increased resistance to a compound that inhibits PPO enzymatic activity. Suitablely, the plant may be transformed with one or more polynucleotides encoding a gene editing system to modify an endogenous plant gene sequence encoding PPO enzyme at one or more positions to produce a modified gene sequence encoding a modified PPO enzyme that provides increased resistance to a compound that inhibits PPO enzymatic activity. Thus, providing a plant or part thereof that expresses a modified PPO enzyme that provides increased resistance to a compound that inhibits PPO enzymatic activity.

[0210] A gene sequence encoding endogenous PPO can be edited in situ by means of gene editing techniques in order to provide a modified PPO enzyme that is at least partially resistant to a compound that inhibits PPO enzymatic activity, such as those described herein, and as such a modified plant as described herein. Such genome editing and / or mutagenesis technologies are well known in the art. Likewise, the introduction can be achieved by any means known in the art, including: introgression, transgenic technology or site-directed nucleases (SDN). In particular, the gene sequence modification is introduced by means of site-directed nuclease (SDN). More particularly, SDN is selected Petition 870250095065, dated 10 / 17 / 2025, page 100 / 375 86 / 346 of: meganuclease, zinc finger, transcription activator-like effector nucleases (TALEN) system, or Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system. DNA is also referred to as genome editing or genome editing with manipulated nucleases (GEEN). This is a type of genetic manipulation in which DNA is inserted, deleted, or replaced in an organism's genome using manipulated nucleases that create site-specific double-strand breaks (DSBs) at desired locations in the genome. The induced double-strand breaks are repaired through non-homologous end joining (NHEJ) or homologous recombination (HR), resulting in targeted mutations (edits). Specifically, SDN can include techniques such as: Meganucleases, Zinc finger nucleases (ZFNs), Transcription activator-like effector (TALEN) based nucleases (Feng et al. 2013 Cell Res. 23, 1229-1232, Sander & Joung Nat. Biotechnol.32, 347-355 2014) and the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR-Cas) system. Gene editing can also be achieved by SDN-2. SDN-2 is similar to SDN, but also provides a small nucleotide template complementary to the break area. The template contains one or more sequence modifications in the genomic DNA that are incorporated to create the mutation in the target gene. In one embodiment, the gene editing system may include a CRISPR-Cas system.

[0211] As used herein, the term guide RNA or gRNA generally refers to an RNA molecule (or a group of RNA molecules collectively) that can bind to a CRISPR system effector, such as a Cas or Cpf1 protein, and Petition 870250095065, dated 10 / 17 / 2025, p. 101 / 375 87 / 346 assists in directing the Cas or Cpfl protein to a specific location within a target polynucleotide (e.g., a DNA). A guide RNA of the invention can be a single RNA molecule (sgRNA), manipulated, where for example sgRNA comprises a crRNA segment and optionally a tracrRNA segment. A guide RNA of the invention can also be a dual guide system, where the crRNA and tracrRNA molecules are physically distinct molecules that then interact to form a duplex for recruiting a CRISPR system effector, such as Cas9, and for directing that protein to the target polynucleotide.

[0212] As used herein, the term crRNA or crRNA segment refers to an RNA molecule or a portion of an RNA molecule that includes a polynucleotide targeting guide sequence, a stem sequence involved in protein binding, and optionally a 3' overhang sequence. The polynucleotide targeting guide sequence is a nucleic acid sequence that is complementary to a sequence in a target DNA (e.g., a gene encoding a PPO enzyme). This polynucleotide targeting guide sequence is also referred to as the protospacer. In other words, the polynucleotide targeting guide sequence of a crRNA molecule interacts with a target DNA in a sequence-specific manner through hybridization (i.e., base pairing).As such, the nucleotide sequence of the guide sequence of the crRNA molecule's polynucleotide targeting can vary and determines the location within the target DNA where the guide RNA and the target DNA will interact. Petition 870250095065, dated 10 / 17 / 2025, p. 102 / 375 88 / 346

[0213] The polynucleotide targeting guide sequence of a crRNA molecule can be modified (e.g., by genetic manipulation) to hybridize with any desired sequence within a target DNA. The polynucleotide targeting guide sequence of a crRNA molecule of the invention can have a length of about 12 nucleotides to about 100 nucleotides. For example, the polynucleotide targeting guide sequence of a crRNA can have a length of about 12 nucleotides (nt) to about 80 nt, about 12 nt to about 50 nt, about 12 nt to about 40 nt, about 12 nt to about 30 nt, about 12 nt to about 25 nt, about 12 nt to about 20 nt, or about 12 nt to about 19 nt. For example, the polynucleotide targeting guide sequence of a crRNA can be about 17 nt to about 27 nts long.

[0214] For example, the polynucleotide targeting guide sequence of a crRNA may have a length of about 19 nt to about 20 nt, about 19 nt to about 25 nt, about 19 nt to about 30 nt, about 19 nt to about 35 nt, about 19 nt to about 40 nt, about 19 nt to about 45 nt, about 19 nt to about 50 nt, about 19 nt to about 60 nt, about 19 nt to about 70 nt, about 19 nt to about 80 nt, about 19 nt to about 90 nt, about 19 nt to about 100 nt, about 20 nt to about 25 nt, from about 20 nt to about 30 nt, from about 20 nt to about 35 nt, from about 20 nt to about 40 nt, from about 20 nt to about 45 nt, from about 20 nt to about 50 nt, from about 20 nt to about 60 nt, from about 20 nt to about 70 nt, about Petition 870250095065, dated 10 / 17 / 2025, page 103 / 375 89 / 346 from 20 nt to about 80 nt, from about 20 nt to about 90 nt, or from about 20 nt to about 100 nt. The nucleotide sequence of the polynucleotide targeting guide sequence of a crRNA can have a length of at least about 12 nt. In some embodiments, the polynucleotide targeting guide sequence of a crRNA is 20 nucleotides in length. In some embodiments, the polynucleotide targeting guide sequence of a crRNA is 19 nucleotides in length.

[0215] The present invention also provides a guide RNA comprising a manipulated crRNA, wherein the crRNA comprises a decoy RNA segment capable of hybridizing with a genomic target sequence. This manipulated crRNA may be a physically distinct molecule, as in a dual guide system.

[0216] As used herein, the term tracrRNA or tracrRNA segment refers to an RNA molecule or portion thereof that includes a protein-binding segment (e.g., the protein-binding segment is capable of interacting with a CRISPR-associated protein, such as a Cas9). The present invention also provides a guide RNA comprising a manipulated tracrRNA, wherein the tracrRNA further comprises a decoy RNA segment that is capable of binding to a donor DNA molecule. The manipulated tracrRNA may be a physically distinct molecule, as in a double guide system, or it may be a segment of an sgRNA molecule.

[0217] Guide RNA, as an sgRNA or as two or more RNA molecules, does not contain a tracrRNA, as it is known in the art that some CRISPR-associated nucleases, such Petition 870250095065, dated 10 / 17 / 2025, p. 104 / 375 90 / 346, such as Cpfl (also known as Casl2a), do not require a tracrRNA for their RNA-mediated endonuclease activity (Qi et al., (2013), Cell, 152: 1173-1183; Zetsche et al., (2015), Cell 163: 759-771). Such a guide RNA of the invention may comprise a crRNA with the decoy RNA functionally attached to the 5' or 3' end of the crRNA. Cpfl also has RNase activity in its cognate precrRNA (Fonfara et al., 2016, Nature, doi.org / 10.1038 / natural!945).

[0218] A guide RNA of the invention may comprise multiple crRNAs that Cpfl possesses for maturing crRNAs. Each of these crRNAs may be operationally linked to a decoy RNA. At least one of these crRNAs may be operationally linked to a decoy RNA. The decoy RNA may be specific to a sequence of interest (SOI) or may be a universal decoy, which has a corresponding universally attached sequence in the donor DNA molecule.

[0219] The present invention also provides a polynucleotide comprising a sequence encoding a guide RNA of the invention. The polynucleotide may be a DNA or RNA molecule. The polynucleotide molecule may be circular or linear. The polynucleotide may be single-stranded, partially double-stranded, or double-stranded. The polynucleotide may be complexed with at least one polypeptide. The polypeptide may have a nucleic acid recognition or nucleic acid binding domain. The polypeptide may be a carrier to mediate the delivery of, for example, a polynucleotide of the invention, a nuclease, and optionally, a donor molecule. The polypeptide may be a Feldan Shuttle (U.S. Patent Publication No. 20160298078, herein incorporated by Petition 870250095065, dated 10 / 17 / 2025, page 105 / 375 91 / 346 reference). The polynucleotide may comprise an expression cassette capable of directing the expression of the polynucleotide. The polynucleotide may additionally comprise additional expression cassettes capable of expressing, for example, a nuclease such as a CRISPR-associated nuclease.

[0220] Suitably, therefore, the plant or part thereof may be provided with, specifically transformed with, a Cas enzyme, or one or more polynucleotides encoding a Cas enzyme, and a polynucleotide sequence encoding a guide RNA. Suitably, the guide RNA is complementary to a PPO gene or regulatory sequences thereof, in the plant or part thereof. Suitably, the guide RNA is operable to target the Cas enzyme to edit the PPO gene and provide the plant or part thereof with increased resistance to a compound that inhibits PPO enzymatic activity relative to an unmodified plant.

[0221] Alternatively, a plant may be modified by providing the plant or part thereof with one or more operable regulatory RNA sequences to target a gene encoding a PPO enzyme or a regulatory sequence thereof. Suitably, the plant or part thereof may be transformed with one or more operable regulatory RNA sequences to target a gene encoding a PPO enzyme or a regulatory sequence thereof. Suitably, the regulatory RNA sequence may be complementary to, and bind to, a gene encoding a PPO enzyme or a regulatory sequence thereof in the plant or part thereof and act to provide increased resistance to compounds that inhibit PPO enzymatic activity. Petition 870250095065, dated 10 / 17 / 2025, p. 106 / 375 92 / 346

[0222] Transformation refers to a process of introducing an exogenous nucleic acid molecule (e.g., a recombinant polynucleotide) into a cell or protoplast, and this exogenous nucleic acid molecule is incorporated into a host cell genome or an organelle genome (e.g., chloroplast or mitochondrion) or is capable of autonomous replication. Transformed or transgenic refers to a cell, tissue, organ, or organism into which a foreign nucleic acid, such as an expression vector or a recombinant nucleic acid molecule, has been introduced. The nucleic acid molecule may be stably integrated into the host genome, or the nucleic acid molecule may also be present as an extrachromosomal molecule. Such an extrachromosomal molecule may be self-replicating. The nucleic acid molecule may also be introduced into the chloroplast genome or mitochondria of a plant cell.

[0223] Methods for transforming plant cells or tissues include, but are not limited to, Agrobacterium-mediated transformation methods and Biolistica-mediated or particle gun-mediated transformation methods. Suitable plant transformation vectors for the purpose of Agrobacterium-mediated transformation include those elements derived from a tumor-inducing plasmid (Ti) of Agrobacterium tumefaciens, for example, right border regions (RB) and left border regions (LB), and others disclosed by Herrera-Estrella et al., Nature 303: 209 (1983); Bevan, Nucleic Acids Res. 12: 8711-8721 (1984); Klee et al., Bio-Technology 3 (7): 637-642 (1985). In addition to transformation vectors of Petition 870250095065, dated 10 / 17 / 2025, p. 107 / 375 93 / 346 plants derived from Agrobacterium Ti or root-inducing (Ri) plasmids, alternative methods can be used to insert the nucleic acid molecules of this invention into plant cells. Such methods may involve, but are not limited to, for example, the use of liposomes, electroporation, chemicals that enhance the uptake of free DNA, delivery of free DNA via microprojectile bombardment, and transformation using viruses or pollen.

[0224] A transgenic or transformed cell or plant also includes the offspring of the cell or plant and the offspring produced from a breeding program employing such a transgenic plant as a parent in a cross and exhibiting an altered phenotype resulting from the presence of the foreign nucleic acid molecule.

[0225] Transgenic plants can be homozygous for the polynucleotide encoding a modified PPO enzyme described herein (i.e., those containing two added genes encoding the enzyme at the same position on each chromosome of the chromosome pair). Homozygous transgenic plants can be obtained by crossing (self-pollination) isolates of independent transgenic plants containing a single added gene, germinating some of the resulting seeds and transforming the resulting plant with the target gene. Plant breeding

[0226] The development or regeneration of transgenic plants containing a nucleic acid molecule encoding recombinant modified PPO enzymes of the invention is well known in the art. The regenerated plants are Petition 870250095065, dated 10 / 17 / 2025, page 108 / 375 94 / 346 self-pollinated plants are used to provide homozygous transgenic plants, as discussed above. Alternatively, pollen obtained from regenerated plants is crossed with plants grown from seeds of agronomically important lines. Conversely, pollen from plants of these important lines is used to pollinate regenerated plants.

[0227] The at least partially resistant plants and the offspring of such plants described herein (such as transformed, modified, or transgenic plants described herein) can be used in methods to prepare at least partially resistant plants, plants having increased tolerance to compounds that inhibit PPO enzymatic activity, and seeds of such plants. Thus, for example, the plants exemplified herein can be used in breeding programs to develop plants at least partially resistant to additional herbicides, such as commercial varieties of such plants. According to such methods, a first parent plant can be used in crosses with a second parent plant, where at least one of the first or second parent plants contains at least one modified PPO enzyme as described herein. One application of the process is in the production of F1 hybrid plants.Another aspect of this process is that it can be used for the development of innovative dihaploid or inbred parent lines. For example, a line of plants as described here could be crossed with any second plant, and the resulting hybrid offspring would self-fertilize and / or reproduce for about 5 to 7 or more generations, thus providing a large... Petition 870250095065, dated 10 / 17 / 2025, p. 109 / 375 95 / 346 number of distinct parent lines. These parent lines could then be crossed with other lines and the resulting hybrid offspring analyzed for beneficial traits. In this way, innovative lines conferring desirable traits could be identified. Several breeding methods can be used, including haploidy, pedigree breeding, single seed offspring, modified single seed offspring, recurrent selection, and backcrossing.

[0228] Plants and their offspring may exhibit a synergistic rather than additive tolerance effect to compounds that inhibit PPO enzymatic activity, whereby the level of tolerance in plants and their offspring comprising multiple mutations is greater than the combined tolerance of plants comprising a single modified PPO enzyme.

[0229] Plant lines containing the modified PPO enzymes of the present invention can be crossed by natural or mechanical techniques. Mechanical pollination can be carried out by controlling the types of pollen that can be transferred to the stigma or by manual pollination.

[0230] Any breeding method can be used in the methods of the present invention. In one example, the resistant plants of the present invention can be created using a haploid method. In such methods, the parents having the genetic basis for the desired complement of characteristics are crossed in a simple or complex cross. Crossing (or cross-pollination) refers to the transfer of pollen from one plant to a different plant. The offspring of Petition 870250095065, dated 10 / 17 / 2025, page 110 / 375 96 / 346 cross is cultured and the microspores (immature pollen grains) are separated and filtered using techniques known to those skilled in the art [(e.g., Swanson, EB et al, (1987) Plant Cell Reports, 6: 94-97, Efficient isolation of microspores and the production of microspore-derived embryos in Brassica napus, L.; and Swanson, EB, (1990) Microspore culture in Brassica, pp. 159-169 in Methods in Molecular Biology, vol. 6, Plant Cell and Tissue Culture, Humana Press]. These microspores exhibit gene segregation. The microspores are cultured in the presence of an appropriate AHAS-inhibiting herbicide, such as imazethapyr (e.g., PURSUIT™) or imazamox (e.g., SOLO™, BEYOND™ and RAPTOR™) or a 50 / 50 mixture of imazethapyr and imazamox (e.g., ODYSSEY™), which kills microspores lacking the mutations responsible for herbicide resistance. Microspores carrying the genes responsible for herbicide resistance survive and produce embryos, which form haploid plants.Their chromosomes are then duplicated to produce duplicated haploids.

[0231] Other breeding methods may also be used in accordance with the present invention. For example, pedigree breeding may be used to improve largely self-pollinating crops such as Brassica and canola. Pedigree breeding begins with the crossing of two genotypes, each of which may have one or more desirable characteristics that the other lacks or that complement each other. If the two original parents do not provide all the desired characteristics, additional parents may be included in the breeding plan. These parents may be crossed in a Petition 870250095065, dated 10 / 17 / 2025, page 111 / 375 97 / 346 simple or complex way to produce a simple or complex F1. An F2 population is produced from F1 by self-fertilization of one or more F1 plants or by intercrossing two F1s (i.e., plants with the same two parents). Selection of the best individuals can begin in the F2 generation and, from the F3 generation onwards, the best families and the best individuals within the best families are selected. Replicated family testing can begin in the F4 generation to improve the effectiveness of selection for traits with low heritability. At an advanced stage of inbreeding (i.e., F6 and F7), the best lines or mixtures of phenotypically similar lines can be tested for potential release as new cultivars.However, the pedigree method takes longer than the haploidy method to develop improved plants that are at least partially resistant to PPO enzyme inhibitor compounds, because the plants exhibit segregation over multiple generations, and the recovery of desirable traits is relatively low.

[0232] The individual seed descent (SSD) procedure can also be used to create improved varieties. The SSD procedure strictly refers to planting a segregated population, collecting a sample of one seed per plant, and using the population of individual seeds to plant the next generation. When the population has progressed from F2 to the desired level of inbreeding, the plants from which the lines are derived will each produce different F2 individuals. The number of plants in a population decreases with each generation due to some seeds failing to germinate or some plants not producing fruit. Petition 870250095065, dated 10 / 17 / 2025, page 112 / 375 98 / 346 at least one seed. As a result, not all plants originally sampled in the F2 population will be represented by offspring when the generation advancement is complete.

[0233] In a multiple seeding procedure, canola breeders commonly collect one or more pods from each plant in a population and thresh them together to form a bulk. Part of the bulk is used to plant the next generation and part is set aside in reserve. The procedure has been referred to as modified single seed descent or bulk pod technique. The multiple seeding procedure has been used to save labor in harvesting. It is considerably faster to thresh the pods with a machine than to manually remove one seed from each for the single seeding procedure. The multiple seeding procedure also allows planting the same number of seeds from a population in each inbreeding generation. Enough seeds are collected to compensate for plants that did not germinate or produce seeds.

[0234] Backcrossing can be used to transfer a gene or genes for a simply inherited, highly heritable trait from one variety or parent line (the donor parent) to another desirable cultivar or inbred line (the recurrent parent). After the initial cross, individuals possessing the phenotype of the donor parent are selected and are repeatedly crossed (backcrossed) with the recurrent parent. When the backcross is complete, the resulting plant is expected to have the attributes of the parent. Petition 870250095065, dated 10 / 17 / 2025, page 113 / 375 99 / 346 recurring and the desirable trait transferred from the donor parent.

[0235] Improved varieties can also be developed through recurrent selection. In this method, a genetically variable population of heterozygous individuals is identified or created by intercrossing several different parents. The best plants are selected based on individual superiority, excellent offspring, or excellent combining ability. The selected plants are crossed to produce a new population in which further cycles of selection are continued.

[0236] At least partially resistant plants can be produced by cross-pollination of a first plant with a second plant, allowing the pollen-accepting plant (either the first or the second plant) to produce seeds from this cross-pollination. The seeds and offspring plants generated from them may have the cross-mutation in the genome of the seed and / or offspring plants. The pollen-accepting plant may be the first or the second plant. The first plant comprises a nucleic acid encoding a modified PPO enzyme as disclosed herein. The second plant may be any compatible plant and may comprise a second of the same modified PPO enzyme or a different modified PPO enzyme. The first and second enzymes may comprise a nucleic acid encoding the same amino acid substitution(s) or different amino acid substitution(s) relative to a wild-type modified PPO enzyme.Seeds or plants from the offspring can be selected. Petition 870250095065, dated 10 / 17 / 2025, page 114 / 375 100 / 346 resulting from the crossbreeding that comprise one or two nucleic acids encoding modified PPO enzymes.

[0237] When the first and second plants are homozygous for the first and second nucleic acid molecules, respectively, each of the resulting offspring plants comprises one copy of each of the first and second nucleic acid molecules and the selection step can be omitted. When at least one of the first and second plants is heterozygous, the offspring plants comprising both nucleic acid molecules can be selected, for example, by analyzing the DNA of the offspring plants to identify offspring plants comprising both the first and second nucleic acid molecules or by testing the offspring plants for increased herbicide tolerance.

[0238] Offspring and / or progeny plants can be evaluated for nucleic acid molecules of the present invention by any method for determining the presence of a specific modified PPO nucleic acid or enzyme.

[0239] Therefore, methods are also provided here for selecting a plant or part thereof that includes a modified PPO enzyme of the invention or a nucleic acid or expression vector encoding the same, by exposing the plant or part thereof to an effective amount of a compound that inhibits sufficient PPO enzymatic activity to prevent or reduce the growth of a plant that does not include at least one modified PPO enzyme of the invention or a nucleic acid or expression vector encoding the same. It can then be determined by the methods described herein whether the plant has been affected (e.g., has reduced growth or Petition 870250095065, dated 10 / 17 / 2025, p. 115 / 375 101 / 346 reduced damage) by the compound. Plants that are not affected by the compound can then be selected.

[0240] Methods for determining whether a plant includes the modified PPO enzyme of the invention or a nucleic acid or expression vector encoding the same and / or is affected by a compound that inhibits PPO enzymatic activity include phenotypic assessments, genotypic assessments, or combinations thereof. Offspring plants can be evaluated in subsequent generations for resistance to the compound and other desirable traits. Resistance to compounds that inhibit PPO enzymatic activity can be evaluated by exposing plants to one or more appropriate compounds and assessing the lesions. Some traits, such as lodging resistance and plant height, can be evaluated by visual inspection of the plants, while precocity of maturity can be evaluated by visual inspection of the seeds within the pods (silicas).Other trace elements, such as oil percentage, protein percentage, and total glucosinolates in seeds, can be evaluated using techniques such as Near-Infrared Spectroscopy and / or liquid chromatography and / or gas chromatography.

[0241] The plants of the present invention can also be identified using any genotypic analysis method. Genotypic evaluation of plants includes the use of techniques such as Isoenzyme Electrophoresis, Restriction Fragment Length Polymorphisms (RFLPs), Randomly Amplified Polymorphic DNAs (RAPDs), Arbitrarily Initiated Polymerase Chain Reaction (AP-PCR), Allele-Specific PCR (AS-PCR), Fingerprinting of Petition 870250095065, dated 10 / 17 / 2025, page 116 / 375 102 / 346 DNA Amplification Factors (DAF), Sequence Characterized Amplified Regions (SCARs), Amplified Fragment Length Polymorphisms (AFLPs), Simple Sequence Repeats (SSRs), which are also referred to as Microsatellites. Additional compositions and methods for analyzing plant genotypes provided herein include those methods disclosed in EUA Publication No. 2004 / 0171027, EUA Publication No. 2005 / 02080506, and EUA Publication No. 2005 / 0283858, the entirety of which are hereby incorporated by reference.

[0242] Evaluation and manipulation (through exposure to one or more appropriate compounds that inhibit PPO enzymatic activity) can occur over several generations. The performance of new lines can be evaluated using objective criteria in comparison with verification varieties. Lines showing the desired trait combinations are crossed with another line or self-pollinated to produce seeds.

[0243] DNA sequencing refers to the determination of the nucleic acid sequence of a piece of DNA, e.g., of a gene. Standard methods and commercial services are known in the art. Basic methods for DNA sequencing include the Maxam-Gilbert method and the strand termination method. High-throughput techniques have also been developed and can be used in the method of the present invention. These high-throughput techniques include, but are not limited to, Massively Parallel Signature Sequencing (MPSS), Polony sequencing, 454 pyrosequencing, Illumina (Solexa) sequencing, probe anchor synthesis. Petition 870250095065, dated 10 / 17 / 2025, p. 117 / 375 103 / 346 combinatorial sequencing (cPAS), SOLiD sequencing, Ion Torrent semiconductor sequencing, DNA nanoball sequencing, Heliscope single-molecule sequencing, Single-molecule real-time sequencing (SMRT), and Nanopore DNA sequencing.

[0244] Sequencing can be carried out using primers that are capable of binding to an isolated polynucleotide of the invention. For example, primers that are complementary to at least a portion of an isolated polynucleotide of the invention.

[0245] As used herein, the term primer refers to an oligonucleotide that is capable of pairing with a polynucleotide target and serving as a starting point for DNA synthesis when placed under conditions in which synthesis of a primer extension product is induced (e.g., in the presence of nucleotides and a polymerization agent such as DNA polymerase and at a suitable temperature and pH). A primer (in some examples an extension primer and in some examples an amplification primer) may be single-stranded for maximum efficiency in extension and / or amplification. The primer may be an oligodeoxyribonucleotide. A primer is typically long enough to initiate the synthesis of extension and / or amplification products in the presence of the polymerization agent. The minimum primer length may depend on many factors, including but not limited to the temperature and composition (A / T vs. G / C content) of the primer.In the context of amplification initiators, these are typically provided as a pair of bidirectional initiators consisting of a forward and a reverse initiator. Petition 870250095065, dated 10 / 17 / 2025, page 118 / 375 104 / 346 or provided as a pair of direct primers, as commonly used in DNA amplification techniques such as PCR amplification.

[0246] As such, it will be understood that the term primer, as used herein, may refer to more than one primer, particularly in cases where there is some ambiguity in the information regarding the terminal sequence(s) of the target region to be amplified. Consequently, a primer may include a collection of primer oligonucleotides containing sequences representing possible variations in the sequence or include nucleotides that allow for typical base pairing. Primers may be prepared by any suitable method known in the art. Methods for preparing sequence-specific oligonucleotides are known in the art and include, for example, cloning and restriction of appropriate sequences and direct chemical synthesis. Chemical synthesis methods may include, for example, the phosphodi- or tri-ester method, the diethylphosphoramidate method, and the solid support method disclosed in U.S. Patent No. 4,458,066.

[0247] Primers can be labeled, if desired, by incorporating detectable fractions, for example spectroscopic, fluorescence, photochemical, biochemical, immunochemical, or chemical fractions. Diagnostic primers (i.e., capable of identifying or selecting based on the presence of nucleic acids encoding modified PPO and the modified PPO enzymes as described herein) for resistance to compounds that inhibit PPO enzymatic activity can be created by any known methods. The PCR method is well described in manuals. Petition 870250095065, dated 10 / 17 / 2025, page 119 / 375 105 / 346 and known to the expert. After PCR amplification, the target polynucleotides can be detected by hybridization with a probe polynucleotide, which forms a stable hybrid with the target sequence under stringent to moderately stringent hybridization and washing conditions. If the probes are expected to be essentially completely complementary (i.e., about 99% or more) to the target sequence, stringent conditions can be used.

[0248] If some mismatch is expected, for example, if variant polynucleotides are expected with the result of the probe not being completely complementary, the stringency of the hybridization can be reduced. In some instances, conditions are chosen to exclude nonspecific / accidental binding. Conditions that affect hybridization and select against nonspecific binding are known in the art and are described in, for example, Sambrook & Russell (2001) Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, United States of America. Generally, hybridization and / or washes with lower salt concentrations and higher temperatures increase the stringency of the hybridization conditions. Seeds

[0249] Also included here are seeds capable of producing a plant or part thereof of the invention. Suitablely, seeds comprising a modified PPO enzyme provided herein or a polynucleotide or expression vector encoding a modified PPO enzyme provided herein, which provides increased resistance to Petition 870250095065, dated 10 / 17 / 2025, p. 120 / 375 106 / 346 is a compound that inhibits the enzymatic activity of PPO compared to an unmodified plant.

[0250] The term seed encompasses seeds and plant propagules of all types including but not limited to true seeds, seed pieces, sprouts, calluses, bulbs, fruit, tubers, grains, cuttings, sprouts from cuttings and the like.

[0251] Seeds can be treated or untreated. For example, seeds can be treated to improve germination, for example, by seed pre-preparation or by disinfection to protect against seed-borne pathogens. In another example, seeds can be coated with any available coating to improve, for example, plantability, seed emergence, and protection against seed-borne pathogens. Seed coating can be any form of seed coating, including, but not limited to, pelleting, film coating, and encrustations.

[0252] The seed can be germinated and used to produce or cultivate a plant or part thereof of the invention. That is, a plant or part thereof including a modified PPO enzyme that provides increased resistance to a compound that inhibits the enzymatic activity of PPO compared to an unmodified plant.

[0253] Also provided herein is a container containing seeds of the invention. A seed container may hold any number, weight or volume of seeds. For example, a container may hold at least, or more than, approximately 10, 25, 50, 75, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 or more. Petition 870250095065, dated 10 / 17 / 2025, p. 121 / 375 107 / 346 seeds. Alternatively, the container may hold at least, or more than, approximately 1 ounce, 5 ounces, 10 ounces, 1 pound, 2 pounds, 3 pounds, 4 pounds, 5 pounds or more of seeds.

[0254] Plant seed containers can be any container available in the art. By way of non-limiting example, a container could be a box, a bag, a packet, a pouch, a roll of tape, a bucket, a sheet of metal or a tube.

[0255] The seeds contained in containers may be treated or untreated seeds. For example, seeds may be treated to improve germination, for example, by seed pre-preparation or by disinfection to protect against seed-borne pathogens. In another example, seeds may be coated with any available coating to improve, for example, plantability, seed emergence and protection against seed-borne pathogens. Seed coating may be any form of seed coating, including, but not limited to, pelleting, film coating and encrustations.

[0256] At least 10% of the seeds within a container may be seeds of the invention. For example, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100% of the seeds in the container may be seeds of the invention.

[0257] The seeds of the invention may be hybrid seeds produced by a method including crossing a first plant according to the invention with a second plant; and obtaining seeds. For example, crossing a plant including a modified PPO enzyme or a polynucleotide or expression vector. Petition 870250095065, dated 10 / 17 / 2025, p. 122 / 375 108 / 346 encoding such a modified PPO enzyme, which provides increased resistance to a compound that inhibits the enzymatic activity of PPO with another plant.

[0258] The term hybrid seed refers to a seed produced by cross-pollination of two plants. Plants grown from hybrid seeds may have improved agricultural characteristics, such as better yield, greater uniformity and / or disease resistance. Hybrid seeds do not reproduce truly, that is, the seed produced by self-fertilization of a hybrid plant (the plant grown from a hybrid seed) does not reliably result in the next generation in an identical hybrid plant. Therefore, new hybrid seeds have to be produced from the parent plant lines for each planting. Since most cultivated plants have both male and female reproductive organs, hybrid seeds can only be produced by preventing self-pollination of the female parent and allowing or facilitating pollination with the desired pollen.There are a variety of methods to prevent self-pollination of the female parent; one method by which self-pollination is prevented is the mechanical removal of the pollen-producing organ before pollen release. Commercial seed production of more hybrid (Zea mays) typically involves planting the desired male and female parent lines, usually in separate rows or blocks in an isolated field, treating the female parent plant to prevent pollen release, ensuring female pollination only by the designated male parent, and collecting hybrid seed only from the female parent. Petition 870250095065, dated 10 / 17 / 2025, page 123 / 375 109 / 346 hybrid seeds can be the result of a single cross (e.g., a first-generation cross between two inbred lines), a modified single cross (e.g., a first-generation cross between two inbred lines, one or the other of which may have been slightly modified by the use of closely related crosses), a double cross (e.g., a first generation of a cross between two single crosses), a three-way cross (e.g., a first generation of a cross between a single cross and an inbred line), a top cross (e.g., the first generation of a cross between an inbred line and an open-pollinated variety or the first generation of a cross between a single cross and an open-pollinated variety), or an open-pollinated variety (e.g., a population of plants selected according to a pattern that may show variation but has characteristics by which a variety can be differentiated from other varieties).

[0259] As used herein, the terms cross or crossbreeding refer to the fusion of gametes through pollination to produce offspring (e.g., cells, seeds, or plants). The term encompasses both sexual crosses (the pollination of one plant by another) and self-fertilization (self-pollination, e.g., when the pollen and ovule are from the same plant). The term crossbreeding refers to the act of fusing gametes through pollination to produce offspring. Nucleic acids

[0260] The invention further provides isolated polynucleotides encoding a PPO enzyme. Petition 870250095065, dated 10 / 17 / 2025, page 124 / 375 110 / 346 modified or a fragment thereof as defined in any aspect or embodiment herein, suitably which may encode a modified PPO enzyme or a fragment thereof as defined in any of the sequences listed herein, suitably which may encode any of the SEQ ID Nos: 1, 2, 4, 151, 153-302 or 305-336. Suitably which may encode any of the SEQ ID Nos: 37-39, 58, 59, 97-99, 118, 119, 125-137, 139-151, 188-190, 209, 210, 248-250, 269, 270, 277-288 or 291-302. As such, modified PPO enzymes or functional fragments thereof are also provided. They can be expressed from such isolated polynucleotides.

[0261] An isolated polynucleotide is substantially separated from other polynucleotide sequences to which the polynucleotide is normally associated, such as from the chromosomal or extrachromosomal DNA of a cell in which the polynucleotide occurs naturally. A polynucleotide may be an isolated polynucleotide when it comprises a transgene or part of a transgene present in the genome of another organism. The term also encompasses polynucleotides that are biochemically purified so as to substantially remove contaminating polynucleotides and other cellular components. Isolated polynucleotides are substantially free of sequences (such as protein-coding sequences) that naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the polynucleotide) in the genomic DNA of the organism from which the polynucleotide is derived.For example, the isolated polynucleotide may contain less than approximately 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of the sequences. Petition 870250095065, dated 10 / 17 / 2025, page 125 / 375 111 / 346 nucleotides that naturally flank the polynucleotide in the genomic DNA of the cell from which the polynucleotide is derived. The isolated polynucleotide may be flanked by its native genomic sequences that control its expression in the cell, for example, the native promoter or native 3' untranslated region. Expression constructs and vectors

[0262] The expression constructs and vectors, which include at least one polynucleotide of the present invention embedded therein, may be any construct or vector capable of delivering the polynucleotide into a host or host cell and enabling the expression of the polynucleotide to provide a functional modified PPO enzyme as described herein or a fragment thereof.

[0263] Such constructs or vectors may contain heterologous polynucleotide sequences, that is, polynucleotide sequences that are not naturally found adjacent to polynucleotides of the present invention and that may be derived from a species different from the species from which the polynucleotide molecule(s) is / are derived. The construct or vector may be RNA or DNA, prokaryotic or eukaryotic, and typically the vector is a virus or a plasmid.

[0264] A number of vectors suitable for stable transfection of plant cells or for the establishment of transgenic plants have been described in, e.g., Pouwels et al., Cloning Vectors: A Laboratory Manual, 1985, sup. 1987; Weissbach and Weissbach, Methods for Plant Molecular Biology, Academic Press, 1989; and Gelvin et al., Handbook of Plant Molecular Biology, Kluwer Academic Publishers, 1990. Petition 870250095065, dated 10 / 17 / 2025, p. 126 / 375 112 / 346 Typically, plant expression vectors include, for example, one or more cloned plant genes under the transcriptional control of 5' and 3' regulatory sequences and a selectable dominant marker. For example, the vector could be pBIN 19 (Bevan, Nucl. Acids Res. (1984)).

[0265] The expression vector of the invention may include one or more regulatory sequences. For example, expression vectors may contain a promoter regulatory region (e.g., a regulatory region controlling inducible or constitutive expression, regulated by the environment or development, or specific to cells or tissues), a transcription initiation start site, a ribosome binding site, an RNA processing signal, a transcription termination site, and / or a polyadenylation signal. Such a portion of an expression vector may be referred to as an expression construct. The expression construct may include one or more regulatory sequences that are functional in plants, thus enabling the expression of the polynucleotide encoding a modified PPO enzyme of the invention in a plant.

[0266] Expression construct as used herein means a nucleic acid sequence capable of directing the expression of a particular nucleic acid sequence in an appropriate host cell, comprising a promoter operationally linked to the polynucleotide sequence of interest that is operationally linked to termination signal sequences. It also typically comprises sequences required for appropriate translation of the polynucleotide sequence. The expression construct comprising the polynucleotide of interest may be chimeric, Petition 870250095065, dated 10 / 17 / 2025, page 127 / 375 113 / 346 meaning that at least one of its components is heterologous with respect to at least one of its other components. The expression construct may also be one that occurs naturally but has been obtained in a recombinant form useful for heterologous expression. Typically, however, the expression construct is heterologous with respect to the host, i.e., the particular polynucleotide of the expression cassette does not occur naturally in the host cell and must have been introduced into the host cell or an ancestor of the host cell by a transformation event. The expression of the polynucleotide sequence in the expression construct may be under the control of, for example, a constitutive promoter or an inducible promoter that initiates transcription only when the host cell is exposed to some particular external stimulus.In the case of a multicellular organism, such as a plant, the promoter may also be specific to a particular tissue or organ or developmental stage.

[0267] The term regulatory element or regulatory sequence as used herein refers to a nucleic acid that is capable of regulating the transcription and / or translation of an operationally linked polynucleotide. Regulatory elements include, but are not limited to, promoters, enhancers, introns, 5' UTRs and 3' UTRs.

[0268] The expression cassettes may include in the 5'-3' direction of transcription a transcriptional and translational initiation region (e.g., a promoter), a polynucleotide sequence encoding a modified PPO enzyme of the invention, and a transcriptional termination region. Petition 870250095065, dated 10 / 17 / 2025, page 128 / 375 114 / 346 translational (e.g., termination region) functional in plants.

[0269] Any promoter may be used in the production of the construct and expression vectors, including such expression constructs as described herein. The promoter may be native or analogous, or foreign or heterologous, to the host plant and / or to the polynucleotide sequences encoding the modified PPO of the invention. Additionally, the promoter may be a naturally occurring sequence or alternatively a synthetic sequence. Where the promoter is foreign or heterologous to the host plant, 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 polynucleotide encoding the modified PPO of the invention, it is intended that the promoter is not the native or naturally occurring promoter for the operationally linked polynucleotide of the invention.

[0270] Although it may be preferable to express the polynucleotide encoding a modified PPO of the invention using heterologous promoters, native promoter sequences can be used in the preparation of expression constructs. Such expression constructs can change the expression levels of the modified PPO enzyme in the plant or plant cell. Thus, the phenotype of the plant or plant cell is altered.

[0271] Any promoter can be used in the preparation of expression constructs to control the expression of the modified PPO-encoding polynucleotide, such as promoters providing constitutive, tissue-preferred, inducible, or other promoters for expression. Petition 870250095065, dated 10 / 17 / 2025, page 129 / 375 115 / 346 in plants. Constitutive promoters include, for example, the nuclear promoter of the Rsyn7 promoter and other constitutive promoters disclosed in WO 99 / 43 838 and U.S. Patent No. 6,072,050; the 35S nuclear promoter of CaMV (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 similar promoters. Other constitutive promoters include, for example, U.S. Patents 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.

[0272] Preferred tissue promoters can be used to direct the expression of the modified PPO enzymes of the invention within a specific plant tissue. Such preferred tissue promoters include, but are not limited to, preferred leaf promoters, preferred root promoters, preferred seed promoters, and preferred stem promoters. Preferred tissue promoters include those described in 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. 112 (3): 13311341; Van Camp et al. (1996) Plant Physiol. 112 (2): 525535; Canevascini et al. (1996) Plant Physiol. 112 (2): 513524; Yamamoto et al. (1994) Plant Cell Physiol. 35 (5): 773Petition 870250095065, of 10 / 17 / 2025, p. 130 / 375 116 / 346 778; Lam (1994) Results Probl. Cell Differ. 20: 181-196; Orozco et al (1993) Plant Mol Biol. 23 (6) : 1129-1138; Matsuoka et al. (1993) Proc Natl. Acad. Sci. USA 90 (20): 9586- 9590; and Guevara-Garcia et al. (1993) Plant J. 4 (3): 495-505.

[0273] Expression constructs may also comprise transcription termination regions. Where transcription termination regions are used, any termination region may be used in the preparation of expression cassettes. For example, the termination region may be native to the transcription initiation region, may be native to the operationally linked polynucleotide of interest, may be native to the host plant, or may be derived from another source (i.e., foreign or heterologous to the promoter, to the polynucleotide of interest encoding modified PPO enzyme, to the host plant, or any combination thereof). Examples of termination regions that are available for use in the expression constructs and vectors of the present invention include those of the A. tumefaciens Ti plasmid, such as the octopine synthase and nopaline synthase termination regions. See also Guerineau et al. (1991) Mol. Gen. Genet. 262: 141-144; 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; Bailas et al. (1989) Nucleic Acids Res. 17: 7891-7903; and Joshi et al. (1987) Nucleic Acids Res. 15: 9627-9639.

[0274] For example, the expression construct may comprise an omega 5' leader of Tomato Mosaic Virus (TMV) and a gene encoding a modified PPO enzyme of interest is excised using Xhol / Kpnl and cloned into pBIN 19 behind a Petition 870250095065, dated 10 / 17 / 2025, page 131 / 375 117 / 346 double-intensified 35S promoter and ahead of a NOS 3' transcription terminator. A suitable exemplary vector comprising such an expression construct is provided here as SEQ ID NO: 3.

[0275] Polynucleotides can be optimized for enhanced expression in a transformed plant. That is, polynucleotides encoding modified PPO enzymes 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 the use of host-preferred codons. Methods in the art are available for synthesizing plant-preferred genes. See, for example, U.S. Patents Nos. 5,380,831 and 5,436,391 and Murray et al., (1989) Nucleic Acids Res. 17: 477-498, incorporated herein by reference.

[0276] Additionally, other sequence modifications can be made to the polynucleotides of the invention. For example, additional sequence modifications that are known to enhance gene expression in a host cell. These include elimination of sequences encoding spurious polyadenylation signals, signals from exon / intron splice sites, transposon-like repeats, and other well-characterized sequences that may be detrimental to gene expression. The GC content of the sequence can also be adjusted to average levels for a target host cell, as calculated by reference to known genes expressed in the host cell. Additionally, the sequence can be modified to avoid predicted hairpin mRNA secondary structures. Petition 870250095065, dated 10 / 17 / 2025, page 132 / 375 118 / 346

[0277] Other polynucleotide sequences may also be used in the preparation of the expression constructs of the present invention, for example to enhance the expression of the modified PPO-encoding polynucleotide sequence. Such polynucleotide sequences include the introns of the Adhl gene, intron 1 (Callis et al. (1987) Genes and Development 1: 1183-1200) and leader sequences (W sequence) of Tobacco Mosaic Virus (TMV), More Chlorotic Spot Virus and Alfalfa Mosaic Virus (Gallie et al. (1987) Nucleic Acid Res. 15: 8693-8711, and Skuzeski et al. (1990) Plant Mol. Biol. 15: 65-79, 1990). It has been shown that the shrunken-1 locus further increases gene expression in chimeric gene constructs. U.S. Patents Nos. 5,424,412 and 5,593,874 disclose the use of specific introns in gene expression constructs, and Gallie et al. (1994) Plant Physiol.106: 929-939) also showed that introns are useful for regulating gene expression on a tissue-specific basis. Plant cells transformed with such modified expression constructs or vectors can then exhibit overexpression or constitutive expression of a polynucleotide of the invention.

[0278] Expression constructs may additionally contain 5' leader sequences. Such leader sequences may act to enhance translation. Translation leaders are known in the art and include: picornavirus leaders, for example, EMCV leader (Encephalomyocarditis 5' non-coding region) (Elroy-Stein et al. (1989) Proc. Natl. Acad. ScL USA 86: 6126-6130); potivirus leaders, for example, TEV leader (Tobacco Etch Virus) (Gallie et al. (1995) Gene 165 (2): 233-238), MDMV leader (Virgo Petition 870250095065, dated 10 / 17 / 2025, page 133 / 375 119 / 346 Mosaic virus of the More Dwarf) (Virology 154: 9-20) and human immunoglobulin heavy chain binding protein (BIP) (Macejak et al. (1991) Nature 353: 90-94); untranslated mRNA leader of alfalfa mosaic virus coat protein (AMV RNA 4) (Jobling et al. (1987) Nature 325: 622-625); tobacco mosaic virus (TMV) leader (Gallie et al. (1989) in Molecular Biology of RNA, ed. CECH (Liss, New York), pp. 237-256); and more chlorotic spot virus (MCMV) leader (Lommel et al. (1991) Virology 81: 382-385). See also Della-Cioppa et al. (1987) Plant Physiol. 84: 965-968.

[0279] In preparing the constructs and expression vectors described herein, the various polynucleotides may be manipulated to provide the polynucleotides in the appropriate orientation and, as appropriate, in the appropriate reading frame. To this end, adapters or linkers may be used to join the nucleic acid molecules, or other manipulations may be involved to provide convenient restriction sites, removal of superfluous polynucleotides, removal of restriction sites, or the like. For this purpose, in vitro mutagenesis, primer repair, restriction, pairing, re-substitutions, e.g., transitions and transversions may be involved.

[0280] Expression vectors may include additional features. For example, gRNA promoters to regulate the expression of at least one gRNA, e.g., prOsU3-01, which is the U3 promoter of Rice for pol III-dependent transcription of non-coding RNAs. Vectors may similarly include additional features such as selectable markers, e.g. Petition 870250095065, dated 10 / 17 / 2025, page 134 / 375 120 / 346 ex., Phosphomannose Isomerase (PMI), and antibiotic resistance genes that can be used to aid in the recovery of stably transformed plants.

[0281] By operationally linked or operationally associated as used herein, it is understood that the indicated elements are functionally related to each other and are also generally physically related. Thus, the term operationally linked or operationally associated, as used herein, refers to polynucleotides in a single nucleic acid molecule that are functionally associated. Thus, a first polynucleotide sequence or nucleic acid molecule that is operationally linked to a second polynucleotide sequence or nucleic acid molecule means a situation where the first polynucleotide sequence or nucleic acid molecule is placed in a functional relationship with the second polynucleotide sequence or nucleic acid molecule.For example, a promoter is operationally associated with a polynucleotide sequence or nucleic acid molecule if the promoter effects the transcription or expression of that polynucleotide sequence or nucleic acid molecule. Skilled in the art will understand that control sequences (e.g., promoter) need not be contiguous to the polynucleotide sequence or nucleic acid molecule to which they are operationally associated, provided that the control sequences function to direct their expression. Thus, for example, untranslated but transcribed intervening sequences may be present between a promoter and a polynucleotide sequence or nucleic acid molecule, and the promoter may still be considered. Petition 870250095065, dated 10 / 17 / 2025, p. 135 / 375 121 / 346 operationally linked or operationally associated with a polynucleotide sequence or nucleic acid molecule. Herbicide Resistance and Methods of Use

[0282] The plants or parts thereof according to the invention are at least partially resistant to inhibition by a compound that inhibits a PPO enzyme (i.e., PPO enzymatic activity). The present invention further relates to a method of controlling the growth of unwanted vegetation in a locus comprising plants having the modified PPO enzymes provided herein in the vicinity thereof, wherein the method comprises applying an effective amount of at least one PPO-inhibiting herbicide.

[0283] The term locus may include soil, seeds, seedlings, field, greenhouse, a cultivated area, as well as established vegetation.

[0284] Appropriately, the compound that inhibits a PPO enzyme is a herbicide. Appropriately therefore, the plants of the invention are resistant to a herbicide that inhibits the enzymatic activity of PPO (referred to herein as a PPO-inhibiting herbicide) and, therefore, such plants can be used in methods where these herbicides are applied. The herbicides can be applied pre-emergence or post-emergence of the crop plant or unwanted vegetation.

[0285] A plant having increased resistance to a compound that inhibits PPO enzyme activity (a PPO-inhibiting herbicide) may be referred to as a herbicide-tolerant or herbicide-resistant plant. Such plants are tolerant or at least partially resistant to at least one compound that inhibits PPO enzyme activity at a level that would normally kill or inhibit growth. Petition 870250095065, dated 10 / 17 / 2025, page 136 / 375 122 / 346 of, a normal, control or wild-type plant lacking the modified PPO enzymes, polynucleotides encoding said enzymes or expression vectors of the invention. The term herbicide is used herein to mean an active ingredient that kills, controls or otherwise adversely modifies plant growth and is typically used to control unwanted vegetation.

[0286] For example, the plants of the invention may have at least a 2-fold increase in resistance to a compound that inhibits the enzymatic activity of PPO, such as the inhibitor herbicides described herein. For example, the plants of the invention may have at least a 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold increase in resistance. In one embodiment, the plants of the invention comprising at least one of the modified PPO enzymes disclosed herein may have at least a 2-fold increase in resistance to any of the compounds A to E described herein, compared to an unmodified plant. In one embodiment, the plants of the invention comprising at least one of the disclosed modified PPO enzymes may have at least a 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold increase in resistance to any of the compounds A to E described herein, compared with an unmodified plant.In one embodiment, the plants of the invention comprising at least one of the disclosed modified PPO enzymes may have at least a 2-fold increase in resistance to any of the compounds A to E described herein, compared to a control plant. In another embodiment, the plants of the invention comprising at least one of the... Petition 870250095065, dated 10 / 17 / 2025, page 137 / 375 123 / 346 disclosed modified PPO enzymes may have at least a 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold increase in resistance to any of the compounds A to E described herein, compared to a control plant. For example, a control plant may be a plant comprising an alternative modified PPO enzyme to those of the invention. For example, a plant comprising a modified PPO having a sequence as presented in SEQ ID NO: 124.

[0287] Resistance to compounds that inhibit PPO enzymatic activity can be determined by any known methods for comparing the growth, damage, or other properties of two plants after application of the compound that inhibits PPO enzymatic activity to one plant. For example, the resistance of a plant of the invention can be determined by comparing the percentage of damage caused to the plant compared to a wild-type or control plant after application of a compound that inhibits PPO enzymatic activity, such as the herbicides described herein.

[0288] Enhanced resistance to a compound that inhibits the enzymatic activity of PPO (a PPO-inhibiting herbicide) refers in the context of the invention to a plant that has been modified to comprise a modified PPO enzyme that has improved or enhanced resistance to a compound that inhibits the enzymatic activity of PPO (the PPO-inhibiting herbicide), relative to an unmodified plant (e.g., wild type) or control plant (e.g., a plant comprising a PPO enzyme according to SEQ ID NO: 124). Suitably, the enhanced resistance may be due to increased activity of the modified PPO enzyme in the plant, in Petition 870250095065, dated 10 / 17 / 2025, page 138 / 375 124 / 346 comparison with the activity of such enzymes in an unmodified plant, when in the presence of at least one compound that is known to interfere with the enzymatic activity of PPO in plants at a concentration or level that is known to inhibit the activity of the wild-type PPO protein. Enhanced resistance means that the plant comprising the enzyme, and the modified PPO enzyme itself, is at least partially resistant to a compound that inhibits the enzymatic activity of PPO (the PPO-inhibiting herbicide). The partially resistant plants of the invention may still have some decreased PPO enzymatic activity when exposed to a compound that inhibits PPO enzymatic activity, such as a maximum decrease of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% in enzymatic activity.Therefore, the modified PPO enzymes used in the invention may be partially resistant and may even have some decrease in enzymatic activity when exposed to a compound that inhibits PPO enzymatic activity. In some embodiments, plants modified to comprise the modified PPO enzymes may have total or near-total resistance to a compound that inhibits PPO enzymatic activity (the PPO-inhibiting herbicide). Therefore, the plants may have 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% resistance to a compound that inhibits PPO enzymatic activity. Therefore, the modified PPO enzymes used in the invention may not have a substantial decrease in enzymatic activity when exposed to a compound that inhibits PPO enzymatic activity. Petition 870250095065, dated 10 / 17 / 2025, page 139 / 375 125 / 346

[0289] However, any decrease in activity is less than a decrease in activity relative to the activity of a wild-type enzyme when in the presence of at least one compound that is known to interfere with PPO enzymatic activity and at a concentration or level of the compound that is known to inhibit wild-type PPO protein activity. A decrease in activity seen for a partially resistant modified PPO enzyme may be a decrease in activity that does not have a negative effect on the growth, propagation, or development of a plant comprising a partially resistant modified PPO enzyme. Furthermore, the activity of such a partially resistant modified PPO enzyme may be referred to herein as herbicide-tolerant or herbicide-resistant enzymes.Plants that are at least partially resistant to at least one compound that inhibits PPO enzymatic activity exhibit few, if any, necrotic, lytic, chlorotic, or other lesions when subjected to the compound at concentrations and rates that are typically employed by the agricultural community to kill unwanted vegetation in the vicinity of the plant, such as a field.

[0290] The modified PPO enzymes described herein can be compared with an unmodified or wild-type reference enzyme, which may otherwise be referred to as a control enzyme or a plant comprising such an enzyme. The term wild type is used to refer to a nucleic acid or protein molecule that can be found in nature as distinct from being artificially produced or mutated by man. A reference or unmodified PPO enzyme may be a PPO-derived enzyme. Petition 870250095065, dated 10 / 17 / 2025, p. 140 / 375 126 / 346 of the same species of origin as the modified enzyme, which does not include any modifications of the invention as described herein. In some embodiments, the reference enzyme may be a wild-type enzyme. However, the use of the term unmodified or reference is not intended to necessarily 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. The reference or unmodified PPO enzymes as mentioned herein may well include other mutations or modifications that do not affect resistance to compounds that inhibit PPO enzymatic activity. For example, a reference enzyme may include mutations or modifications to enhance or alter the expression, translation, or targeting of the control enzyme to specific tissues, organs, or cells.

[0291] In some examples, the reference to the control enzyme (or plant including said enzyme) may be a PPO enzyme that includes alternative modifications that increase resistance to compounds that inhibit the enzymatic activity of PPO to those of the invention.

[0292] An unmodified wild-type reference PPO enzyme may be an enzyme encoded by SEQ ID NO: 1. An unmodified wild-type reference PPO enzyme may be a PPO enzyme encoded by SEQ ID NO: 2. A wild-type or control PPO enzyme may be an enzyme encoded by SEQ ID NO: 124.

[0293] Many herbicides that inhibit PPO enzymatic activity are known in the art and include a structurally diverse range of chemistries. Accordingly, the expert Petition 870250095065, dated 10 / 17 / 2025, p. 141 / 375 127 / 346 will understand that a wide range of PPO-inhibiting herbicides are useful in the present invention.

[0294] In one embodiment, the PPO-inhibiting herbicide is selected from the group consisting of butafenacil, carfentrazone-ethyl, cyclopyranil, epirifenacil (Herbicide A), flufenoximecil, flumioxazin, fomesafen, oxyfluorfen, piraflufen-ethyl, saflufenacil (Herbicide E), sulfentrazone, thiafenacil, trifludimoxazin (Herbicide B), compounds disclosed in WO2016 / 095768, for example ethyl ester of 3-(2-chloro-4-fluoro-5-(3-methyl-2,6-dioxo-4-trifluoromethyl-3,6-dihydropyrimidin-1(2H)-yl)phenyl)-5-methyl-4,5-dihydroisoxazole-5-carboxylic acid (Herbicide C), compounds described in WO2017 / 202768, for example 2—[[3—[[3 — chloro-5-fluoro-6-[3-methyl-2,6-dioxo-4(trifluoromethyl)pyrimidin-1-yl]-2-pyridi1]oxy]ethyl acetate, compounds disclosed in WO2020 / 239607, for example (5S)-3-[2-chloro-5-(3,5-dimethyl-2,6-dioxo-4-thioxo-1,3,5-triazinan-1-yl)-4-fluorophenyl]-5-methyl-4H-isoxazol-5-carboxylate ethyl (Herbicide D), compounds disclosed in WO2021 / 013799,for example pyridin-2-ylmethyl-[ (3-(2-chloro4-fluoro-5-[3-methyl-2,6-dioxo-4-(trifluoromethyl)-3, 6-dihidropyrimidin-l (2H)-yl]phenoxy)pyridine-2-yl)oxy]acetate, 2-Methoxyethyl-[(3-(2-chloro-4-fluoro-5-[3-methyl-2,6-dioxo-4(trifluoromethyl)-3,6-di-hidropyrimidin-l(2H)yl]phenoxy)pyridine-2-yl)oxy]acetate, 2-Methoxyethyl-[ (3 - (2cyan-4-fluoro-5-[3-methyl-2,6-dioxo-4-(trifluoromethyl)-3, 6di-hidro-pyrimidin-1(2H)-yl]phenoxy)pyridine-2yl)oxy]acetate and cyanomethyl-[(3-(2-bromo-4-fluoro-5-[3methyl-2,6-dioxo-4-(trifluoromethyl)-3,6-di-hydropyrimidin1(2H)-yl]phenoxy)pyridine-2-yl)oxy]acetate, compound, Petition 870250095065, 17 / 10 / 2025, pág. 142 / 375 128 / 346 disclosed in WO2021 / 143677, for example 3-[2-chloro-5-[3,6-dihydro-3-methyl-2,6-dioxo-4-(trifluoromethyl)-1(2H)pyrimidinyl]-4-fluorophenyl]-3a,4,5,6-tetrahydro-6-methyl6aH-cyclopent[d]isoxazol-6a-carboxylate methyl, compounds disclosed in WO2021 / 175689, for example 2—[2—[2—bromo-4-fluoro-5-[3-methyl-2,6-dioxo-4(trifluoromethyl)pyrimidin-1-yl]phenoxy]phenoxy]-2-methoxyacetate methyl, compounds disclosed in WO2022 / 138633, for example 1-[2-chloro-4-fluoro-5-[3-methyl-2,6-dioxo-4(trifluoromethyl)pyrimidin-lyl]phenoxy]cyclopropanecarboxylate of (2-ethoxy-2-oxo-ethyl) and compounds disclosed in WO2023 / 044364 including, where applicable, enantiomers (including enantiomer-enriched mixtures) of said compounds and, where applicable, agrochemically acceptable salts and / or esters of all previously mentioned compounds.

[0295] The compound that inhibits PPO enzymatic activity may be any combination of one or more such inhibitory compounds, suitably one or more of the compounds listed above. For example, 1, 2, 3, 4, 5 or more of the inhibitory compounds as described herein.

[0296] The plants of the invention, which include a modified PPO enzyme of the invention, can be used in methods of controlling unwanted vegetation in the vicinity of the plant. The methods may include applying an effective amount of at least one compound, such as those listed above, that inhibits the enzymatic activity of PPO to the unwanted vegetation and the plant.

[0297] Additionally, the plants of the invention, which include a modified PPO enzyme of the invention, can be used in Petition 870250095065, dated 10 / 17 / 2025, p. 143 / 375 129 / 346 methods of enhancing plant growth by controlling unwanted vegetation in the vicinity of the plant. The methods may include applying an effective amount of at least one compound, such as those listed above, that inhibits PPO enzymatic activity to the unwanted vegetation and the plant.

[0298] Suitablely, certain modified PPO enzymes may perform better when used with certain compounds that inhibit PPO enzymatic activity. Suitablely, certain modified PPO enzymes may provide better resistance to certain compounds that inhibit PPO enzymatic activity compared to other compounds that inhibit PPO enzymatic activity. Suitablely, therefore, certain combinations of PPO modifications and compounds may be ideal for use in the methods of the invention.

[0299] In one embodiment of such methods, the plant of the invention may comprise a PPO enzyme having a mutation at position 362 of SEQ ID NO: 1 or a residue corresponding thereto and is for use with any PPO-inhibiting herbicide disclosed herein, including for example compound D.In one embodiment of such methods, the plant of the invention may comprise a PPO enzyme having a mutation at position 362 of SEQ ID NO: 1 or a residue corresponding thereto and is for use with any PPO inhibitor herbicide disclosed herein, including for example compound B. In one embodiment of such methods, the plant of the invention may comprise a PPO enzyme having a mutation at position 365 of SEQ ID NO: 1 or a residue corresponding thereto and is for use with any PPO inhibitor herbicide disclosed herein, including for example compound B. In one embodiment of such methods, a. Petition 870250095065, dated 10 / 17 / 2025, p. 144 / 375 130 / 346 The plant of the invention may comprise a PPO enzyme having a mutation at position 365 of SEQ ID NO: 1 or a residue corresponding thereto and is for use with any PPO inhibitor herbicide disclosed herein, including for example compound E. In one embodiment of such methods, the plant of the invention may comprise a PPO enzyme having a mutation at position 479 of SEQ ID NO: 1 or a residue corresponding thereto and is for use with any PPO inhibitor herbicide disclosed herein, including for example compound B. In one embodiment of such methods, the plant of the invention may comprise a PPO enzyme having a mutation at position 479 of SEQ ID NO: 1 or a residue corresponding thereto and is for use with any PPO inhibitor herbicide disclosed herein, including for example compound D.In one embodiment of such methods, the plant of the invention may comprise a PPO enzyme having a mutation at position 479 of SEQ ID NO: 1 or a residue corresponding thereto and is for use with any PPO inhibitor herbicide disclosed herein, including for example compound E.

[0300] In one embodiment of such methods, the plant of the invention may comprise a PPO enzyme having a mutation at positions 305 and 426 of SEQ ID NO: 1 or corresponding residues thereto, wherein the mutation at position 305 is S305L and the mutation at position 426 is Y426V or corresponding residues thereto (i.e., SEQ ID NO 125 or 139) and is for use with any PPO inhibitor herbicide disclosed herein, including for example compound A. In one embodiment of such methods, the plant of the invention may comprise a PPO enzyme having a mutation at positions 305 and 426 of SEQ ID NO: 1 or corresponding residues thereto, wherein the mutation at position 305 is Petition 870250095065, dated 10 / 17 / 2025, p. 145 / 375 131 / 346 S305L and at position 426 is Y426V or corresponding residues thereof and is for use with any PPO inhibitor herbicide disclosed herein, including, for example, compound C. In one embodiment of such methods, the plant of the invention may comprise a PPO enzyme having a mutation at positions 305 and 426 of SEQ ID NO: 1 or corresponding residues thereof, wherein the mutation at position 305 is S305L and at position 426 is Y426V or corresponding residues thereof and is for use with any PPO inhibitor herbicide disclosed herein, including, for example, compound D. In another embodiment of such methods, the plant of the invention may comprise a PPO enzyme having a mutation at positions 305 and 426 of SEQ ID NO: 1 or corresponding residues thereof wherein the mutation at position 305 is S305L and at position 426 is Y426V or corresponding residues thereof and is for use with any PPO inhibitor herbicide disclosed herein, including, for example, compound D. For example, compound E.

[0301] In one embodiment of such methods, the plant of the invention may comprise a PPO enzyme having a mutation at positions 365, 426 and 479 of SEQ ID NO: 1 or residues corresponding thereto (e.g., SEQ ID NO 126) and is for use with any PPO inhibitor herbicide disclosed herein, including for example compound A. In one embodiment of such methods, the plant of the invention may comprise a PPO enzyme having a mutation at positions 365, 426 and 479 of SEQ ID NO: 1 or residues corresponding thereto and is for use with any PPO inhibitor herbicide disclosed herein, including for example compound C. In one embodiment of such methods, the plant of the invention may comprise a PPO enzyme having a mutation at positions 365, 426 and 479 of Petition 870250095065, dated 10 / 17 / 2025, p. 146 / 375 132 / 346 SEQ ID NO: 1 or residues corresponding thereto and is for use with any PPO inhibitor herbicide disclosed herein, including for example compound E. In one embodiment of such methods, the plant of the invention may comprise a PPO enzyme having a mutation at positions 365, 426 and 479 of SEQ ID NO: 1 or residues corresponding thereto and is for use with any PPO inhibitor herbicide disclosed herein, including for example compound E.

[0302] In one embodiment of such methods, the plant of the invention may comprise a PPO enzyme having a mutation at positions 305, 404, 426, 431 and 479 of SEQ ID NO: 1 or residues corresponding thereto (e.g., SEQ ID NO 127) and is for use with any PPO inhibitor herbicide disclosed herein, including for example compound A. In one embodiment of such methods, the plant of the invention may comprise a PPO enzyme having a mutation at positions 305, 404, 426, 431 and 479 of SEQ ID NO: 1 or residues corresponding thereto and is for use with any PPO inhibitor herbicide disclosed herein, including for example compound C. In one embodiment of such methods, the plant of the invention may comprise a PPO enzyme having a mutation at positions 305, 404, 426, 431 and 479 of SEQ ID NO: 1 or residues corresponding thereto and It is for use with any PPO-inhibiting herbicide disclosed here, including for example compound E.In one embodiment of such methods, the plant of the invention may comprise a PPO enzyme having a mutation at positions 305, 404, 426, 431 and 479 of SEQ ID NO: 1 or residues corresponding thereto and is for use with any PPO inhibitor herbicide disclosed herein, including for example compound E. Petition 870250095065, dated 10 / 17 / 2025, p. 147 / 375 133 / 346

[0303] In one embodiment of such methods, the plant of the invention may comprise a PPO enzyme having a mutation at positions 305 and 365 of SEQ ID NO: 1 or residues corresponding thereto (e.g., SEQ ID NO 128) and is for use with any PPO inhibitor herbicide disclosed herein, including for example compound C. In one embodiment of such methods, the plant of the invention may comprise a PPO enzyme having a mutation at positions 305 and 365 of SEQ ID NO: 1 or residues corresponding thereto and is for use with any PPO inhibitor herbicide disclosed herein, including for example compound E. In one embodiment of such methods, the plant of the invention may comprise a PPO enzyme having a mutation at positions 305 and 365 of SEQ ID NO: 1 or residues corresponding thereto and is for use with any PPO inhibitor herbicide disclosed herein, including for example compound E.

[0304] In one embodiment of such methods, the plant of the invention may comprise a PPO enzyme having a mutation at positions 305, 362 and 404 of SEQ ID NO: 1 or residues corresponding thereto (e.g., SEQ ID NO 129) and is for use with any PPO inhibitor herbicide disclosed herein, including for example compound A. In one embodiment of such methods, the plant of the invention may comprise a PPO enzyme having a mutation at positions 305, 362 and 404 of SEQ ID NO: 1 or residues corresponding thereto and is for use with any PPO inhibitor herbicide disclosed herein, including for example compound B. In one embodiment of such methods, the plant of the invention may comprise a PPO enzyme having a mutation at positions 305, 362 and 404 of SEQ ID NO: 1 or residues corresponding thereto and is for Petition 870250095065, dated 10 / 17 / 2025, p. 148 / 375 134 / 346 use with any PPO-inhibiting herbicide disclosed herein, including for example compound C. In one embodiment of such methods, the plant of the invention may comprise a PPO enzyme having a mutation at positions 305, 362 and 404 of SEQ ID NO: 1 or residues corresponding thereto and is for use with any PPO-inhibiting herbicide disclosed herein, including for example compound E. In one embodiment of such methods, the plant of the invention may comprise a PPO enzyme having a mutation at positions 305, 362 and 404 of SEQ ID NO: 1 or residues corresponding thereto and is for use with any PPO-inhibiting herbicide disclosed herein, including for example compound E.

[0305] In one embodiment of such methods, the plant of the invention may comprise a PPO enzyme having a mutation at positions 305, 361, 365, 431 and 479 of SEQ ID NO: 1 or residues corresponding thereto (e.g., SEQ ID NO 130) and is for use with any PPO inhibitor herbicide disclosed herein, including for example compound A. In one embodiment of such methods, the plant of the invention may comprise a PPO enzyme having a mutation at positions 305, 361, 365, 431 and 479 of SEQ ID NO: 1 or residues corresponding thereto and is for use with any PPO inhibitor herbicide disclosed herein, including for example compound B. In one embodiment of such methods, the plant of the invention may comprise a PPO enzyme having a mutation at positions 305, 361, 365, 431 and 479 of SEQ ID NO: 1 or residues corresponding to the same and is for use with any PPO-inhibiting herbicide disclosed here, including for example compound E.In one embodiment of such methods, the plant of the invention may comprise a PPO enzyme having a mutation at positions 305, 361, 365, 431. Petition 870250095065, dated 10 / 17 / 2025, p. 149 / 375 135 / 346 and 479 of SEQ ID NO: 1 or residues corresponding to them and is for use with any PPO inhibitor herbicide disclosed herein, including for example compound E.

[0306] In one embodiment of such methods, the plant of the invention may comprise a PPO enzyme having a mutation at positions 305, 426 and 479 of SEQ ID NO: 1 or residues corresponding thereto (e.g., SEQ ID NO 131) and is for use with any PPO inhibitor herbicide disclosed herein, including for example compound A. In one embodiment of such methods, the plant of the invention may comprise a PPO enzyme having a mutation at positions 305, 426 and 479 of SEQ ID NO: 1 or residues corresponding thereto and is for use with any PPO inhibitor herbicide disclosed herein, including for example compound B. In one embodiment of such methods, the plant of the invention may comprise a PPO enzyme having a mutation at positions 305, 426 and 479 of SEQ ID NO: 1 or residues corresponding thereto and is for use with any PPO inhibitor herbicide disclosed herein, including for example compound C.In one embodiment of such methods, the plant of the invention may comprise a PPO enzyme having a mutation at positions 305, 426 and 479 of SEQ ID NO: 1 or residues corresponding thereto and is for use with any PPO inhibitor herbicide disclosed herein, including for example compound E. Petition 870250095065, dated 10 / 17 / 2025, p. 150 / 375 136 / 346

[0307] In one embodiment of such methods, the plant of the invention may comprise a PPO enzyme having a mutation at positions 426, 461 and 479 of SEQ ID NO: 1 or residues corresponding thereto (e.g., SEQ ID NO 132) and is for use with any PPO inhibitor herbicide disclosed herein, including for example compound A. In one embodiment of such methods, the plant of the invention may comprise a PPO enzyme having a mutation at positions 426, 461 and 479 of SEQ ID NO: 1 or residues corresponding thereto and is for use with any PPO inhibitor herbicide disclosed herein, including for example compound B. In one embodiment of such methods, the plant of the invention may comprise a PPO enzyme having a mutation at positions 426, 461 and 479 of SEQ ID NO: 1 or residues corresponding thereto and is for use with any PPO inhibitor herbicide disclosed herein, including for example compound C.In one embodiment of such methods, the plant of the invention may comprise a PPO enzyme having a mutation at positions 426, 461 and 479 of SEQ ID NO: 1 or residues corresponding thereto and is for use with any PPO inhibitor herbicide disclosed herein, including for example compound E.

[0308] In one embodiment of such methods, the plant of the invention may comprise a PPO enzyme having a mutation at positions 305, 361 and 365 of SEQ ID NO: 1 or residues corresponding thereto (e.g., SEQ ID NO 133) and is for use with any PPO inhibitor herbicide disclosed herein, including for example compound A. In one embodiment of such methods, the plant of the invention may comprise a PPO enzyme having a mutation at positions 305, 361 and 365 of SEQ ID NO: 1 or residues corresponding thereto and is for Petition 870250095065, dated 10 / 17 / 2025, p. 151 / 375 137 / 346 use with any PPO-inhibiting herbicide disclosed herein, including for example compound B. In one embodiment of such methods, the plant of the invention may comprise a PPO enzyme having a mutation at positions 305, 361 and 365 of SEQ ID NO: 1 or residues corresponding thereto and is for use with any PPO-inhibiting herbicide disclosed herein, including for example compound C. In one embodiment of such methods, the plant of the invention may comprise a PPO enzyme having a mutation at positions 305, 361 and 365 of SEQ ID NO: 1 or residues corresponding thereto and is for use with any PPO-inhibiting herbicide disclosed herein, including for example compound E. In one embodiment of such methods, the plant of the invention may comprise a PPO enzyme having a mutation at positions 305, 361 and 365 of SEQ ID NO: 1 or residues corresponding thereto and is for use with any PPO-inhibiting herbicide disclosed herein, including for example compound E.

[0309] In one embodiment of such methods, the plant of the invention may comprise a PPO enzyme having a mutation at positions 305, 361, 362, 426 and 479 of SEQ ID NO: 1 or residues corresponding thereto (e.g., SEQ ID NO 134) and is for use with any PPO inhibitor herbicide disclosed herein, including for example compound A. In one embodiment of such methods, the plant of the invention may comprise a PPO enzyme having a mutation at positions 305, 361, 362, 426 and 479 of SEQ ID NO: 1 or residues corresponding thereto and is for use with any PPO inhibitor herbicide disclosed herein, including for example compound C. In one embodiment of such methods, the plant of the invention may comprise a PPO enzyme having a mutation at positions 305, 361, 362, 426 Petition 870250095065, dated 10 / 17 / 2025, p. 152 / 375 138 / 346 and 479 of SEQ ID NO: 1 or residues corresponding thereto and is for use with any PPO inhibitor herbicide disclosed herein, including for example compound E. In one embodiment of such methods, the plant of the invention may comprise a PPO enzyme having a mutation at positions 305, 361, 362, 426 and 479 of SEQ ID NO: 1 or residues corresponding thereto and is for use with any PPO inhibitor herbicide disclosed herein, including for example compound E.

[0310] In one embodiment of such methods, the plant of the invention may comprise a PPO enzyme having a mutation at positions 361 and 365 of SEQ ID NO: 1 or residues corresponding thereto (e.g., SEQ ID NO 135) and is for use with any PPO inhibitor herbicide disclosed herein, including for example compound C. In one embodiment of such methods, the plant of the invention may comprise a PPO enzyme having a mutation at positions 361 and 365 of SEQ ID NO: 1 or residues corresponding thereto and is for use with any PPO inhibitor herbicide disclosed herein, including for example compound B. In one embodiment of such methods, the plant of the invention may comprise a PPO enzyme having a mutation at positions 361 and 365 of SEQ ID NO: 1 or residues corresponding thereto and is for use with any PPO inhibitor herbicide disclosed herein, including for example compound E.In one embodiment of such methods, the plant of the invention may comprise a PPO enzyme having a mutation at positions 361 and 365 of SEQ ID NO: 1 or residues corresponding thereto and is for use with any PPO inhibitor herbicide disclosed herein, including for example compound E. Petition 870250095065, dated 10 / 17 / 2025, p. 153 / 375 139 / 346

[0311] In one embodiment of such methods, the plant of the invention may comprise a PPO enzyme having a mutation at positions 365, 404 and 479 of SEQ ID NO: 1 or residues corresponding thereto (e.g., SEQ ID NO 136) and is for use with any PPO inhibitor herbicide disclosed herein, including for example compound B. In one embodiment of such methods, the plant of the invention may comprise a PPO enzyme having a mutation at positions 365, 404 and 479 of SEQ ID NO: 1 or residues corresponding thereto and is for use with any PPO inhibitor herbicide disclosed herein, including for example compound C. In one embodiment of such methods, the plant of the invention may comprise a PPO enzyme having a mutation at positions 365, 404 and 479 of SEQ ID NO: 1 or residues corresponding thereto and is for use with any PPO inhibitor herbicide disclosed herein, including for example compound E.In one embodiment of such methods, the plant of the invention may comprise a PPO enzyme having a mutation at positions 365, 404 and 479 of SEQ ID NO: 1 or residues corresponding thereto and is for use with any PPO inhibitor herbicide disclosed herein, including for example compound E.

[0312] In one embodiment of such methods, the plant of the invention may comprise a PPO enzyme having a mutation at positions 305, 404 and 479 of SEQ ID NO: 1 or residues corresponding thereto (e.g., SEQ ID NO 137) and is for use with any PPO inhibitor herbicide disclosed herein, including for example compound A. In one embodiment of such methods, the plant of the invention may comprise a PPO enzyme having a mutation at positions 365, 404 and 479 of SEQ ID NO: 1 or residues corresponding thereto and is for Petition 870250095065, dated 10 / 17 / 2025, p. 154 / 375 140 / 346 use with any PPO-inhibiting herbicide disclosed herein, including for example compound B. In one embodiment of such methods, the plant of the invention may comprise a PPO enzyme having a mutation at positions 365, 404 and 479 of SEQ ID NO: 1 or residues corresponding thereto and is for use with any PPO-inhibiting herbicide disclosed herein, including for example compound C. In one embodiment of such methods, the plant of the invention may comprise a PPO enzyme having a mutation at positions 365, 404 and 479 of SEQ ID NO: 1 or residues corresponding thereto and is for use with any PPO-inhibiting herbicide disclosed herein, including for example compound E. In one embodiment of such methods, the plant of the invention may comprise a PPO enzyme having a mutation at positions 365, 404 and 479 of SEQ ID NO: 1 or residues corresponding thereto and is for use with any PPO-inhibiting herbicide disclosed herein, including for example compound E.

[0313] Undesirable vegetation may include, for example, dicotyledonous and monocotyledonous weeds. Dicotyledonous weeds include, but are not limited to, weeds of the genera: Sinapis, Lepidium, Galium, Slellaria, Matricaria, Anthemis, Galinsoga, Chenopodium, Urtica, Senecio, Amaranthus, Portulaca, Xanthium, Convolvulus, Ipomoea, Polygonum, Sesbania, Ambrosia, Cirsium, Carduus, Sonchus, Solanum, Rorippa, Rotala, Lindernia, Lamium, Veronica, Abutilon, Emex, Datura, Viola, Galeopsis, Papaver, Centaurea, Trifolium, Ranunculus and Taraxacum. Monocotyledonous weeds include, but are not limited to, weeds from the genera: Echinochloa, Setaria, Panicum, Digitaria, Phleum, Poa, Petition 870250095065, dated 10 / 17 / 2025, page 155 / 375 141 / 346 Festuca, Eleusine, Brachiaria, Lolium, Bromus, Avena, Cyperus, Sorghum, Agropyron, Cynodon, Monochoria, Fimbristyslis, Sagittaria, Eleocharis, Scirpus, Paspalum, Ischaemum, Sphenoclea, Dactyloctenium, Agrostis, Alopecurus and Apera.

[0314] Additionally, unwanted vegetation may include, for example, crop plants that are growing in an unwanted location. For example, a volunteer more plant that is in a field that predominantly comprises soybean plants may be considered a weed if the more plant is unwanted in the soybean field.

[0315] An effective amount or effective concentration refers to an amount and concentration, respectively, of a compound that inhibits the enzymatic activity of PPO (a PPO-inhibiting herbicide), which is sufficient to kill or inhibit the growth of a wild-type and / or unwanted plant, plant tissue, plant cell, microspore, or host cell, but which said amount does not so severely kill or inhibit the growth of plants, parts thereof, plant tissues, plant cells, and seeds having the modified PPOs described herein. Typically, the effective amount is an amount that is routinely used in agricultural production systems to kill unwanted vegetation of interest. Such an amount is known to those skilled in the art or can be readily determined using methods known in the art.Furthermore, it is recognized that the effective quantity in an agricultural production system could be substantially different from an effective quantity for a cropping system such as, for example. Petition 870250095065, dated 10 / 17 / 2025, page 156 / 375 142 / 346 For example, the microspore culture system. Thus, the quantity may be small enough to simply retard or suppress the growth or development of a given weed or unwanted vegetation, or the quantity may be large enough to irreversibly destroy a given weed or unwanted vegetation. Additionally, the quantity may be any quantity in between.

[0316] An effective quantity can be at least 1 gram of active compound per hectare (g of ai / ha). For example, a compound that inhibits the enzymatic activity of PPO can be applied at a concentration of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 405, 410, 415, 420, 425, 430, 435, 440, 445, 450, 455, 460, 465, 470, 475, 480, 485, 490, 495, 500 grams of active compound per hectare.

[0317] It should be understood that in the methods mentioned above the compound can be applied to the vicinity of the plant pre-emergence of the crop and / or post-emergence of the crop - a so-called over-the-top application.

[0318] Pre-emergent refers to a compound that is applied to the vicinity of at least partially resistant plant of the invention (e.g., a field or cultivated area) before the plant visibly emerges from the soil and / or before seed germination. Post-emergent refers to a Petition 870250095065, dated 10 / 17 / 2025, p. 157 / 375 143 / 346 compound that is applied to the vicinity of at least partially resistant plant of the invention after a plant has visibly emerged from the soil. In some cases, the terms pre-emergent and post-emergent are used with reference to a weed or unwanted vegetation in the vicinity of at least partially resistant plant of the invention and, in some cases, these terms are used with reference to a crop plant in the vicinity of at least partially resistant plant of the invention. When used with reference to a weed or unwanted vegetation, these terms may only be applied to a particular type of weed or species of weed or unwanted vegetation that is present or believed to be present in the area of ​​interest.Although any compound that inhibits PPO enzymatic activity can be applied in a pre-emergent and / or post-emergent treatment, some such compounds are known to be more effective in controlling a weed or unwanted plants when applied pre-emergence or post-emergence. The compound can be applied by pre-planting incorporation, which involves incorporating the compound into the soil before planting.

[0319] Application rates of a compound that inhibits PPO enzymatic activity can vary within wide limits and depend on the nature of the soil, the application method (pre-emergence; post-emergence; seed furrow application; no soil treatment application, etc.), the plant, the unwanted vegetation to be controlled, the prevailing climatic conditions and other factors governed by the application method, the timing of Petition 870250095065, dated 10 / 17 / 2025, page 158 / 375 144 / 346 application and by the target plant. The compound that inhibits the enzymatic activity of PPO can be applied at a rate of at least 1 L / ha. For example, at least 1, 2, 3, 4, 5, 6, R. 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, R. 360, 365, 370, 375, 380, 385, 390, 395, 400, 405, 410, 415, 420, 425, 430, 435, 440, 445, 450, 455, 460, 465, 470, 475, 480, 485, 490, 495, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900, 1950, 2000 L / ha. In some examples, the compound may be applied at a rate 200 L / ha.

[0320] Application is usually done by spraying the compound, typically by tractor-mounted sprayer for large areas, but other methods such as dusting (for powders), drip irrigation or irrigation can also be used.

[0321] As discussed elsewhere herein, plants and plant parts and seeds are provided having stably incorporated into their genome a modified PPO provided herein, as well as an additional polynucleotide conferring tolerance to at least one additional herbicide, including, for example, herbicides comprising glyphosate, glufosinate, HPPD herbicides and others. As such, the application of an effective amount of a PPO-inhibiting herbicide can be combined with the application of other Petition 870250095065, dated 10 / 17 / 2025, page 159 / 375 145 / 346 herbicides to which the crop is naturally tolerant or to which the crop is resistant through the expression of one or more modified herbicide resistance genes.

[0322] One or more other herbicides, which are applied in combination with the PPO inhibitor herbicide, may be applied sequentially (e.g., in succession) or simultaneously without affecting the yield or growth of the herbicide-resistant plant. This includes applying one or more other herbicides according to a schedule based on the application of the PPO inhibitor herbicide. When applied simultaneously, an effective amount of each herbicide is applied to the plant at the same time (e.g., at the same plant growth stage and / or as constituents of a herbicide composition comprising one or more herbicides and the HPPD inhibitor herbicide). When applied sequentially, an effective amount of each herbicide is applied in succession to the plant.

[0323] According to the present invention, enhancing plant growth means improving plant vigor, improving plant quality, improving tolerance to stress factors and / or improving resource use efficiency.

[0324] The invention described herein also relates to a case comprising a container and instructions for use, the container comprising a compound that inhibits the enzymatic activity of PPO, and the instructions comprising directions for applying the compound to a modified plant comprising a modified PPO enzyme that provides the plant or part thereof with increased resistance to said compound. Petition 870250095065, dated 10 / 17 / 2025, page 160 / 375 146 / 346

[0325] Appropriately, the compound may be any of those as defined above. Appropriately, the direction to apply the compound may comprise the direction to apply the compound in an effective amount as defined above. Appropriately, the direction to apply the compound may comprise the direction to apply the compound at a particular rate as described above. Appropriately, the direction to apply the compound may comprise the direction to apply the compound by a particular method, such as by spraying, as defined above. Appropriately, the direction to apply the compound may comprise the direction to apply the compound at a particular time, such as pre-emergence of the crop and / or post-emergence of the crop, or during a particular season or month.

[0326] Appropriately, the instructions may additionally include directions for preparing the compound so that it can be applied in the desired effective quantity and by the desired method. Appropriately, such directions may include directions for diluting the compound appropriately in a solvent, such as water, appropriately to an effective concentration.

[0327] Compositions are further provided comprising a modified PPO enzyme provided herein (as any of the SEQ ID Nos: 1, 2, 4 - 151, 153 302 or 305 - 336 or SEQ ID Nos: 37- 39, 58, 59, 97 - 99, 118, 119, 125 - 137, 139 - 151, 188 - 190, 209, 210, 248 250, 269, 270, 277 - 288 or 291 - 302 or an active variant thereof) and a PPO inhibitor herbicide, including but not limited to the herbicides disclosed herein as herbicide A, B, C, D or E. Screening Methods Petition 870250095065, dated 10 / 17 / 2025, pp. 161 / 375 147 / 346

[0328] Several screening methods are additionally provided for plants having increased resistance to a compound that inhibits PPO enzymatic activity, for the modified PPO enzyme comprising increased resistance to a compound that inhibits PPO enzymatic activity, and also for compounds that inhibit PPO enzymatic activity.

[0329] In one aspect of the invention, a method is provided for selecting a plant or organism (including, for example, a prokaryotic organism such as a bacterium) having increased resistance to a compound that inhibits PPO enzymatic activity compared to an unmodified plant or organism, comprising: (a) providing a plant or organism; (b) optionally mutagenizing the plant or organism; (c) exposing the plant or organism to an effective amount of a compound that inhibits PPO enzymatic activity; and (d) selecting the plant or organism if the plant or organism exhibits resistance to the compound.

[0330] In one embodiment, the plant may be a plant according to the first aspect of the invention and may comprise any of the features defined in relation to such a plant described herein.

[0331] Appropriately, step (b) may comprise mutagenizing the plant or organism by any known means such as EMS or other chemical treatment or by X-rays or other radiation treatment. Appropriately to induce modifications, appropriately one or more mutations, in the PPO gene.

[0332] Properly following step (b), the plant comprises one or more modifications to the endogenous PPO gene or Petition 870250095065, dated 10 / 17 / 2025, p. 162 / 375 148 / 346 heterologous, suitably one or more mutations to the endogenous or heterologous PPO gene. Suitably, the modified PPO gene may encode a modified PPO enzyme, suitably as described herein. Suitably therefore, after step (b), the plant or organism may comprise a modified PPO enzyme as described herein, suitably having one or more mutations as described herein.

[0333] Appropriately, step (c) comprises exposing the plant or organism to an effective amount of a compound as described above. Appropriately to determine whether one or more modifications to the endogenous or heterologous PPO gene, and the resulting modified PPO enzyme created in step (b), confer resistance to the compound.

[0334] Appropriately selecting the plant or organism if the plant or organism exhibits resistance to the compound may comprise, for example, for plants selecting those that do not exhibit any signs of PPO deficiency. Appropriately, such signs may include stunted growth, wilting, necrosis, discoloration and the like. For organisms, such as bacteria, resistance to the compound may comprise the ability to grow in the presence of the herbicide.

[0335] A method for identifying a modified PPO enzyme is further provided comprising increased resistance to a compound that inhibits the enzymatic activity of PPO, comprising: (a) generating a library of modified PPO-encoding polynucleotides; (b) screening a population of the resulting modified PPO-encoding polynucleotides by expression of each of said polynucleotides in a bacterium, a plant or a part of Petition 870250095065, dated 10 / 17 / 2025, page 163 / 375 149 / 346 plant and expose the bacterium, plant or part thereof to an effective amount of a compound that inhibits the enzymatic activity of PPO; (c) select modified PPO-encoding polynucleotides that provide the bacterium, plant or part thereof with increased resistance to said compound compared with a reference bacterium, plant or part thereof containing an unmodified or control PPO-encoding polynucleotide.

[0336] Appropriately, generating a library of modified PPO-encoding polynucleotides can be carried out by any known technique for generating a gene library comprising different modifications, appropriately mutations along the gene sequence. Appropriately, these methods can be random or directed. Such methods may include a step of exposing the PPO-encoding polynucleotides to a mutagen, such as a chemical or radiation, or carrying out error-prone PCR on the PPO-encoding polynucleotides, for example. Other molecular techniques may include performing DNA scrambling or staggered extension processes on PPO-encoding polynucleotides.

[0337] Properly expressing the modified polynucleotides in a bacterium, a plant or a plant part, such that the modified PPO enzymes are expressed, comprises transforming a bacterium, a plant or a plant part with each of the modified polynucleotides. Suitable transformation methods are described elsewhere herein, as are suitable constructs for expression of a PPO-encoding polynucleotide. Petition 870250095065, dated 10 / 17 / 2025, page 164 / 375 150 / 346

[0338] Properly exposing the bacterium, plant or part thereof to an effective amount of a compound that inhibits PPO enzymatic activity comprises using an effective amount as described above of a suitable compound as described above.

[0339] Appropriately selecting modified PPO-encoding polynucleotides that provide the same bacterium, plant, or plant part with increased resistance to said compound comprises selecting those bacteria, plants, or plant parts of the same hosts that exhibit resistance to the compound, appropriately selecting those that do not exhibit any signs of PPO deficiency. Appropriately, such signs in plants may include stunted growth, wilting, necrosis, discoloration, and the like. Appropriately, such signs in bacteria may include stunted growth or death. In one embodiment, the sign is reduced growth compared to an unmodified or control bacterium or unmodified or control plant.

[0340] A method is also provided for identifying a compound that inhibits PPO enzymatic activity comprising: (a) generating a modified plant or part thereof as described herein, (b) applying a test compound to the plant or part thereof from step (a) and to an unmodified reference plant; (c) selecting the test compounds that confer reduced growth to the unmodified reference plant compared with the growth of the modified plant or part thereof.

[0341] In one embodiment, the modified plant or plant part is a plant having increased resistance to Petition 870250095065, dated 10 / 17 / 2025, pp. 165 / 375 151 / 346 compounds that inhibit the enzymatic activity of PPO, as defined according to the first aspect of the invention, and may comprise any of the features defined in relation to such plant mentioned herein.

[0342] Appropriately, the test compound may be any compound that may have, or is expected to have, an inhibitory effect on a plant PPO enzyme. Appropriately, the test compound may be related to, derived from, or synthesized from, a compound as defined herein, which may be a known compound that inhibits PPO enzymatic activity, such as a known herbicidal compound.

[0343] Suitablely, the test compound may be applied in a test quantity, suitably the test quantity may be the same as any known effective quantity for such herbicidal compounds, suitable quantities are defined beforehand.

[0344] In another aspect of the invention, one or more compounds identified by such a method are provided, optionally for use as herbicides. FIGURES The invention will now be described with reference to the following figures in which:

[0345] Figure 1 shows a plasmid map of the pBin TMV AtPPOL transformation vector. EXAMPLES Example 1 - Expression and assay of a library of Arabidopsis thaliana PPO1 site saturation variants in E. coli Petition 870250095065, dated 10 / 17 / 2025, page 166 / 375 152 / 346

[0346] A pET24a plasmid containing a DNA sequence encoding an Arabidopsis thaliana-derived PPO1 variant (AtPPOl), with codon optimization for expression in E. coli and under the control of a T7 promoter, was synthesized by Twist Bioscience (SEQ ID NO:3). This plasmid was used as the basis of a Site Saturation Variant Library (SSVL) that replaced codons at 352 amino acid positions with codons encoding one of the other 19 amino acids or removed the codon completely, resulting in a translation product with a deletion at that position. The 352 positions that were mutated are listed in Table 1. Tabela 1: Positions of amino acids from AtPPOl mutadas com referência to SEQ ID NO 1 161 L131 R2 0 0 P271 L357 Y407 P463 E526 V62 K132 G203 Q272 S358 S408 L464 V527 G63 D133 D204 G273 K359 S409 K465 G64 D134 E205 Q274 L360 S410 L4 6 6 G65 L135 V2 0 6 T275 Y361 L411 G467 16 6 V136 F207 V2 7 6 Y362 F412 V468 S67 L137 E208 G277 P363 P413 R4 6 9 G68 G138 R2 0 9 S278 P364 N414 V4 7 0 L69 D139 L210 F279 V365 R415 W471 C70 P140 1211 R2 8 0 A3 6 6 A416 P472 171 T141 E212 K2 81 A367 P417 Q473 A72 A142 P213 G282 V368 P418 A4 7 4 Q73 P143 F214 L283 S369 G419 1475 A7 4 R144 0215 R2 8 4 1370 R420 P476 V8 8 F145 S216 M2 8 5 S371 1421 Q477 T89 V146 G217 L286 Y372 L422 F478 Petition: 870250095065, on 10 / 17 / 2025, page. 167 / 375 153 / 346 E90 L147 V218 P287 P373 L423 L479 A91 W148 Y219 E288 K374 L424 V4 8 0 K92 N149 A220 A2 8 9 E375 N425 G481 D93 G150 G221 1290 A376 Y426 H482 R94 K151 D222 S291 1377 1427 F483 V95 L152 P223 A2 92 R378 G428 D484 G96 R153 S224 R2 93 T379 G429 1485 G97 P154 K225 L294 E380 S430 L486 N98 V155 L226 V298 C381 T431 D487 199 P156 S227 K2 9 9 L382 N432 T488 1100 S157 M228 L300 1383 T433 A4 8 9 T101 K158 K229 S301 D384 G434 L503 R102 L159 A2 3 0 W302 G385 1435 G504 E103 T160 A2 31 K303 E386 L436 G505 F107 D161 F232 L304 L387 S437 N5 0 6 L108 L162 G233 S305 K388 E442 Y507 W109 P163 K234 E320 G389 L443 V5 0 8 E110 F164 V235 T321 F390 A4 4 6 A5 0 9 Elll F165 W2 3 6 V333 G391 V4 4 7 G510 G112 Dl 6 6 L238 V334 Q392 D448 V511 P113 L167 1246 M335 L393 R4 4 9 A512 N114 M168 G248 T336 H394 D450 L513 S115 S169 T249 V337 P395 L451 G514 F116 G172 F250 P338 R396 R452 R515 Q117 K173 K2 51 S339 T397 K453 C516 P118 A176 A252 H340 Q398 M4 54 V517 S119 G179 1253 V341 G399 L455 E518 D120 S193 Q254 A342 V4 0 0 1456G519 P121 V194 D265 S343 E401 K457 A520 Petition 870250095065, dated 10 / 17 / 2025, page 168 / 375 154 / 346 M122 E195 P2 6 6 G344 T402 P458 Y521 L123 E196 R2 67 A353 L403 N459 E522 T124 F197 L268 A354 G404 S460 T523 V126 V198 P269 N355 T405 T461 A524 V127 R199 K2 7 0 A356 1406 D462 1525

[0347] SSVL was batch-transformed by electroporation into a BL21 (DE3) hemG strain of E. coli with the hemG gene substituted by CmR conferring chloramphenicol resistance and spread in LA supplemented with kanamycin at 50 pg / mL, chloramphenicol at 25 pg / mL, and hematin at 20 pg / mL. Each batch consisted of all plasmids containing variants at two loci. Batch-transformed colonies were inoculated into 500 pL of Terrific broth supplemented with kanamycin at 50 pg / mL, chloramphenicol at 25 pg / mL, and hematin at 20 pg / mL in 96-well deep blocks. Cultures were grown for 24 hours at 37 °C with agitation at 250 rpm. The overnight cultures were centrifuged and the cell pellets resuspended with 500 µL of Terrific broth.Resuspended cultures were used at a ratio of 1:100 to inoculate a fresh 96-well deep block containing 500 pL of Terrific broth supplemented with 0.1 mM IPTG, 50 pg / mL kanamycin, 25 pg / mL chloramphenicol, and 6.25 ppm of herbicide D or E, or 0.78 ppm of herbicide B, or DMSO. Assay cultures were grown for 24 hours at 37 °C with agitation at 250 rpm, with OD600 measured 24 hours post-inoculation. Strains that grew in both DMSO and herbicide were determined to be herbicide-tolerant, with the percentage of growth inhibition calculated as follows: OD600 DMSO-OD600 herbicide / OD600 DMSO x 100 Petition 870250095065, dated 10 / 17 / 2025, page 169 / 375 155 / 346

[0348] Plasmids were isolated from herbicide-tolerant strains and sequenced to identify the causal mutation in the AtPPOl gene carried by pET24a in each strain. All identified herbicide-tolerant variants are in Table 2.

[0349] Note throughout the examples: Herbicides A to E are as described above. Table 2: AtPPOl variants that have been identified as being resistant to one or more of herbicides B, D, or E. The table shows the percentage of growth inhibition in the presence of herbicides B, D, or E compared to growth in DMSO. SEQ ID Variante de AtPPOl Herbicida B (0,78 ppm) Herbicida D (6, 25 ppm) Herbicida E (6,25 ppm) SEQ ID NO:1 WT 61 58 64 SEQ ID NO:4 G64A 36 38 34 SEQ ID NO:5 V8 8I 32 43 66 SEQ ID NO:6 E111D 23 30 69 SEQ ID NO:7 E111F 27 29 66 SEQ ID NO:8 V127I 28 23 69 SEQ ID NO:9 V127L 31 29 65 SEQ ID NO:10 P154Y 34 38 68 SEQ ID NO:11 K173H 29 34 55 SEQ ID NO:12 A176L 35 42 61 SEQ ID NO:13 V198W 30 47 51 SEQ ID NO:14 V2 0 6G 32 19 58 SEQ ID NO:15 F207Y 29 19 66 SEQ ID NO:16 E212Y 25 27 65 Petição 870250095065, de 17 / 10 / 2025, pág. 170 / 375 156 / 346 SEQ ID NO:17 F214Y 26 34 63 SEQ ID NO:18 V218K 38 31 50 SEQ ID NO:19 Y219S 28 33 49 SEQ ID NO:20 A220C 15 33 66 SEQ ID NO:21 P271C 24 24 66 SEQ ID NO:22 Q272H 27 28 64 SEQ ID NO:23 Q272I 24 29 63 SEQ ID NO:24 Q272T 28 23 64 SEQ ID NO:25 K281C 35 33 54 SEQ ID NO:26 K281E 37 33 61 SEQ ID NO:27 K2 81N 39 37 56 SEQ ID NO:28 R284G 35 37 60 SEQ ID NO:29 S305L 26 14 39 SEQ ID NO:30 L360C 25 21 33 SEQ ID NO:31 Y361D 20 33 14 SEQ ID NO:32 Y361F 31 37 50 SEQ ID NO:33 Y361P 28 36 38 SEQ ID NO:34 Y361Q 19 32 0 SEQ ID NO:35 Y361R 36 43 56 SEQ ID NO:36 Y361T 37 44 37 SEQ ID NO:37 Y362F 34 13 43 SEQ ID NO:38 V365L 11 55 27 SEQ ID NO:39 V365M 3 42 22 SEQ ID NO:40 G404A 12 4 2 SEQ ID NO:41 P413G 16 32 59 SEQ ID NO:42 P418M 12 25 64 SEQ ID NO:43 L424V 19 33 66 SEQ ID NO:44 Y426C 2 34 4 SEQ ID NO:45 Y426L 15 25 12 Petição 870250095065, de 17 / 10 / 2025, pág. 171 / 375 157 / 346 SEQ ID NO: 46 Y426M 7 25 17 SEQ ID NO: 47 Y426T 8 12 11 SEQ ID NO: 48 Y426V 5 16 7 SEQ ID NO: 49 T431R 25 28 56 SEQ ID NO: 50 V447C 34 32 53 SEQ ID NO: 51 V447I 35 36 61 SEQ ID NO: 52 D448H 31 26 57 SEQ ID NO: 53 D448L 38 38 54 SEQ ID NO: 54 D448N 40 38 63 SEQ ID NO: 55 R4 4 9K 35 37 68 SEQ ID NO: 56 T461Q 31 22 35 SEQ ID NO: 57 F478R 23 30 61 SEQ ID NO: 58 L479M 29 4 28 SEQ ID NO: 59 L479N 23 3 31 SEQ ID NO: 60 G510N 24 31 70 SEQ ID NO: 61 V511L 18 18 46 SEQ ID NO: 62 V511N 20 22 48 SEQ ID NO: 63 C516Q 18 28 51 SEQ NO:305 ID K270M 28 23 29 SEQ NO:306 ID Y361C 36 15 35 SEQ NO:307 ID Y362C 37 43 58 SEQ NO:308 ID T431F 41 0 37 SEQ NO:309 ID L464Y 0 12 5 Petição 870250095065, de 17 / 10 / 2025, pág. 172 / 375 158 / 346 SEQ NO:310 ID G481D 40 20 16 SEQ NO:311 ID G481K 10 12 0 SEQ NO:312 ID G481N 44 14 4 Exemplo 2 - Triagem de biblioteca de variantes combinatórias de AtPPOl.

[0350] A subset of herbicide tolerance mutations identified from Example 1 was selected to be incorporated into a combinatorial variant library (produced by Twist Bioscience). The selected mutations and their assigned frequency within the combinatorial library are shown in Table 3. Table 3: AtPPOL mutations selected for inclusion in a combinatorial library. A percentage frequency of each mutation within the library is shown. Amino acid position in AtPPOl (SEQ ID NO: 1) WT VARIANT 1 VARIANT 2 VARIANT 3 VARIANT 4 270 K (95%) M (5%) 305 S (75%) L (25%) 360 L (95%) C (5%) 361 Y (75%) C (6.25%) D (6.25%) Q (6.25%) T (6.25%) 362 Y (95%) F (5%) 365 V (75%) L (12.5%) M (12.5%) Petition 870250095065, dated 10 / 17 / 2025, page 173 / 375 159 / 346 404G (75%) (2.5%) 481 G (75%) D (8.33%) K (8.33%) N (8.33%)

[0351] The combinatorial library was transformed into E. coli BL21(DE3) hemG and selected for increased herbicide tolerance as described in Example 1 except for herbicide concentrations being increased to 25 ppm (herbicides B and D) and 50 ppm (herbicide E). Plasmids were isolated from herbicide-tolerant strains and sequenced to identify the causal mutation in the AtPPOl gene carried by the pET24 plasmid in each strain. All identified herbicide-tolerant variants are in Table 4. Table 4: AtPPOL variants with multiple mutations selected based on strong growth in the presence of herbicide. SEQ ID NO:124 S305L Y426M (comparator) SEQ ID NO:125 S305L Y426V AtPPOl V030 SEQ ID NO:126 V365L Y426V L479N AtPPOl V004 SEQ ID NO:127 S305L G404A Y426L Y426L L73N V036 SEQ ID NO:128 S305L V365M AtPPOl V055 SEQ ID NO:129 S305L Y362F G404A AtPPOl V018 Petition 870250095065, of 17 / 10 / 2025, p. 174 / 375 160 / 346 SEQ ID NO:130 S305L Y361T V365L T431R L479M AtPPOl V020 SEQ ID NO:131 S305L Y426V L479M AtPPOl V002 SEQ ID NO:132 Y426M T461Q L479M AtPPOl V SEQ ID NO: 2013 Q3023 Y361D V365M AtPPOl V052 SEQ ID NO:134 S305L Y361C Y362F Y426M L479M AtPPOl V031 SEQ ID NO:135 Y361D V365L AtPPOL V035 SEQ ID NO:136 V361D V365L AtPPOL V035 SEQ ID NO:136 V365 AtPPOL G402 LQl2 ID NO:137 S305L G404A L479N AtPPOl V019 Example 3 - Expression and assay of selected Arabidopsis thaliana PPO1 variants and inhibition percentage values ​​versus herbicides A to E.

[0352] A pET24 plasmid containing a DNA sequence encoding each identified AtPPOl variant mutant derived from Arabidopsis thaliana, with codon optimization for expression in E. coli and under the control of a T7 promoter, was synthesized by Twist Bioscience. This plasmid was expressed in E. coli BL21 (DE3) hemG with selection at kanamycin at 50 pg / mL and chloramphenicol at 25 pg / mL. Overnight cultures grown at 37 °C were used to inoculate 150 mL of AIM (AIM - Terrific Broth Base including Trace Elements, Formedium) into 500 mL shake flasks at a 1:100 ratio. The cultures were maintained at 37 °C, 200 rpm for 3 hours and then overnight at 26 °C. The cells were collected by centrifugation at 4 °C for 20 minutes at 600 rpm. The cells were stored at -80 °C until extraction.0.5-1 g of cell pellets were thawed and resuspended at a 1:1 ratio in lysis buffer (20 mM Tris pH 8, 500 mM sodium chloride, 10% glycerol, IX Complete Protease Inhibitor EDTA-Free Tablet (Roche)). The resuspension was sonicated to 50% using 10-20. Petition 870250095065, dated 10 / 17 / 2025, page 175 / 375 161 / 346 cycles of 10 seconds, 20 seconds off. After sonication, Triton X-100 was added to the lysates at a final concentration of 0.5%. The lysates were centrifuged at 13,000 rpm for 10 min at 4 °C. The impure extracts (i.e., supernatants) were stored at -80 °C.

[0353] Protein concentration was measured and calculated using absorbance at 280 nm. Extracts were diluted 1 in 4 and 10 uL were analyzed by SDS PAGE.

[0354] Impure extracts of the variants were evaluated in a Protoporphyrinogen Oxidase fluorescent assay in 384-well black plates with a transparent bottom. The total volume of the reaction mixture was 100 µL and contained 100 mM K2HPO4 pH 7.2, 1 mM NazEDTA, 5 mM DTT, 20% (v / v) glycerol, and 0.025% (v / v) Tween 20. Reactions were initiated with the addition of a final concentration of 1 µM in the protoporphyrinogen IX assay. Fluorescence was monitored over 30 min using a Tecan plate reader with an excitation wavelength of 405 nm, an emission wavelength of 635 nm, a gain of 90, and 15 flashes. 10 µL of serial dilution of 3 times the impure extract in 100 mM K2HPO4 pH 7.2, 1 mM Na2EDTA, 5 mM DTT, 20% (v / v) glycerol, and 0.05% (v / v) Tween 20 were tested in the assay against 1% DMSO and 100 ppm herbicide B to determine the correct amount of impure extract to add to the assay to observe the activity.10 µL of the appropriate impure extract dilution were then tested in the Protoporphyrinogen Oxidase fluorescent assay against DMSO and a 10-fold dose response of each compound (herbicides A to E) with a maximum dose of 100 ppm. Petition 870250095065, dated 10 / 17 / 2025, page 176 / 375 162 / 346

[0355] The percentage of inhibition was calculated as the activity in the presence of one of four concentrations of each herbicide compound A to E (note that the compounds labeled A to E are identified earlier in the relevant section of the description) as a percentage of the enzymatic activity in DMSO (Table 5). Table 5: Petition 870250095065, dated 10 / 17 / 2025, page 177 / 375 163 / 346 Herbicide ES 00T 8 a 8 oot 100 s ft 100 30 8 100 oot 8 O 00T 001 s P1- in ft S rJ 00 <3 to 8 Πί 00 to 00 oot ft 00 rJ 1-J rJ $ i—1 m Πί υί 00 rJ to rJ S σι Πί ft σι Πί to to σ' s 00 Πί o O iH o υί ΟΙ O ο ο ο ο o o ο rJ rJ Herbicide D 100 0OT 100 ΟΙ 00 00 ΟΙ rJ 3Ι oot to ΟΙ rJ ΟΙ ft 100 oot 100 100 oot 100 s 8 100 8 a K2 8 sa SB 8 8 8 ώ 8 8 8 Lfl 00 sm Πί 3 υί «=Τ rJ to *0- <Τί 00 to Πί Οί to Πί 00 Πί o' sm υί o rJ s S Οί fM 00 Πί ft ο fM rJ Herbicida C 100 m Οί 100 & Πί Οί tfl <Ti 100 100 ft 100 100 100 100 100 100 100 O 00 ΟΊ 100 00 rJ 00 ΠΊ 00 ϋΊ ΟΊ ΠΊ ΟΊ -0Ί -0Ί 8 ϋΊ r-J ΟΊ ΟΊ ΟΊ -3Ί -3Ί 100 s 00 00 ft ft to to 00 <ΤΊ ϋΊ a to ϋΊ ϋΊ to a 8 8 5· s ft o *3- ft ft a +J C a 8 8 3 8 3 8 Herbicida B 100 001 0OT 0OT 001 OCT O0T OOI 00 οοτ ΟΟΤ οοτ οοτ 0OT οοτ ΟΟΤ o <Ti 100 100 100 100 υΊ <Ti ft ft 100 100 100 100 100 100 100 i—1 & 100 ΟΊ σι 100 100 ft ΠΊ σι 100 to σι 100 100 100 100 100 00 σι 11 ft οοτ 00 00 3Ί ft οοτ ¢3 ~rj σι 3Ί 100 to ΟΊ οοτ r-J 00 Herbicida A 001 100 100 00 800 00 ft 100 100 100 100 100 rJ ΟΙ to ΟΙ 100 100 s & 100 saa 8 £α ft a S3 8 100 100 i—l 001 rJ 00 3 Πί υΙ a 00 -0Ί to rJ υΙ a Πί Πί Πί Πί σΙ σ O o O ΟΙ ft Λ_ιΙ rJ υΙ ο s ΟΙ ο to ΟΙ 00 Πί a O Ci 100 1—1 m O tO oo rJ ο τ—1 ο t—1 ΟΙ ο eoo ηΙ 00 ο Q υΙ ο υΙ 'tt o ο ΤΙΙ 1H ooo O o rJ Πί P*· Ο Γ**· Ο o ο 5 ooo 1—1 oa ο P*· Ο Ρ-« τ—1 ο o υΙ P-« WT(SEQID N0.1) S30&L Y426M (SEQ ID NO.124) 530&LY426V (SEQ ID NO.125) V365LY426V L479N (SEQ ID NO.126) ft Q Ξ Ci 2 tn 5 Cu rn a δ rJ 1 s tTl S30&L V365M (SEQ ID NO. 128) σ rJ d Ξ Ο Cf lU tn I LL rJ 8 a tn J d Ξ Ci 2 S- -0Ί s □Í —J υΙ to > 8 8 tn S30&L Y426V L479M fSEQ ID NO.131) Υ426Μ T461Q L479M (SEQ ID NO.132) ΟΊ' 3 d Ξ Ο CÍ L±JS· Ξ ΔΊ to > a $ tn § d Ξ Ci 3 tn s Ξ to rJ g rJ 8 —ID 8 a tn ín 3 d Ξ □ Cf LU tn, _| DI to > Cl d 3 V365L G404A L479N (SEQ ID NO.136) S30&L G404A L479N (SEQ ID NO.137) Petition 870250095065, de 17 / 10 / 2025, pág. 178 / 375 164 / 346

[0356] The results show that the selected variants showed improved tolerance to at least one of the PPO inhibitor herbicides A to E compared to the AtPPOl progenitor gene (SEQ ID NO:1) or previously known PPO1 variant (SEQ ID NO:2). In some cases, such as for AtPPOl S305L Y426V (SEQ ID NO:125), improved herbicide tolerance was seen for all herbicides tested. In other examples, differential tolerances are seen for the herbicides tested, such as with AtPPOl Y361D V365L (SEQ ID NO:135) where improvement over SEQ ID NO:124 was seen for some of the herbicides (i.e., B, C, D, and E) but not for others (i.e., herbicide A).

[0357] A pET24 plasmid containing a DNA sequence encoding AtPPOl variants derived from Arabidopsis thaliana with cTPs removed and both a 6xHis tag and a TEV protease cleavage tag added to the N-terminus, with codon optimization for expression in E. coli and under the control of a T7 promoter, was synthesized by Twist Bioscience. The following variants were synthesized: WT AtPPOl (SEQ ID NO: 370), AtPPOl L479M (SEQ ID NO: 371), AtPPOl L479N (SEQ ID NO: 372), AtPPOl S305L Y426V (SEQ ID NO: 374) and AtPPOl S305L Y426V L479M (SEQ ID NO: 373). This plasmid was expressed in E. coli BL21 (DE3) with selection at kanamycin at 50 pg / mL. 10 mL of overnight cultures grown at 37 °C were used to inoculate 1 L of AIM (AIM - Terrific Broth Base including Trace Elements, Formedium) into 2 L shaker flasks. The cultures were grown at 37 °C, 200 rpm for 3 hours and then overnight at 26 °C.The cells were collected by centrifugation. 5-15 g of cell pellet were lysed and... Petition 870250095065, dated 10 / 17 / 2025, page 179 / 375 165 / 346 resuspended at a 1:1.5 ratio in Use buffer (20 mM Tris pH 8, 500 mM sodium chloride, 10% glycerol, 10 mM EDTA-free Complete Protease Inhibitor Tablet (Roche), 10 mM imidazole). The resuspension was sonicated to 75% using 10 cycles of 30 seconds, 40 seconds off. After sonication, Triton X-100 was added to the lysates at a final concentration of 0.5%. The lysates were centrifuged at 20,000 rpm for 25 min at 4 °C. The clarified lysates were loaded onto His GraviTrap (Cytiva) columns, washed with 20 mL of lysis buffer, and eluted in elution buffer (20 mM Tris pH 8, 500 mM sodium chloride, 10% glycerol, 200 mM imidazole, 0.03% (v / v) Triton X-100). The eluate was desalted on a PD10 column, and 25-3.2 mL were collected. Protein concentration was measured and calculated using absorbance at 280 nm. Extracts were diluted 1 in 4, and 10 µL were analyzed by SDS PAGE.

[0358] Partially purified protoporphyrinogen oxidase enzymes were evaluated for activity using fluorescence adapted from the Shepherd & Dailey method (Anal Biochem. 2005 344 (1) 115-121). The assay was performed in 384-well black plates with a transparent bottom, and the total volume of the reaction mixture was 91 µL. The final assay mixture contained 100 mM K2HPO4 pH 7.2, 1 mM NazEDTA, 5 mM DTT, 20% (v / v) glycerol, 0.05% (v / v) Tween 20, 1.1% (v / v) DMSO, 1 µM protoporphyrinogen IX, and 1.5 nM enzyme. The compounds were dissolved in 1 mM DMSO, and a 4-fold serial dilution was carried out to give 8 rates. 1 µL was added to the assay mixture. This gave final compound concentrations of 11, 2.7, 0.69, 0.17, 0.04, 0.01, 0.002, 0.0007 µM. The enzyme and the inhibitor Petition 870250095065, dated 10 / 17 / 2025, page 180 / 375 166 / 346 were pre-incubated for 5 minutes before initiating the reaction with the addition of the substrate. Fluorescence was monitored over 30 min at room temperature using a Tecan Infinite M200PRO plate reader with an excitation wavelength of 405 nm, emission wavelength of 635 nm, gain of 100, and 15 flashes. The % inhibition relative to the uninhibited control was calculated for each compound at each concentration during the linear phase of the reaction. IC50s were calculated using GraphPad Prism 7.05 nonlinear regression (Table 6). Each compound was tested in duplicate.

[0359] The IC50s of AtPPOl V002 (SEQ ID NO: 373) were higher than AtPPOl V030 (SEQ ID NO: 374) for all tested herbicides without fomesafen. These variants share two mutations, but AtPPOl V002 (SEQ ID NO: 373) has an additional L479M substitution that AtPPOl V030 (SEQ ID NO: 374) lacks. One mechanism by which substitutions at L479 may enhance tolerance is by increasing the enzymatic activity rate of PPO1 (Table 7). Table 6: IC50 (nM) of Protoporphyrinogen Oxidase variants treated with different PPO inhibitors. <1.0 is below the assay detection limit. His- His- His- His- His-TEV- TEV- TEV- TEV- TEV- AtPPOl AtPPOl AtPPOl AtPPOl AtPPOl AcTP AcTP AcTP AcTP AcTP V030 (SEQ L479N L479M V002 (SEQ ID ID NO: (SEQ (SEQ (SEQ NO: 374) 370) ID NO: ID NO: ID NO: 372) 371) 373) Petition 870250095065, dated 10 / 17 / 2025, p. 181 / 375 167 / 346 Herbicide A 0.7 0.9 0.8 446.7 323.2 Herbicide B 0.7 1.0 0.8 2.1 1.3 Herbicide C 0.7 0.9 0.8 121.0 92.0 Herbicide D 0.5 0.8 0.6 335.1 268.9 Herbicide E 2.1 2.6 2.4 997.6 546.9 Azafenadine 0.8 0.8 0.7 148.7 117.0 Butafenacil 0.6 0.9 0.7 12.5 11.0 Flufenoximecil 0.6 0.8 0.7 863.1 717.5 Flumioxazin 0.8 1.0 1.0 187.4 47.2 Fomesaphen 1.8 1.4 1.2 0.5 0.7 Oxadiazone 19.0 6.1 11.2 5620.0 4604.0 Piraclonil 2.5 1.5 2.3 5558.0 4351.0 Pyraflufen-ethyl 0.6 0.8 0.7 24.2 18.6 Table 7 - Activity rates of variants of Protoporphyrinogen Oxidase in an in vitro assay. His- His- His- His- His- TEV- TEV- TEV- TEV- TEV- AtPPOl AtPPOl AtPPOl AtPPOl AtPPOl AcTP AcTP AcTP AcTP AcTP (SEQ L479N L479M V002 V030 ID NO: (SEQ (SEQ (SEQ (SEQ 370) ID NO: ID NO: ID NO: ID NO: 372) 371) 373) 374) Rate (rfu second^pmole-1) 5.1 13.2 9.9 7.6 5.3 * rfu = relative fluorescence units Example 4 - PPO1 sequences and expression in tobacco plants

[0360] PPO1 of Arabidopsis thaliana or orthologs thereof, for example SEQ ID NOs: 1 and 124 to 137 or PPO1 of Setaria italica or orthologs thereof, for example SEQ ID NOs: 2 and 138-151, are Petition 870250095065, dated 10 / 17 / 2025, p. 182 / 375 168 / 346 expressed in transgenic tobacco. The DNA sequences encoding these polypeptides (optimized for tobacco or, optionally, with codon optimization according to a target crop such as soybean or corn) are synthetically prepared. Each sequence is designed to include a 5' fusion with the TMV omega 5' leader sequence and to be flanked at the 5' end with Xhol and at the 3' end with Kpnl to facilitate direct cloning into a suitable binary vector for Agrobacterium-based plant transformation.

[0361] In one example, the expression cassette, comprising the 5' omega leader of TMV and a PPO1-encoding gene of interest, is excised using Xhol / Kpnl and cloned onto similarly digested pBIN 19 (Bevan, Nucleic Acids Res. 12: 87118721 (1984)) to create a binary vector, e.g., the vector in Figure 1 (SEQ ID NO: 152) behind a doubly enhanced 35S promoter, ahead of a NOS 3' transcription terminator, and then transformed into competent E. coli DH5 alpha cells. The DNA recovered from E. coli is used to transform Agrobacterium tumefaciens LBA4404, and the transformed bacteria are screened in media containing rifampicin and kanamycin. Tobacco tissue is subjected to Agrobacterium-mediated transformation using methods well described in the art or as described here.For example, a main plate of Agrobacterium tumefaciens containing the binary vector expressing PPO1 is used to inoculate 10 mL of LB (L broth) containing rifampicin at 100 mg / L plus kanamycin at 50 mg / L using a single bacterial colony. This is incubated overnight at 28 °C shaking at 200 rpm. This entire culture maintained overnight is used. Petition 870250095065, dated 10 / 17 / 2025, page 183 / 375 169 / 346 to inoculate a 50 mL volume of LB containing the same antibiotics. Again, this is cultured overnight at 28 °C shaking at 200 rpm. Agrobacterium cells are pelleted by centrifugation at 3000 rpm for 15 minutes and then resuspended in MS medium (Murashige and Skoog) containing 30 g / L sucrose, pH 5.9 to an OD (600 nM) = 0.6. This suspension is dispensed into 25 mL aliquots into Petri dishes.

[0362] Clonally micropropagated tobacco bud cultures are used to excise young leaves (not yet fully expanded). The middle rib and outer margins of the leaf are removed and discarded, and the remaining blade is cut into 1 cm squares. These are transferred to Agrobacterium suspension for 20 minutes. The explants are then removed, moistened on sterile filter paper to remove excess suspension, then transferred to solid NBM medium (MS medium containing 30 g / L sucrose, 1 mg / L BAP (benzylaminopurine) and 0.1 mg / L NAA (naphthaleneacetic acid) at pH 5.9 and solidified with 8 g / L Plantagar), with the abaxial surface of each explant in contact with the medium. Approximately 7 explants are transferred per plate, which are then sealed and kept in an incubator illuminated at 25 °C during a 16-hour photoperiod for 3 days.

[0363] The explants are then transferred to NBM medium containing kanamycin at 100 mg / L plus antibiotics to prevent further growth of Agrobacterium (timentin at 200 mg / L and carbenicillin at 250 mg / L). Additional subculture in this same medium was then performed every 2 weeks. Petition 870250095065, dated 10 / 17 / 2025, page 184 / 375 170 / 346

[0364] As shoots begin to regenerate from callus leaf explants, these are removed to Shoot Elongation Medium (MS medium, sucrose at 30 g / L, Plantagar at 8 g / L, kanamycin at 100 mg / L, timentine at 200 mg / L, carbenicillin at 250 mg / L, pH 5.9). Stable transgenic plants root rapidly within 2 weeks. To provide multiple plants per event to ultimately allow more than one herbicide test per transgenic plant, all rooting shoots are micropropagated to generate 3 or more rooted clones.

[0365] Putative transgenic plants that are rooting and showing vigorous shoot growth in medium incorporating kanamycin are analyzed by PCR using primers that amplified a specific 500 bp fragment of the AtPPOl transgene of interest. Evaluation of this same primer set in untransformed tobacco conclusively showed that these primers would not amplify any sequences from the native tobacco genome.

[0366] The transformed shoots are divided into 2 or 3 clones and regenerated from kanamycin-resistant callus. The shoots are rooted on MS agar containing kanamycin. The surviving rooted explants are re-rooted to provide approximately 40-50 kanamycin-resistant and PCR-positive events for each event.

[0367] Once rooted, the seedlings are transferred from agar and potted in 50% peat moss, 50% John Innes Soil No. 3 with slow-release fertilizer in 3-inch round pots and left to water regularly to Petition 870250095065, dated 10 / 17 / 2025, pp. 185 / 375 171 / 346 are established for 8-12 days in the greenhouse. Greenhouse conditions are approximately 24-27°C day; 18-21°C night and a photoperiod of approximately 14 hours. Humidity is adjusted to ~65% and light levels used are up to 2000 mmol / m2 at bench level.

[0368] Transgenic populations of approximately forty tobacco plants comprising a gene encoding a full-length Arabidopsis thaliana PPO1 gene (SEQ ID NO: 1) or variants (SEQ ID NO: 1 and 124 to 137) or encoding a full-length Setaria italica PPO1 gene (SEQ ID NO: 2) or variants (SEQ ID NO: 138-151) are thus produced. Plants are selected based on similar size of each population and ELISA or Mass Western blot tests are carried out to monitor the expression levels of the transgenic PPO protein. The highest expression T1 lines are selected to be directed to self-fertilization and to generate the T1 seed and T2 lines and seed in the normal manner.Seeds from the highest expression lines are tested for germination on agar plates containing a range of concentrations of PPO1 inhibitor herbicides as taught for example here, and resistant plant lines are selected as showing the least damage to root or leaf growth and morphology at the highest herbicide concentrations. Resistant plant lines exhibit a dose response to herbicide damage by PPO1 inhibitors that is shifted to the right compared to similarly grown and treated wild-type and null segregating plants. Petition 870250095065, dated 10 / 17 / 2025, page 186 / 375 172 / 346 Example 5 - Herbicide tolerance assay in transgenic tobacco plants expressing heterologous AtPPOl variants and heterologous SiPPOl variants.

[0369] In order to determine whether overexpression of the genes detailed in Example 3, the PPOls of Setaria italica (SEQ ID NO: 2 or 138-151) or any of the SEQ ID NO: 1, 2, 4, 151, 155-302 or 305-336, confers resistance to PPO-inhibiting compounds when expressed in plants, GM tobacco lines are produced and tested with PPO-inhibiting compounds. Transgenic tobacco plants expressing the PPO1 gene from Arabidopsis thaliana and variants thereof (SEQ ID NOs 1 and 124-137) are produced and transgenic tobacco plants expressing the PPO1 gene from Setaria italica and variants thereof (SEQ ID NOs 2 and 138-151) are produced.

[0370] Transgenic tobacco populations comprising 20-30 transgenic events per plant transformation construct are generated as described in Example 4. These lines were propagated clonally and 1 clone per event was sprayed with PPO herbicides to confirm herbicide resistance.

[0371] Herbicide damage is visually assessed across the entire population and a herbicide damage score is given at 7 and 14 days. A score of 0 indicates no visible damage or stunting, while a score of 100 indicates complete plant death. 20 transgenic events (i.e., individual transgenic plants) from each GM tobacco row are transplanted into the soil 1 week before spraying with PPO inhibitor compounds. WT tobacco is included as a control. Table 8 below shows the treatment regimens used. Petition 870250095065, dated 10 / 17 / 2025, page 187 / 375 173 / 346 Example 5A - Herbicide tolerance assay in transgenic tobacco plants expressing heterologous AtPPOL variants

[0372] Data for herbicide tolerance in transgenic tobacco plants were generated according to Example 5 above and are shown in Tables 11-24. Herbicide damage was visually assessed across the entire population and a herbicide damage score was given at 7 and 14 days. A score of 0 indicates no visible damage or stunting, while a score of 100 indicates complete plant death. 20 transgenic events (i.e., individual transgenic plants) from each GM tobacco row were transplanted into the soil 1 week before spraying with PPO inhibitor compounds. WT tobacco is included as a control. Table 8 below shows the treatment regimes used. Table 8: Treatment regimens for testing PPO inhibitor resistance No. of treatment Herbicide Application rate (g of active ingredient / ha) Formulation and Adjuvant 1 B 120 Genapol X080 at 0.2% 2 C 120 Genapol X080 at 0.2% 3 D 120 Genapol X080 at 0.2% Petition 870250095065, dated 10 / 17 / 2025, pp. 188 / 375 174 / 346 Table 9: Plants transformed with variants of the Arabidopsis PPO gene. Name Protein Mutations SEQ ID NO DNA SEQ ID NO pBin TMV AtPPOl Type selvagem SEQ ID NO: 1 SEQ ID NO: 3 pBin TMV AtPPOl V030 S305L Y426V SEQ ID NO:125 SEQ ID NO: 366 pBin TMV AtPPOl V004 V365L Y426V L479N SEQ ID NO:126 SEQ ID NO:364 pBin TMV AtPPOl V036 S305L G404A Y426V T431R L479N SEQ ID NO:127 SEQ ID NO: 360 pBin TMV AtPPOl V055 S305L V365M SEQ ID NO:128 SEQ ID NO: 361 pBin TMV AtPPOl V018 S305L Y362F G404A SEQ ID NO:129 SEQ ID NO: 356 pBin TMV AtPPOl V020 S305L Y361T V365L T431R L479M SEQ ID NO:130 SEQ ID NO: 363 pBin TMV AtPPOl V002 S305L Y426V L479M SEQ ID NO:131 SEQ ID NO: 365 pBin TMV AtPPOl V025 Y426M T461Q L479M SEQ ID NO:132 SEQ ID NO: 358 pBin TMV AtPPOl V052 S305L Y361D V365M SEQ ID NO:133 SEQ ID NO: 368 pBin TMV AtPPOl V031 S305L Y361C Y362F Y426M L479M SEQ ID NO:134 SEQ ID NO: 367 pBin TMV AtPPOl V035 Y361D V365L SEQ ID NO:135 SEQ ID NO: 359 pBin TMV AtPPOl V022 V365L G404A L479N SEQ ID NO:136 SEQ ID NO: 357 Petition 870250095065, 17 / 10 / 2025, pág. 189 / 375 175 / 346 pBin TMV AtPPOl V019 S305L G404A L479N SEQ ID NO:137 SEQ ID NO: 362

[0373] The results are shown in Tables 11 to 24 below. A control population of wild-type Samsun tobacco was evaluated for herbicide damage, with results shown in Table 10. Wild-type Samsun plants show significant damage at evaluation time days 7 and 14. Table 10: Results of the percentage of damage to WT tobacco plants Wild Type Samsun Tobacco Herbicide C Herbicide D Herbicide B Plant WT 7 DAT 14 DAT 7 DAT 14 DAT 7 DAT 14 DAT 1 100 100 98 100 100 100 2 100 100 98 100 100 100 3 100 100 98 100 100 100 4 100 100 98 100 100 100 5 100 100 100 100 100 100 6 100 100 100 100 100 100 7 100 100 100 100 100 100 8 100 100 98 100 100 100 Table 11: Results of the percentage of damage to tobacco plants containing pBin TMV AtPPOl. pBin TMV AtPPOl Herbicide C Herbicide D Herbicide B Event number 7 DAT 14 DAT 7 DAT 14 DAT 7 DAT 14 DAT 0150 100 100 99 100 100 100 0154 100 100 100 100 100 100 Petition 870250095065, dated 10 / 17 / 2025, pp. 190 / 375 176 / 346 0167 100 100 100 100 100 100 0169 95 90 95 95 99 100 0173 100 100 99 100 100 100 0177 95 100 97 99 99 100 0179 100 100 99 100 100 100 0184 100 100 99 100 100 100 0185 100 100 99 100 100 100 0188 100 100 100 100 100 100 0190 94 95 65 75 87 80 0193 95 97 95 100 100 100 0194 100 100 99 100 100 100 0195 100 100 100 100 100 100 0197 97 99 92 97 97 98 0215 100 100 99 100 100 100 0224 100 100 99 100 100 100 0225 97 100 92 96 99 99 0226 100 100 99 100 100 100 0231 100 100 98 99 100 100 Table 12: Results of the percentage of damage to tobacco plants containing pBin TMV AtPPOl V022 (V365L G404A L479N mutations) pBin TMV AtPPOl V022 Herbicide C Herbicide D Herbicide B 7 DAT 14 DAT 7 DAT 14 DAT 7 DAT 14 DAT 9981 90 95 85 80 99 100 9984 99 100 90 95 98 100 9990 10 5 5 3 85 85 9992 100 100 99 100 100 100 0005 100 100 100 100 100 100 Petition 870250095065, dated 10 / 17 / 2025, pp. 191 / 375 177 / 346 0008 100 100 99 100 100 100 0009 98 100 95 96 100 100 0016 98 100 95 100 99 100 0017 100 100 100 100 100 100 0018 100 100 98 100 100 100 0019 10 5 3 2 85 95 0026 100 100 99 100 100 100 0032 100 100 99 100 100 100 0033 65 60 20 5 80 70 0045 7 3 5 3 75 70 0052 99 100 99 100 99 100 0054 5 2 3 0 90 95 0055 99 100 99 100 100 100 0062 100 100 100 100 100 100 0063 100 100 99 100 100 100 Table 13: Results of the percentage of damage to tobacco plants containing pBin TMV AtPPOl V025 (Y426M T461Q L479M mutations) pBin TMV AtPPOl V025 Herbicide C Herbicide D Herbicide B 7 DAT 14 DAT 7 DAT 14 DAT 7 DAT 14 DAT 0319 65 45 55 30 99 100 0324 0 0 0 0 85 80 0326 100 100 100 100 100 100 0331 95 97 90 85 100 100 0337 85 90 75 75 99 100 0341 85 80 85 70 100 100 0346 10 5 35 5 95 98 0351 35 20 35 5 97 100 Petition 870250095065, dated 10 / 17 / 2025, page 192 / 375 178 / 346 0356 96 98 95 97 99 100 0359 99 100 100 100 100 100 0364 100 100 100 100 100 100 0367 98 100 95 99 100 100 0370 10 0 10 5 99 100 0373 35 10 50 15 97 100 0387 75 95 75 95 99 100 0390 100 100 100 100 100 100 0392 0 0 1 0 98 95 0397 35 20 70 45 99 100 0398 20 15 40 30 99 100 0400 50 20 35 15 99 100 Table 14: Results of the percentage of damage to tobacco plants containing pBin TMV AtPPOl V035. (Y361D V365L mutations) pBin TMV AtPPOl V035 Herbicide C Herbicide D Herbicide B 7 DAT 14 DAT 7 DAT 14 DAT 7 DAT 14 DAT 0245 98 100 99 100 100 100 0250 98 100 99 100 100 100 0251 50 10 80 75 99 100 0252 100 100 99 100 100 100 0254 100 100 100 100 100 100 0258 99 100 99 100 100 100 0260 45 40 80 80 100 100 0264 100 100 99 100 100 100 0267 100 100 100 100 100 100 0268 99 100 99 100 100 100 0271 99 100 99 99 100 100 0273 99 100 99 100 99 100 Petition 870250095065, dated 10 / 17 / 2025, pp. 193 / 375 179 / 346 0276 80 85 85 85 99 100 0286 80 80 90 85 99 100 0287 100 100 97 99 99 100 0292 60 35 75 60 85 70 0298 80 80 80 80 100 100 0306 95 96 90 95 99 100 0310 100 100 100 100 100 100 0313 30 30 50 70 97 95 Table 15: Results of the percentage of damage to tobacco plants containing pBin TMV AtPPOl V055. (S305L V365M mutations) pBin TMV AtPPOl V055 Herbicide C Herbicide D Herbicide B 7 DAT 14 DAT 7 DAT 14 DAT 7 DAT 14 DAT 9897 65 60 90 95 99 100 9899 95 98 99 100 100 100 9902 25 5 30 5 99 100 990 9 10 5 30 5 90 70 9918 55 40 50 55 99 100 9923 35 10 45 25 99 100 9927 100 100 100 100 100 100 9931 5 5 20 8 90 80 9935 99 100 99 100 100 100 9936 97 99 99 100 98 99 9938 95 95 96 97 100 100 9942 20 5 85 70 98 99 9943 35 30 80 85 92 85 9947 90 96 96 99 99 100 9953 6 5 25 10 99 100 9955 20 8 25 30 95 95 Petition 870250095065, dated 10 / 17 / 2025, pp. 194 / 375 180 / 346 9959 100 100 100 100 100 100 9962 10 5 30 8 85 50 9973 100 100 100 100 100 100 9977 5 3 30 10 98 95 Table 16: Results of the percentage of damage to tobacco plants containing pBin TMV AtPPOl V019. (Mutations S305L G404A L479N) pBin TMV AtPPOl V019 Herbicide C Herbicide D Herbicide B 7 DAT 14 DAT 7 DAT 14 DAT 7 DAT 14 DAT 0067 35 30 15 25 75 50 0072 65 60 15 20 80 65 0080 75 90 30 40 80 80 0086 65 55 30 25 90 85 0096 75 75 30 10 80 85 0097 55 25 20 5 60 30 0102 55 20 25 2 85 90 0103 75 85 25 25 100 100 0104 85 95 60 80 98 100 0105 40 30 20 15 75 55 0106 55 50 25 20 80 50 0107 100 100 100 100 100 100 0108 97 100 95 95 97 100 0115 55 50 40 30 85 75 0126 100 100 99 100 100 100 0131 70 70 35 35 80 55 0137 97 100 95 96 99 100 0140 100 100 100 100 100 100 0141 100 100 99 100 100 100 Petition 870250095065, dated 10 / 17 / 2025, pp. 195 / 375 181 / 346 0143 70 85 35 30 85 75 Table 17: Results of the percentage of damage to tobacco plants containing pBin TMV AtPPOl V020. (S305L Y361T mutations) V365L T431R L479M) pBin TMV AtPPOl V020 Herbicide C Herbicide D Herbicide B 7 DAT 14 DAT 7 DAT 14 DAT 7 DAT 14 DAT 0407 100 100 97 100 100 100 0411 95 99 95 98 99 100 0414 90 95 90 85 99 100 0420 100 100 100 100 100 100 0425 100 100 100 100 100 100 0428 100 100 99 100 100 100 0430 100 100 99 100 100 100 0433 99 100 99 100 100 100 0438 99 100 97 100 100 100 0442 85 80 85 90 99 100 0443 100 100 100 100 100 100 0444 100 100 100 100 100 100 0447 75 80 75 80 92 90 0449 90 97 90 96 99 99 0451 100 100 99 100 100 100 0460 95 95 90 90 99 100 0471 99 100 99 100 100 100 0478 95 96 90 90 100 100 0484 100 100 100 100 100 100 0485 100 100 100 100 100 100 Petition 870250095065, dated 10 / 17 / 2025, pp. 196 / 375 182 / 346 Table 18: Results of the percentage of damage to tobacco plants containing pBin TMV AtPPOl V004. (Mutations V365L Y426V L479N) pBin TMV AtPPOl V004 Herbicide C Herbicide D Herbicide B 7 DAT 14 DAT 7 DAT 14 DAT 7 DAT 14 DAT 9567 100 100 100 100 100 100 9568 25 25 10 15 85 75 9571 0 0 0 0 80 65 9575 0 0 0 0 90 65 9577 30 25 20 0 99 100 9578 0 0 0 0 68 50 9580 65 70 70 60 99 100 9582 0 0 0 0 30 15 9585 85 90 85 85 100 100 9587 100 100 100 100 100 100 9588 15 0 20 5 97 95 9592 80 85 85 75 100 100 9599 5 0 3 0 85 65 9603 0 0 5 5 70 25 9604 0 0 0 0 99 100 9608 0 0 0 0 90 75 9625 98 100 96 96 100 100 9626 20 10 20 15 99 100 9631 100 100 100 100 100 100 9634 40 20 45 10 99 100 Petition 870250095065, dated 10 / 17 / 2025, page 197 / 375 183 / 346 Table 19: Results of the percentage of damage to tobacco plants containing pBin TMV AtPPOl V036. (Mutations S305L G404A Y426V T431R L479N) pBin TMV AtPPOl V036 Herbicide C Herbicide D Herbicide B 7 DAT 14 DAT 7 DAT 14 DAT 7 DAT 14 DAT 9654 65 70 65 75 75 75 9655 100 100 100 100 100 100 9660 100 100 100 100 100 100 9668 65 75 70 75 75 70 9670 65 75 60 70 65 65 9671 55 70 55 50 65 50 9679 100 100 100 100 100 100 9681 50 55 55 65 60 50 9683 70 90 75 80 85 98 9684 100 100 100 100 100 100 9686 100 100 100 100 100 100 9688 98 100 95 99 96 100 9692 65 75 65 70 75 70 9 6 94 70 70 75 70 70 60 9697 100 100 100 100 100 100 9698 100 100 100 100 100 100 9703 60 45 94 95 85 85 9709 50 10 50 15 40 5 9712 100 100 100 100 100 100 9717 40 30 35 20 35 20 Table 20: Results of the percentage of damage to tobacco plants containing pBin TMV AtPPOl V002. (mutations S305L Y426V L479M) Petition 870250095065, dated 10 / 17 / 2025, pp. 198 / 375 184 / 346 Bin TMV AtPPOl V002 Herbicide C Herbicide D Herbicide B 7 DAT 14 DAT 7 DAT 14 DAT 7 DAT 14 DAT 9898 90 75 30 10 100 100 9901 80 75 30 10 100 100 9903 0 0 10 20 15 10 9907 15 5 5 0 100 100 9910 25 10 5 0 99 100 9911 0 2 0 0 85 65 9914 100 100 100 100 100 100 9918 100 100 100 100 100 100 9919 90 85 65 25 100 100 9922 96 100 85 90 100 100 9923 98 100 93 97 100 100 9924 0 0 0 0 25 20 9928 0 0 0 0 95 92 9929 100 100 100 100 100 100 9930 5 0 5 0 85 65 9939 0 0 0 0 50 35 9940 0 10 0 5 60 55 9941 0 0 0 0 70 50 9943 98 96 80 90 100 100 9953 100 100 100 100 100 100 Table 21: Results of the percentage of damage to tobacco plants containing pBin TMV AtPPOl V030. (S305L Y426V mutations) pBin TMV AtPPOl V030 Herbicide C Herbicide D Herbicide B 7 DAT 14 DAT 7 DAT 14 DAT 7 DAT 14 DAT 9816 85 70 45 40 100 100 Petition 870250095065, dated 10 / 17 / 2025, page 199 / 375 185 / 346 9818 0 0 0 0 95 85 9821 25 0 10 0 85 50 9823 0 0 8 2 85 65 9826 10 10 15 5 99 100 9829 90 95 85 85 99 100 9830 0 0 0 0 75 25 9836 96 97 85 85 99 100 9838 100 100 100 100 100 100 9839 5 0 0 0 95 90 9840 90 90 80 75 100 100 9843 0 0 0 0 85 75 9851 100 100 100 100 100 100 9855 0 0 0 0 85 70 9868 2 0 0 0 85 80 9871 40 55 30 5 100 100 9885 98 100 90 90 100 100 9890 0 0 0 0 75 50 9893 0 0 0 0 80 50 9894 0 0 0 0 85 65 Table 22: Results of the percentage of damage to tobacco plants containing pBin TMV AtPPOl V052. (Mutations S305L Y361D V365M) pBin TMV AtPPOl V052 D4562 Herbicide C Herbicide D Herbicide B 7 DAT 14 DAT 7 DAT 14 DAT 7 DAT 14 DAT 9732 65 80 92 85 100 100 9743 99 100 100 100 100 100 9744 60 70 90 90 98 100 9754 100 100 100 100 100 100 Petition 870250095065, dated 10 / 17 / 2025, pp. 200 / 375 186 / 346 9755 99 100 99 100 100 100 9756 60 35 80 70 96 97 9758 80 45 90 75 100 100 9761 75 35 85 55 100 100 9774 75 30 90 70 99 100 9778 100 100 100 100 100 100 9779 99 100 100 100 100 100 9793 95 90 97 95 100 100 9795 80 50 90 70 100 100 9801 100 100 100 100 100 100 9802 100 100 100 100 100 100 9805 96 96 96 97 99 100 9807 100 100 100 100 100 100 9808 97 98 99 100 100 100 9809 90 70 85 60 100 100 9812 100 100 99 100 100 100 Table 23: Results of the percentage of damage to tobacco plants containing pBin TMV AtPPOl V031. (Mutations S305L Y361C Y362F Y426M L479M) pBin TMV AtPPOl V031 Herbicide C Herbicide D Herbicide B 7 DAT 14 DAT 7 DAT 14 DAT 7 DAT 14 DAT 0487 60 45 50 30 100 100 0490 0 0 0 0 96 90 0491 95 98 98 100 100 100 0495 100 100 98 100 100 100 0500 85 95 95 95 100 100 0507 5 0 5 5 85 70 0509 100 100 98 100 100 100 Petition 870250095065, dated 10 / 17 / 2025, page 201 / 375 187 / 346 0518 0 0 0 5 96 100 0520 0 0 0 0 95 100 0521 0 0 0 0 95 90 0522 0 0 0 0 75 60 0527 100 100 98 100 100 100 0535 0 0 0 0 85 75 0540 100 100 98 100 100 100 0543 100 100 98 100 100 100 0551 90 90 95 90 100 100 0553 0 0 0 0 80 85 0557 0 0 0 0 80 75 0559 0 0 0 0 70 40 0561 100 100 98 100 100 100 Table 24: Results of the percentage of damage to tobacco plants containing pBin TMV AtPPOl V018. (Mutations S305L Y362F G404A) pBin TMV AtPPOl V018 Herbicide C Herbicide D Herbicide B 7 DAT 14 DAT 7 DAT 14 DAT 7 DAT 14 DAT 0571 80 85 80 70 90 95 0573 60 70 80 90 85 80 0575 100 100 95 100 100 100 0576 100 100 100 100 100 100 0579 95 100 90 95 98 100 0582 85 90 90 90 95 98 0587 100 100 98 100 100 100 0593 100 100 98 100 100 100 0595 100 100 98 100 100 100 0598 100 100 100 100 100 100 Petition 870250095065, dated 10 / 17 / 2025, page 202 / 375 188 / 346 0600 100 100 100 100 100 100 0605 100 100 98 100 100 100 0611 100 100 98 100 100 100 0612 50 75 35 30 80 85 0617 100 100 98 100 100 100 0618 75 80 60 65 97 100 0620 100 100 98 100 100 100 0624 100 100 98 100 100 100 0628 100 100 98 100 100 100 0644 100 100 98 100 100 100 Example 6: Corn transformation with AtPPOL or SiPPOL variants.

[0374] Transgenic maize lines expressing AtPPOl and variants (SEQ ID N0:1 and 124-137) and SiPPOl and variants (SEQ ID N0:2 and 138-151) are created using methods known in the art.

[0375] The transformation of more immature embryos is carried out essentially as described in Negrotto et al. (Plant Cell Reports (2000) 19: 798-803). Briefly, the Agrobacterium LBA4404 strain (pSBl) comprising an expression vector expressing AtPPOl and variants (SEQ ID NO: 1 and 124-137) and SiPPOl and variants (SEQ ID NO: 2 and 138-151) disclosed is cultured in solid YEP medium (yeast extract (5 g / L), peptone (10 g / L), NaCl (5 g / L), agar at 15 g / L, pH 6.8) for 2-4 days at 28 °C. Approximately 0.8 x 10⁹ Agrobacterium cells are suspended in LS-inf medium supplemented with As at 100 μM. The bacteria are pre-induced in this medium for approximately 30–60 minutes.

[0376] Immature embryos from a more inbred line are excised from ears at 8-12 days of age for LS-inf + As Petition 870250095065, dated 10 / 17 / 2025, pp. 203 / 375 189 / 346 at 100 μM liquid. The embryos are rinsed once with fresh infection medium. The Agrobacterium solution is then added, and the embryos are vortexed for 30 seconds and allowed to sediment with the bacteria for 5 minutes. The embryos are then transferred with the scutellum facing upwards to LSA medium and cultured in the dark for two to three days. Subsequently, approximately 20 to 25 embryos per Petri dish are transferred to LSDc medium supplemented with cefotaxime (250 mg / L) and silver nitrate (1.6 mg / L) and cultured in the dark at approximately 28 °C for 10 days.

[0377] Immature embryos, producing embryogenic callus, are transferred to LSD1M0.5S medium. Cultures are selected in this medium for approximately 6 weeks with a subculture step at about 3 weeks. Surviving calluses are transferred to Reg1 medium supplemented with mannose. After light culture (16-hour light / 8-hour dark regime), green tissues are then transferred to Reg2 medium without growth regulators and incubated for about 1-2 weeks. Plantlets are transferred to Magenta GA-7 boxes (Magenta Corp, Chicago Ill.) containing Reg3 medium and grown under light. After about 2-3 weeks, plants are tested for the presence of PPO genes released by PCR. PCR-positive plants are transferred to an incubator for further evaluation.

[0378] The plants are treated with PPO-inhibiting herbicides to confirm herbicide resistance. Example 7: Transformation of Soybeans with AtPPOL or SiPPOL variants. Petition 870250095065, dated 10 / 17 / 2025, pp. 204 / 375 190 / 346

[0379] Transgenic soybean lines expressing AtPPOl and variants (SEQ ID NO:1 and 124-137) and SiPPOl and variants (SEQ ID NO:2 and 138-151) are created.

[0380] Soybean plant material can be suitably transformed and fertile plants can be regenerated by many methods that are well known to those skilled in the art. For example, morphologically normal fertile transgenic soybean plants can be obtained by: 1) production of somatic embryogenic tissue from, for example, cotyledon, hypocotyl or other suitable immature tissue; 2) transformation by particle bombardment or infection with Agrobacterium; and 3) plant regeneration. In one example, as described in U.S. Patent No. 5,024,944, cotyledon tissue is excised from immature soybean embryos, optionally with the embryonic axis removed, and is cultured in hormone-containing medium so as to form somatic embryogenic plant material.This material is transformed using, for example, direct DNA methods, bombardment with DNA-coated microprojectiles, or infection with Agrobacterium, is grown in a suitable selection medium, and is regenerated, optionally also in the continued presence of a selection agent, into fertile transgenic soybean plants. Selection agents can be antibiotics such as kanamycin, hygromycin, or herbicides, or alternatively, selection can be based on the expression of a visualizeable marker gene such as GUS. Target tissues for transformation include meristematic tissue, somaclonal embryogenic tissue, and flower or flower-forming tissue. Other examples of soybean transformation include physical methods of DNA delivery, such as particle bombardment (see, ...). Petition 870250095065, dated 10 / 17 / 2025, pp. 205 / 375 191 / 346 for example. Finer & McMullen, In Vitro Cell Dev. Biol, 1991, 27P: 175-182; McCabe et al., Bio / technology, 1998, 6: 923-926), capillary crystal (Khalafalla et al., African J. of Biotechnology, 2006, 5: 1594-1599), aerosol beam injection (U.S. Patent No. 7,001,754), or by Agrobacterium-mediated delivery methods (Hinchee et al., Bio / Technology, 1988, 6: 915-922; U.S. Patent No. 7,002,058; U.S. Patent Application Publications Nos. 20040034889 and 20080229447; Paz et al.

[0381] Transgenic soybean plants can be generated with a binary vector containing AtPPOl and variants (SEQ ID NO:1 and 124-137) or SiPPOl and variants (SEQ ID NO:2 and 138-151) using any available transformation method. Optionally, the PPO gene can provide the means of selection and identification of transgenic tissue. For example, a vector is used to transform immature seed targets as described to generate transgenic PPO soybean plants directly using PPO inhibitor as the selection agent. A PPO gene may be present in the polynucleotide in conjunction with other sequences that provide additional means of selection / identification of transformed tissue including, for example, known genes that provide resistance to kanamycin, hygromycin, phosphinothricin, butafenacil or glyphosate. For example, different binary vectors containing the selectable marker genes PAT or EPSPS are known in the art (see, e.g.(U.S. Patent Application Publication No. 20080229447). Alternatively, selectable marker sequences may be present on separate polynucleotides and one is used. Petition 870250095065, dated 10 / 17 / 2025, pp. 206 / 375 192 / 346 process of, for example, cotransformation and coselection. A scoreable marker gene such as GUS can also be used to identify transformed tissue.

[0382] TO plants are collected from tissue culture and taken to the greenhouse where they are transplanted into water-saturated soil (REDI-EARTH® Plug and Seedling Mix, Sun Gro Horticulture, Bellevue, WA, or Fafard Germinating Mix) mixed with 1% granular MARATHON® (Olympic Horticultural Products, Co., Mainland, PA) at 5-10 g / gal of soil in 2 square pots. The plants are covered with humidity domes and placed in a Conviron chamber (Pembina, ND) with the following environmental conditions: 24°C day; 20°C night; photoperiod of 16-23 hours of light - 1-8 hours of darkness; 80% relative humidity.

[0383] After the plants have established themselves in the soil and new growth has appeared (-1-2 weeks), the plants are sampled and tested for the presence of the desired transgene by TAQMAN® analysis using probes appropriate for PPO genes or promoters. Positive plants are transplanted into 4-liter square pots containing Fafard #3 soil. Sierra 17-6-12 slow-release fertilizer is incorporated into the soil at the recommended rate. The plants are then repositioned in a standard greenhouse to acclimatize (~1 week). Environmental conditions were: 27 °C day; 21 °C night; 14-hour photoperiod (with supplemental light); ambient humidity. After acclimatization (~1 week), the plants are sampled and tested in detail for the presence and number of copies of inserted transgenes. Transgenic soybean plants are grown to maturity for T1 seed production. Petition 870250095065, dated 10 / 17 / 2025, page 207 / 375 193 / 346

[0384] TI plants are grown and tested for the presence of PPO genes disclosed by PCR. Plants that test positive in the PCR assay are transferred to an incubator for further evaluation.

[0385] After TAQMAN® analysis, homozygous plants were grown for seed production. Transgenic seeds and offspring plants are used to further evaluate their herbicide tolerance performance and molecular characteristics.

[0386] The plants are treated with PPO-inhibiting herbicides to confirm herbicide resistance. Example 8: Generation of Transgenic Corn and Soybean Plants Expressing Heterologous Variants of AtPPOl and SiPPOl

[0387] The transformation methods described in Examples 6 and 7 were used to transform corn and soybeans with AtPPOL and SiPPOL variants disclosed here.

[0388] At least 6 PPO1 events / variants shown in Table 25 and Table 26 were selected to advance to the T1 screening. For each event, 36-54 seeds were sown per event. Ten days after sowing, leaf tissue from all successfully germinated seeds was sampled for TAQMAN® as noted in Example 7, and zygosity was determined. Chi-square analyses were performed on all zygosity data to determine the segregation pattern of each event. The Chi-square null hypothesis was defined as a 1:2:1 ratio of Null:Heterozygous:Homozygous. A p-value cutoff of <=0.05 was used to reject the null hypothesis. Table 25: PPO1 variants expressed in corn AtPPol-VO Petition 870250095065, dated 10 / 17 / 2025, pp. 208 / 375 194 / 346 AtPP01-V2 AtPP01-V4 AtPP01-V18 AtPP01-V19 AtPP01-V22 AtPP01-V25 AtPPOL-V30 AtPP01-V31 AtPP01-V35 AtPP01-V36 AtPP01-V52 AtPP01-V55 S1PP01-V4 S1PP01-V18 S1PPO1-V30 S1PPO1-V55 Table 26: PP01 variants expressed in soybeans AtPPol-VO AtPPOL-V2 AtPPOL-V4 AtPPOL-V18 AtPPOL-V19 AtPPOL-V20 AtPP01-V22 AtPP01-V25 AtPPOL-V30 AtPPOl-V31 AtPP01-V35 AtPP01-V36 Petition 870250095065, dated 10 / 17 / 2025, page 209 / 375 195 / 346 AtPP01-V52 AtPP01-V55 SiPPOL-VO S1PPO1-V2 S1PPO1-V4 S1PPO1-V18 SIPPO1-V30 SIPPO1-V36 SIPPO1-V55 TABLES OF DETAILED DESCRIPTION Table 27 - Modified PPO Reasons Modified residue (with reference to SEQ ID NO: 1) Reason (modified residue shown in bold) SEQ ID NO S305 S; (K / N / R); (L / V); K; L; St; W; (E / K / T); L; L; (G / S); I; (I / K / S / T); K 337 S305 -2 K; L; St; W; (E / K / T); L; L; (G / S); I 346 Y361 L; (TO THE); (I / K / N / Q / R / S); (F / L); (D / F / P / Q / R / T); Y; P; P; V; (A / G); (TO THE); V 338 Y361 -2 (D / F / P / Q / R / T) ; Y; P; P; V; (A / G); (TO THE); V 347 Y362 L; (TO THE); (I / K / N / Q / R / S); (F / L) ; (D / F / H / Y); F; P; P; V; (A / G); (TO THE); V 339 Petition 870250095065, dated 10 / 17 / 2025, pp. 210 / 375 196 / 346 Y362-2 (F / L) ; (D / F / H / Y) ; F; P; P; V; (A / G); (A / S); V 348 V365 L; (A / S); (I / K / N / Q / R / S) ; (F / L) ; (D / F / H / Y); Y; P; P; (L / M); (A / G); (A / S); V 340 V365-2 Y; P; P; (L / M); (A / G); (A / S); V 349 G404 G; (I / L / V); E; T; L; A; (S / T); I; Y; S; S; S; L; F; P 341 G404-2 E; T; L; A; (S / T); I; Y; S; S; S; L; F; P 350 Y426 L; F; P; (G / N) ; R; A; P; (A / D / K / N / P / S) ; G; (Q / R); (l / T / V); (M / L) ; (I / L); L; (N / S); (C / L / M / T / V) ; I; G; G; (A / S); (K / L / T); N 342 Y426-2 L; (N / S); (C / L / M / T / V); I; G; G; (A / S); (K / L / T); N 351 T431 L; F; P; (G / N) ; R; A; P; (A / D / K / N / P / S); G; (Q / R); (l / T / V); (M / L); (I / L); L; (N / S); (F / Y); I; G; G; (A / S); R; N 343 T431-2 L; (N / S); (F / Y); I; G; G; (A / S); R; N 352 T461 (G / P / S); (D / K / N / R / S / T); (A / S); Q; (D / E); P; (F / L / R / S); (A / T / V); (L / T / V) ; (G / S) ; V; (K / R) ; V; W; (P / R); (K / Q / R); A; I; P; Q; F; L; (I / V); G 344 Petição 870250095065, de 17 / 10 / 2025, pág. 211 / 375 197 / 346 T461-2 A / S); Q; (D / E); P; (F / L / R / S); (A / T / V); (L / T / V); (G / S); V; (K / R) ; V; W; (P / R) ; (K / Q / R) ; A; I; P; Q; F; L 353 L479 (G / P / S); (D / K / N / R / S / T); (A / S); (A / E / G / K / Q / V) ; (D / E) ; P; (F / L / R / S); (A / T / V); (L / T / V); (G / S) ; V; (K / R) ; V; W; (P / R) ; (K / Q / R); A; I; P; Q; F; (M / N) ; (l / V); G 345 L479 - 2 (G / P / S); (D / K / N / R / S / T); (A / S); (A / E / G / K / Q / V / T) ; (D / E) ; P; (F / L / R / S); (A / T / V / K); (L / T / V); (G / S) ; V; (K / R) ; V; W; (P / R) ; (K / Q / R); A; I; P; Q; F; (M / N) ; (I / V); G 354 L479 - 3 AIPQF[M / N][I / V]G 355 Petição 870250095065, de 17 / 10 / 2025, pág. 212 / 375 198 / 346 SEQUÊNCIAS PP01 de Arabidopsis thaliana (com peptídeo de trânsito) (SEQ ID NO:1) MELSLLRPTTQSLLPSFSKPNLRLNVYKPLRLRCSVAGGPTVGS S KIE GGGGT TIT T DC VIVGGGISGLCIAQALATKHPDAAPNLIVTEAKDRVGGNIITREENGFLWEEGPNSFQP SDPMLTMWDSGLKDDLVLGDPTAPRFVLWNGKLRPVPSKLTDLPFFDLMSIGGKIRAG FGALGIRPSPPGREESVEEFVRRNLGDEVFERLIEPFCSGVYAGDPSKLSMKAAFGKVW KLEQNGGSIIGGTFKAIQERKNAPKAERDPRLPKPQGQTVGSFRKGLRMLPEAISARLG SKVKLSWKLSGITKLESGGYNLTYETPDGLVSVQSKSWMTVPSHVASGLLRPLSESAA NALSKLYYPPVAAVSISYPKEAIRTECLIDGELKGFGQLHPRTQGVETLGTIYSSSLFP NRAPPGRILLLNYIGGSTNTGILSKSEGELVEAVDRDLRKMLIKPNSTDPLKLGVRVWP QAIPQFLVGHFDILDTAKSSLTSSGYEGLFLGGNYVAGVALGRCVEGAYETAIEVNNFM SRYAYK* PPO1 de Setaria italica (com peptideo de tránsi) (SEQ ID NO: 2) MVAAAMATAPSAGVPPLRGTRGPARFRIRGVSVRCAAVAGGAAEAPASAGARVSADCW VGGGISGLCTAQALATKHGVGDVLVTEARARPGGNITTVERPDEGYLWEEGPNSFQPSD PVLTMAVDSGLKDDLVFGDPNAPRFVLWEGKLRPVPSKPADLPFFDLMSIPGKLRAGFG ALGIRPPPGREESVEE FVRRNL GAEVFE RLIE P FC S GVYAGDP S KL SMKAAFGKVWRL EEAGGSIIGGTIKTIQERGKNPKPPPRPDRLPTPKGQTVASFRKGLAMLPNAITSSLGSK VKLSWKLTSITKSDGMGYVLVYETPEGWSVQAKSVIMTIPSYVASDILRPLSSDAADA LSRFYYPPVAAVTISYPKEAIRKECLIDGELQGFGQLHPRSQGVETLGTIYSSSLFPNR APAGRVLLLNYIGGATNTGIVSKSASELVEAVDRDLRKMLINPSAVDPLVLGVRVWPQA IPQFLVGHLDLLEAAKSSLDRGGYDGLFLGGNYVAGVALGRCVEGAYESASQISDFLTK YAYK* pET24A AtPPO1 (SEQ ID NO:3) GCGGCCGCACTCGAGCACCACCACCACCACCACTGAGATCCCGGCTGCTAACAAAGCCCG AAAGGAAGCTGAGTTGGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGG CCCTTAAACGGGTCTTGAGGGGTTTTTGCTGAAAGGAGGAACTATATCCGGATTGGCG AATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGTGTGGTGGTTACGCGGCAGC Petition 870250095065, de 17 / 10 / 2025, pág. 213 / 375 199 / 346 GTGACCGCTACACTTGCCAGCGCCCTAGCCCGCTCCTTTCGCTTTCTTCCCTTCCTT TCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGT TCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCA CGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTT CTTTAATAGTGGACTCTTTGTCCAAACTGGAACACACTCAACCCTATCTCGGTCTATT CTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAATGAGACTGAATTTGAATTTGGCCTAAT TATTATT TAACGT T TACAAT TT CAGGT GGCAC TTTTCGGGGAAATGCGCGGAACCCCTATTTGTTTTTTTCTAAATACATTCAAATA TGTATCCGCTCAT GAAT TAAT TCT TAGAAAAAC TCATCGAGCAT CAAAT GAAACT GCAA TT TAT T CATAT CAGGAT CATACGT CATT CATTT CATTT CATAT TTCT GTAAT GAAG GAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATT CCGACTCGTCCAACATCAATACAACCTATTTCCCCTCGTCAAAAATAAGGTTATC AAGTGAGAAATCACCATGAGTGACTGACTGAATCCGGAGAGAATGAGCAAGTTGATT TTTCTTTCCAGACTTGTCCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCA TCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCG CATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTC CCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGAT GGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACAT CATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCA TACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCC ATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTAGAGCAAGACGTTTCCCGTT GAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTT CATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAA AGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACA AAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTT TCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGC CGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTA ATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTC AAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACAC Petição 870250095065, de 17 / 10 / 2025, pág. 214 / 375 200 / 346 AGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGA GAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGT CGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTC CTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGG CGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTG GCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTA CCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCA GTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGG TATTTCACACCGCATATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTT AAGCCAGTATACACTCCGCTATCGCTACGTGACTGGGTCATGGCTGCGCCCCGACACCC GCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGAC AAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAA CGCGCGAGGCAGCTGCGGTAAAGCTCATCAGCGTGGTCGTGAAGCGATTCACAGATGTC TGCCTGTTCATCCGCGTCCAGCTCGTTGAGTTTCTCCAGAAGCGTTAATGTCTGGCTTC TGATAAAGCGGGCCATGTTAAGGGCGGTTTTTTCCTGTTTGGTCACTGATGCCTCCGTG TAAGGGGGATTTCTGTTCATGGGGGTAATGATACCGATGAAACGAGAGAGGATGCTCAC GATACGGGTTACTGATGATGAACATGCCCGGTTACTGGAACGTTGTGAGGGTAAACAAC TGGCGGTATGGATGCGGCGGGACCAGAGAAAAATCACTCAGGGTCAATGCCAGCGCTTC GTTAATACAGATGTAGGTGTTCCACAGGGTAGCCAGCAGCATCCTGCGATGCAGATCCG GAACATAATGGTGCAGGGCGCTGACTTCCGCGTTTCCAGACTTTACGAAACACGGAAAC CGAAGACCATTCATGTTGTTGCTCAGGTCGCAGACGTTTTGCAGCAGCAGTCGCTTCAC GTTCGCTCGCGTATCGGTGATTCATTCTGCTAACCAGTAAGGCAACCCCGCCAGCCTAG CCGGGTCCTCAACGACAGGAGCACGATCATGCGCACCCGTGGGGCCGCCATGCCGGCGA TAATGGCCTGCTTCTCGCCGAAACGTTTGGTGGCGGGACCAGTGACGAAGGCTTGAGCG AGGGCGTGCAAGATTCCGAATACCGCAAGCGACAGGCCGATCATCGTCGCGCTCCAGCG AAAGCGGTCCTCGCCGAAAATGACCCAGAGCGCTGCCGGCACCTGTCCTACGAGTTGCA TGATAAAGAAGACAGTCATAAGTGCGGCGACGATAGTCATGCCCCGCGCCCACCGGAAG GAGCTGACTGGGTTGAAGGCTCTCAAGGGCATCGGTCGAGATCCCGGTGCCTAATGAGT GAGCTAACTTACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGT CGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGG Petition 870250095065, dated 10 / 17 / 2025, pp. 215 / 375 201 / 346 CGCCAGGGTGGTTTTTCTTTTCACCAGTGAGACGGGCAACAGCTGATTGCCCTTCACCG CCTGGCCCTGAGAGAGTTGCAGCAAGCGGTCCACGCTGGTTTGCCCCAGCAGGCGAAAA TCCTGTTTGATGGTGGTTAACGGCGGGATATAACATGAGCTGTCTTCGGTATCGTCGTA TCCCACTACCGAGATATCCGCACCAACGCGCAGCCCGGACTCGGTAATGGCGCGCATTG CGCCCAGCGCCATCTGATCGTTGGCAACCAGCATCGCAGTGGGAACGATGCCCTCATTC AGCATTTGCATGGTTTGTTGAAAACCGGACATGGCACTCCAGTCGCCTTCCCGTTCCGC TATCGGCTGAATTTGATTGCGAGTGAGATATTTATGCCAGCCAGCCAGACGCAGACGCG CCGAGACAGAACTTAATGGGCCCGCTAACAGCGCGATTTGCTGGTGACCCAATGCGACC AGATGCTCCACGCCCAGTCGCGTACCGTCTTCATGGGAGAAAATAATACTGTTGATGGG TGTCTGGTCAGAGACATCAAGAAATAACGCCGGAACATTAGTGCAGGCAGCTTCCACAG CAATGGCATCCTGGTCATCCAGCGGATAGTTAATGATCAGCCCACTGACGCGTTGCGCG AGAAGATTGTGCACCGCCGCTTTACAGGCTTCGACGCCGCTTCGTTCTACCATCGACAC CACCACGCTGGCACCCAGTTGATCGGCGCGAGATTTAATCGCCGCGACAATTTGCGACG GCGCGTGCAGGGCCAGACTGGAGGTGGCAACGCCAATCAGCAACGACTGTTTGCCCGCC AGTTGTTGTGCCACGCGGTTGGGAATGTAATTCAGCTCCGCCATCGCCGCTTCCACTTT TTCCCGCGTTTTCGCAGAAACGTGGCTGGCCTGGTTCACCACGCGGGAAACGGTCTGATAAGAGACACCGGCATACTCTGCGACATCGTATAACGTTACTGGTTTCACATTCACCACC CTGAATTGACTCTCTTCCGGGCGCTATCATGCCATACCGCGAAAGGTTTTGCGCCATTC GATGGTGTCCGGGATCTCGACGCTCTCCTTATGCGACTCCCTGCATTAGGAAGCAGCCC AGTAGTAGGTTGAGGCCGTTGAGCACCGCCGCGCCAAGGAATGGTGCATGCAAGGAGAT GGCGCCCAACAGTCCCCCGGCCACGGGCCTGCCACCATACCCACGCCGAAACAAGCGC TCATGAGCCCGAAGTGGCGAGCCCGATCTTCCCCATCGGTGATGTCGGCCGGGGGGGG CCAGCAACCGCACCTGTGGCGCCGGTGATGCCGGCCACGATGCGTCCGGCGTAGGAT CGAGATCTCGATCCCGCGAAATTACGACTCACTATAGGGGAATTTGTGAGCGGATAA CAATTCCCCTCTAGAAATAATTTGTTAACTTTAAGAAGGAGATATACATATGGAACT TTCTTTGTTGCGCCCGACTACACAATCCCTGCTGCCCTCTTTTAGCAAGCCGAATCTGC GTCTGAATGTGTATAAGCCACTGCGCCTGCGCTGTTCTGTGGCAGGCGGCCGACGGTG GGGTCGTCGAAGATCGAGGGTGGCGGCGGCACTACGATTACCGACTGCGTTATCGT GGGCGGGGCATCAGCCGGCTTGTGCATCGCAAGCCCTGGCCACGAAACATCCCGATG CAGCACCCAATCTGATCGTAACGGAAGCGAAGGATCGCGTGGGTGGGAACATTACA Petition 870250095065, of 17 / 10 / 2025, p. 216 / 375 202 / 346 CGCGAAGAGAATGGTTTTCTGTGGGAAGAAGGGCCTAATTCCTTTCAGCCAAGCGATCC GATGTTAACCATGGTAGTGGACTCAGGCCTGAAAGATGACCTCGTGCTCGGCGACCCGA CCGCACCGCGTTTCGTCCTGTGGAATGGCAAACTTCGCCCGGTGCCGTCTAAACTGACC GACCTGCCGTTCTTTGACCTCATGTCCATCGGGGGTAAAATCCGTGCTGGCTTTGGCGC TCTCGGGATTCGTCCGTCTCCGCCGGGTCGCGAAGAAAGTGTAGAAGAGTTCGTCCGCC GTAATCTGGGCGATGAAGTCTTTGAGCGTCTGATCGAGCCATTCTGTAGTGGTGTTTAT GCTGGTGATCCGAGTAAACTGTCCATGAAGGCCGCGTTTGGCAAGGTGTGGAAACTGGA GCAGAATGGAGGTAGCATCATCGGGGGTACATTCAAAGCAATTCAAGAACGGAAAAATG CTCCCAAAGCAGAACGTGATCCGCGGCTCCCGAAACCGCAAGGTCAGACCGTTGGTTCC TTCCGCAAAGGACTTCGCATGCTGCCGGAAGCCATCAGCGCGCGTTTGGGAAGCAAAGT AAAATTGTCTTGGAAACTGTCGGGTATTACGAAACTGGAATCGGGGGGTTATAATTTGA CATATGAAACCCCAGATGGTTTGGTGTCAGTGCAGAGCAAGAGCGTTGTCATGACCGTG CCCTCACATGTCGCCTCCGGGTTGTTGCGTCCGCTGAGCGAAAGCGCAGCGAACGCCCT CTCTAAACTTTACTATCCGCCTGTTGCCGCCGTGAGCATTAGTTATCCTAAGGAAGCTA TTCGCACCGAATGTCTGATTGATGGTGAGCTCAAGGGTTTTGGTCAGCTGCACCCGCGG ACTCAGGGCGTCGAGACTTTAGGTACCATCTACTCCTCTTCTCTGTTTCCGAACCGCGCACCGCCGGGCCGCATTCTGCTGCTGAATTATATTGGTGGTAGTACGAATACCGGAATTT TATCGAAAAGTGAAGGTGAATTGGTCGAAGCCGTCGATCGTGACCTTCGCAAAATGCTG ATTAAACCGAACTCGACCGACCCGCTGAAGTTAGGCGTGCGTGTGTGGCCCCAAGCGAT CCCACAGTTTCTGGTTGGTCACTTCGATATTCTGGATACCGCCAAATCCAGCCTGACAA GTAGCGGCTATGAAGGGCTGTTCCTGGGCGGGAACTA...

Claims

1. Modified Protoporphyrinogen Oxidase (PPO) enzyme or functional fragment thereof, characterized in that the modified Protoporphyrinogen Oxidase or functional fragment thereof comprises at least one mutation in one or more amino acid residues selected from residues 479, 365 and / or 362 of SEQ ID NO:1 or residues corresponding thereto.

2. Modified PPO enzyme or functional fragment thereof, according to claim 1, characterized in that the modified Protoporphyrinogen Oxidase or functional fragment thereof comprises the motif: (G / P / S); (D / K / N / R / S / T); (A / S); (A / E / G / K / Q / V / T); (D / E); P; (F / L / R / S); (A / T / V / K); (L / T / V); (G / S); V; (K / R); V; W; (P / R); (K / Q / R); A; I; P; Q; F; (M / N); (I / V); G (SEQ ID NO: 354); or AIPQF[M / N][I / V]G (SEQ ID NO: 356).

3. Modified PPO enzyme or functional fragment thereof, according to any one of claims 1 or 2, characterized in that it comprises a combination of mutations in residues: (a) 365, 426 and 479 of SEQ ID NO:1 or residues corresponding thereto; optionally where the mutations comprise the substitutions 365L, 426V and 479N of SEQ ID NO:1 or substitutions corresponding thereto; (b) 305, 426 and 479 of SEQ ID NO:1 or residues corresponding thereto; optionally where the mutations comprise the substitutions 305L, 426V and 479M of SEQ ID NO:1 or substitutions corresponding thereto; or Petition 870250095065, dated 10 / 17 / 2025, p. 362 / 375 2 / 11 (c) 305, 361, 362, 426 and 479 of SEQ ID NO:1 or residues corresponding thereto; optionally wherein the mutations comprise the substitutions 305L, 361C, 362F, 426M and 479M of SEQ ID NO:1 or substitutions corresponding thereto.

4. Modified PPO enzyme or functional fragment thereof, according to any one of claims 1, 2 or 3, characterized in that the modified PPO enzyme or functional fragment thereof comprises an amino acid sequence having at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 98% or at least 99% sequence identity with: any of the following SEQ ID NOs: 58, 59, 126, 127, 130, 131, 132, 134, 136, 137, 209, 210, 277, 278, 281, 282, 283, 285, 287, 288, 118, 119, 140, 141, 144, 145, 146, 148, 150, 151, 269, 270, 291, 292, 295, 296, 297, 299, 301 or 302; or any of the SEQ ID NOS 126, 131, 134, 140, 145, 148, 277, 282, 285, 291, 296 or 299.

5. Modified protoporphyrinogen oxidase (PPO) enzyme or functional fragment thereof, characterized in that the modified protoporphyrinogen oxidase or functional fragment thereof comprises mutations in amino acid residues 305 and 426 of SEQ ID NO:1 or residues corresponding thereto, wherein the mutations are the 305L and 426V substitutions.

6. Modified PPO enzyme or functional fragment thereof, according to any of claims 1, 2, 3, 4 or 5, Petition 870250095065, dated 10 / 17 / 2025, p.363 / 375 3 / 11 characterized in that the modified PPO enzyme or functional fragment thereof is a PPO1 enzyme; optionally wherein the modified PPO enzyme or functional fragment thereof comprises increased resistance to a compound that inhibits a PPO enzyme relative to a control PPO enzyme and / or has a decreased percentage of inhibition in response to a compound that inhibits a PPO enzyme relative to a control PPO enzyme; additionally optionally wherein the increased resistance to the compound comprises an increase of at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold in resistance to the compound compared to an unmodified reference plant or part thereof; additionally optionally wherein the control PPO enzyme is an unmodified PPO enzyme or a PPO enzyme modified according to SEQ ID NO:

124.

7. Modified PPO enzyme or functional fragment thereof, according to any one of claims 1, 5 or 6, characterized in that the modified PPO enzyme or functional fragment thereof comprises: (a) an amino acid sequence having at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 98% or at least 99% sequence identity with SEQ ID NO: 1 and at least one mutation in one or more amino acid residues selected from residues 362, 365 and / or 479 of SEQ ID NO: 1 or residues corresponding to the same: or Petition 870250095065, of 10 / 17 / 2025, page 364 / 375 4 / 11 (b) an amino acid sequence having at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 70%,at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 98% or at least 99% sequence identity with SEQ ID NO: 2 and at least one mutation in one or more amino acid residues selected from residues 360, 363 and / or 477 of SEQ ID NO: 2 or residues corresponding to them; or (c) an amino acid sequence having at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 98% or at least 99% sequence identity with SEQ ID NO: 153 and at least one mutation in one or more amino acid residues selected from residues 328,331 and / or 445 of SEQ ID NO: 153 or residues corresponding thereto; or (d) an amino acid sequence having at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 98% or at least 99% sequence identity with SEQ ID NO: 154 and at least one mutation in one or more amino acid residues selected from residues 325, 328 and / or 442 of SEQ ID NO: 154 or residues corresponding thereto. Petition 870250095065, dated 10 / 17 / 2025, pp. 365 / 375 5 / 11, 8. Modified PPO enzyme or functional fragment thereof: (i) according to any one of claims 1, 6 or 7, characterized in that the modified PPO enzyme or functional fragment thereof further comprises one or more additional mutations in one or more amino acid residues selected from residues 305, 361, 404, 426, 431 and / or 461 of SEQ ID NO: 1 or residues corresponding thereto; or (ii) of any one of claims 5, 6 or 7, characterized in that the modified PPO enzyme or functional fragment thereof further comprises one or more additional mutations in one or more amino acid residues selected from residues 361, 362, 365, 404, 431, 461 and / or 479 of SEQ ID NO: 1 or residues corresponding thereto; optionally where: (a) the mutation at residue 305 of SEQ ID NO: 1 or residues corresponding to it comprises a substitution for a leucine;(b) the mutation at residue 361 of SEQ ID NO: 1 or residues corresponding thereto comprises a substitution for cysteine, aspartic acid, glutamine or threonine; (c) the mutation at residue 404 of SEQ ID NO: 1 or residues corresponding thereto comprises a substitution for alanine; (d) the mutation at residue 426 of SEQ ID NO: 1 or residues corresponding thereto comprises a substitution for leucine, cysteine, methionine, threonine or valine; Petition 870250095065, dated 10 / 17 / 2025, pp. 366 / 375 6 / 11 (e) the mutation at residue 431 of SEQ ID NO: 1 or residues corresponding thereto comprises a substitution for alanine, phenylalanine or arginine; and / or (f) the mutation at residue 461 of SEQ ID NO: 1 or residues corresponding thereto comprises a glutamine substitution.; 9. Modified PPO enzyme or functional fragment thereof: (i) according to any one of claims 1 or 6 or 7 or claim 8(i), characterized in that; (a) the mutation at residue 362 of SEQ ID NO: 1 or residues corresponding thereto comprises a substitution for a cysteine ​​or phenylalanine; (b) the mutation at residue 365 of SEQ ID NO: 1 or residues corresponding thereto comprises a substitution for a methionine or a leucine; and / or (c) the mutation at residue 479 of SEQ ID NO: 1 or residues corresponding thereto comprises a substitution for a methionine or asparagine; or (ii) according to claim 8(ii), wherein: (a) the mutation at residue 362 of SEQ ID NO: 1 or residues corresponding thereto comprises a substitution for a cysteine ​​or phenylalanine; (b) the mutation at residue 365 of SEQ ID NO: 1 or residues corresponding thereto comprises a substitution for a methionine or a leucine;and / or (c) the mutation at residue 479 of SEQ ID NO: 1 or residues corresponding thereto comprises a substitution for a methionine or asparagine.; 10. Modified PPO enzyme or functional fragment thereof, according to any one of claims 1 or 6, 7, 8. Petition 870250095065, dated 10 / 17 / 2025, p.367 / 375 7 / 11 or 9, characterized in that it comprises a combination of mutations in residues: (a) 365, 426 and 479 of SEQ ID NO:1 or residues corresponding thereto; (b) 305, 404, 426, 431 and 479 of SEQ ID NO:1 or residues corresponding thereto; (c) 305 and 365 of SEQ ID NO:1 or residues corresponding thereto; (d) 305, 362 and 404 of SEQ ID NO:1 or residues corresponding thereto; (e) 305, 361, 365, 431 and 479 of SEQ ID NO:1 or residues corresponding thereto; (f) 305, 426 and 479 of SEQ ID NO:1 or residues corresponding thereto; (g) 426, 461 and 479 of SEQ ID NO:1 or waste corresponding to them; (h) 305, 361 and 365 of SEQ ID NO:1 or waste corresponding to them; (i) 305, 361, 362, 426 and 479 of SEQ ID NO:1 or waste corresponding to them; (j) 361 and 365 of SEQ ID NO:1 or waste corresponding to them; (k) 365, 404 and 479 of SEQ ID NO:1 or waste corresponding to them; or (1) 305, 404 and 479 of SEQ ID NO:1 or waste corresponding to them.

11. Modified PPO enzyme or functional fragment thereof: (a) according to any one of claims 1 or 6, 7, 8, 9 or 10, characterized in that the modified PPO enzyme or functional fragment thereof comprises or Petition 870250095065, dated 10 / 17 / 2025, pp. 368 / 375 8 / 11 consists of a sequence according to SEQ ID Nos: 188 - 190, 209, 210, 248 - 250, 269, 270, 277 - 288 or 291 302; or (b) any of claims 5, 6, 7, 8, 9 or 10, characterized in that the modified PPO enzyme or functional fragment thereof comprises or consists of a sequence according to SEQ ID NOS: 276 or 290.

12. Modified PPO enzyme or functional fragment thereof, according to any one of claims 1 or 6, 7, 8, 9, 10 or 11 or claims 5, 6, 7, 8, 9, 10 or 11, characterized in that the modified PPO enzyme further comprises a transit peptide; optionally wherein the transit peptide comprises a mitochondrial transit peptide and / or a chloroplastic transit peptide; additionally optionally wherein the modified PPO enzyme comprises a sequence having at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 98% or at least 99% sequence identity with a sequence according to any of the SEQ ID NOs: 1, 2, 4 - 151 or 305 - 320;additionally optionally, wherein the modified PPO enzyme comprises or consists of a sequence in accordance with the SEQ ID Nos: 37-39, 58, 59, 97-99, 118, 119, 125-137 or 139-151. Petition 870250095065, dated 10 / 17 / 2025, pp. 369 / 375 9 / 11; 13. Nucleic acid, characterized in that it comprises a polynucleotide encoding a modified PPO enzyme or a functional fragment thereof, as defined in any one of claims 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12; or a recombinant vector comprising nucleic acid.

14. A plant or part thereof, characterized in that it comprises a modified PPO enzyme or a functional fragment thereof, as defined in any one of claims 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12, or a recombinant nucleic acid or vector, as defined in claim 13; optionally wherein the plant or part thereof comprises increased resistance to a compound that inhibits a PPO enzyme relative to a control plant and / or has a decreased percentage of inhibition in response to a compound that inhibits a PPO enzyme relative to a control plant; additionally optionally wherein the control plant is a wild-type plant or a plant comprising a modified PPO enzyme according to SEQ ID NO:

124.

15. A method for producing a plant or part thereof according to claim 14, characterized in that it comprises: modifying the plant or part thereof to comprise a PPO enzyme, as defined in any one of claims 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12; optionally wherein modifying the plant or part thereof comprises transforming the plant or part thereof with a recombinant nucleic acid or vector, as defined in claim 10.

16. A method for controlling unwanted vegetation in the vicinity of a plant or part thereof, or at a locus for plant growth or part thereof, characterized in that the method comprises applying an effective amount of at least one PPO-inhibiting herbicide to the vicinity of the plant, to the locus for plant growth, or to the plant or part thereof, wherein said plant or part thereof comprises the plant or part thereof as defined in claim 14, wherein the effective amount of said PPO-inhibiting herbicide controls the unwanted vegetation in said vicinity or at said locus.

17. Use, characterized in that it is a compound that inhibits the enzymatic activity of PPO in combination with a plant or part thereof, as defined in claim 14.

18. Modified PPO enzyme, according to any one of claims 6, 7, 8, 9, 10, 11 or 12, the plant or part thereof of claim 14 or the method of controlling unwanted vegetation of claim 16 or the use of claim 17, characterized in that the compound is a herbicide selected from the group consisting of: butafenacil, carfentrazone-ethyl, cyclopyranil, epirifenacil (Herbicide A), flufenoximecil, flumioxazin, fomesafen, oxyfluorfen, pyraflufen-ethyl, saflufenacil (Herbicide E), sulfentrazone, thiafenacil, trifludimoxazin (Herbicide B); ethyl ester of 3-(2-chloro-4-fluoro-5-(3-methyl-2,6-dioxo-4-trifluoromethyl-3,6-dihydropyrimidin-1(2H)-yl)phenyl)-5-methyl-4,5-dihydroisoxazole-5-carboxylic acid (Herbicide C); 2-[[3-[[3-chloro-5-fluoro-6-[3-methyl-2,6-dioxo-4-(trifluoromethyl)pyrimidinyl-yl]-2-pyridyl]oxy] ethyl acetate;(5S)-3-[2-chloro-5(3,5-dimethyl-2,6-dioxo-4-tioxo-l,3,5-triazinan-l-yl)-4fluoro-phenyl]-5-methyl-4H-isoxazole-5-carboxylate de ethyl (Herbicida D); pyridin-2-ylmethyl-[(3-(2-chloro-4-fluoro-5-[3-methyl-2,6-dioxo-4-(trifluoromethyl)-3,6-dihydropyrimidin-1(2H)-yl]phenoxy)pyridine-2-yl)oxy]acetate, 2-Methoxyethyl-[(3-(2-chloro-4-fluoro-5-[3-methyl-2,6-dioxo-4(trifluoromethyl)-3,6-dihydropyrimidin-1(2H)yl]phenoxy)pyridine-2-yl)oxy]acetate, 2-Methoxyethyl-[(3 - (2cyano-4-fluoro-5-[3-methyl-2,6-dioxo-4-(trifluoromethyl)-3,6-dihydropyrimidin-1(2H)-yl]phenoxy)pyridine-2-yl)oxy]acetate and cyanomethyl-[(3-(2-bromo-4-fluoro-5-[3-methyl-2,6-dioxo-4(trifluoromethyl)-3,6-di-hydropyrimidin-l(2H)yl]phenoxy)pyridine-2-yl)oxy]acetate; 3-[2-chloro-5-[3,6-dihydro-3-methyl-2,6-dioxo-4-(trifluoromethyl)-1(2H)pyrimidinyl]-4-fluorophenyl]-3a,4,5,6-tetra-hydro-6-methyl6aH-cyclopent[d]isoxazole-6a-carboxylate de methyl; 2—[2—[2—bromo-4-fluoro-5-[3-methyl-2,6-dioxo-4(trifluoromethyl)pyrimidin-l-yl]phenoxy]phenoxy]-2-methoxyacetate de methyl;1-[2-chloro-4-fluoro-5-[3-methyl-2,6-dioxo-4-(trifluoromethyl)pyrimidin-lyl]phenoxy]cyclopropanecarboxylate of (2-ethoxy-2-oxo-ethyl), enantiomers (including mixtures enriched with enantiomers) of said compounds and / or agrochemically acceptable salts and / or esters thereof.

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