WO2025006490A2

WO2025006490A2 - Plants with increased herbicide tolerance and methods of production and use thereof

Info

Publication number: WO2025006490A2
Application number: PCT/US2024/035450
Authority: WO
Inventors: Dean Edward RIECHERS; Olivia Augusta LANDAU
Original assignee: University of Illinois at Urbana Champaign; University of Illinois System
Current assignee: University of Illinois at Urbana Champaign; University of Illinois System
Priority date: 2023-06-26
Filing date: 2024-06-25
Publication date: 2025-01-02
Anticipated expiration: 2025-12-26
Also published as: WO2025006490A3; WO2025006490A9

Abstract

Provided herein are newly identified genes encoding proteins involved in herbicide detoxification processes in plants. It is demonstrated that these genes and their encoded proteins are involved in herbicide detoxification processes in plants and increased expression of these genes can increase herbicide tolerance in plants. Accordingly, methods for generating a plant with increased herbicide tolerance is provided, including gene-editing and transformation methods that increase the level of the one or more herbicide detoxification genes or gene products in plants. Also provided are the plants, seeds, plant cells, or plant parts generated by or during these methods.

Description

PLANTS WITH INCREASED HERBICIDE TOLERANCE AND METHODS OF PRODUCTION

AND USE THEREOF

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No. 63/523,127, filed June 26, 2023, which is herein incorporated by reference in its entirety.

FIELD

The present disclosure generally relates to the field of transformed or gene-edited plants, particularly plants with increased herbicide resistance.

SEQUENCE LISTING

The nucleic and amino acid sequences described herein and listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and single letter code for amino acids, as defined in 37 C.F.R. 1 .822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand. The Sequence Listing is submitted as an XML file in the form of the file named “7950-112089-01_SEQ_ST.26.xml” (156,552 bytes), which was created on June 25, 2024, which is incorporated by reference herein.

BACKGROUND

Halauxifen-methyl (HM) is a member of the picolinic carboxylic acid subclass of synthetic auxins (Epp et al., 2016; Schmitzer et al., 2015). Like other synthetic auxins, HM mimics indole-3-acetic acid (natural auxin), which regulates almost every aspect of plant growth and development (McSteen, 2010; Zazimalova et al., 2014), and it provides selective post-emergence dicot weed control in cereal crops (Epp et al., 2016; Mithila et aL, 2011). HM was initially developed as a herbicide mixing partner for postemergence weed control in cereal crops at low application rates (5.0 to 7.5 g ha ¹), but it can also be used as a burndown treatment prior to soybean planting at even lower rates (1.0 to 2.0 g ha ¹) due to its short soil half-life of 10-25 days and little soil residual activity (Epp et al., 2016). HM is typically applied postemergence to wheat in tank mixtures with other herbicides and herbicide safeners (Epp et al., 2018).

Allohexaploid bread wheat (Triticum aestivum L., 2n = 6x = 42; AABBDD) comprises three genomes denoted as the A, B, and D genomes, which results in three homeologous sets of seven chromosomes (Glover et al., 2016; IWGSC, 2018). Like other cereal crops, wheat is naturally tolerant to the synthetic auxin herbicides, which are commonly utilized for selective postemergence dicot weed control (Grossmann, 2010; Mithila et al., 2011). The primary mechanism behind this selectivity is qualitative and quantitative differences in detoxification of these herbicides between grasses and dicots (Grossmann, 2010). For example, grasses possess cytochrome P450s (CYPs) that catalyze irreversible ring-hydroxylation, O- demethylation, or dealkylation reactions of synthetic auxin herbicides, forming a less toxic compound and predispose the herbicide to glucose conjugation by UDP-dependent glucosyltransferase (UGTs) and subsequent sequestration to the vacuole by ATP-binding cassette transport proteins (Davies & Caseley, 1999; Gaines et al., 2020; Yuan et al., 2007). By contrast, dicot metabolism mainly consists of reversible reactions, such as amino acid or glucose conjugation of the carboxylic acid, which results in some level of the biologically active form of the herbicide remaining in the plant cell (Mithila et al., 2011; Sterling & Hall, 1997).

Allohexaploid bread wheat (Triticum aestivum L.) achieves tolerance to HM through rapid detoxification of its biologically active form, halauxifen acid (HA) (Dzikowski et al., 2016). More specifically, once HM is de-esterified to HA by esterases, the HA is (9-demethylated by CYPs, and subsequently conjugated with glucose, thereby becoming a non-phytotoxic, polar metabolite (Figure 1) (Dzikowski et al., 2016). Herbicide safeners, such as cloquintocet-mexyl (CM), are often applied in wheat to increase the expression of the genes encoding herbicide-detoxifying enzymes, which results in enhanced herbicide tolerance. One common wheat herbicide utilized with CM is the synthetic auxin, halauxifen- methyl (HM).

CYPs are a superfamily of hemethiolate enzymes that are present in all kingdoms of life and catalyze reactions in numerous primary and secondary metabolite synthesis pathways, and xenobiotic detoxification (Bak et al., 201 1 ; Hansen et al., 2021 ; Nelson, 2009; Nelson et al., 2004). In plants, CYPs are essential for the biosynthesis and modification of primary (sterols and fatty acids) and secondary metabolites (phenylpropanoids, glucosinolates, and carotenoids), and they are also responsible for the synthesis and catabolism of hormones, including gibberellins, jasmonic acid, abscisic acid, brassinosteroids, and strigolactones (Mizutani & Ohta, 2010; Mizutani & Sato, 2011; Wakabayashi et al., 2019). The crosskingdom nomenclature for CYPs is based on amino acid sequence similarity with 40%, 55%, and 97% sequence identities used as cut-offs for family, subfamily, and allelic variant designations, respectively (Dimaano & Iwakami, 2021; Nelson, 2009). The total number of CYPs among species varies, but in general plants contain more CYPs than animals, which is thought to be a consequence of their sessile nature and their need to produce a vast number of secondary metabolites to adapt to abiotic and biotic stresses and communicate to other organisms (Bak et al., 2011). When it comes to herbicide-metabolizing CYPs, they are most often identified within Poaceae, of which there are currently 15 identified herbicide-detoxifying CYPs (Dimaano & Iwakami, 2021; Han et al., 2020; Pan et al., 2022; Zheng et aL, 2022).

Alien substitution and nullisomic-tetrasomic (NT) lines lacking chromosome 5A displayed significantly higher sensitivity to HM, compared to the unaltered hexapioid wheat cultivar, ‘Chinese Spring’ (CS) (Obenland & Riechers, 2020). Additionally, NT lines and alien substitution lines retaining 5A or tetrasomic for 5A maintained tolerance to HM similar to CS (Obenland & Riechers, 2020).

While metabolism of HM in wheat has been characterized, the genes encoding the HA-detoxifying enzymes have not been identified. SUMMARY

The present disclosure provides methods for generating a plant with increased tolerance to a herbicide. In some aspects, the methods include increasing expression of one or more herbicide detoxification genes, and/or increasing activity of one or more proteins encoded by the one or more herbicide detoxification genes in a plant, thereby generating the plant with increased tolerance to a herbicide, wherein the one or more herbicide detoxification genes include any of SEQ ID NOs: 1-7 (CYP81A-5A) or a nucleic acid sequence that includes at least 80% identity to any of SEQ ID NOs: 1-7; any of SEQ ID NOs: 8- 13 (CYP81A-5B) or a nucleic acid sequence that includes at least 80% identity to any of SEQ ID NOs: 8-13; and/or any of SEQ ID NOs: 14-19 (CYP81A-5D) or a nucleic acid sequence that includes at least 80% identity to any of SEQ ID NOs: 14-19; or wherein the one or more herbicide detoxification genes encode a protein including a sequence of any of SEQ ID NOs: 99-104, or a sequence that includes at least 80% identify to any of SEQ ID NOs: 99-104.

In other aspects, the methods include increasing expression of one or more herbicide detoxification genes, and/or increasing activity of one or more proteins encoded by the one or more herbicide detoxification genes in a plant cell or plant part, and growing the plant cell or plant part into a plant, thereby generating the plant with increased tolerance to a herbicide, wherein the one or more herbicide detoxification genes include any of SEQ ID NOs: 1 -7 (CYP81A-5A) or a nucleic acid sequence that includes at least 80% identity to any of SEQ ID NOs: 1-7; any of SEQ ID NOs: 8-13 CYP81A-5B) or a nucleic acid sequence that includes at least 80% identity to any of SEQ ID NOs: 8-13; and/or any of SEQ ID NOs: 14-19 (CYP81A-5D) or a nucleic acid sequence that includes at least 80% identity to any of SEQ ID NOs: 14-19; or wherein the one or more herbicide detoxification genes encode a protein including a sequence of any of SEQ ID NOs: 99-104, or a sequence that includes at least 80% identify to any of SEQ ID NOs: 99-104.

In some examples, increasing expression of one or more herbicide detoxification genes, and/or increasing activity of one or more proteins encoded by the one or more herbicide detoxification genes includes introducing one or more exogenous nucleic acid molecules into the plant, thereby generating a transformed plant, or into the plant part or plant cell, thereby generating a transformed plant part or plant cell, wherein the one or more exogenous nucleic acid molecules include the one or more herbicide detoxification genes, increase expression of the one or more herbicide detoxification genes, and/or increase activity of the one or more proteins.

In some examples, the one or more exogenous nucleic acid molecules generates one or more gain- of-function mutations in the one or more herbicide detoxification genes, wherein the one or more herbicide detoxification genes are endogenous to the plant, plant part, or plant cell. In some examples, the one or more gain-of-function mutations are in the promoter region of the one or more herbicide detoxification genes, and increase expression of the one or more herbicide detoxification genes in response to a herbicide safener or herbicide. In some examples, the gain-of-function mutation is a single nucleotide insertion about 256 bp upstream of TATA box. In some examples, the single nucleotide insertion is a G insertion is immediately after a position corresponding to position 137 of SEQ ID NO: 1. In some examples, the one or more exogenous nucleic acid molecules generate a mutated herbicide detoxification gene that includes the nucleic acid sequence of any of SEQ ID NOs: 20-22, or a nucleic acid sequence that includes at least 90% identity to any of SEQ ID NOs: 20-22 and has a G at a position corresponding to position 138 of SEQ ID NO: 20.

In some examples, the one or more exogenous nucleic acid molecules includes one or more guide nucleic acid molecules that are complementary to one or more regions of the one or more herbicide detoxification genes, wherein the one or more herbicide detoxification genes are endogenous to the plant, plant part, or plant cell. In some examples, the one or more exogenous nucleic acid molecules further includes a nucleic acid molecule encoding a Cas protein, or the method further includes introducing one or more Cas proteins into the plant, plant part, or plant cell.

In some examples, the transformed plant, plant cell, or plant part includes one or more gain-of- function mutations in the one or more herbicide detoxification genes, wherein the one or more herbicide detoxification genes are endogenous to the plant, plant part, or plant cell. In some examples, the transformed plant, plant part, or plant cell includes the one or more exogenous nucleic acid molecules including the one or more herbicide detoxification genes, wherein the one or more exogenous herbicide detoxification genes are integrated or not integrated into a genome of the transformed plant, plant part, or plant cell.

Also provided are transformed plant, plantlet, plant part, plant tissue, plant cell, or seed generated by or during the above methods.

The present disclosure also provides a transformed plant, plant part, plant cell, or seed, including one or more gain-of-function mutations in one or more endogenous herbicide detoxification genes, or including one or more exogenous herbicide detoxification genes integrated into a genome of the transformed plant, plant part, plant cell, or seed, wherein the one or more herbicide detoxification genes include any of SEQ ID NOs: 1-7 (CYP8IA-5A) or a nucleic acid sequence that includes at least 80% identity to any of SEQ ID NOs: 1-7; any of SEQ ID NOs: 8-13 (CYP81A-5B) or a nucleic acid sequence that includes at least 80% identity to any of SEQ ID NOs: 8-13; and/or any of SEQ ID NOs: 14-19 (CYP81A- 5D) or a nucleic acid sequence that includes at least 80% identity to any of SEQ ID NOs: 14-19; or wherein the one or more herbicide detoxification genes encode a protein including a sequence of any of SEQ ID NOs: 99-104, or a sequence that includes at least 80% identify to any of SEQ ID NOs: 99-104.

In some examples, the gain-of-function mutation increases binding affinity of the promoter region to a transcription factor or decrease binding affinity to a repressor. In some examples, the gain-of-function mutation is a single G insertion about 256 bp upstream of TATA box. In some examples, the single G insertion is immediately after a position corresponding to position 137 of SEQ ID NO: 1.

In some examples, the transformed plant, plant part, plant cell, or seed includes any of SEQ ID NOs: 20-22, or a nucleic acid sequence that includes at least 90% identity to any of SEQ ID NOs: 20-22 and has a G at a position corresponding to position 138 of SEQ ID NO: 20. In some examples, the transformed plant has tolerance to a herbicide increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 75%, at least 90%, at least 100%, at least 200%, or at least 400% as compared to a control plant. In some examples, the herbicide is an ALS inhibitor, such as sulfonylamino carbonyl triazolinones, such as propoxycarbazone-sodium (PROP) or a derivative or analog thereof. In some examples, the tolerance to a herbicide is tolerance to a herbicide in presence of a herbicide safener. In some examples, the herbicide safener is cloquintocet-mexyl (CM) or a derivative or analog thereof.

In some examples, the transformed plant, plant part, plant cell, or seed does not include a transgene used to generate the one or more gain-of-function mutations. In some examples, the transformed plant, plant part, plant cell, or seed is transgene-free. In other examples, the transformed plant, plant part, plant cell, or seed includes one or more transgenes.

In some examples, the transformed plant is a monocot or dicot, and in some examples, from the Poaceae family, and in some examples a cereal grass, and in some examples, wheat.

The present disclosure further provides methods of growing the transformed plant, plant part, plant cell, or seed of in the presence of a herbicide. In some examples, the herbicide is an acetolactate synthase (ALS) inhibitor. In some examples, the ALS inhibitor is a sulfonylamino carbonyl triazolinone. In some examples, the sulfonylamino carbonyl triazolinone is propoxycarbazone-sodium (PROP) or an analog or derivative thereof.

In some examples, the method further includes growing the transformed plant, plant part, plant cell, or seed in the presence of a herbicide safener. In some examples, the herbicide safener is cloquintocet- mexyl (CM) or a derivative or analog thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D: Structures of halauxifen-methyl and its metabolites in wheat. FIG. 1A: halauxifen- methyl (HM); FIG. IB: halauxifen acid (HA); FIG. 1C: O-demethylated HA; FIG. ID: HA-glucose conjugate.

FIGS. 2A-2C: Photographs showing wheat ‘Chinese Spring’ (CS; FIG. 2A) and nullisomic- tetrasomic (NT) lines N5A-T5D (FIG. 2B) and N5D-T5A (FIG. 2C after treatment with HM. Plants were photographed 20 days following treatments (one day before harvest). Seedlings at Zadoks stages 11-12 were treated with 1.25% methylated seed oil, 2.5% ammonium sulfate, and 60 g a.e. ha ¹ HM in the treatment group, or treated with 1.25% methylated seed oil, 2.5% ammonium sulfate, and adjuvant in the control group.

FIG. 3: A bar graph showing mean normalized peak abundance of HA per g fresh weight in CS, Aegilops searsii (AS), and wheat group 5 alien substitution lines for 5A, 5B, and 5D. Means were determined from seven biological replicates. Error bars represent the standard error of the mean. An asterisk indicates a significant difference from CS at the given time point based on Fisher’s LSD (a = 0.05). FIG. 4: A bar graph showing mean normalized peak abundance of HA per g fresh weight in CS and NT lines N5A-T5D and N5D-T5A. Means were determined from five biological replicates. Error bars represent the standard error of the mean. An asterisk indicates a significant difference from CS at the given time point based on Fisher’s LSD (a = 0.05).

FIG. 5: A mean-difference plot showing the log2 fold change and mean abundance of each transcript in log2 counts per million (CPM). Genes that were significantly induced (Up) and repressed (Down) by cloquintocet-mexyl (CM) are highlighted in red and blue, respectively. Genes that were not significantly differentially expressed (Non-DE) are in black.

FIG. 6: A tree map of functional annotations assigned to significant differentially expressed genes identified by RNA-Seq. The main categories (bolded and underlined) include Phase I (light green), Phase II (orange), and Phase III (grey) metabolism, Amino Acid Metabolism (light blue), Transcription Factors (TFs; yellow), Proteins with only Domain Info (dark blue), and Stress/Defense Related (dark green). The number of identified genes is listed in parentheses or after the comma. Abbreviations: 2OG = 2-oxoglutarate; 50- PORs = progesterone 5-beta-reductase; ABC = ATP-binding cassette; ADH = alcohol dehydrogenase; AzoR = azoreductase; CCR = cinnamoyl-CoA reductase 4; CSE = cystathionine gamma-lyase; CYP = cytochrome P450; GST = glutathione 5-transferase; GT = glycosyltransferase; IP = inhibitor protein; LAC = laccase; MTOX = N- methyl -L-tryptophan oxidase; OPR = 12-oxophytodienoate reductase; PRP = pathogen -related protein; SBP = selenium-binding protein; SCP = serine carboxypeptidase; SDR = short chain dehydrogenase/reductase.

FIG. 7: Complete molecular function results of agriGO v2.0. The color of the box indicates the significance level of the false discovery rate (reported in parentheses), with the yellow indicating relatively low significance and the gradation intensifies towards red to indicate higher significance. At the bottom of each significant box, the first fraction represents number of significant differentially expressed genes assigned the GO term (out of 99), and the second fraction indicates the number of genes in the Triticum aestivum L. reference background with the same GO annotation.

FIG. 8: Results of agriGO v2.0 related to transferase activity. The color of the box indicates the significance level of the false discovery rate (reported in parentheses), with the yellow indicating relatively low significance and the gradation intensifies towards red to indicate higher significance. At the bottom of each significant box, the first fraction represents number of significant differentially expressed genes assigned the GO term (out of 99), and the second fraction indicates the number of genes in the Triticum aestivum L. reference background with the same GO annotation.

FIG. 9: Results of agriGO v2.0 related to oxidoreductase activity. The color of the box indicates the significance level of the false discovery rate (reported in parentheses), with the yellow indicating relatively low significance and the gradation intensifies towards red to indicate higher significance. At the bottom of each significant box, the first fraction represents number of significant differentially expressed genes assigned the GO term (out of 99), and the second fraction indicates the number of genes in the Triticum aestivum L. reference background with the same GO annotation. FIG. 10: A bar graph showing mean fold induction of significant cytochrome P450s (CYPs) and UDP-dependent glucosyltransferase (UGTs) located on the group 5 wheat chromosomes identified by RNA- Seq. Green bars represent UGTs and blue bars represent CYPs. Genes were induced by 15 g a.i. ha^-1 of cloquintocet-mexyl relative to untreated controls. Error bars indicate standard error of the mean.

FIG. 11: A bar graph showing mean fold changes for CYP81A-5A, CYP81A-5B, and CYP81A-5D in response to CM, HM, and CM + HM. Treatments included CM (15 g a.i. ha^-1 of CM), HM (5 g a.e. ha ¹ of HM), or CM+HM (15 g a.i. ha of CM and 5 g a.e. ha ¹ of HM). All treatments also included 0.1% nonionic surfactant (NIS). Untreated control included 0.1% nonionic surfactant (NIS). Within each timepoint values that share the same letter are not significantly different (a = 0.05). Fold inductions for each gene at each time point were calculated by 2 ^AACt) with P-tubulin (P-TUB) as a reference gene. Error bars represent standard error of the mean.

FIGS. 12A-12C: Bar graphs showing mean cycle threshold (Ct) value for P-tubulin P-TUB). CYP81A-5A, CYP81A-5B, and CYP81A-5D at 3 (FIG. 12A), 6 (FIG. 12B), and 12 (FIG. 12C) hours after treatment (HAT) in response to CM, HM, and CM + HM. Treatments included CM (15 g a.i. ha^-1 of CM), HM (5 g a.e. ha ¹ of HM), or CM+HM (15 g a.i. ha^-1 of CM and 5 g a.e. ha ¹ of HM). All treatments also included 0.1% NIS. Untreated control (UT) included 0.1% NIS. Within each timepoint, an asterisk indicates the mean for a given gene is significantly different from its respective UT mean (a = 0.05). Error bars represent standard error of the mean.

FIG. 13: A bar graph showing mean cycle threshold (Ct) value for P-tubulin (fi-TUB), CYP81A-5A, CYP81A-5B, and CYP81A-5D at 3, 6, and 12 hours after treatment (HAT) in untreated tissue. Untreated plants were subjected to 0.1% NIS. Within each timepoint, values that share the same letter are not significantly different (a = 0.05). Error bars represent standard error of the mean.

FIG. 14: Gene model of CYP81A-5A. Blue arrows represent the binding sites of guide RNAs (sgRNAs). The table indicates sequences of the CB037 wild type (WT; SEQ ID NO: 105) and mutant alleles (SEQ ID NO: 106), and the insertion in the mutant allele is highlighted in orange. The insertion is 262 bp from the TATA box. The protospacer adjacent motif sequence is underlined and italicized.

FIG. 15: A bar graph showing mean fold change of CYP81A-5A expression in response to CM in unaltered wheat (CB) and wheat that is homozygous for the mutant allele in CYP81A-5A (CBE). An asterisk indicates a significant difference (a = 0.01), and error bars represent the standard error of the mean.

FIG. 16: A bar graph showing mean cycle threshold (Ct) value for -tubulin (P-TUB) and CYP81A- 5A in response to CM in leaf tissue from unaltered wheat (CB) and wheat that is homozygous for the mutant allele in CYP81A-5A (CBE). CM treatments included 15 g a.i. ha of CM and 0.1% NIS; untreated control (UT) included 0.1% NIS. An asterisk indicates a significant difference (a = 0.01). Error bars represent standard error of the mean.

FIG. 17 : A bar graph showing mean dry biomass represented as a percent of the untreated control (UT) in unaltered wheat (CB), wheat that is homozygous for the mutant allele of CYP81A-5A (CBE), and Aegilops searsii (AS). UT plants were treated with a solution of 1% solution of methylated seed oil (MSO) and 2.5% ammonium sulfate (AMS), halauxifen-methyl(HM)-treated plants were treated with 40 g a.e. ha ¹ HM, florasulam(FLOR)-treated plants were treated with 10 g a.i. ha ¹ FLOR, propoxycarbazone- sodium(PROP)-treated plants were treated with 5.5 g a.i. ha'¹ PROP, and fenoxaprop-P-ethyl(FEN)-treated plants were treated with 46.2 g a.i. ha^-1 FEN. All herbicide rates included 1% solution of MSO and 2.5% AMS. An asterisk indicates a significant difference from CB for the given herbicide treatment (a = 0.01). Error bars represent standard error of the mean.

FIG. 18: Photograph showing phenotypes of unaltered wheat (CB), wheat homozygous for the mutant allele of CYP81A-5A (CBE), and Aegilops searsii (AS) in response to propoxycarbazone-sodium (PROP), compared to control plants. Control plants were treated with a solution of 1% methylated seed oil (MSO) and 2.5% ammonium sulfate (AMS), and treated plants were exposed to 5.5 g a.i. ha ¹ PROP with 1% MSO and 2.5% AMS.

DETAILED DESCRIPTION

I. Summary of Terms

Unless otherwise noted, technical terms are used according to conventional usage. Definitions of many common terms in molecular biology may be found in Krebs et al. (eds.), Lewin’s genes XII, published by Jones & Bartlett Learning, 2017. As used herein, the singular forms “a,” “an,” and “the,” refer to both the singular as well as plural, unless the context clearly indicates otherwise. For example, the term “a cell” includes singular or plural cells and can be considered equivalent to the phrase “at least one cell.” As used herein, the term “comprises” means “includes.” For example, reference to “comprising a cell” includes one or a plurality of such cells. It is further to be understood that any and all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for descriptive purposes, unless otherwise indicated. Although many methods and materials similar or equivalent to those described herein can be used, particularly suitable methods and materials are described herein. In case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

In some examples, the numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth, used to describe and claim certain embodiments are to be understood as being modified in some instances by the term “about” or “approximately.” Accordingly, in some embodiments, the numerical parameters set forth herein are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some examples are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable. The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range.

Whenever a range is given in the specification, for example, a temperature range, a time range, or a composition or concentration range, all intermediate ranges and subranges, as well as all individual values included in the ranges given are intended to be included in the disclosure. It will be understood that any subranges or individual values in a range or subrange that are included in the description herein can be excluded from the aspects herein.

All molecules (e.g., proteins and nucleic acids) described herein, unless indicated otherwise, include any forms of the molecules, and include isolated, recombinantly produced, and manufactured molecules (e.g., by synthetic methods and recombinant technologies), and molecules in their natural environment.

To facilitate review of the various aspects, the following explanations of terms are provided:

Acetolactate synthase (ALS) inhibitor: Inhibitors for acetolactate synthase, a protein found in plants and micro-organisms that catalyzes the first step in the synthesis of branched-chain amino acids. Inhibitors of ALS are used as herbicides that slowly starve affected plants of these amino acids. The ALS inhibitor family includes sulfonylureas (such as bensulfuron-methyl, chlorimuron-ethyl, chlorsulfuron, foramsulfuron, halosulfuron-methyl, mesosulfuron-methyl, metsulfuron-methyl, nicosulfuron, primisulfuron-methyl, prosulfuron, rimsulfuron, sulfometuron-methyl, sulfosulfuron, thifensulfuron-methyl, tribenuron-methyl, trifloxysulfuron-sodium, triflusulfuron-methyl, and triasulfuron); imidazolinones (such as imazapyr, imazamox, imazethapyr, imazapic, imazaquin, and imazamethabenz-methyl); triazolopyrimidines (such as cloransulam-methyl, diclosulam, florasulam, flumetsulam, penoxsulam, and pyroxsulam); pyrimidinyloxybenzoates (such as bispyribac-sodium, and pyribenzoxim); pyrimidinylthiobenzoates (such as pyrithiobac-sodium); and sulfonylamino carbonyl triazolinones (such as propoxycarbazone-sodium (PROP), flucarbazone-sodium, and thiencarbazone-methyl).

Analog/Derivative/Mimetic: An analog is a molecule that is structurally similar to a parent molecule, differing slightly by addition, subtraction, substitution, or alteration of one or more atoms or groups of atoms. For example, analogs include molecules that differ by an increment or decrement in the chemical structure, such as a difference in the length of an alkyl chain; molecules that differ by one or more functional groups, molecules that differ by a change in ionization; and a molecular fragment. Structural analogs are often found using quantitative structure activity relationships (QSAR), with techniques such as those disclosed in Remington (The Science and Practice of Pharmacology, 19th Edition (1995), chapter 28). A derivative is a molecule that is chemically modified from a parent molecule by a specific chemical reaction. A mimetic is a molecule that mimics the activity of another molecule, such as a biologically active molecule.

Backcross: The mating of a hybrid to one of its parents. For example, hybrid progeny, for example a first generation hybrid (Fi), can be crossed back one or more times to one of its parents. Backcrossing can be used to introduce one or more single locus conversions (such as one or more desirable traits) from one genetic background into another.

Cell: Cell as used herein includes a plant cell, whether isolated, in tissue culture or being part of a plant or plant part. In some examples a cell is altered or gene-edited, e.g., it includes a nucleic acid and/or protein sequence not found in nature. In some examples a cell is recombinant/transformed/transgenic, e.g., it includes an exogenous nucleic acid molecule. Codon optimization: When the codon usage of a DNA or RNA is adapted to that of a cell or organism of interest to improve the transcription rate of the recombinant nucleic acid in the cell (such as a plant cell) or organism of interest (such as a plant). A target nucleic acid can be modified at one position due to the codon degeneracy, whereas this modification will still lead to the same amino acid sequence at that position after translation, which is achieved by codon optimization to take into consideration the species-specific codon usage of a target cell or organism.

Complementarity: The ability of a nucleic acid to form hydrogen bond(s) with another nucleic acid sequence by either traditional Watson-Crick base pairing or other non-traditional types. A percent complementarity indicates the percentage of residues in a nucleic acid molecule which can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100% complementary). "Perfectly complementary" means that all the contiguous residues of a nucleic acid sequence will form hydrogen bonds with the same number of contiguous residues in a second nucleic acid sequence. "Substantially complementary" as used herein refers to a degree of complementarity that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, or more nucleotides, or refers to two nucleic acids that hybridize under stringent conditions.

Control: Refers to a plant, plant part, or plant cell that has a similar (or the same) genetic makeup and/or phenotypic traits as a treated plant, plant part, or plant cell before receiving the treatment. The treatment, for example, can include gene editing resulting in one or more modifications in one or more genes, or expression of an exogenous gene. In some aspects, the control plant, plant part, or plant cell is wild-type with respect to the gene(s) being modified by the treatment, or is wild-type. In other examples, the treatment includes treatment with one or more herbicides and/or safeners and the control may be a plant that is untreated.

Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated (Cas) system: CRISPR-Cas is an adaptive immune system existing in most bacteria and archaea, preventing them from being infected by phages, viruses and other foreign genetic elements. It includes CRISPR repeat-spacer arrays, which upon transcription generates CRISPR RNA (crRNA) and optionally trans-activating CRISPR RNA (tracrRNA), and a set of Cas genes which encode Cas proteins with endonuclease activity. CRISPR-Cas systems can be classified into two classes (Class 1 and Class 2), six types (I to VI) and several subtypes, with multi-protein effector complexes in Class 1 systems (Type I, III, and IV) and a single effector protein in Class 2 systems (Type II, V, and VI). CRISPR/Cas systems can be used for nucleic acid (DNA and RNA) targeting or editing, for example to detect a target nucleic acid, or cut or modify a target nucleic acid at any desired location.

The CRISPR repeat-spacer array (or CRISPR array) is a defining feature of CRISPR-Cas systems. The term “CRISPR” refers to the architecture of the array which includes constant direct repeats (DRs) interspaced with the variable spacers. In some examples, a CRISPR array includes at least a DR-spacer-DR- spacer. CRISPR spacer sequences are transcribed into short RNA sequences (“CRISPR RNAs” or “crRNAs”) capable of guiding Cas proteins to matching sequences of DNA.

Cas proteins provide the enzymatic machinery required for acquiring new spacers targeting invading elements and cleaving these elements upon subsequent encountering. Cas proteins that have endonuclease activity include Cas9, Casl2 (Cpfl), and Casl3.

Cas9 cleaves DNA and possesses two nuclease domain (HNH and RuvC), each cleaving one strand of the target double-stranded DNA. Cas9 nucleic acid and protein sequences are publicly available. For example, GenBank® Accession Nos. nucleotides 796693..800799 of CP012045.1 and nucleotides 1100046..1104152 of CP014139.1 disclose Cas9 nucleic acids, and GenBank® Accession Nos. AMA70685.1 and AKP81606.1 disclose Cas9 proteins. In some examples, Cas9 comprises at least 80% sequence identity, for example at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to such sequences, and retains the ability to cut DNA.

In some examples, Cas9 can be catalytically inactive or deactivated (dCas9), such as one that is nuclease deficient. In some examples, dCas9 includes one or more of the following point mutations: D10A, 5 H840A, and N863A. In some examples, dCas9 comprises a sequence as shown in GenBank® Accession Nos. AKA60242.1 and KR011748.1, or comprises at least 80% sequence identity, for example at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to such sequences.

Cross: Synonymous with hybridize or crossbreed.; includes the mating of genetically different individual plants, such as the mating of two parent plants.

Cross-pollination: Fertilization by the union of two gametes from different plants.

CYP81A-5A/5B/5D: Includes both genes and proteins, and all homologs, variants, and fragments of the genes and proteins.

Endogenous: With reference to a nucleic acid molecule and/or protein, referring to any such substance as found in a plant in its natural form, synonymous with “native” as used herein. Endogenous genes include any naturally occurring alleles, and include those that have been modified at some point by traditional plant breeding methods and/or next generation plant breeding methods. Endogenous genes can be edited or mutated according to any methods known or described herein.

Exogenous: With reference to a nucleic acid molecule, protein, vector, plasmid, and/or construct referring to any such substance that does not naturally occur in a cell or plant but is introduced into the cell or plant through human intervention. In some examples, an exogenous nucleic acid may be a guide nucleic acid (such as one specific for a region of a CYP81 A gene). In some examples, an exogenous nucleic acid may be a gene carried by a vector for expression in the cell or plant to which it is introduced (optionally integrated into the genome of the cell or plant), wherein the gene can be a copy or variant of a gene naturally occurring in the cell or plant, or can be a gene not naturally occurring in the cell or plant (such as a CYP gene introduced into a dicot plant who does not naturally possess the gene). In some examples, an exogenous nucleic acid or vector or plasmid may be a CRISPR/Cas construct (such as a CRISPR/Cas9 construct) encoding the components of a CRISPR/Cas system, such as one specific for a CYP81A gene. In some examples, an exogenous construct may be a preassembled Cas protein (such as Cas9)-gRNA ribonucleoproteins .

Expression: Refers to the production of a functional gene product, e.g., an mRNA or a protein (precursor or mature).

Fi hybrid: The first-generation progeny of the cross of two stable parents that are nonisogenic or isogenic plants.

Fragment: A portion of the parent sequence (nucleic acid or protein) having the minimal size characteristics of such sequences, or any larger fragment of the full-length molecule, up to and including the full-length molecule. A fragment may be a C-terminal fragment, N-terminal fragment, or an internal fragment that lies anywhere between the C-terminal and N-terminal amino acids. In some aspects, a fragment of a gene (e.g., a CYP8 I A gene) comprises no more than 1000, 950, 900, 850, 800, 750, 700, 650, 600, 550, 500, 450, 400, 350, 300, 250, 200, 150, 140, 130, 120, 110, or 100, and/or no less than 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 200, 250, or 300 contiguous nucleic acids of a full-length gene (such as one that comprises a DNA sequence set forth in any of SEQ ID NOs: 1-22). In some aspects, a fragment of a gene is a N-terminal fragment including the N-terminal nucleic acid of a full-length gene. In some aspects, a fragment of a gene is a C-terminal fragment including the C-terminal nucleic acid of a full- length gene. In some aspects, a fragment of a gene is an internal fragment including neither the C-terminal nor N-terminal nucleic acid of a full-length gene. In some aspects, a fragment of a gene may encode a biologically active portion of a full-length protein (such as any of those set forth in SEQ ID NOs: 99-104). In some aspects, a fragment of a gene encodes no more than 350, 340, 330, 320, 310, 300, 290, 280, 270, 260, 250, 240, 230, 220, 210, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 40, or 30 and/or no less than 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, or 80 contiguous amino acids of a full-length protein. In some aspects, a biologically active portion of a full-length protein encoded by a fragment of a gene is a C-terminal, N-terminal, or internal fragment of the full-length protein. A functional fragment is a fragment that retains one or more functions or activities of the corresponding full-length nucleic acid or protein at a desirable level (e.g., at least 60%, 70%, 80%, 90%, 95%, 99%, or 100% of the level provided by the full-length molecule).

Gain-of-function mutation: A mutation in a gene or protein that results in increased transcription of the gene or increased activity of the protein. Such mutation includes mutation in the regulatory region of a gene, and coding region of a gene, and includes insertion, deletion, substitution, etc.

Gene editing: Modifying a genome of an organism, including mutating one or more genomic nucleotides, deleting one or more genomic nucleotides, adding one or more nucleotides into the genome, replacing a genomic sequence with an exogenous sequence, inserting an exogenous sequence into the genome, and any combination thereof. Gene editing can be achieved, for example, by using engineered nucleases, which create site-specific double-strand breaks (DSBs) at desired locations in the genome, and whose improper repair by endogenous natural mechanisms results in an altered/non-native genomic sequence. The induced DSBs may be repaired through nonhomologous end-joining (NHEJ) or homologous recombination (HR), resulting in targeted mutations or deletions of a genomic sequence, or insertion of an exogenous sequence into the genome. Thus, the resulting genome is one that does not occur in nature.

In some examples, gene editing results in the introduction of an exogenous transgene into the genome of a plant, plant part, or plant cell. In other examples, a plant, plant part, or plant cell is edited by an exogenous nucleic acid molecule (e.g., a CRISPR/Cas vector) specific for an endogenous gene, thereby altering the endogenous sequence of the gene, but the exogenous nucleic acid molecule is not integrated into the genome of the gene-edited plant, plant part, or plant cell. In either case, such edited plants, plant parts, and plant cells are referred to as gene-edited plants, gene-edited plant parts, and gene-edited plant cells, respectively. In some examples, the gene-edited plants, plant parts or plant cells are transgene-free. Gene editing in a plant can be used, for example, to confer a desirable trait to the plant, such as increased tolerance to a herbicide.

Genome: All genetic material of a cell (such as a plant cell) or an organism (such as a plant), including nuclear genome and organelle genome and excluding artificially introduced nucleic acid molecules not integrated into a chromosome.

Genotype: The genetic constitution (e.g., the specific allele makeup) of a cell (e.g., a plant cell) or an organism (e.g., a plant) usually with reference to a specific character under consideration.

Growing or regeneration: Growing a whole, differentiated plant from a seed, a plant cell, a protoplast, a group of plant cells, callus, a plant part, a plant tissue, etc. In some examples, regeneration refers to the development of a plant from tissue culture. The cells may, or may not have been genetically modified. Plant tissue culture relies on the fact that plant cells have the ability to generate a whole plant (totipotency). Single cells (protoplasts), pieces of leaves, or roots can often be used to generate a new plant on culture media given the required nutrients and plant hormones.

Guide nucleic acid: Including RNA molecules comprising a sequence that has sufficient complementarity with a target polynucleotide sequence to hybridize with the target sequence (guide sequence) and a sequence that assists binding of a nuclease (such as a DNA endonuclease, such as a Cas protein); and DNA molecules from which such RNA molecules are transcribed. Guide nucleic acid as used herein can refer to the final products (e.g., guide RNAs or gRNAs) that bind with a nuclease and hybridize with a target sequence, any nucleic acid intermediates/precursors that can be processed into the final product, and/or the DNA molecules from which the final products or the intermediate/precursor are transcribed.

A single guide nucleic acid includes a single nucleic acid sequence comprising two parts, one being or encoding for a CRISPR RNA (crRNA), a sequence complementary to the target DNA (also referred to as guide sequence), the other being or encoding for a trans-activating CRISPR RNA (tracrRNA), serving as a binding scaffold for a nuclease (e.g., Cas9). A single guide nucleic acid also includes a single nucleic acid sequence comprising or encoding a crRNA (that functions together with a Cpfl). In some examples, a guide nucleic acid is a single guide RNA (sgRNA). A guide nucleic acid can include modified bases or chemical modifications (e.g., see Latorre et al., Angewandte Chemie 55:3548-50, 2016).

In some examples, the degree of complementarity between a guide sequence and its corresponding target sequence, when optimally aligned using a suitable alignment algorithm, is about or more than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more. Optimal alignment may be determined with the use of any suitable algorithm for aligning sequences, non-limiting example of which include the Smith- Waterman algorithm, the Needleman- Wunsch algorithm, algorithms based on the Burrows- Wheeler Transform (e.g., the Burrows Wheeler Aligner), ClustalW, Clustal X, BLAT, Novoalign (Novocraft Technologies, ELAND (Illumina, San Diego, Calif.), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net). In some examples, a guide sequence is about, or at least about, 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75, or more nucleotides in length. In some examples, a guide sequence is less than about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12, or fewer nucleotides in length. In some examples, a guide sequence is 15-25 nucleotides (such as 18- 22 or 18 nucleotides).

The ability of a guide sequence to direct sequence- specific binding of a CRISPR complex to a target sequence may be assessed by a suitable assay. For example, the components of a CRISPR system sufficient to form a CRISPR complex, including the guide sequence to be tested, may be provided to a host cell having the corresponding target sequence, such as by transfection with vectors encoding the components of the CRISPR sequence, followed by an assessment of preferential cleavage within the target sequence, such as by Surveyor assay. Similarly, cleavage of a target polynucleotide sequence may be evaluated in a test tube by providing the target sequence, components of a CRISPR complex, including the guide sequence to be tested and a control guide sequence different from the test guide sequence, and comparing binding or rate of cleavage at the target sequence between the test and control guide sequence reactions.

Herbicide: Substances used to control undesired plants, such as weeds. Selective herbicides control specific weed species while leaving the desired plant relatively unharmed, while non-selective herbicides kill plants indiscriminately. Preplant herbicides are nonselective herbicides applied to the soil before planting. Preemergence herbicides are applied before the weed seedlings emerge through the soil surface. Postemergence herbicides are applied after weed seedlings have emerged through the soil surface. Herbicides interfere with the biochemical machinery that supports plant growth. In some examples, herbicides mimic natural plant hormones, enzyme substrates, and cofactors. Herbicides are often classified according to their site of action because as a general rule, herbicides within the same site of action class produce similar symptoms on susceptible plants.

Herbicdes can be classified into: acetyl coenzyme A carboxylase (ACCase) inhibitors (which affect cell membrane production in the meristems); acetolactate synthase (ALS) inhibitors; enolpyruvylshikimate 3-phosphate synthase enzyme (EPSPS) inhibitors (which inhibit synthesis of amino acids tryptophan, phenylalanine and tyrosine); auxin-like herbicides (which mimic actions of the plant hormone auxin); photosystem II inhibitors (which reduce electron flow from water to NADP+ at the photochemical step in photosynthesis); photosystem I electron diverters (which produce excessive oxidation reactions and reactive oxygen species); and 4-hydroxyphenylpyruvate dioxygenase (HPPD) inhibitors (which inhibit tyrosine catabolism).

Some general commercial herbicide families (and representative active ingredients) are: sulfonylureas (ALS or Group 2 (according to the mode of action classification system proposed by the Herbicide Resistance Action Committee) (e.g. metsulfuron-methyl, nicosulfuron, foramsulfuron, chlorimuron); sulfonylamino-carbonyl-triazolinones (ALS or Group 2) (e.g. propoxycarbazone-sodium, thiencarbazone-methyl); triazolopyrimidines (ALS or Group 2) (e.g. cloransulam-methyl, penoxsulam); pyrimidinyl-benzoates (ALS or Group 2) (e.g. bispyribac, pyrithiobac); aryl triazinones (Protox or Group 14) (e.g. carfentrazone-ethyl, sulfentrazone); A'-phenyl-irnides (Protox or Group 14) (e.g. saflufenacil, trifludimoxazin, flumioxazin); triketones (HPPD or Group 27) (e.g. mesotrione, tembotrione); pyrazoles (HPPD or Group 27) (e.g. topramezone, pyrasulfotole, tolpyralate); isoxazoles (HPPD or Group 27) (e.g. isoxaflutole); cyclohexanediones (ACCase or Group 1) (e.g. clethodim, sethoxydim, tralkoxydim); aryloxyphenoxyproprionates (ACCase or Group 1) (e.g. fenoxaprop-ethyl, quizalofop-ethyl, cyhalofop- butyl); phenylpyrazoline (ACCase or Group 1) (e.g. pinoxaden); phytoene desaturase (PDS) or Group 12 (e.g. fluridone, norflurazon, diflufenican); dinitroanilines (mitotic disrupters or Group 3) (e.g. pendimethalin, trifluralin, ethalfluralin); acetamides (VLCFAE or Group 15) (e.g. 5-metolachlor, acetochlor, dimethenamid-p, flufenacet); isoxazolines (VLCFAE or Group 15) (e.g. pyroxasulfone); alkylazines (cellulose synthase or Group 29) (e.g. indaziflam); dihydroorotate dehydrogenase inhibitor or Group 28 (e.g. tetflupyrolimet); pyridine carboxylates (synthetic auxins or Group 5) (e.g. halauxifen-methyl, florpyrauxifen-benzyl, aminopyralid, fluroxypyr, clopyralid); homogentisate solanesyltransferase inhibitor (Group 33) (e.g. cyclopyrimorate); solanesyl diphosphate synthase inhibitor (Group 32) (e.g. aclonifen).

Herbicide safener: Substances used to reduce the effect of a herbicide on desired plants. In some examples, a herbicide safener exerts the protective effect through increasing the expression of one or more genes involved in herbicide detoxification (such as a CYP) in the desired plants. Herbicide safeners include cloquintocet-mexyl (CM), isoxadifen-ethyl, copper(I) thiophene-2-carboxylate, dichlormid, benoxacor, mefenpyr-diethyl, furilazole, fluxofenim, fenclorim, fenchlorazole-ethyl, oxabetrinil, flurazole, dichlormid, cyprosulfamide, and analogs or derivatives thereof.

Heterologous: A substance coming from some source or location other than its native source or location. A heterologous nucleic acid can refer to a nucleic acid sequence that is not naturally found in the particular organism. Two nucleic acid sequences are heterologous to one another if the sequences are derived from separate organisms, whether or not such organisms are of different species, as long as the sequences do not naturally occur together in the same arrangement in the same organism. In some examples, a heterologous promoter refers to a promoter that has been taken from one source organism and utilized in another organism, in which the promoter is not naturally found. In some examples, a heterologous promoter refers to a promoter that is from within the same source organism, but is used at a novel location, in which the promoter is not normally located. Heterologous gene sequences can be introduced into a cell (such as a plant cell) by using an expression vector, which can be an eukaryotic expression vector, for example a plant expression vector. Methods used to construct vectors are known and described in various publications. In particular, techniques for constructing suitable vectors, including selecting and organizing the functional components such as promoters, enhancers, termination and poly adenylation signals, selection markers, origins of replication, and splicing signals, are known.

Homologs: With reference to a gene or gene product, nucleic acids and proteins thought, believed, or known to be functionally related. A functional relationship may be indicated by, for example (a) degree of sequence identity and/or (b) the same or similar biological function. Homology can be determined using software programs readily available in the art, such as those discussed in Current Protocols in Molecular Biology (F.M. Ausubel et al., eds., 1987) Supplement 30, section 7.718, Table 7.71. Some alignment programs are MacVector (Oxford Molecular Ltd, Oxford, U.K.) and ALIGN Plus (Scientific and Educational Software, Pennsylvania). Other non- limiting alignment programs include Sequencher (Gene Codes, Ann Arbor, Michigan), AlignX, and Vector NTI (Invitrogen, Carlsbad, CA). Homologous genes/proteins arise in evolution in two possible ways: separation of two populations with the ancestral gene into two species, and duplication of the ancestral gene within a lineage. Homologous genes/proteins separated by speciation are also called orthologs. Homologous genes/proteins separated by speciation and brought back together in a single species by allopolyploidization are also called homeologs. Homologous genes/proteins arise from gene duplication events within a species are also called paralogs. Homologs include orthologs, homeologs, and paralogs.

Increase or decrease: A statistically significant positive or negative change, respectively, in quantity from a control value. An increase is a positive change, such as an increase of at least 50%, at least 100%, at least 200%, at least 300%, at least 400%, or at least 500% as compared to the control value. A decrease is a negative change, such as a decrease of at least 20%, at least 25%, at least 50%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 100% decrease as compared to a control value. In some examples, the control value is a value or range of values expected for the same plant that is not gene-edited (e.g., a wild-type plant), or not gene-edited with respect to the gene in question (e.g., a wild-type plant with respect to a particular gene).

Insertion: Addition of one or more nucleotides to a nucleic acid sequence. Insertions can vary in size, ranging from small insertions of a single or a few nucleotides to large insertions of, for example, an entire coding region.

Isolated: Altered by the hand of human from the natural state. For example, a polynucleotide or a polypeptide as present in an organism is not isolated, but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is isolated.

Next generation plant breeding: Refers to plant breeding tools and methodologies that are available to a plant breeder. One distinguishing feature of next generation plant breeding is that the breeder is no longer confined to relying upon observed phenotypic variation, in order to infer underlying genetic causes for a given trait. Rather, next generation plant breeding can include the utilization of molecular markers and marker assisted selection (MAS), such that the breeder can directly observe movement of alleles and genetic elements of interest from one plant in the breeding population to another, and is not confined to merely observing phenotypes. Further, next generation plant breeding methods are not confined to utilizing natural genetic variation found within a plant population. Rather, the breeder utilizing next generation plant breeding methodology can access modern genetic engineering tools that directly alter/change/edit the plant’s underlying genetic architecture in a targeted manner, in order to bring about a phenotypic trait of interest. In some aspects, the plants bred with a next generation plant breeding methodology are indistinguishable from a plant that was bred in a traditional manner, as the resulting end product plant could theoretically be developed by either method. In particular aspects, a next generation plant breeding methodology may result in a plant that comprises a genetic modification (e.g., a deletion or insertion of any size; a substitution of one or more base pairs; an introduction of nucleic acid sequences from within the plant’ s natural gene pool (e.g. any plant that could be crossed or bred with a plant of interest) or from editing of nucleic acid sequences in a plant to correspond to a sequence known to occur in the plant’ s natural gene pool); and offspring of the plant.

Naturally occurring: As applied to a substance (e.g., nucleic acid, polypeptide/protein, etc.), cell, or organism, referring to a substance, cell, or organism that is found in nature, without any intentional human intervention in its existence or evolvement.

Non-naturally occurring or engineered: Indicating involvement of the hand of human. In some examples, the terms, when referring to a substance (e.g., a nucleic acid molecule, or a polypeptide/protein) or a cell, indicate that the substance or cell is at least substantially free from at least one other substances or cells with which it is naturally associated or found together in nature. In some examples, a non-naturally occurring or engineered sequence (e.g., of a nucleic acid molecule, polypeptide/protein, etc.) refers to a sequence that is at least partially different from naturally occurring sequences, and the difference is achieved by synthesis, recombinant technology, gene editing, or any other production or intervention means that are developed by human.

Offspring: Refers to any plant resulting as progeny from a vegetative or sexual reproduction from one or more parent plants or descendants thereof. For instance, an offspring plant may be obtained by cloning or selfing of a parent plant (or a plant of Fl, F2, or still further generations), or by crossing two parent plants (or a plant of Fl, F2, or still further generations). An offspring of Fl generation is a first- generation offspring produced from parents. Subsequent generations, denoted as F2, F3, and so forth, arise from selfing or crossing within the preceding generation. In some examples, an Fl may be (and usually is) a hybrid resulting from a cross between two true breeding parents (true breeding referring to homozygous for a trait), while an F2 may be (and usually is) an offspring resulting from self-pollination of the Fl hybrids.

Operably linked: Two nucleic acid sequences are operably linked if the nature of the linkage does not interfere with the normal functions of the sequences. A first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. In some examples, a promoter is operably linked to a nucleic acid sequence (such as a guide nucleic acid sequence or a coding sequence) if the promoter controls the transcription or expression of the nucleic acid sequence. In some examples, operably linked DNA sequences are contiguous and, where necessary join two protein-coding regions in the same reading frame. In some examples, coding sequences can be operably linked to regulatory sequences in a sense or antisense orientation.

Plant: Includes reference to an immature or mature whole plant, including a plant from which seed, roots, or leaves have been removed. Seeds or embryos that will produce a plant is also considered to be the plant. In some examples, the plants (including seeds and embryos) can include one or more exogenous nucleic acid molecules. Any commercially or scientifically valuable plant can be used in accordance with this disclosure. Exemplary plants include plants belonging to the super family Viridiplantae, such as monocotyledonous and dicotyledonous plants including a fodder or forage legume, ornamental plant, food crop, tree, or shrub, such as Acacia spp., Acer spp., Actinidia spp., Aesculus spp.. Agathis australis, Albizia amara. Alsophila tricolor. Andropogon spp., Arachis spp, Areca catechu, Asteliafragrans, Astragalus cicer, Baikiaea plurijuga. Betula spp.. Brassica spp., Bruguiera gymnorrhiza, Burkea africana, Butea frondosa, Cadaba farinosa, Calliandra spp. Camellia sinensis, Canna indica. Capsicum spp., Cassia spp., Centroema pubescens, Chacoomeles spp., Cinnamomum cassia, Coffea arabica, Colophospermum mopane, Coronillia varia. Cotoneaster serotina, Crataegus spp., Cucumis spp., Cupressus spp., Cyathea dealbata, Cydonia oblonga, Cryptomeria japonica, Cymbopogon spp., Cynthea dealbata, Cydonia oblonga, Dalbergia monetaria, Davallia divaricata, Desmodium spp., Dicksonia squarosa, Dibeteropogon amplectens, Dioclea spp, Dolichos spp., Dorycnium rectum, Echinochloa pyramidalis, Ehraffia spp., Eleusine coracana, Eragrestis spp., Erythrina spp., Eucalyptus spp., Euclea schimperi, Eulalia vi/losa, Pagopyrum spp., Feijoa sellowlana, Fragaria spp., Flemingia spp, Freycinetia banksli. Geranium thunbergii, GinAgo biloba, Glycine javanica, Gliricidia spp, Gossypium hirsutum, Grevillea spp., Guibourtia coleosperma, Hedysarum spp., Hemaffhia altissima, Heteropogon contoffus, Hordeum vulgare, Hyparrhenia rufa, Hypericum erectum, Hypeffhelia dissolute, Indigo incamata, Iris spp., Leptarrhena pyrolifolia, Lespediza spp., Lettuca spp., Leucaena leucocephala, Loudetia simplex, Lo tonus bainesli, Lotus spp., Macro tyloma axillare, Malus spp., Manihot esculenta, Medicago saliva, Metasequoia glyptostroboides, Musa sapientum, Nicotianum spp., Onobrychis spp., Ornithopus spp., Oryza spp., Peltophorum africanum, Pennisetum spp., Persea gratissima, Petunia spp., Phaseolus spp., Phoenix canadensis, Phormium cookianum, Photinia spp., Picea glauca. Pinus spp., Pisum sativum. Podocarpus totara, Pogonarthria fleckii, Pogonaffhria squarrosa, Populus spp., Prosopis cineraria, Pseudotsuga menziesii, Pterolobium stellatum, Pyrus communis, Quercus spp., Rhaphiolepsis umbellata. Rhopalostylis sapida, Rhus natalensis, Ribes grossularia, Ribes spp.. Robinia pseudoacacia, Rosa spp., Rubus spp., Salix spp., Schyzachyrium sanguineum, Sciadopitys vefficillata, Sequoia sempervirens, Sequoiadendron giganteum, Sorghum bicolor, Spinacia spp., Sporobolus fimbriatus, Stiburus alopecuroides, Stylosanthos humilis, Tadehagi spp, Taxodium distichum, Themeda triandra, Trifolium spp., Triticum spp., Tsuga heterophylla, Vaccinium spp.. Vicia spp., Vitis vinifera, Watsonia pyramidata, Zantedeschia aethiopica, Zea mays, amaranth, artichoke, asparagus, broccoli, Brussels sprouts, cabbage, canola, carrot, cauliflower, celery, collard greens, flax, kale, lentil, oilseed rape, okra, onion, potato, rice, soybean, straw, sugar beet, sugar cane, sunflower, tomato, squash tea, maize, wheat, barley, rye, oat, peanut, pea, lentil and alfalfa, cotton, rapeseed, canola, pepper, sunflower, tobacco, eggplant, switchgrass, Miscanthus, Setaria, fescue, eucalyptus, a tree, an ornamental plant, a perennial grass and a forage crop. In a specific aspect the plant is a pennycress plant, such as Thlaspi arvense. In a specific aspect the plant is a soybean plant, such as Glycine max. In another aspect the plant is a canola plant, such as Brassica napus. In another aspect the plant is a rice plant, such as a plant of the genus Oryza, or such as Oryza sativa. In another aspect, the plant is a sorghum or great millet plant, such as Sorghum bicolor. In some examples, the plant is from the Poaceae family. In some examples, the plant is a cereal grass. In some examples, the plant is wheat. When a plant family is referred to, all genus within the family is included. When a plant genus is referred to, all species within the genus, all cultivars within each species, and all varieties within each cultivar are included.

Plant cell: Includes a single plant cell or a plurality of plant cells; includes any cell that constitutes a plant; includes protoplasts, gamete producing cells, and cells that can regenerate into a whole plant, embryos, and callus tissue; includes cells from seeds, suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen and microspores.

Plant part: Includes protoplasts, leaves, stems, roots, root tips, anthers, pistils, seeds, embryos, pollens, stamens, ovules, microspores, sporophytes, gametophytes, cotyledons, hypocotyls, flowers, shoots, fruits, tissues, petioles, cells, meristematic cells, and the like; includes differentiated and undifferentiated tissues (which may be in a plant, a plant organ, or a tissue or cell culture); includes plant cells of a tissue culture from which plants can be regenerated. In some examples, a plant part is one or more plant cells (e.g., single cells, protoplasts, embryos, and callus tissue).

Poaceae: a family of monocotyledonous flowering plants, a division of the order Poales. A cereal grass is a member of the Poaceae family that is cultivated for its edible grain. A cereal grass includes barley, corn/maize, goat grass, millet, oat, rice, rye, sorghum, and wheat. Wheat (Triticum) species include T. aestivum, T. aethiopicum, T. araraticum, T. boeoticum, T. carthlicum, T. compactum, T. dicoccoides, T. dicoccon, T. durum, T. ispahanicum, T. karamyschevii, T. monococcum, T. polonicum, T. spelta, T. thaoudar, T. timopheevii, T. turanicum, T. turgidum, T. Urartu, T. vavilovii, and T. zhukovskyi.

Polynucleotide/nucleic acid molecule/nucleotide sequence: These terms are used interchangeably herein and refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides, or analogs thereof. Includes double-stranded (such as sense and antisense) and single-stranded (such as sense or antisense) DNA, double- and single-stranded RNA, as well as multistranded DNA or RNA. Includes genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases. Also includes modified nucleic acids such as methylated and/or capped nucleic acids, nucleic acids containing modified bases, backbone modifications, and the like.

Oligonucleotide generally refers to polynucleotides of between about 5 and about 100 nucleotides of single- or double-stranded DNA. However, for the purposes of this disclosure, there is no upper limit to the length of an oligonucleotide. Oligonucleotides are also known as “oligomers” or “oligos” and may be isolated from genes, or chemically synthesized by methods known in the art.

Progeny: Offspring; descendants.

Promoter: A nucleic acid sequence, or an array of nucleic acid sequences, that direct or control transcription of a nucleic acid (e.g., a coding sequence). A promoter includes a necessary nucleic acid sequence near the start site of transcription. A promoter also optionally includes distal enhancer or repressor elements. In some examples, a promoter used for recombinant expression of a nucleic acid molecule is not naturally occurring in the cell into which it is introduced, is not native to the nucleic acid molecule to which it is attached, or both. In one example, a promoter used is not endogenous (i.e., is exogenous) to the plant in which it is introduced. In some examples, promoter is about 80-120 base pairs long and located upstream of the initiation site of a gene, to which RNA polymerase may bind and initiate correct transcription. There can be associated additional transcription regulatory sequences which provide on/off regulation of transcription and/or which enhance (increase) expression of the downstream coding sequence.

Protein/peptide/polypeptide: These terms are used interchangeably herein, and refer to a polymeric form of amino acids of any length, which can include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones.

Recombinant: Of or resulting from new combinations of genetic material.

A recombinant protein refers to a protein produced by the use of recombinant DNA technology, which involves the combination of genetic material from different sources to create a new (non-naturally occurring) DNA sequence, which is then introduced into a host organism (such as bacteria, yeast, or mammalian cells) to produce the desired protein.

A recombinant nucleic acid or a recombinant construct refers to an artificial combination of nucleic acid sequences, e.g., regulatory and coding sequences that are not found together in nature. For example, a recombinant construct may include regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that found in nature. Such a construct may be used by itself or may be incorporated into a vector or plasmid to form a recombinant vector or plasmid. Different independent transformation events of a recombinant construct or vector can result in different levels and patterns of expression (Jones et al., (1985) EMBO J. 4:2411-2418; De Almeida et aL, (1989) Mol. Gen. Genetics 218:78-86). Lines displaying the desired expression level and pattern can be screened. Such screening may be accomplished, for example, by Southern analysis of DNA, Northern analysis of mRNA expression, immunoblotting analysis of protein expression, or phenotypic analysis, among others.

A recombinant or host cell refers to a cell that has been genetically altered, or is capable of being genetically altered, by introduction of an exogenous polynucleotide, such as a recombinant construct, plasmid or vector. In some examples, the exogenous polynucleotide may express a protein or guide RNA molecule that leads to increased expression or activity of one or more of CYP81A genes or proteins. Typically, a host cell is a cell in which a vector can be propagated and its nucleic acid expressed. In specific examples, such cells are plant cells, such as from a monocot or dicot. The term also includes any progeny of the subject host cell. It is understood that all progenies may not be identical to the parental cell since there may be mutations that occur during replication. However, such progenies are included when the term “host cell” is used.

Ribonucleoprotein (RNP): A complex of ribonucleic acid and DNA-binding protein. In some examples, the RNP includes one or more, such as 2, 3, 4, or 5 different ribonucleic acids, such as guide RNAs specific for different targets. In some examples, the DNA-binding protein is a Cas protein, such as a native or mutant Cas9 protein.

Self-pollination: The transfer of pollen from the anther to the stigma of the same plant.

Sequence identity/similarity: The similarity between proteins, or between nucleic acid molecules can be characterized by similarity between the amino acid sequences or nucleotide sequences, otherwise referred to as sequence identity. Sequence identity is frequently measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar the two sequences are.

Methods of alignment of sequences for comparison are well known. Various programs and alignment algorithms are described in: Smith and Waterman, Adv. Appl. Math. 2:482, 1981 : Needleman and Wunsch, J. Mol. Biol. 48:443, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A. 85:2444, 1988; Higgins and Sharp, Gene 73:237, 1988; Higgins and Sharp, CABIOS 5: 151, 1989; Corpet et al., Nucleic Acids Research 16: 10881, 1988; and Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A. 85:2444, 1988. Altschul et al., Nature Genet. 6: 119, 1994, presents a detailed consideration of sequence alignment methods and homology calculations.

The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215:403, 1990) is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, MD) and on the internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx. A description of how to determine sequence identity using this program is available on the NCBI website on the internet.

Variants of protein sequences known and disclosed herein are typically characterized by possession of at least about 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity counted over the full-length alignment with the amino acid sequence using the NCBI Blast 2.0, gapped blastp set to default parameters. For comparisons of amino acid sequences of greater than about 30 amino acids, the Blast 2 sequences function is employed using the default BLOSUM62 matrix set to default parameters, (gap existence cost of 11, and a per residue gap cost of 1). When aligning short peptides (fewer than around 30 amino acids), the alignment should be performed using the Blast 2 sequences function, employing the PAM30 matrix set to default parameters (open gap 9, extension gap 1 penalties). Proteins with even greater similarity to the reference sequences will show increasing percentage identities when assessed by this method, such as at least 95%, at least 98%, or at least 99% sequence identity. When less than the entire sequence is being compared for sequence identity, homologs and variants will typically possess at least 80% sequence identity over short windows of 10-20 amino acids, and may possess sequence identities of at least 85% or at least 90% or at least 95% depending on their similarity to the reference sequence. Methods for determining sequence identity over such short windows are available at the NCBI website on the internet. These sequence identity ranges are provided for guidance only; it is entirely possible that strongly significant homologs could be obtained that fall outside of the ranges provided.

Variants of the disclosed nucleic acid sequences are typically characterized by possession of at least about 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity counted over the full-length alignment with the nucleic acid sequence using the NCBI Blast 2.0, gapped blastn set to default parameters. One of skill in the art will appreciate that these sequence identity ranges are provided for guidance only; it is possible that sequences coding for the disclosed proteins could be obtained that fall outside of the ranges provided.

Tissue culture: A composition that includes isolated cells of the same or a different type or a collection of such cells organized into parts of a plant. In some examples, the tissue culture includes a homogenous population of plant cells. In some examples, the tissue culture includes a callus tissue. In some examples, the tissue culture includes an anther culture or apical stem tip meristem culture. In some examples, the tissue culture includes a hairy root culture.

Tolerance to a herbicide: Refers to the ability of a plant to survive and continue growing even after being exposed to a herbicide. The ability can be measured by the biomass (e.g., as measured by fresh weight or dry weight, aboveground biomass or total biomass, etc.) accumulated in the presence of a herbicide. Increased tolerance to a herbicide refers to the ability is increased (e.g., more biomass is accumulated in the presence of a herbicide) as compared to an appropriate control. Tolerance to a herbicide can be inherent in some plant species or can be developed through selective breeding or genetic engineering. Inherent tolerance can occur naturally in some plant species that have developed mechanisms to detoxify or evade the effects of the herbicide. Such tolerance can be further increased by gene editing. Plants that do not have natural tolerance can be genetically modified to express one or more of a herbicide detoxification genes (such as a CPY81A-5A, 5B or 5D gene), which endow herbicide tolerance.

Herbicide resistance is used when referring to the ability of weeds or unwanted plants or populations to evolve and adapt to withstand herbicides that previously controlled the growth of most plants in the unwanted species effectively at labeled rates. Resistance typically results from the repeated and widespread use of the same herbicide, leading to selection pressure that favors resistant individuals.

Traditional plant breeding: Refers to the utilization of natural variation found within a plant population as a source for alleles and genetic variants that impart a trait of interest to a given plant. Traditional breeding methods make use of crossing procedures that rely largely upon observed phenotypic variation to infer causative allele association. That is, traditional plant breeding relies upon observations of expressed phenotype of a given plant to infer underlying genetic cause. These observations are utilized to inform the breeding procedure in order to move allelic variation into germplasm of interest. Further, traditional plant breeding has also been characterized as comprising random mutagenesis techniques, which can be used to introduce genetic variation into a given germplasm. These random mutagenesis techniques may include chemical and/or radiation-based mutagenesis procedures. Consequently, one feature of traditional plant breeding is that the breeder does not utilize a genetic engineering tool that directly alters/changes/edits the plant’s underlying genetic architecture in a targeted manner, in order to introduce genetic diversity and bring about a phenotypic trait of interest.

Transformation: The introduction of exogenous material, such as nucleic acid (e.g., guide nucleic acids or vectors providing for such, vectors comprising coding sequences, etc.) into a cell, such as a plant cell. Exemplary mechanisms for introducing nucleic acids into plant cells include electroporation, microprojectile bombardment, Agrobacterium-mediated transformation, and direct DNA uptake by protoplasts.

Transformed: A transformed plant, plant part or plant cell is a plant, plant part or plant cell that has taken up an exogenous nucleic acid (including a linear or circular DNA, a vector, a plasmid, a guide RNA molecule, etc.), regardless of whether the exogenous nucleic acid is integrated into the genome of the plant, plant part or plant cell, and regardless of whether the exogenous nucleic acid alters the genome of the plant, plant part or plant cell. Thus, transformed plants, plant parts or plant cells include transgenic plants, plant parts or plant cells; gene-edited plants, plant parts or plant cells; as well as plants, plant parts or plant cells that have taken up the exogenous nucleic acid but with an unaltered genome.

Transgene: An exogenous gene or other nucleic acid material that has been integrated into the genome of a plant, plant part or plant cell, for example by transformation or genetic engineering methods. In some examples, a transgene describes a segment of DNA containing a gene sequence or a random noncoding sequence and is integrated into the genome of a plant, plant part or plant cell. This non-native segment of DNA may retain the ability to produce RNA or protein in the transgenic plant, or it may alter the normal function of the transgenic plant’s genetic code. In some examples, a transgene is incorporated into the plant’s germ line.

Transgene-free: Not containing any transgene. Many strategies have been developed to remove or prevent the integration of a transgene (such as a gene editing construct), thereby generating a transgene-free plant, plant cell, plant part, or plant seed. Such strategies include elimination of a transgene via genetic segregation; transient expression by DNA vectors; and DNA-independent editor delivery, such as delivery of RNA or preassembled Cas9 protein-gRNA ribonucleoproteins (Gu, Xiaoyong et al. “Transgene-free Genome Editing in Plants.” Frontiers in genome editing vol. 3 805317. 2 Dec. 2021, doi: 10.3389/fgeed.2021.805317).

Under conditions sufficient for: Referring to any combinations of factors or environmental conditions that permit a desired activity.

Upstream or downstream: With reference to a nucleic acid molecule, upstream refers to the direction towards the 5' end of molecule, downstream refers to the direction towards the 3' end of the molecule. Vector: A nucleic acid molecule capable of carrying a nucleic acid molecule of interest and permitting its expression and/or integration in a host cell. A vector may also be capable of replicating in a host cell (e.g., along with or independent of the host genome replication during cell division), for example, by including a nucleic acid sequence (such as an origin of replication) that permits its replication. A vector may also include one or more selectable marker genes and other genetic elements known in the art. An integrating vector is capable of integrating itself or the nucleic acid molecule of interest it carries into a host nucleic acid. An expression vector is a vector that contains necessary regulatory sequences to allow transcription and translation of the nucleic acid molecule of interest (e.g., one or more genes encoding a protein), without integration with a host nucleic acid. Vectors include, but are not limited to, nucleic acid molecules that are single-stranded, double-stranded, or partially double-stranded; nucleic acid molecules that comprise one or more free ends, or no free ends e.g., circular); nucleic acid molecules that include DNA, RNA, other varieties of polynucleotides known in the art, or any combination thereof.

In some examples, a vector is not native to the cell into which it is introduced. In some examples, a vector includes a guide nucleic acid (e.g., specific for one or more of a CYP81A genes) operably linked to a promoter sequence, which can be non-native (e.g., promoter that does not occur naturally in the plant into which the vector is introduced) or native (e.g., a promoter found in the plant). In some examples, the vector further includes coding sequences for proteins participating in gene editing (e.g., an endonuclease). In some examples, a vector includes a nucleic acid that encodes one or more of a CYP81 A genes operably linked to a promoter sequence, which can be non-native or native.

One type of vector is a "plasmid," which refers to a circular double stranded DNA loop into which additional DNA segments can be inserted, such as by standard molecular cloning techniques.

Another type of vector is a viral vector, wherein virally derived DNA or RNA sequences are present in the vector for packaging into a virus (e.g., retroviruses, replication defective retroviruses, adenoviruses, herpes simplex viruses, baculoviruses, replication defective adenoviruses, and adeno-associated viruses). Viral vectors also include recombinant plant viruses, such as TMV-mediated (transient) transfection into tobacco (Tuipe, T-H et al (1993), J. Virology Meth, 42: 227-239), ssDNA genomes viruses (e.g., family Gemini viridae), reverse transcribing viruses (e.g., families Caulimoviridae, Pseudoviridae, and Metaviridae), dsNRA viruses (e.g., families Reoviridae and Partitiviridae), (-) ssRNA viruses (e.g., families Rhabdoviridae and Bunyaviridae), (+) ssRNA viruses (e.g., families Bromoviridae, Closteroviridae, Comoviridae, Luteoviridae, Potyviridae, Sequiviridae and Tombusviridae) and viroids (e.g., families Pospiviroldae and Avsunviroidae). Detailed classification information of plant viruses can be found in Fauquet et al. (2008, "Geminivirus strain demarcation and nomenclature". Archives of Virology 153:783-821, incorporated herein by reference in its entirety), and Khan et al. (Plant viruses as molecular pathogens; Publisher Routledge, 2002, ISBN 1560228954, 9781560228950).

Vectors also include phagemids, cosmids, artificial/mini-chromosomes (e.g., ACE), bacteriophages, pro-viruses, phagemids, transposons, artificial chromosomes, polyamine derivatives of DNA, and the like, that replicate autonomously or can integrate into a chromosome of a host cell. A vector can also be a naked RNA polynucleotide, a naked DNA polynucleotide, a polynucleotide composed of both DNA and RNA within the same strand, a poly-lysine-conjugated DNA or RNA, a peptide-conjugated DNA or RNA, a liposome-conjugated DNA, or the like, that is not autonomously replicating.

Eukaryotic expression vectors in some examples also contain prokaryotic sequences that facilitate the propagation of the vector in bacteria such as an origin of replication and antibiotic resistance genes for selection in bacteria. A variety of eukaryotic expression vectors, containing a cloning site into which a polynucleotide can be operatively linked, are well known and some are commercially available from companies such as Stratagene, La Jolla, Calif.; Invitrogen, Carlsbad, Calif.; Promega, Madison, Wis. or BD Biosciences Clontech, Palo Alto, Calif.

II. Cytochrome P450 Monooxygenases

A. Cytochrome P450 Monooxygenases and their Roles in Herbicide Metabolism

Cytochrome P450 monooxygenases (CYPs) are named in reference to the 450 nm absorption band of their carbon-monoxide bound form (Werck-Reichhart & Feyereisen, 2000). They are a superfamily of hemethiolate enzymes present in all kingdoms of life and catalyze reactions in numerous biosynthetic and xenobiotic pathways, including carbon assimilation, xenobiotic detoxification, secondary metabolite synthesis (Bak et al., 2011; Hansen et al., 2021; Nelson, 2009; Nelson et al., 2004). Within plants, CYPs are essential to the biosynthesis and modification of primary (sterols and fatty acids) and secondary metabolites (phenylpropanoids, glucosinolates, and carotenoids), and are also responsible for the synthesis and catabolism of hormones, including gibberellins, jasmonic acid, abscisic acid, brassinosteroids, and strigolactones (Mizutani & Ohta, 2010; Mizutani & Sato, 2011; Wakabayashi et al., 2019). Members of the CYP superfamily share four structural characteristics: a proline-rich membrane hinge, an I-helix involved in oxygen binding, a cysteine within the heme-binding domain, and two motifs forming the ERR triad, which is involved in positioning and stabilizing the heme pocket (Bak et al., 2011; Rupasinghe & Schuler, 2006; Werck-Reichhart et aL, 2002). However, only the ERR triad and the cysteine residue within the hemebinding domain are conserved among all plant CYPs (Bak et al., 2011; Werck-Reichhart et al., 2002).

The cross-kingdom nomenclature for CYPs is based on amino acid sequence similarity with 40%, 55%, and 97% sequence identities used as cut-offs for categorizing family, subfamily, and allelic variant designations, respectively (Dimaano & Iwakami, 2021; Nelson, 2009). The family is denoted by a number, and in the case of plant CYPs these numbers include, 51, 71-99, 701-999, and 7001 onward (Nelson, 2009). Following this first number is a letter designating the subfamily, which is followed by another number designating a specific protein (Nelson, 2009). For example, CYP81A12 and CYP81A21 are two different CYPs that are members of the same family and subfamily. In contrast, CYP81D5 and CYP81A12 are two different CYPs that are members of the same family but different subfamilies. Different CYP families are organized into clans, which represent the deepest clades that reproducibly appear in multiple phylogenetic trees and are named after their lowest numbered family member (Nelson, 2009; Nelson et al., 1996). Over the course of evolution, lineage-specific CYP subfamilies evolved in plants with CYPs within the same family or subfamily, catalyzing the same or similar reactions (Nelson & Werck-Reichhart, 2011).

The total number of CYPs among species varies, but in general plants contain more CYPs than animals, which is thought to be a consequence of their sessile nature and need to produce a vast number of secondary metabolites to defend and adapt to abiotic and biotic stresses and communicate to other organisms (Bak et al., 2011). Polyploidy is also common in plants, which likely contributes to the higher amount of CYPs present in plants (Dimaano & Iwakami, 2021). For example, there are 1,285 CYP annotations in hexapioid wheat, 622 CYPs in tetrapioid cotton, 103 CYPs in mice, and 57 CYPs in humans (Bak et al., 2011; Li & Wei, 2020; Zhang et al., 2015).

Medical and pharmaceutical xenobiotic metabolism research from the 1950s inspired similar examination of plant CYPs, resulting in the first demonstration of CYP herbicide-detoxifying function in 1968 with the herbicide monuron being detoxified by CYPs from microsomes of cotton seedlings (Frear, 1968; Frear et al., 1969). While CYPs catalyze a diverse range of reactions, these reactions are based on monooxygenase/hydroxylase reactions where molecular oxygen is cleaved, resulting in the insertion of one oxygen atom in the organic substrate while the other oxygen atom is reduced to water (Bak et al., 2011; Werck-Reichhart & Feyereisen, 2000). CYP-catalyzed reactions in plants that are relevant to herbicide metabolism, include alkyl-hydroxylation, 7V-demethylation, O-demethylation, aryl-hydroxylation (i.e. NIH- shift that causes intramolecular migration of a hydrogen on the aromatic ring), and, more rarely, (ei-1)- hydroxylation (Dimaano & Iwakami, 2021; Imaishi & Matumoto, 2007). In contrast, mammalian CYPs are capable of catalyzing more diverse detoxification reactions, such as O-deethy lation. Wdeethylation, and N- deisopropylation, and cleavage (Inui & Ohkawa, 2005).

Herbicide metabolism in plants occurs in four parts or phases: Phase I, Phase II, Phase III, and Phase IV. Phase I involves oxidation, reduction, or hydrolysis of phytotoxic parent molecules, with most reactions being oxidations catalyzed by CYPs or hydrolyses catalyzed by carboxylesterases (Gaines et al., 2020; Riechers et al., 2010). These CYP-mediated reactions serve to make hydrophobic molecules more reactive and water soluble (Dimaano & Iwakami, 2021). Phase II reactions involve conjugation reactions of phytotoxic parent molecules or Phase I metabolites with endogenous substrates, such as glucose, reduced glutathione, amino acids, or malate (Gaines et al., 2020). These Phase II metabolites are subsequently transported to the vacuole or cell wall by ATP binding-cassette (ABC) transporter proteins during Phase III, and within the vacuole they are further degraded and compartmentalized during Phase IV, which results in conjugated metabolites being bound to lignin biopolymers in the cell wall or sequestered within the vacuole (Davies & Caseley, 1999; Gaines et al., 2020; Yuan et al., 2007).

CYPs catalyze Phase I reactions of herbicide metabolism and are responsible for crop tolerance and weed resistance mechanisms to numerous herbicides. Among plant families, the Poaceae has approximately 15 reported herbicide-detoxifying CYPs, which is the highest number reported for any plant family (Dimaano & Iwakami, 2021; Han et al., 2020; Pan et al., 2022; Zheng et al., 2022). Some examples of herbicide-detoxifying CYPs and corresponding traits include wheat tolerance to diclofop-methyl (an acetyl- CoA carboxylase (ACCase) inhibitor) and florasulam (an acetolactate synthase (ALS) inhibitor) due to ring hydroxylation and subsequent glucose conjugation (DeBoer et al., 2006; Tanaka et al., 1990; Zimmerlin & Durst, 1990, 1992), rice CYP71A31 conferring tolerance to an ALS inhibitor (bispyribac-sodium) (Saika et al., 2014), and evidence of CYP involvement in chlorotoluron (a photosystem II (PS II) inhibitor) detoxification (Mougin et al., 1990). Additionally, an in vitro yeast assay using wheat CYP71C6V1 demonstrated metabolism of several ALS -inhibiting herbicides, including chlorsulfuron, triasulfuron, metsulfuron-metyl, bensulfuron-metyl, and tribenuron-metyl, but its function in planta has not been verified (Dimaano & Iwakami, 2021; Xiang et al., 2006).

With the exception of CYP71C6V1, the genes encoding herbicide-detoxifying enzymes in wheat have not been identified. This lack of knowledge could be explained by the size (approximately 16 Gb) and redundancy of the wheat polyploid genome coupled with the inherently large size of plant CYP families (approximately 300-400 genes per diploid genome), making it more difficult to identify and characterize specific genes. Additionally, the lack of a reference genome until 2018 (IWGSC, 2018) likely hindered characterization relative to other sequenced grass crop genomes, such as corn, rice, barley and sorghum (Goff et al., 2002; Mayer et al., 2012; Paterson et al., 2009; Schnable et al., 2009; Yu et al., 2002).

B. Herbicide-Detoxifying CYPs: CYP81As

Members of the CYP81 A sub-family are frequently reported in both crop and weed species for endowing herbicide tolerance and resistance, respectively (Dimaano & Iwakami, 2021), which belongs to the CYP71 clan (Nelson et al., 2004). This sub-family is found exclusively in Poaceae and its endogenous substrates have yet to be determined (Dimaano & Iwakami, 2021).

While CYP-mediated herbicide resistance has been frequently noted in weeds, their roles in herbicide resistance is poorly understood, which is a consequence of a lack of well- annotated genomes for many weed species and the difficulty of studying numerous CYPs in a given species (Dimaano & Iwakami, 2021). CYPs endowing herbicide resistance were first reported in an Echinochloa phyllopogan population resistant to molinate and thiobencarb (very-long-chain fatty acid elongase inhibitors) and fenoxaprop-ethyl (an ACCase inhibitor) in rice fields (Fischer et al., 2000). Knowledge of CYP81A6 involvement with bensulfuron-methyl in rice inspired CYP81A investigation in E. phyllopogan, which resulted in the identification of CYP81A12 and CYP81A21 conferring the concomitant cross-resistance to bensulfuron- methyl, penoxsulam (an ALS inhibitor), diclofop-methyl, pinoxaden, tralkoxydim, and clomazone (a 1- deoxy-D-xylulose 5-phosphate synthase (DOXPS) inhibitor) (Guo et al., 2019; Iwakami et al., 2014, 2019). These two CYPs are likely homoeologs catalyzing almost identical herbicide-metabolizing functions (Dimaano et al., 2020). However, CYP81A12 and CYP81A21 varied in their binding affinities for propyrisulfuron (an ALS inhibitor) (Ha et al., 2022).

Another example is CYP81A10v7 from Lolium rigidum that endows resistance to several herbicides, including diclofop-methyl, tralkoxydim, chlorsulfuron, mesotrione, atrazine (a PSII inhibitor), chlorotoluron, and trifluralin (microtubule assembly inhibitor) (Han et al., 2020). Additionally, there is evidence of CYP81 A involvement with resistance to chlorotoluron and fenoxaprop-P-ethyl (an ACCase inhibitor) in Alopecurus myosuroides (Franco-Ortega et aL, 2021). More recent examples include CYP81A69 from Cynodon dactylon endowing resistance to nicosulfuron, penoxsulam, mesotrione, 2,4-D (a synthetic auxin) and dicamba, and bentazon (Zheng et aL, 2022), and CYP81A68 from Echinochloa crus-galli endowing resistance to penoxsulam, cyhalofop-butyl (an ACCase inhibitor) and metamifop (an ACCase inhibitor), due to O-demethylation (Pan et aL, 2022).

Due to the demonstrated substrate promiscuity of CYP81As, a single CYP is potentially capable of endowing resistance to multiple herbicides, which is concerning when trying to prevent the development and spread of resistant weed populations. Preventing resistant populations from arising, especially target-site based resistance, typically involves rotating herbicides with different modes of action or utilizing tank mixtures of herbicides that each target different sites of action (Evans et aL, 2016; Gressel & Segel, 1990). Given that one CYP can detoxify herbicides with differing sites of action, it would likely be more valuable to rotate herbicide applications based on the enzymes that mediate detoxification to avoid selecting for weeds with these promiscuous, herbicide-detoxifying enzymes endowing resistance to current and future herbicides (Dimaano & Iwakami, 2021). A recent study found the synthetic auxin, florpyrauxifen-benzyl, and penoxsulam are both CYP substrates in E. crus-galli, and resistance to florpyrauxifen-benzyl likely evolved prior to its commercialization in 2018 as the result of previous utilization of penoxsulam in rice fields (Takano et aL, 2023). identification of CYP81 As is essential not only for crop improvement but also for preventing weed resistance.

III. Newly Identified CYP81A Genes and Proteins

Provided herein are newly identified plant herbicide detoxification genes and proteins, namely CYP81A-5A, CYP81 A-5B, and CYP81A-5D genes and proteins and homologs thereof. As used herein, CYP81A-5A, CYP81A-5B, and CYP81A-5D include all homologous genes and proteins.

Exemplary CYP81A-5A DNA sequences include those set forth in SEQ ID NOs: 1-7, and nucleic acid sequences that comprise at least 80% (e.g., 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%, at least 98%, at least 99%, or at least 99.5%) identity to SEQ ID NOs: 1-7. Exemplary CYP81A-5A DNA sequences also include those that encode any of the protein sequences set forth in SEQ ID NOs: 99-100, and protein sequences that comprise at least 80% (e.g., 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%, at least 98%, at least 99%, or at least 99.5%) identity to SEQ ID Nos: 99- 100.

Exemplary CYP81A-5B DNA sequences include those set forth in SEQ ID NOs: 8-13, and nucleic acid sequences that comprise at least 80% (e.g., 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%, at least 98%, at least 99%, or at least 99.5%) identity to SEQ ID NOs: 8-13. Exemplary CYP81A-5B DNA sequences also include those that encode any of the protein sequences set forth in SEQ ID NOs: 101-102, and protein sequences that comprise at least 80% (e.g., 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%, at least 98%, at least 99%, or at least 99.5%) identity to SEQ ID Nos: 101- 102.

Exemplary CYP81A-5D DNA sequences include those set forth in SEQ ID NOs: 14-19, and nucleic acid sequences that comprise at least 80% (e.g., 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%, at least 98%, at least 99%, or at least 99.5%) identity to SEQ ID NOs: 14-19. Exemplary CYP81 A-5D DNA sequences also include those that encode any of the protein sequences set forth in SEQ ID NOs: 103-104, and protein sequences that comprise at least 80% (e.g., 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%, at least 98%, at least 99%, or at least 99.5%) identity to SEQ ID Nos: 103- 104.

Exemplary CYP81 A-5A, CYP81 A-5B, and CYP81 A-5D DNA sequences that have increased rate of transcription include those set forth in SEQ ID NOs: 20-22, and nucleic acid sequences that comprise at least 80% (e.g., 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%, at least 98%, at least 99%, or at least 99.5%) identity to SEQ ID NOs: 20- 22 and has a G at a position corresponding to position 138 of SEQ ID NO: NO: 20 and/or about 200-300 (such as about 200-290, 200-280, 200-270, 200-260, 210-300, 220-300, 230-300, 240-300, 250-300, 240- 260, 250-260, or 256 bp) upstream of TATA box.

Exemplary CYP81A-5A protein sequences include those set forth in SEQ ID NOs: 99-100, and protein sequences that comprise at least 80% (e.g., 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%, at least 98%, at least 99%, or at least 99.5%) identity to SEQ ID NOs: 99-100.

Exemplary CYP81A-5B protein sequences include those set forth in SEQ ID NOs: 101-102, and protein sequences that comprise at least 80% (e.g., 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%, at least 98%, at least 99%, or at least 99.5%) identity to SEQ ID NOs: 101-102.

Exemplary CYP81A-5D protein sequences include those set forth in SEQ ID NOs: 103-104, and protein sequences that comprise at least 80% (e.g., 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%, at least 98%, at least 99%, or at least 99.5%) identity to SEQ ID NOs: 103-104.

Guide nucleic acids (or vectors providing for such), hybridization probes, PCR primers, etc. can be generated based on the above sequences or any fragment thereof, or sequences upstream or downstream of these sequences (such as regulatory sequences). Exemplary guide nucleic acid target regions include SEQ ID NOs: 84, 87, 90, 93, and 96. Exemplary guide sequence include SEQ ID NOs: 85, 88, 91, 94, and 97. Exemplary sgRNAs include SEQ ID NOs: 86, 89, 92, 95, and 98. The above sequences can also be used to study protein-protein interactions and protein-DNA interactions, thereby identifying CYP81A-5A, CYP81A- 5B, and/or CYP81A-5D regulators (e.g., transcription factors, repressors, etc.).

The disclosure also contemplates using variants of the disclosed sequences. Nucleic acid and protein variants can be naturally occurring, such as allelic variants (located at the same locus), paralogs (located at different locus), homeologs (located at different genomes in a polyploid organism), and orthologs (in different organism), or can be non-naturally occurring. Naturally occurring variants can be identified with the use of well-known molecular biology techniques, as, for example, with polymerase chain reaction (PCR) and hybridization techniques as known in the art. Non-naturally occurring variants can be created by gene editing, synthetic techniques, or mutagenesis techniques (such as site-directed mutagenesis), including those applied to polynucleotides, cells, or organisms. Variants also include fragments, such as functional fragments, of sequences disclosed herein. Generally, variants of a particular nucleotide or amino acid sequence of the disclosure have at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to that particular nucleotide or amino acid sequence as determined by sequence alignment programs described elsewhere herein using default parameters.

Variants can contain nucleotide substitutions, deletions, inversions, and insertions. Variation can occur in either or both the coding and non-coding regions. When variation occurs in non-coding regions, such as a regulatory region (e.g., a promoter, enhancer, silencer, insulator, upstream regulatory element (URE), 5’ untranslated region (5’ UTR), 3’ UTR, etc.), transcription may remain unaffected or be increased or decreased. When variation occurs in a coding region, the variation may be silent in terms of the amino acid sequence being produced because of degeneracy of the genetic code, or can produce both conservative and non-conservative amino acid changes.

In some examples, variants comprise a gain-of-function mutation. In some examples, variation is in a regulatory region, such as a promoter. In some examples, the variation increases transcription of the gene. In some examples, the variation increases transcription of the gene in response to a herbicide (such as an ALS inhibitor, such as PROP) or herbicide safener (such as CM). In some examples, the variation is in the promoter of a gene. In some examples, the variation is a single G insertion immediately after a position corresponding to position 137 of SEQ ID NO: 1. In some examples, the variation is a single G insertion at a position corresponding to position 138 of SEQ ID NO: NO: 20 and/or about 200-300 (such as about 200- 290, 200-280, 200-270, 200-260, 210-300, 220-300, 230-300, 240-300, 250-300, 240-260, 250-260, or 256 bp) upstream of TATA box. In some examples, the variation results in increased binding affinity for a transcription factor, and/or reduced binding affinity for a repressor. In some examples, the variation is in a coding region, which results in a protein with increased activity, such as an enzymatic or catalytic activity, such as an activity of catalyzing ring-hydroxylation, O-demethylation, and/or dealkylation reactions. In some examples, such variants are generated by CRISPR/Cas technologies to edit genomic DNA or RNA.

In some examples, gain-of-function CYP81A-5A, 5B, or 5D gene variants have a transcription level increased by at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99%, compared to that of a corresponding wild-type gene. In some examples, gain-of-function CYP81A- 5A, 5B, or 5D gene variants encode a protein that has an activity increased by at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99%, compared to that of a corresponding wild-type protein.

Variants also include sequences derived from mutagenic or recombinant procedures such as “DNA shuffling” which can be used for swapping domains in a polypeptide of interest with domains of other polypeptides. With DNA shuffling, one or more different CYP81A-5A, CYP81A-5B, and CYP81A-5D coding sequences can be manipulated to create new CYP81 A-5A, CYP81A-5B, and CYP81 A-5D coding sequences that produce proteins with desired properties. In this procedure, libraries of recombinant polynucleotides are generated from a population of related polynucleotides comprising sequence regions that have substantial sequence identity and can undergo homologous recombination in vitro or in vivo. For example, using this approach, sequence motifs encoding a domain of interest may be shuffled between a CYP81A-5A, CYP81A-5B, or CYP81A-5D gene of the disclosure and other CYP81A-5A, CYP81A-5B, or CYP81A-5D genes to obtain a new gene coding for a protein with an increased activity or function of interest, such as increased tolerance to a herbicide. Strategies for DNA shuffling are known from e.g., Stemmer (1994, Proc. Natl. Acad. Sci. USA 91: 10747-10751; 1994, Nature 370:389-391); Crameri et al. (1997, Nature Biotech. 15:436-438); Moore et al. (1997, J. Mol. Biol. 272:336-347); Zlang et al. (1997 Proc. Natl. Acad. Sci. USA 94:450-44509); Crameri et al. (1998, Nature 391:288-291); and U.S. Pat. Nos. 5,605,793 and 5,837,458.

In some examples, variant (including fragments) are functional variants, which are variants that retain one or more activities or functions of the reference CYP81A protein at a desirable level. It is recognized that the gene or cDNA encoding a protein can be mutated without materially altering one or more of the protein’s functions. First, the genetic code is degenerate, and thus different codons encode the same amino acids. Second, even where an amino acid substitution is introduced, the mutation can be conservative and have no material impact on the essential function(s) of a protein. See, e.g., Stryer Biochemistry 3rd Ed., 1988. Third, part of a protein chain can be deleted without impairing or eliminating all of its functions. Fourth, insertions or additions can be made in the protein chain for example, adding epitope tags, without impairing or eliminating its functions (Ausubel et al. J. Immunol. 159(5): 2502-12, 1997). Other modifications that can be made without materially impairing one or more functions of a protein can include, for example, in vivo or in vitro chemical and biochemical modifications or the incorporation of unusual amino acids. Such modifications include, but are not limited to, for example, acetylation, carboxylation, phosphorylation, glycosylation, ubiquination, labelling, e.g., with radionucleotides, and various enzymatic modifications, as will be readily appreciated by those well skilled in the art. A variety of methods for labelling polypeptides, and labels useful for such purposes, are well known in the art, and include radioactive isotopes such as 32P, ligands which bind to or are bound by labelled specific binding partners (e.g., antibodies), fluorophores, chemiluminescent agents, enzymes, and antiligands. Functional fragments and variants can be of varying length. For example, some fragments have at least 10, 25, 50, 75, 100, 200, or even more amino acid residues.

Variants of CYP81A-5A, 5B, and 5D proteins can have “conservative” changes, or “nonconservative” changes as described above, such as an addition or deletion that does not alter a protein function significantly. Conservative amino acid substitutions are those substitutions that, when made, least interfere with the properties of the original protein, that is, the structure and especially the function of the protein is conserved and not significantly changed by such substitutions. Conservative substitutions generally maintain (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. Further information about conservative substitutions can be found, for instance, in Ben Bassat et al. (J. Bacteriol., 169:751 757, 1987), O’Regan et al. (Gene, 77:237 251 , 1989), Sahin Toth et al. (Protein Sci., 3:240247, 1994), Hochuli et al. (Bio/Technology, 6:1321 1325, 1988) and in widely used textbooks of genetics and molecular biology. The Blosum matrices are commonly used for determining the relatedness of polypeptide sequences. The Blosum matrices were created using a large database of trusted alignments (the BLOCKS database), in which pairwise sequence alignments related by less than some threshold percentage identity were counted (Henikoff et al., Proc. Natl. Acad. Sci. USA, 89: 10915-10919, 1992). A threshold of 90% identity was used for the highly conserved target frequencies of the BLOSUM90 matrix. A threshold of 65% identity was used for the BLOSUM65 matrix. Scores of zero and above in the Blosum matrices are considered “conservative substitutions” at the percentage identity selected. Table 1 shows exemplary conservative amino acid substitutions.

Table 1. Exemplary conservative amino acid substitutions

In some examples, variants can have no more than 3, 5, 10, 15, 20, 25, 30, 40, 50, or 100 conservative amino acid changes (such as very highly conserved or highly conserved amino acid substitutions). In other examples, one or several hydrophobic residues (such as Leu, lie, Vai, Met, Phe, or Trp) in a variant sequence can be replaced with a different hydrophobic residue (such as Leu, lie, Vai, Met, Phe, or Trp) to create a variant functionally similar to the disclosed amino acid sequences.

In some examples, variants may differ from the disclosed sequences by alteration of the coding region to fit the codon usage bias of the particular organism into which the molecule is to be introduced. In other examples, the coding region may be altered by taking advantage of the degeneracy of the genetic code to alter the coding sequence such that, while the nucleotide sequence is substantially altered, it nevertheless encodes a protein having an amino acid sequence substantially similar to the disclosed amino acid sequences.

In some examples, functional fragments derived from CYP81A-5A, 5B, or 5D proteins of the present disclosure are provided. In some examples, the functional fragments include one or more conserved region shared by two or more CYP81 A proteins. The conserved regions can be determined by any suitable computer program, such as NCBI protein BLAST program and NCBI Alignment program, or equivalent programs. In some examples, the functional fragments are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more amino acids shorter compared to a full-length CYP81A-5A, 5B, or 5D protein. In some examples, the functional fragments are made by deleting one or more amino acids of a full-length CYP81A-5A, 5B, or 5D protein of the present disclosure. In some examples, the functional fragments share at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more identity to a full-length CYP81A-5A, 5B, or 5D protein of the present disclosure. Nucleic acids encoding the functional protein fragments described herein are also provided.

The disclosure also encompasses conserved regions of CYP81A-5A, 5B, or 5D proteins or genes. The conserved regions can be determined by any suitable computer program, such as NCBI protein BLAST program and NCBI Alignment program, or equivalent programs. Sequences of conserved regions can be used to design guide nucleic acids that simultaneously edit multiple genes.

The disclosure also encompasses isolated or substantially purified nucleic acid or protein compositions. “Isolated” or “substantially purified” means substantially or essentially free from components that normally accompany or interact with the nucleic acid molecule or protein as found in its natural environment or during its manufacture. Thus, an isolated or purified polynucleotide or polypeptide is substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. In some examples, an isolated polynucleotide is free of sequences that naturally flank the polynucleotide (i.e., sequences located at the 5' and 3' ends of the polynucleotide) in the genomic DNA of the cell or plant from which the polynucleotide was derived. In some examples, the isolated polynucleotide can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequences that naturally flank the polynucleotide in genomic DNA of the cell or plant from which the polynucleotide was derived. A polypeptide that is substantially free of cellular material includes preparations of protein having less than about 30%, 20%, 10%, 5%, (by dry weight) of contaminating protein. When a protein (including its functional fragments) of the disclosure is recombinantly produced, in some examples the culture medium suitably represents less than about 30%, 20%, 10%, or 5% (by dry weight) of chemical precursors or non- protein-of-interest chemicals.

In accordance with the present disclosure, increasing expression of one or more of CYP81A-5A, CYP81A-5B, and CYP81A-5B genes or activity of proteins encoded by these genes, can be used to generate plants that have increased herbicide tolerance. The present disclosure provides exemplary nucleic acid and protein sequences for CYP81A-5A, 5B, or 5D. Thus, the provided sequences can be used in breeding programs, for example by designing appropriate guide nucleic acid molecules, or other nucleic acid molecules that mutate one or more of CYP81A-5A, 5B, and 5D genes, or one or more CYP81A-5A, 5B, or 5D regulator genes, for increasing herbicide tolerance of a plant.

IV. Increasing Herbicide Tolerance By Gene Editing and/or Transformation

CYP81A-5A, 5B, or 5D expression/activity can be increased, for example by editing an endogenous CYP81A-5A, 5B, and/or 5D gene in a plant cell, or by introducing an exogenous nucleic acid comprising one or more of CYP81A-5A, 5B, and 5D genes into a plant cell.

In some aspects, a gene editing system is used that includes one or more nucleic acid (e.g., DNA or RNA)-binding domains or components and one or more nucleic acid (e.g., DNA or RNA)-modifying domains or components, or isolated nucleic acids, e.g., one or more vectors, encoding the nucleic acid (e.g., DNA or RNA)-binding and nucleic acid (e.g., DNA or RNA)-modifying domains or components. Gene editing systems can be used for modifying a coding sequence of a target gene and/or for modulating the expression of a target gene, e.g., by modifying a non-coding/regulatory sequence (e.g., promoter, operator, etc.) of the gene, or by modifying the coding sequence/expression of a regulator (e.g., repressor or activator) of the gene. In some examples, the one or more nucleic acid (e.g., DNA or RNA)-hinding domains or components are associated with the one or more nucleic acid (e.g., DNA or RNA)-modifying domains or components, such that the one or more nucleic acid (e.g., DNA or RNA)-binding domains target the one or more nucleic acid (e.g., DNA or RNA)-modifying domains or components to a specific nucleic acid site. Methods and compositions for enhancing gene editing is known. See example, U.S. Patent Application Publication No. 2018/0245065. The one or more nucleic acid (e.g., DNA or RNA)-binding domains can be protein domains or nucleic acids that are engineered to recognize target sequences.

Exemplary gene editing systems include but are not limited to, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), CRISPR/ Cas systems, meganuclease systems, Fokl restriction endonuclease systems, and viral vector-mediated gene editing. In some aspects, CRISPR/Cas-based gene editing methods are used to genetically modify the genome of plant species of the present disclosure in order to increase tolerance to an herbicide.

A. CRISPR/Cas Systems

CRISPR and Cas were originally discovered as adaptive immunity systems evolved by bacteria and archaea to protect against viral and plasmid invasion. Naturally occurring CRISPR/Cas systems in bacteria are composed of one or more Cas genes and one or more CRISPR arrays consisting of short palindromic repeats of base sequences separated by genome-targeting sequences acquired from previously encountered viruses and plasmids (called spacers) (Wiedenheft, B., et. al. Nature. 2012; 482:331; Bhaya, D., et. al., Annu. Rev. Genet. 2011; 45:231; and Terms, M.P. et. al., Curr. Opin. Microbiol. 2011; 14:321). Bacteria and archaea possessing one or more CRISPR loci respond to viral or plasmid challenge by integrating short fragments of foreign sequence (protospacers) into the host chromosome at the proximal end of the CRISPR array. Transcription of CRISPR loci generates a library of CRISPR-derived RNAs (crRNAs) containing sequences complementary to previously encountered invading nucleic acids (Haurwitz, R.E., et. al., Science. 2012:329; 1355; Gesner, E.M., et. Al., Nat. Struct. Mol. Biol. 2001, 18:688; Jinek, M., et. AL, Science. 2012:337; 816-21). Target recognition by crRNAs occurs through complementary base pairing with target DNA, which directs cleavage of foreign sequences by means of Cas proteins (Jinek et. AL 2012 “A Programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.” Science. 2012:337; 816-821).

There are at least five main CRISPR system types (Type I, II, III, IV and V) and at least 16 distinct subtypes (Makarova, K.S., et al., Nat Rev Microbiol. 2015. Nat. Rev. Microbiol. 13, 722-736). CRISPR systems are also classified based on their effector proteins. Class 1 systems possess multi-subunit crRNA- effector complexes, whereas in Class 2 systems, all functions of the effector complex are carried out by a single protein (e.g., Cas9 or Cpfl). In some examples, the present disclosure provides using type II and/or type V single-subunit effector systems.

As CRISPR systems occur in many different types of bacteria, the exact arrangements and structures of CRISPR, function and number of Cas genes and their product differ somewhat from species to species (Haft et al. (2005) PloS Comput. Biol. 1: e60; Kunin et al. (2007) Genome Biol. 8: R61; Mojica et al. (2005) J. Mol. Evol. 60: 174-182; Bolotin et al. (2005) Microbiol. 151 : 2551 -2561 ; Pourcel et al. (2005) Microbiol. 151: 653-663; and Stern et al. (2010) Trends. Genet. 28: 335-340.). For example, in E. coli K12, the CRISPR/cas system comprises eight cas genes: Cas3 (predicted HD-nuc lease fused to a DEAD-box helicase), five genes designated casABCDE, casl (predicted integrase), and the endoribonuclease gene Cas2. After transcription of the CRISPR, a complex of Cas proteins (casABCDE) termed Cascade cleaves a CRISPR RNA precursor in each repeat and retains the cleavage products containing the virus-derived sequence. Assisted by the helicase Cas3, these mature CRISPR RNAs then serve as small guide RNAs that enable Cascade to interfere with virus proliferation (Brouns et al. (2008) Science 321: 960-964). In other prokaryotes, Cas6 processes the CRISPR transcript. The CRISPR-based phage inactivation in E. coli requires Cascade and Cas3, but not Casl or Cas2. The Cmr (Cas RAMP module) proteins in Pyrococcus furiosus and other prokaryotes form a functional complex with small CRISPR RNAs that recognizes and cleaves complementary target RNAs. A simpler CRISPR system relies on the protein Cas9, which is a nuclease with two active cutting sites, one for each strand of the double helix. Combining Cas9 and modified CRISPR locus RNA can be used in a system for gene editing (Pennisi (2013) Science 341: 833- 836). a. CRISPR/Cas9

Provided are methods of gene editing using a Type II CRISPR system. Type II systems rely on i) a single endonuclease protein, ii) a transactivating crRNA (tracrRNA), and iii) a crRNA wherein a ~20- nucleotide portion of the 5’ end of the crRNA is complementary to a target nucleic acid. The region of a CRISPR crRNA strand that is complementary to its target DNA protospacer is referred to as “guide sequence.” In some aspects, the tracrRNA and crRNA components of a Type II system can be replaced by a single guide RNA (sgRNA), also known as a guide RNA (gRNA). The sgRNA can include, for example, an at least 12-20 nucleotide sequence complementary to the target DNA sequence (guide sequence) and can include a common scaffold RNA sequence at its 3' end. As used herein, “a common scaffold RNA” refers to any RNA sequence that mimics the tracrRNA sequence or any RNA sequences that function as a tracrRNA.

Cas9 endonucleases produce blunt end DNA breaks, and are recruited to target DNA by a combination of a crRNA oligo and a tracrRNA oligo, which tether the endonuclease via complementary hybridization of the RNA CRISPR complex. The HNH and RuvC nuclease domains of Cas9 in a type II CRISPR-Cas system are responsible for cleaving the DNA strand complementary to the guide sequence and the non-target strand, respectively, which creates a double- stranded break in the DNA utilized to introduce modifications by non-homologous end joining (NHEJ) or homology -directed repair (HDR) (Gao et al., 2017; Jiang & Doudna, 2017; Jinek et al., 2012; Symington & Gautier, 2011). HDR is more precise and requires a donor DNA template to repair the double-strand breaks, whereas NHEJ does not require a repair template (Puchta, 2005; Puchta et al., 1996). Due to its comparative simplicity, NHEJ is a more common method to disrupt genes in plants, especially in wheat (Li et al., 2021), by inducing small indels (insertions/deletions) in target genes, while HDR can precisely introduce specific point mutations and insert or replace sequences into the target DNA (Li et al., 2013a).

In some examples, DNA recognition by the crRNA/tracrRNA/endonuclease (or sgRNA/endonuclease) complex uses additional complementary base-pairing with a protospacer adjacent motif (PAM) (e.g., 5’-NGG-3 ) located in a 3’ portion of the target DNA, downstream from the target protospacer (Jinek, M., et. AL, Science. 2012, 337:816-821). In some examples, the PAM motif recognized by a Cas9 varies for different Cas9 proteins.

Cas9 proteins that can be used in the methods and systems described herein include any naturally occurring and artificially obtained variants. In some examples, Cas9 can include one or more of the mutations described in the literature, including but not limited to the functional mutations described in: Fonfara et al. Nucleic Acids Res. 2014 Feb;42(4):2577-90; Nishimasu H. et al. Cell. 2014 Feb 27,156(5):935-49; Jinek M. et al. Science. 2012 337:816-21; and Jinek M. et al. Science. 2014 Mar 14, 343(6176) (which are hereby incorporated by refernece). See also U.S. Pat. Nos. 10,266,850; 8,697,359; 8,771,945; 8,795,965; 8,865,406; 8,871,445; 8,889,356; 8,895,308; 8,906,616; 8,932,814; 8,945,839; 8,993,233; and 8,999,641. Thus, in some examples, the systems and methods disclosed herein can be used with the wild type Cas9 protein having double-stranded nuclease activity, Cas9 mutants that act as single stranded nickases, or other mutants with modified nuclease activity.

Cas9 proteins that can be used in the methods and systems described herein include Cas9 proteins (or variant thereof) of a variety of species, e.g., .S’, pyogenes, S. thermophilus, Staphylococcus aureus, and Neisseria meningitidis. Additional Cas9 species include those from: Acidovorax avenae, Actinobacillus pleuropneumoniae, Actinobacillus succinogenes, Actinobacillus suis, Actinomyces sp., eye liphilus denitrificans, Aminomonas paucivorans, Bacillus cereus, Bacillus smithii, Bacillus thuringiensis, Bacteroides sp., Blastopirellula marina, Bradyrhiz obium sp., Brevibacillus latemsporus, Campylobacter coli, Campylobacter jejuni, Campylobacter lad, Candidatus Puniceispirillum, Clostridi cellulolyticum, Clostridium perfringens, Corynebacterium accolens, Corynebacterium diphtheria, Corynebacterium matruchotii, Dinoroseobacter sliibae, Eubacterium dolichum, gamma proteobacterium, Gluconacetobacler diazotrophicus, Haemophilus parainfluenzae, Haemophilus sputorum, Helicobacter canadensis, Helicobacter cinaedi, Helicobacter mustelae, Ilyobacler polytropus, Kingella kingae, Lactobacillus crispatus, Listeria ivanovii, Listeria monocytogenes, Listeriaceae bacterium, Methylocystis sp., Methylosinus trichosporium, Mobiluncus mulieris, Neisseria bacilliformis, Neisseria cinerea, Neisseria flavescens, Neisseria lactamica, Neisseria sp., Neisseria wadsworthii, Nitrosomonas sp., Parvibaculum lavamentivorans , Pasteurella multocida, Phascolarctobacterium succinatutens, Ralstonia syzygii, Rhodopseudomonas palustris, Rhodovulum sp., Simonsiella muelleri, Sphingomonas sp., Sporolactobacillus vineae, Staphylococcus lugdunensis, Streptococcus sp., Subdoligranulum sp., Tislrella mobilis, Treponema sp., or Verminephrobacter eiseniae.

Cas9 proteins that can be used in the methods and systems described herein also include SpyCas9, SaCas9, and StlCas9. See for example, Song et al. (2016), The Crop Journal 4:75-82; Mali et al. (2013) Science 339: 823-826; Ran et al. (2015) Nature 520: 186-191; Esvelt et al. (2013) Nature methods 10(11): 1 1 16-1 121.

Editing a single base pair in the genome without introducing double-strand breaks can also be achieved by utilizing an engineered Cas9-based editors comprising a dead Cas9 domain fused to a cytidine deaminase enzyme, and a sgRNA, which can convert G to A and C to T (Komor et al., 2016). The same base conversions can also be achieved with a Cas9 fused with a transfer RNA adenosine deaminase (Gaudelli et al., 2017). The main benefit of these techniques is they induce point mutations without generating excess undesired editing by-products, such as off-target editing. These techniques have been used to edit genes in maize, rice, wheat, etc. (Rees & Liu, 2018; Zong et al., 2017). b. CRISPR/Cpfl

In some aspects, a Type V CRISPR system is used to edit a plant genome. In some examples, the Cpfl CRISPR system from Prevotella, Francisella, Acidaminococcus, Lachnospiraceae, or Moraxella is used.

The Cpfl CRISPR systems can include i) a single endonuclease protein, and ii) a crRNA, wherein a portion of the 3’ end of crRNA contains the guide sequence complementary to a target nucleic acid. In this system, the Cpfl nuclease is directly recruited to the target DNA by the crRNA. In some embodiments, guide sequences for Cpfl are at least 12 nt, 13 nt, 14 nt, 15 nt, or 16 nt in order to achieve detectable DNA cleavage, and a minimum of 14 nt, 15nt, 16 nt, 17 nt, or 18 nt to achieve efficient DNA cleavage.

The Cpfl system differs from the Cas9 system in some ways. First, unlike Cas9, Cpfl does not require a separate tracrRNA for cleavage. In some examples, Cpfl crRNAs can be as short as about 42-44 nt long — of which about 23-25 nt is guide sequence and about 19 nt is the constitutive direct repeat sequence. In contrast, in some examples, the combined Cas9 tracrRNA and crRNA synthetic sequences can be about 100 nt long.

Second, certain Cpfl systems prefer a “TTN” PAM motif that is located 5' upstream of its target. This is in contrast to the “NGG” PAM motifs located on the 3’ of the target DNA for common Cas9 systems such as Streptococcus pyogenes Cas9 system. In some examples, the uracil base immediately preceding the guide sequence cannot be substituted (Zetsche, B. et al. 2015. “Cpfl Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System” Cell 163, 759-771, which is hereby incorporated by reference in its entirety for all purposes).

Third, the cut sites for Cpfl are staggered by about 3-5 nt, which create “sticky ends” (Kim et aL, 2016. “Genome-wide analysis reveals specificities of Cpfl endonucleases in human cells” published online June 06, 2016). These sticky ends with 3-5 nt overhangs are thought to facilitate NHEJ-mediated-ligation, and improve gene editing of DNA fragments with matching ends. The cut sites are in the 3' end of the target DNA, distal to the 5' end where the PAM is. The cut positions usually follow the 18th nt on the nonhybridized strand and the corresponding 23rd nt on the complementary strand hybridized to the crRNA.

Fourth, in Cpfl complexes, the “seed” region is located within the first 5 nt of the guide sequence. Cpfl crRNA seed regions are highly sensitive to mutations, and even single base substitutions in this region can drastically reduce cleavage activity (see Zetsche B. et al. 2015 “Cpfl Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System” Cell 163, 759-771). Critically, unlike the Cas9 CRISPR target, the cleavage sites and the seed region of Cpfl systems do not overlap. Additional guidance on designing Cpfl crRNA targeting oligos is available on Zetsche B. et al. 2015 (“Cpfl Is a Single RNA- Guided Endonuclease of a Class 2 CRISPR-Cas System” Cell 163, 759-771). c. CRISPR/Cas Applied in Wheat (Triticum aestivum L.)

Any CRSPR/Cas system can be used to edit a plant in the methods and systems disclosed herein. In some examples, a type II CRISPR-Cas system of Streptococcus pyogenes (Cunningham, 2000; Garneau et al., 2010; Rosinski-Chupin et al., 2019).

In some examples, to edit a plant gene of interest, one or more sgRNAs are designed to target certain regions of the gene by complementarity. A CRISPR/Cas construct (e.g., CRSPR/Cas9) (a vector, including a plasmid) is introduced into a plant cell, which includes a sequence that encodes the one or more sgRNAs, a sequence (optionally codon optimized) that encode a Cas endonuclease (e.g., Cas9), a constitutive or inducible promoter, a transcription terminator, and an antibiotic or herbicide resistance marker for selection purposes (Fauser et al., 2014; Li et al., 2013a; Shimatani et al., 2017). The CRISPR/Cas construct can be delivered to a plant cell by polyethylene glycol mediated delivery, Agrobacterium-mediated transformation, and bombardment/biolistic transformation.

In some examples, Agrobacterium-mediated transformation is used to deliver the vector. In an exemplary method, the vector is introduced into A. tumefaciens (or Rhizobium rhizogenes if the plant species not susceptible to A. tumefaciens), the colonies of A. tumefaciens (or Rhizobium rhizogenes) containing the CRISPR/Cas are used to transform a plant cell or plant tissues (e.g., callus, leaf or floral organs), and then first-generation transgenic plants are identified by antibiotic or herbicide selection (El-Mounadi et aL, 2020; Li et al., 2013a; Pyott et aL, 2016; Veillet et aL, 2019). Subsequent sequencing of the target gene may be performed to identify the mutations introduced by genome editing. Due to the presence of the CRISPR/Cas cassette, plants are considered transgenic and subject to corresponding biosafety regulations (Callaway, 2018; Eckerstorfer et aL, 2019; Garcia Ruiz et aL, 2018). However, the CRISPR/Cas transgene can be eliminated by crossing and Mendelian segregation when dealing with a sexually propagated plant species (Zhang et aL, 2019a), which results in removal of the transgene in the third or subsequent generations in which the mutation in the gene of interest is maintained (Pyott et aL, 2016). These transgene-free, edited plants more closely resemble plants with mutations induced naturally or chemical mutagenesis (Lellis et aL, 2002; Pyott et aL, 2016).

There are many advantages and benefits of CRISPR/Cas9 that give it immense potential for crop breeding and the development of sustainable agriculture, including simplicity, efficiency, low cost, the possibility to target multiple genes, allowing faster genetic modification than other techniques, modifying previously neglected crops, and providing resistance to viruses and other pathogens (including those lacking natural resistance options) (Cong et al., 2013; Garcia-Ruiz, 2018; Luria et aL, 2017; Mali et aL, 2013; Toda et aL, 2019; Wamaitha et aL, 2018; Wurtzel et aL, 2019; Zhang et aL, 2017a; Zhang et aL, 2019b). CRTSPR/Cas9 is a valuable tool successfully implemented to enhance metabolic pathways, tolerance to biotic (fungal, bacterial or viral pathogens) and abiotic stresses (cold, drought, salt), improve nutritional content, increase yield and grain quality, obtain haploid seeds, obtain domesticated traits in wild species, and endow herbicide resistance (Chandrasekaran et aL, 2016; Endo et aL, 2016; Jia et aL, 2016; Jiang et al., 2017; Li et al., 2018a; Li et aL, 2016; Li et aL, 2018b; Nekrasov et aL, 2017; Ortigosa et aL, 2019; Peng et aL, 2017; Sun et al., 2016, 2017; Wang et aL, 2016a; Wang et aL, 2017; Yao et aL, 2018; Zhang et aL, 2019a; Zhou et aL, 2015; Zsbgbn et aL, 2018).

Other advanced technologies for wheat genome editing include platforms for multiplex genome editing, gene replacement through HDR, simultaneous HDR and base editing, site-directed artificial evolution, de novo domestication of wild relatives, and genotype-independent technologies (Li et aL, 2021).

B. Guide Nucleic Acids

In some examples, a guide nucleic acid (e.g., RNA or DNA) of the present disclosure includes two regions, being or encoding for crRNA and tracrRNA, respectively. The crRNA is complementary to a target, and the tracrRNA is responsible for binding with a Cas protein. In some examples, the two regions are provided as separate molecules. In some examples, the guide RNA is a single guide RNA (sgRNA) (a crRNA/tracrRNA hybrid), produced from transcribing a corresponding DNA or synthetically produced. In some examples, the guide RNA is a crRNA for a Cpfl endonuclease.

Unless otherwise noted, all references to a single guide nucleic acid (e.g., sgRNA or sgDNA) in the present disclosure can be read as referring to a guide nucleic acid (e.g., gRNA or gDNA). Therefore, examples described in the present disclosure which refer to a single guide nucleic acid (e.g., sgRNA or sgDNA) will also be understood to refer to a guide nucleic acid (e.g., gRNA or gDNA). Guide nucleic acids are designed to recruit the CRISPR endonuclease to a target nucleic acid region. Such methods are known in the art. Software programs can be used to identify candidate CRISPR target sequences on both strands of an input DNA sequence based on desired guide sequence length and a CRISPR motif sequence (e.g., PAM) for a specified CRISPR enzyme. For example, target sites for Cpfl from Francisella novicida U112, with PAM sequences TTN, may be identified by searching for 5'-TTN- 3' both on the input sequence and on the reverse-complement of the input. The target sites for Cpfl from Lachnospiraceae bacterium and Acidaminococcus sp., with PAM sequences TTTN, may be identified by searching for 5’-TTTN-3’ both on the input sequence and on the reverse complement of the input. Likewise, target sites for Cas9 of S. thermophilus CRISPR, with PAM sequence NNAGAAW, may be identified by searching for 5'-Nx-NNAGAAW-3' both on the input sequence and on the reverse-complement of the input. The PAM sequence for Cas9 of S. pyogenes is 5’-NGG-3’.

Since multiple occurrences in the genome of the DNA or RNA target site may lead to nonspecific genome editing, after identifying all potential sites, sequences may be filtered out based on the number of times they appear in the relevant reference genome or modular CRISPR construct. For those CRISPR enzymes for which sequence specificity is determined by a “seed” sequence (such as the first 5 nt of the guide sequence for Cpfl -mediated cleavage) the filtering step may also account for any seed sequence limitations.

In some aspects, algorithmic tools identify potential off target sites for a particular guide sequence. For example, Cas-Offinder can be used to identify potential off target sites for Cpfl (see Kim et al., 2016. Nature Biotechnology 34, 863-868). Any other publicly available CRISPR design/identification tool may also be used, including for example the Zhang lab crispr.mit.edu tool (see Hsu, et al. 2013 “DNA targeting specificity of RNA guided Cas9 nucleases” Nature Biotech 31, 827-832).

In some aspects, the user can choose the length of the seed sequence. The user can specify the number of occurrences of the seed and PAM sequence in a genome for purposes of passing the filter. The default is to screen for unique sequences. Filtration level is altered by changing both the length of the seed sequence and the number of occurrences of the sequence in the genome. The program may in addition or alternatively provide the sequence of a guide sequence complementary to the reported target sequence(s) by providing the reverse complement of the identified target sequence(s).

In some aspects, the transgenic plant, plant part, plant cell, or plant tissue culture taught herein includes a recombinant construct, which includes at least one nucleic acid sequence encoding a guide RNA. In some examples, the nucleic acid is operably linked to a promoter. In some examples, a recombinant construct further comprises a nucleic acid sequence encoding a CRISPR endonuclease. In some examples, the guide RNA or DNA is capable of forming a complex with the CRISPR endonuclease, and the complex is capable of binding to and creating a double-strand break in a target nucleic acid sequence of the plant genome. In some examples, the CRISPR endonuclease is Cas9. In some examples, the CRISPR endonuclease is Cpfl. In some examples, the CRISPR endonuclease is Casl3d. In some aspects, the target sequence is within a CYP81A-5A, CYP81A-5B, or CYP81A-5D gene (including the regulatory regions). C. Promoters

Nucleic acid molecules (such as coding sequences or guide sequences) included in expression vectors are typically driven by a nucleotide sequence comprising a regulatory element, for example, a promoter. In some examples, promoter sequences can be operably linked to a nucleic acid molecule that increases CYP81A-5A, CYP81A-5B, and/or CYP81A-5D activity, such as a nucleic acid sequence encoding any of these proteins, or nucleic acid sequences encoding components of a gene editing systems, which will generate a gain-of-function mutation in these genes and proteins. Exemplary promoters include a plant promoter, such as one from Arabidopsis or other plants (e.g., constitutive promoter from the Arabidopsis serine carboxypeptidase-like gene AtSCPL30, PD1 from Arabidopsis, HVA22E, PLDdelta, AtSl, and AtS3). In some examples, the promoter is heterologous to the plant into which it is introduced. In some examples, the promoter is heterologous to the sequence to which it is operably linked.

Promoter includes a region of DNA upstream from the start of transcription and involved in recognition and binding of RNA polymerase and other proteins to initiate transcription. A plant promoter is a promoter capable of initiating transcription in plant cells. Examples of promoters under developmental control include promoters that preferentially initiate transcription in certain tissues, such as leaves, roots, seeds, fibers, xylem vessels, tracheids, or sclerenchyma. Such promoters are referred to as “tissuepreferred.” Promoters that initiate transcription only in a certain tissue are referred to as “tissue-specific”. A “cell-type specific” promoter primarily drives expression in certain cell types in one or more organs, for example, vascular cells in roots or leaves. An “inducible” promoter is a promoter that is under environmental control. Examples of environmental conditions that may affect transcription by inducible promoters include anaerobic conditions or the presence of light. Tissue-specific, tissue-preferred, cell-type specific, and inducible promoters constitute the class of “non-constitutive” promoters. A “constitutive” promoter is a promoter that is active under most environmental conditions, and in most cell types. a. Constitutive Promoters

In some examples, a constitutive promoter is operably linked to a gene or coding sequence (including one that can generate a guide RNA) for expression in a plant. In some examples, the gene or coding sequence includes those that encode a CYP81A-5A, 5B, and/or 5D protein. In some examples, the gene or coding sequence includes those that encode components of a CRIPSR system.

Any suitable constitutive promoters can be used in the present methods and systems. Exemplary constitutive promoters include, but are not limited to, the promoters from plant viruses such as the 35S promoter from CaMV (Odell et al., Nature 313:810-812 (1985)) and the promoters from such genes as rice actin (McElroy et aL, Plant Cell 2: 163-171 (1990)); ubiquitin (Christensen et al., Plant Mol. Biol. 12:619- 632 (1989) and Christensen et aL, Plant Mol. Biol. 18:675-689 (1992)); pEMU (Last et aL, Theor. Appl. Genet. 81:581-588 (1991)); MAS (Velten et aL, EMBO J. 3:2723-2730 (1984)) and maize H3 histone (Lepetit et aL, Mol. Gen. Genetics 231:276-285 (1992) and Atanassova et aL, Plant Journal 2 (3): 291-300 (1992)). The ALS promoter, Xbal/Ncol fragment 5' to the Brassica napus ALS3 structural gene (or a nucleotide sequence similarity to said Xbal/Ncol fragment), represents a particularly useful constitutive promoter. See WO 96/30530.

In a specific example the constitutive promoter is CaMV-35S, CaMV-35Somega, UBQ10 from Arabidopsi, Ubil from maize/rice, or barley leaf thionin BTH6 promoter. b. Inducible Promoters

In some examples, an inducible promoter is operably linked to a gene or coding sequence (including one that can generate a guide RNA) for expression in a plant. In some examples, the gene or coding sequence includes those that encode a CYP81A-5A, 5B, and/or 5D protein. In some examples, the gene or coding sequence includes those that encode components of a CRIPSR system. With an inducible promoter the rate of transcription increases in response to an inducing agent.

Any suitable inducible promoters can be used in the present methods and systems. See Ward et al., Plant Mol. Biol. 22:361-366 (1993). Exemplary inducible promoters include, but are not limited to, that from the ACEI system which responds to copper (Mett et al., PNAS 90:4567-4571 (1993)); In2 gene from maize which responds to benzenesulfonamide herbicide safeners (Hershey et al., Mol. Gen Genetics 227:229-237 (1991) and Gatz et al., Mol. Gen. Genetics 243:32-38 (1994)) or Tet repressor from TnlO (Gatz et al., Mol. Gen. Genetics 227:229-237 ( 1991 )). One exemplary inducible promoter is a promoter that responds to an inducing agent to which plants do not normally respond. An exemplary inducible promoter is the inducible promoter from a steroid hormone gene, the transcriptional activity of which is induced by a glucocorticosteroid hormone (Schena et al., Proc. Natl. Acad. Sci. USA 88:0421 (1991)). c. Tissue-Specific or Tissue-Preferred Promoters

In some examples, a tissue-specific or tissue -preferred promoter is operably linked to a gene or coding sequence (including one that can generate a guide RNA) for expression in a plant. In some examples, the gene or coding sequence includes those that encode a CYP81A-5A, -5B, and/or -5D protein. In some examples, the gene or coding sequence includes those that encode components of a CRIPSR system.

Plants transformed with a gene or coding sequence operably linked to a tissue-specific promoter produce the product of the gene or coding sequence exclusively, or preferentially, in a specific tissue.

Any suitable tissue-specific or tissue-preferred promoter can be used in the present methods and systems. Exemplary tissue-specific or tissue-preferred promoters include, but are not limited to, a rootpreferred promoter such as that from the phaseolin gene (Murai et al., Science 23:476-482 (1983) and Sengupta-Gopalan et al., Proc. Natl. Acad. Sci. USA 82:3320-3324 (1985)); a leaf-specific and light-induced promoter such as that from cab or rubisco (Simpson et al., EMBO J. 4(11):2723-2729 (1985) and Timko et al., Nature 318:579-582 (1985)); an anther-specific promoter such as that from LAT52 (Twell et al., Mol. Gen. Genetics 217:240-245 (1989)); a pollen-specific promoter such as that from Zml3 (Guerrero et al., Mol. Gen. Genetics 244: 161-168 (1993)) or a microspore -preferred promoter such as that from apg (Twell et al., Sex. Plant Reprod. 6:217-224 (1993)). In some examples, a tissue-specific or tissue -preferred promoter is a native promoter of FACT gene, HORST gene, ASFT gene, GPAT5 gene, RALPH gene, and/or MYB84 gene.

D. Exemplary methods of CYP81A Upregulation

Provided herein are methods (e.g., gene-editing and/or transformation methods) of increasing transcription of one or more of CYP81A-5A, CYP81A-5B, and CYP81A-5D genes, or increasing activity of one or more of CYP81A-5A, CYP81A-5B, and CYP81A-5D proteins. Increasing activity of one or more CYP81A proteins includes increasing the level or amount of these proteins, and/or enhancing the function or activity per protein.

Such upregulation methods include, for example: i) creating a mutation in the regulatory region of one or more of endogenous CYP81A-5A, CYP81A-5B, and CYP81A-5D genes, wherein the mutation increases transcription rate of the gene(s); ii) creating a mutation in the coding region(s) of one or more of endogenous CYP81A-5A, CYP 1A-5B, and CYP81A-5D genes, wherein the mutation increases one or more activities/functions of the proteins (e.g., enhancing the protein’s efficiency in catalyzing a reaction); iii) introducing one or more exogenous nucleic acid molecules encoding one or more of CYP81A-5A, CYP81A-5B, and CYP81 A-5D genes into a plant cell, optionally integrating them into the genome.

In some examples, gene edited plants are generated using gene editing technologies, for example using a guide nucleic acid molecule specific for one or more of CYP81 A-5A, CYP81 A-5B, and CYP81 A- 5D genes, that can mutate the target, resulting in its increased expression and/or activity of the protein encoded. In some examples, a CRISPR/Cas system is used.

Unlinked transgenic sequences (including the gRNA, the Cas9 cassette and the Kan^R cassette) will naturally segregate away from any gene-edited site in (4 of the Ti generation. Thus, it is possible for plants to segregate out the gRNA/Cas9 transgenes in subsequent generations, thus producing transgene-free, gene- mutated plants. In one example, the use of recombinant DNA in the construction of gene-edited plants is avoided, and instead plant leaf tissues are transformed using pre-assembled gRNA and Cas9 RNP complexes. In one example, polyethylene glycol (PEG)-transformation of protoplasts (e.g., see Woo et al., Nat. BiotechnoL, 2015. 33(11): 1162-4) or gene gun bombardment of immature embryos with RNPs (e.g., see Zhang et al., Nat Commun, 2016. 7: 12617; Liang et aL, Nat Commun, 2017. 8:14261) is used. gRNAs can be produced using commercial kits, such as the Invitrogen GeneArt™ Precision gRNA Synthesis Kit. To produce more gRNAs, a DNA template can be assembled by PCR with forward and reverse overlapping oligonucleotides that contain the target DNA sequence, together with the T7 promoter and universal reverse primers supplied with the kit. In vitro transcripts can be produced by T7 RNA polymerase and purified by phenol/chloroform extraction and ethanol precipitation. The RNP complexes of 1-5 pg gRNA and 1 pg GeneArt™ Platinum™ Cas9 nuclease with nuclear-targeting signal (Invitrogen) can be assembled and incubated for 10 min at room temperature. The RNP complexes can be mixed with 1 mg 0.6 pm gold particles sterilized by 70% ethanol for gene gun bombardment. Seeds can be sterilized by 10% bleach for 15 min and rinsed three times with sterile water. Seeds can then be germinated. Leaf bases from the first true leaves of 3-week old plants or callus generated from embryos can be used as explants for bombardment. The bombarded plant tissues can be cultured on MS medium supplemented with Gamborg vitamins, 3% sucrose and 16.8 M thidiazuron (TDZ) until shoot formation. Regenerated shoots can be transferred onto MS medium without TDZ but containing 1 j-ig/1 indole-3-butyric acid (IBA) to induce root formation. Fully regenerated plantlets can be transferred to soil and allowed to produce seeds under isolated conditions.

Since CRISPR-mediated gene editing occurs in To plants, the integration of the gRNA/Cas cassettes into the plant genome can be examined by PCR on To plants. Cas9 expression can be validated using a tag (such as a 3X FLAG antibody) to detect the epitope-tagged Cas9 protein in Western blot analysis.

In some examples, the gene editing method is free of recombinant technology and does not involve T-DNA, Ti-plasmids (or other plasmids), Agrobacterium or other pathogenic microbes. Once the gRNA/Cas9 RNP complex is delivered into leaf tissue, it can be rapidly degraded and lost from cells. Gene edited plants without any transgene can be produced immediately from edited plant cells. In some example, editing of more than one gene at a time (e.g., multiple paralogs and/or homeologs of CYP81A-5A, 5B, and/or 5D) can be achieved by bombarding leaf tissues with two or more gRNA/Cas9 RNP complexes. Since there is no selectable marker delivered into leaf tissues, regenerated plantlets can be screened for geneediting individually.

In some examples, transformed (or transgenic) plant cells, plant parts or plants are generated (which, for example, include an exogenous nucleic acid molecule comprising coding sequences for one or more of CYP81A-5A, 5B, and 5D proteins, operably linked to a promoter, or include a construct for gene editing) by plant transformation methods. Portions or all of the exogenous nucleic acid molecule or the gene-editing construct may or may not be integrated into the plant genome.

Plant transformation methods include, but are not limited to, electroporation; microinjection; microprojectile bombardment, also known as particle acceleration or biolistic bombardment; direct incubation with germinating pollen; viral-mediated transformation; and Agrobacterium-mediated transformation. See, for example, U.S. Patent Nos. 5,405,765; 5,472,869; 5,538,877; 5,538,880; 5,550,318; 5,641,664; 5,736,369 and 5,736,369; International Patent Application Publication Nos. W02002/038779 and WO/2009/ 117555; Lu et al., (Plant Cell Reports, 2008, 27:273-278); Watson et al., Recombinant DNA, Scientific American Books (1992); Hinchee et al., Bio/Tech. 6:915-922 (1988); McCabe et al., Bio/Tech. 6:923-926 (1988); Toriyama et al., Bio/Tech. 6: 1072-1074 (1988); Fromm et al., Bio/Tech. 8:833-839 (1990); Mullins et al., Bio/Tech. 8:833-839 (1990); Hiei et al., Plant Molecular Biology 35:205-218 (1997); Ishida et al., Nature Biotechnology 14:745-750 (1996); Zhang et al., Molecular Biotechnology 8:223-231 (1997); Ku et aL, Nature Biotechnology 17:76-80 (1999); and, Raineri et al., Bio/Tech. 8:33-38 (1990)). In some examples, an expression construct can be introduced into embryogenic callus of any plant genus or species and the resulting transformed cells can be regenerated into plants. The transgenic plants are expected to have expression of nucleic acid sequences carried by the construct. “Embryogenic callus cell” used herein refers to an embryogenic cell contained in a cell mass produced in vitro. A transgenic plant formed using Agrobacterium transformation methods typically contains a single gene on one chromosome, although multiple copies are possible. Such transgenic plants can be referred to as being hemizygous for the added gene. A more accurate name for such a plant is an independent segregant, because each transformed plant represents a unique T-DNA integration event (U.S. Patent No. 6,156,953). A transgene locus is generally characterized by the presence and/or absence of the transgene. A heterozygous genotype in which one allele corresponds to the absence of the transgene is also designated hemizygous (U.S. Patent No. 6,008,437).

For efficient plant transformation, a selection method is used such that whole plants are regenerated from a single transformed cell and every cell of the transformed plant carries the nucleic acid of interest. These methods can employ positive selection, whereby a foreign nucleic acid is supplied to a plant cell that allows it to utilize a substrate present in the medium that it otherwise could not use, such as mannose or xylose (for example, refer US 5767378; US 5994629). Negative selection can be used, utilizing selective agents such as herbicides or antibiotics that either kill or inhibit the growth of non-transformed plant cells and reducing the possibility of chimeras. Resistance genes that are effective against negative selective agents are provided on the introduced foreign nucleic acid used for the plant transformation. For example, kanamycin, together with the resistance gene neomycin phosphotransferase (nptll), which confers resistance to kanamycin and related antibiotics (see, for example, Messing & Vierra, Gene 19: 259-268 (1982); Bevan et al., Nature 304: 184-187 (1983)) can be used. However, many different antibiotics and antibiotic resistance genes can be used for transformation purposes (refer US 5034322, US 6174724 and US 6255560). In addition, several herbicides and herbicide resistance genes have been used for transformation purposes, including the bar gene, which confers resistance to the herbicide phosphinothricin (White et al., Nucl Acids Res 18: 1062 (1990), Spencer et al., Theor Appl Genet 79: 625-631(1990), US 4795855, US 5378824 and US 6107549). In addition, the dhfr gene, which confers resistance to the anticancer agent methotrexate, has been used for selection (Bourouis et al., EMBO J. 2(7): 1099-1104 (1983).

The expression control elements used to regulate the expression of a given nucleic acid (such as one or more of a CYP81A genes, or a sequence coding for a Cas protein and a guide RNA) can either be the expression control element that is normally found associated with the coding sequence (homologous expression element) or can be a heterologous expression control element. A variety of homologous and heterologous expression control elements are known and can readily be used to make expression units for use in the present disclosure. Transcription initiation regions, for example, can include any of the various opine initiation regions, such as octopine, mannopine, nopaline and the like that are found in the Ti plasmids of Agrobacterium tumefaciens. Alternatively, plant viral promoters can also be used, such as the cauliflower mosaic virus 19S and 35S promoters (CaMV 19S and CaMV 35S promoters, respectively) to control gene expression in a plant (U.S. Patent Nos. 5,352,605; 5,530,196 and 5,858,742 for example). Enhancer sequences derived from the CaMV can also be utilized (U.S. Patent Nos. 5,164,316; 5,196,525; 5,322,938; 5,530,196; 5,352,605; 5,359,142; and 5,858,742 for example). Plant promoters such as prolifera promoter, fruit specific promoters, Ap3 promoter, heat shock promoters, seed specific promoters, etc. can also be used. A gamete-specific promoter, a constitutive promoter (such as the CaMV or Nos promoter), an organ specific promoter (such as the E8 promoter from tomato), or an inducible promoter can be ligated to the nucleic acid to be expressed. The expression unit may be further optimized by employing supplemental elements such as transcription terminators and/or enhancer elements.

Thus, for expression in plants, the expression units typically contain, in addition to the nucleic acid to be expressed, a plant promoter region, a transcription initiation site and a transcription termination sequence. Unique restriction enzyme sites at the 5' and 3' ends of the expression unit are typically included to allow for easy insertion into a pre-existing vector.

In some examples, the promoter is positioned about the same distance from the heterologous transcription start site as it is from the transcription start site in its natural setting. However, some variation in this distance can be accommodated without loss of promoter function.

In addition to a promoter sequence, the expression cassette can also contain a transcription termination region downstream of the nucleic acid to be expressed to provide for efficient termination. The termination region may be obtained from the same gene as the promoter sequence or may be obtained from different genes. If the mRNA encoded by the nucleic acid to be expressed is to be efficiently processed, DNA sequences which direct polyadenylation of the RNA are also commonly added to the vector construct. Polyadenylation sequences include, but are not limited to the Agrobacterium octopine synthase signal (Gielen et al., EMBO J 3:835 846 (1984)) or the nopaline synthase signal (Depicker et al., Mol. and Appl. Genet. 1:561 573 (1982)). The resulting expression unit is ligated into or otherwise constructed to be included in a vector that is appropriate for higher plant transformation. One or more expression units may be included in the same vector. The vector typically contains a selectable marker gene expression unit by which transformed plant cells can be identified in culture. Usually, the marker gene will encode resistance to an antibiotic, such as G418, hygromycin, bleomycin, kanamycin, or gentamicin or to an herbicide, such as glyphosate (Round-Up) or glufosinate (BASTA) or atrazine. Replication sequences, of bacterial or viral origin, can be included to allow the vector to be cloned in a bacterial or phage host; in one example a broad host range for prokaryotic origin of replication is included. A selectable marker for bacteria may also be included to allow selection of bacterial cells bearing the desired construct. Suitable prokaryotic selectable markers include resistance to antibiotics such as ampicillin, kanamycin or tetracycline. Other DNA sequences encoding additional functions may also be present in the vector. For instance, in the case of Agrobacterium transformations, T DNA sequences can be included for subsequent transfer to plant chromosomes.

Several approaches can be utilized to co-express multiple polynucleotides in plant cells. Each nucleic acid molecule to be expressed (e.g., one or more of a CYP81A gene; or a Cas gene, a guide RNA- encoding sequence, and optionally a selectable marker) can be separately introduced into a plant cell by using separate nucleic acid constructs, or be introduced together using a single nucleic acid construct. Such a single construct can be designed with a single promoter sequence, which can transcribe a polycistronic message RNA including the nucleic acid molecules to be expressed. To enable co-translation of multiple polynucleotide sequences, the polynucleotide sequences can be inter-linked via an internal ribosome entry site (IRES) sequence which facilitates translation of polynucleotide sequences positioned downstream of the IRES sequence. In this case, a transcribed polycistronic RNA molecule can be translated from both the capped 5' end and the internal IRES sequence of the polycistronic RNA molecule to thereby express each nucleic acid molecule.

In some examples, the two or more nucleic acid sequences to be expressed are translationally fused via a protease recognition site cleavable by a protease expressed by the cell to be transformed. In this case, a chimeric polypeptide translated will be cleaved by a cell-expressed protease to thereby generate the plurality of polypeptides.

In other examples, a nucleic acid construct includes multiple promoter sequences each capable of directing transcription of a specific polynucleotide sequence.

Suitable promoters which can be used include constitutive, inducible, or tissue-specific promoters.

Exemplary constitutive promoters include, for example, CaMV 35S promoter (Odell et al., Nature 313:810-812, 1985); maize Ubi 1 (Christensen et al., Plant Sol. Biol. 18:675-689, 1992); rice actin (McElroy et al., Plant Cell 2: 163-171, 1990); pEMU (Last et al., Theor. Appl. Genet. 81:581-588, 1991); and Synthetic Super MAS (Ni et al., The Plant Journal 7: 661-76, 1995). Other constitutive promoters include those in U.S. Pat. Nos. 5,659,026, 5,608,149; 5,608, 144; 5,604,121 ; 5,569,597: 5,466,785; 5,399,680; 5,268,463; and 5,608,142.

Suitable inducible promoters can be pathogen-inducible promoters such as, for example, the alfalfa PR10 promoter (Coutos-Thevenot et al., Journal of Experimental Botany 52: 901-910, 2001 and the promoters described by Marineau et al., Plant Mol. Biol. 9:335-342, 1987; Matton et al. Molecular Plant- Microbe Interactions 2:325-331, 1989; Somsisch et al., Proc. Natl. Acad. Sci. USA 83:2427-2430, 1986: Somsisch et al., Mol. Gen. Genet. 2:93-98, 1988; and Yang, Proc. Natl. Acad. Sci. USA 93:14972-14977, 1996.

Suitable tissue-specific promoters include, but not limited to, leaf-specific promoters such as described, for example, by Yamamoto et aL, Plant J. 12:255-265, 1997; Kwon et al., Plant Physiol. 105:357- 67, 1994; Yamamoto et al., Plant Cell Physiol. 35:773-778, 1994; Gotor et al., Plant J. 3:509-18, 1993; Orozco et al., Plant Mol. Biol. 23: 1129-1138, 1993; and Matsuoka et al., Proc. Natl. Acad. Sci. USA 90:9586-9590, 1993.

A nucleic acid construct can also include at least one selectable marker such as nptll. In one example, the nucleic acid construct is a shuttle vector, which can propagate both in E. coll (wherein the construct comprises an appropriate selectable marker and origin of replication) and be compatible for propagation in cells. In some examples, a construct can be, for example, a plasmid, a bacmid, a phagemid, a cosmid, a phage, a virus or an artificial chromosome.

Following transformation, the transformed cells can be micropropagated to provide a rapid, consistent reproduction of the transformed material. Micropropagation is a process of growing new generation plants from a single piece of tissue that has been excised from a selected parent plant or cultivar. This process permits the mass reproduction of plants having the preferred tissue expressing the fusion protein. The new generation plants which are produced are genetically identical to, and have all of the characteristics of, the original plant. Micropropagation allows mass production of quality plant material in a short period of time and offers a rapid multiplication of selected cultivars in the preservation of the characteristics of the original transgenic or transformed plant. The advantages of cloning plants are the speed of plant multiplication and the quality and uniformity of plants produced.

Micropropagation is a multi-stage procedure that utilizes alteration of culture medium or growth conditions between stages. The micropropagation process involves four basic stages: stage one, initial tissue culturing; stage two, tissue culture multiplication; stage three, differentiation and plant formation; and stage four, greenhouse culturing and hardening. During stage one, initial tissue culturing, the tissue culture is established and certified contaminant-free. During stage two, the initial tissue culture is multiplied until a sufficient number of tissue samples are produced to meet production goals. During stage three, the tissue samples grown in stage two are divided and grown into individual plantlets. At stage four, the transformed plantlets are transferred to a greenhouse for hardening where the plants’ tolerance to light is gradually increased so that it can be grown in the natural environment.

To introduce a nucleic acid to be expressed by conventional methods requires a sexual cross between two lines, and then repeated back-crossing between hybrid offspring and one of the parents until a plant with the desired characteristics is obtained. This process, however, is restricted to plants that can sexually hybridize, and genes in addition to the desired gene will be transferred.

Recombinant DNA techniques circumvent these limitations by enabling introduction of specific genes for desirable traits to plants. Once the foreign genes have been introduced into a plant (such as a CYP81 A gene), that plant can then be used in plant breeding schemes (e.g., pedigree breeding, single-seed- descent breeding schemes, reciprocal recurrent selection) to produce progeny which also contain the gene of interest.

Genes can be introduced in a site directed fashion using homologous recombination. Homologous recombination permits site-specific modifications in endogenous genes and thus inherited or acquired mutations may be corrected, and/or novel alterations may be engineered into the genome. Homologous recombination and site-directed integration in plants are discussed in, for example, U.S. Patent Nos. 5,451,513; 5,501,967 and 5,527,695.

Integration of an exogenous nucleic acid molecule in the genome of the transformed plants can be determined using standard molecular biology techniques, such as PCR and Southern blot hybridization.

In some examples the transformation is stable. In some examples the transformation is transient.

In one example, transformation is by viral infection. Viruses useful for the transformation of plant hosts include CaMV, TMV and BV. Transformation of plants using plant viruses is described in U.S. Pat. No. 4,855,237 (BGV), EP-A 67,553 (TMV), Japanese Published Application No. 63-14693 (TMV), EPA 194,809 (BV), EPA 278,667 (BV); and Gluzman et al. (Communications in Molecular Biology: Viral Vectors, Cold Spring Harbor Laboratory, New York, pp. 172-189, 1988). Pseudovirus particles for use in expressing an exogenous nucleic acid molecule in many hosts, including plants, is described in WO 87/06261.

Suitable modifications can be made to a DNA virus. Alternatively, the virus can first be cloned into a bacterial plasmid for ease of constructing the desired viral vector with the exogenous nucleic acid molecule. The virus can then be excised from the plasmid. If the virus is a DNA virus, a bacterial origin of replication can be attached to the viral DNA, which is then replicated by the bacteria. Transcription and translation of this DNA will produce the coat protein which will encapsidate the viral DNA.

If the virus is an RNA virus, the virus is generally cloned as a cDNA and inserted into a plasmid. The plasmid is then used to make all of the constructions. The RNA virus is then produced by transcribing the viral sequence of the plasmid and translation of the viral genes to produce the coat protein(s) which encapsidate the viral RNA.

In one embodiment, a plant viral nucleic acid is provided in which the native coat protein coding sequence has been deleted from a viral nucleic acid, a non-native plant viral coat protein coding sequence and a non-native promoter, such as the subgenomic promoter of the non-native coat protein coding sequence, capable of expression in the plant host, packaging of the recombinant plant viral nucleic acid, and ensuring a systemic infection of the host by the recombinant plant viral nucleic acid, has been inserted. Alternatively, the coat protein gene may be inactivated by insertion of the exogenous nucleic acid molecule within it, such that a product is produced. The recombinant plant viral nucleic acid may contain one or more additional non-native subgenomic promoters. Each non-native subgenomic promoter can transcribe or express adjacent genes or nucleic acid sequences in the plant host and incapable of recombination with each other and with native subgenomic promoters. Exogenous nucleic acid molecules can be inserted adjacent the native plant viral subgenomic promoter or the native and a non-native plant viral subgenomic promoters if more than one nucleic acid sequence is included. The exogenous nucleic acid sequences are transcribed or expressed in the host plant under control of the subgenomic promoter to produce the desired products.

In some examples, the native coat protein coding sequence is placed adjacent one of the non-native coat protein subgenomic promoters instead of a non-native coat protein coding sequence.

In some examples, a recombinant plant viral nucleic acid is provided in which the native coat protein gene is adjacent its subgenomic promoter and one or more non-native subgenomic promoters have been inserted into the viral nucleic acid. The inserted non-native subgenomic promoters are capable of transcribing or expressing adj cent genes in a plant host and are incapable of recombination with each other and with native subgenomic promoters. Exogenous nucleic acid molecules can be inserted adjacent the non- native subgenomic plant viral promoters such that the sequences are transcribed or expressed in the host plant under control of the subgenomic promoters to produce the desired product.

In some examples, a recombinant plant viral nucleic acid is provided in which the native coat protein coding sequence is replaced by a non-native coat protein coding sequence.

The viral vectors can be encapsidated by the coat proteins encoded by the recombinant plant viral nucleic acid to produce a recombinant plant virus. The recombinant plant viral nucleic acid or recombinant plant virus can be used to infect appropriate host plants. The recombinant plant viral nucleic acid can be capable of replication in the host, systemic spread in the host, and transcription or expression of foreign gene(s) (isolated nucleic acid) in the host to produce the desired product.

In some examples, the exogenous nucleic acid sequences can also be introduced into a chloroplast genome thereby enabling chloroplast expression.

In one example, gene edited plants are generated using Agrobacterium-mediated transformation, which stably integrates a single copy of an exogenous nucleic acid into plant genomes (e.g., see Deschamps and Simon, Plant Cell Rep., 2002. 21:359-364; Phippen and Simon, Cell. Dev. BioL, 2000. 36: 250-4) to produce gene-edited plants. Seeds can be germinated and the leaf tissues taken as explant for Agrobacterium inoculation for 30 min. The EHA 105 strain of Agrobacterium can be transformed with a CRISPR-editing vector. The infected plant tissues can be cultured on MS medium supplemented with Gamborg vitamins, 3% sucrose and 16.8 pM thidiazuron (TDZ) for 3 days, after which plant tissues can be transferred to the same medium containing 300 pg/ml cefotaxime to inhibit the further growth of Agrobacterium and 50 pg/ ml kanamycin to select transformed tissues and regenerate transgenic shoots. Regenerated transgenic shoots can be transferred onto MS medium without TDZ but containing 25 pg/ml kanamycin and 1 pg/1 indole-3-butyric acid (IBA) to induce root formation. Fully regenerated transgenic plantlets can be transferred to soil and allowed to produce seeds. To transgenic plants can be examined for the integration of the transgenes by PCR analysis and the mutation of genes, such as CYP81 A-5A, 5B, and/or 5D genes.

E. Exemplary Methods of Screening Gene- Edited or Transgenic Plants

Gene-edited and transgenic plants generated using the provided methods can be screened to identify or confirm the presence of a mutation or gene introduced.

PCR primers can be used to amplify a genomic DNA segment spanning the selected target sites. Restriction enzyme digestion can be carried out on the PCR products. In some examples, restriction enzyme sites are included at the target sites (before editing occurs), and undigested PCR products in the presence of the restriction enzyme can thus indicate a gene-edited plant. The undigested PCR fragments can also be sequenced to confirm the presence and nature of any mutations or added sequences. RFLP methods can be used to screening large numbers of candidate mutant plants.

A T7E1 assay can be used to screen regenerated mutant plants. This assay allows mutated, edited sites to be detected based on their incomplete hybridization to the WT sequence (due to a mismatch between the WT and edited hybridized DNA strands at the edited site). PCR fragments spanning the mutation sites can be denatured at 95 °C and cooled down to 22 °C slowly using a thermal cycler. Annealed PCR products can be incubated with T7 endonuclease 1 (NEB) at 37°C for 20 min and analyzed by electrophoresis in a 1- 2% agarose gel.

A TaqMan probe-based qPCR analysis can be used. TaqMan probes can be designed for each of the WT target sites and synthesized with fluorescence labeling on the 5’ end and minor groove binder- nonfluorescent quencher (e.g., MGB-NFQ) on the 3' end. In qPCR analysis, the biallelic mutant will not produce any fluorescent signal, while the WT plant will produce double the signal compared to the monoallelic mutant (e.g., see Li et ah, Plant PhysioL, 2015. 169(2): 960-70). This TaqMan-qPCR method in the 96-well format used by the StepOnePlus qPCR System (Applied Biosystems) can be used to screen a large number of regenerated plants, produced by the gene gun bombardment with RNP complexes. This method generates gene edited plants that do not carry selectable marker genes.

Mutations from biallelic To mutants are expected to be inherited in the next generations. For transgenic mutant plants produced by Agrobacterium-mediated transformation, gene-specific PCR assays can be used to screen for T i plants that have segregated out the Cas9 and Kan^R genes. The monoallelic To mutants are expected to segregate according to the Mendelian law with a 1:2: 1 ratio.

V. Exemplary Breeding Methods

In one example, open-pollinated methods are used for crops such as rye, many maizes and sugar beets, herbage grasses, legumes such as alfalfa and clover, and tropical tree crops such as cacao, coconuts, oil palm and some rubber.

Population improvement methods fall into two groups, those based on purely phenotypic selection, normally called mass selection, and those based on selection with progeny testing. Interpopulation improvement utilizes the concept of open breeding populations; allowing genes for flow from one population to another. Plants in one population (cultivar, strain, ecotype, or any germplasm source) are crossed either naturally (e.g., by wind) or by hand or by bees (commonly Apis mellifera L. or Megachile rotundata F.) with plants from other populations. Selection is applied to improve one (or sometimes both) population(s) by isolating plants with desirable traits from both sources.

In one example, a population is changed en masse using a selection procedure. The outcome is an improved population that is indefinitely propagable by random-mating within itself in isolation. Second, the synthetic variety attains the same end result as population improvement but is not itself propagable as such; it has to be reconstructed from parental lines or clones These plant breeding procedures for improving open- pollinated populations are known and comprehensive reviews of breeding procedures routinely used for improving cross-pollinated plants are provided in numerous texts and articles, including: Allard, Principles of Plant Breeding, John Wiley & Sons, Inc. (1960); Simmonds, Principles of Crop Improvement, Longman Group Limited (1979); Hallauer and Miranda, Quantitative Genetics in Maize Breeding, Iowa State University Press (1981); and, Jensen, Plant Breeding Methodology, John Wiley & Sons, Inc. (1988). For population improvement methods specific for soybean see, e.g., J.R. Wilcox, editor (1987) SOYBEANS: Improvement, Production, and Uses, Second Edition, American Society of Agronomy, Inc., Crop Science Society of America, Inc., and Soil Science Society of America, Inc., publishers, 888 pages.

In one example, mass selection methods are used. In mass selection, desirable individual plants are chosen, harvested, and the seed composited without progeny testing to produce the following generation. Since selection is based on the maternal parent only, and there is no control over pollination, mass selection amounts to a form of random mating with selection. The purpose of mass selection is to increase the proportion of superior genotypes in the population.

In one example, a synthetic variety is produced by crossing inter se a number of genotypes selected for good combining ability in all possible hybrid combinations, with subsequent maintenance of the variety by open pollination. Whether parents are (more or less inbred) seed-propagated lines, as in some sugar beet and beans (Vicia) or clones, as in herbage grasses, clovers and alfalfa, makes no difference in principle. Parents are selected on general combining ability, sometimes by test crosses or topcrosses, more generally by polycrosses. Parental seed lines may be deliberately inbred (e.g. by selfing or sib crossing). However, even if the parents are not deliberately inbred, selection within lines during line maintenance ensure that some inbreeding occurs. Clonal parents will, of course, remain unchanged and highly heterozygous.

Whether a synthetic can go straight from the parental seed production plot to the farmer or first undergoes one or two cycles of multiplication depends on seed production and the scale of demand for seed. Generally, grasses and clovers are generally multiplied once or twice and are thus considerably removed from the original synthetic.

In some examples, progeny testing is used for polycrosses, because of their operational simplicity and relevance to the objective, namely exploitation of general combining ability in a synthetic.

The number of parental lines or clones that enters a synthetic can vary. In some examples, numbers of parental lines range from 10 to several hundred, with 100-200 being the average. Broad based synthetics formed from 100 or more clones can be more stable during seed multiplication than narrow based synthetics.

In some examples, hybrids are generated. A hybrid is an individual plant resulting from a cross between parents of differing genotypes. Commercial hybrids are used in many crops, including corn (maize), sorghum, sugar beet, sunflower and broccoli. Hybrids can be formed, for example by crossing two parents directly (single cross hybrids), by crossing a single cross hybrid with another parent (three-way or triple cross hybrids), or by crossing two different hybrids (four-way or double cross hybrids).

Most individuals in an out breeding (i.e., open-pollinated) population are hybrids, but the term is usually reserved for cases in which the parents are individuals whose genomes are sufficiently distinct for them to be recognized as different species or subspecies. Hybrids may be fertile or sterile depending on qualitative and/or quantitative differences in the genomes of the two parents. Heterosis, or hybrid vigor, is usually associated with increased heterozygosity that results in increased vigor of growth, survival, and fertility of hybrids as compared with the parental lines that were used to form the hybrid. Maximum heterosis is usually achieved by crossing two genetically different, highly inbred lines.

The production of hybrids can include the isolated production of both the parental lines and the hybrids which result from crossing those lines. For a detailed discussion of the hybrid production process, see, e.g., Wright, Commercial Hybrid Seed Production 8: 161-176, In Hybridization of Crop Plants.

In some examples, bulk segregation analysis (BSA) is used. BSA, a.k.a. bulked segregation analysis, or bulk segregant analysis, is described by Michelmore et al. (Michelmore et al., 1991, Proceedings of the National Academy of Sciences, USA, 99:9828-9832) and Quarrie et al. (Quarrie et al., 1999, Journal of Experimental Botany, 50(337): 1299-1306). For BSA of a trait of interest, parental lines with certain different phenotypes are chosen and crossed to generate F2, doubled haploid or recombinant inbred populations with QTE analysis. The population is then phenotyped to identify individual plants or lines having high or low expression of the trait. Two DNA bulks are prepared, one from the individuals having one phenotype (e.g., increased phellem size, periderm size, and/or suberin production), and the other from the individuals having reversed phenotype (e.g., average or decreased phellem size, periderm size, and/or suberin production), and analyzed for allele frequency with molecular markers. Only a few individuals are required in each bulk (e.g., 10 plants each) if the markers are dominant (e.g., RAPDs). More individuals are needed when markers are co-dominant (e.g., RFLPs). Markers linked to the phenotype can be identified and used for breeding or QTE mapping.

In some examples, gene pyramiding is used to combine into a single genotype a series of target genes identified in different parents. The first part of a gene pyramiding breeding is called a pedigree and is aimed at cumulating one copy of all target genes in a single genotype (called root genotype). The second part is called the fixation steps and is aimed at fixing the target genes into a homozygous state, that is, to derive the ideal genotype (ideotype) from the root genotype. Gene pyramiding can be combined with marker assisted selection (MAS) or marker based recurrent selection (MBRS).

VI. Exemplary Plants for Use with the Disclosed Methods

The present disclosure teaches plants transformed with a plant transformation construct or vector, and gene-edited and transgenic plants. The methods for creating such plants can be used to confer desired traits on essentially any plant. A wide variety of plants and plant cell systems may be engineered for the desired physiological and agronomic characteristics described herein using the nucleic acid constructs of the present disclosure and the various transformation methods. In some examples, the plant for the transformation is a monocotyledonous plant (monocot) or a dicotyledonous plant (dicot).

Monocots are flowering plants having embryos with one cotyledon or seed leaf, parallel leaf veins, and flower parts in multiples of three. Examples of monocots that can be used for transformation or geneediting include, but are not limited to turfgrass, corn/maize, rice, oat, annual ryegrass, wheat, barley, sorghum, orchid, iris, lily, onion, and palm. Examples of turfgrass include, but are not limited to Agrostis spp. (bentgrass species including colonial bentgrass and creeping bentgrasses), Poa pratensis (Kentucky bluegrass), Lolium spp. (ryegrass species including annual ryegrass and perennial ryegrass), Festuca arundinacea (tall fescue) Festuca rubra commutata (Chewings fescue), Cynodon dactylon (bermudagrass), Pennisetum clandestinum (kikuyu grass), Stenotaphrum secundatum (St. Augustine grass), Zoysia japonica (zoysia grass), and Dichondra micrantha.

Other exemplary plants that can be used for transformation or gene-editing include, but are not limited to angiosperm and gymnosperm plants such as acacia, alfalfa, amaranth, apple, apricot, artichoke, ash tree, asparagus, avocado, banana, barley, beans, beet, birch, beech, blackberry, black raspberry, blueberry, broccoli, Brussel's sprouts, cabbage, cane berry, canola, cantaloupe, carrot, cassava, cauliflower, cedar, a cereal, celery, chestnut, cherry, Chinese cabbage, citrus, Clementine, clover, coffee, corn, cotton, cowpea, cucumber, cypress, eggplant, elm, endive, eucalyptus, fennel, figs, fir, geranium, grape, grapefruit, groundnuts, ground cherry, gum hemlock, hickory, kale, kiwifruit, kohlrabi, larch, lettuce, leek, lemon, lime, locust, pine, maidenhair, maize, mango, maple, melon, millet, mushroom, mustard, nuts, oak, oats, oil palm, okra, onion, orange, an ornamental plant or flower or tree, papaya, palm, parsley, parsnip, pea, peach, peanut, pear, peat, pepper, persimmon, pigeon pea, peach, pine, pineapple, plantain, plum, pomegranate, potato, pumpkin, radicchio, radish, rapeseed, raspberry, rice, rye, sorghum, safflower, sallow, soybean, spinach, spruce, squash, strawberry, sugar beet, sugarcane, sunflower, sweet potato, sweet corn, tangerine, tea, tobacco, tomato, trees, triticale, turf grasses, turnips, vine, walnut, watercress, watermelon, wheat, wild strawberry, yams, yew, and zucchini.

In some aspects, plants and plant cells for transformation or gene -editing include, but are not limited to, those monocotyledonous and dicotyledonous plants, such as crops including grain crops (e.g., wheat, maize, rice, millet, barley), fruit crops (e.g., tomato, apple, grape, peach, pear, plum, raspberry, black raspberry, blackberry, cane berry, cherry, avocado, strawberry, wild strawberry, orange), forage crops (e.g., alfalfa), root vegetable crops (e.g., carrot, potato, sugar beets, yam), leafy vegetable crops (e.g., lettuce, spinach); flowering plants (e.g., petunia, rose, chrysanthemum), conifers and pine trees (e.g., pine fir, spruce); plants used in phytoremediation (e.g., heavy metal accumulating plants); oil crops (e.g., sunflower, rape seed) and plants used for experimental purposes (e.g., Arabidopsis). In some embodiments, fruit crops such as tomato, apple, peach, pear, plum, raspberry, black raspberry, blackberry, cane berry, cherry, avocado, strawberry, wild strawberry, grape and orange.

In some aspects, plants and plant cells for transformation, genetic engineering or gene-editing include, but are not limited to, Canola Brassica napus), Soybean (Glycine max), Cotton Gossypium hirsutum), Rice (Oryza sativa), Lotus (Lotus japonicus), Radish (Raphanus sativus), Setaria (Setaria italica), Sorghum (Sorghum bicolor), Pennycress (Thlaspi arvense), Southern cattail (Typha domingensis), Wheat (Triticum aestivum), and Maize (Zea mays).

In some aspects, the plant, plant part, or plant cell for transformation, genetic engineering or geneediting is a dicot. In some examples, the plant, plant part, or plant cell is a species selected from Arabidopsis genus, Brassica genus, Glycine genus, Gossypium genus, Raphanus genus, and Thlaspi genus. In some aspects, the plant, plant part, or plant cell for transformation, genetic engineering or gene-editing is a monocot. In some examples, the plant, plant part, or plant cell is a species selected from Typha genus, Triticum genus, Hordeum genus, Avena genus, Oryza genus, Setaria genus, Sorghum genus, and Zea genus.

In some aspects, the plant, plant part, or plant cell is Arabidopsis thaliana.

In some aspects, the plant, plant part, or plant cell is from Triticum genus, such as 7. aestivum, T. aethiopicum, T. araraticum, T. boeoticum, T. carthlicum, T. compactum, T. dicoccoides, T. dicoccon, T. durum, T. ispahanicum, T. karamyschevii, T. monococcum, T. polonicum, T. spelta, T. thaoudar, T. timopheevii, T. turanicum, T. turgidum, T. urartu, T. vavilovii, or T. zhukovskyi. In some aspects, the plant, plant part, or plant cell is from Oryza genus, such as Oryza australiensis, Oryza barthii, Oryza brachyantha, Oryza coarctata, Oryza eichingeri, Oryza glaberrima, Oryza grandiglumis, Oryza latifolia, Oryza longiglumis, Oryza longistaminata, Oryza meyeriana, Oryza minuta, Oryza neocaledonica, Ory a officinalis, Oryza punctata, Oryza ridleyi, Oryza rufipogon, Oryza sativa, or Oryza schlechteri.

In some aspects, the plant, plant part, or plant cell is from Hordeum genus, such as Hordeum aegiceras, Hordeum arizonicum, Hordeum bogdanii, Hordeum brachyantherum, Hordeum brachyatherum, Hordeum brevisubulatum, Hordeum bulbosum, Hordeum californicum, Hordeum capense, Hordeum chilense, Hordeum comosum, Hordeum cordobense, Hordeum depressum, Hordeum distichon, Hordeum erectifolium, Hordeum euclaston, Hordeum flexuosum, Hordeum fuegianum, Hordeum guatemalense, Hordeum halophilum, Hordeum intercedens, Hordeum jubatum, Hordeum x lagunculciforme, Hordeum lechleri, Hordeum marinum, Hordeum murinum, Hordeum muticum, Hordeum parodii, Hordeum patagonicum, Hordeum x pavisii, Hordeum procerum, Hordeum pubiflorum, Hordeum pusilium, Hordeum roshevitzii, Hordeum secalinum, Hordeum spontaneum, Hordeum stenostachys, Hordeum tetraploidum, Hordeum vulgare.

In some aspects, the plant, plant part, or plant cell is from Zea genus, such as Zea diploperennis, Zea luxurious, Zea mays L., Zea nicaraguensis, or Zea perennis.

In some aspects, the plant, plant part, or plant cell is from Avena genus, such as Avena sativa, Avena abyssinica, Avena byzantina, Avena nuda, Avena strigosa, Avena aemulans, Avena barbata, Avena brevis, Avena chinensis, Avena clauda, Avena eriantha, Avena fatua, Avena longiglumis, Avena maroccana, Avena murphyi, Avena prostrata, Avena saxatilis, Avena sterilis, Avena vaviloviana, Avena ventricosa, or Avena volgensis.

In some aspects, the plant, plant part, or plant cell is from the Brassica genus, such as Brassica balearica (Mallorca cabbage), Brassica carinata (Abyssinian mustard or Abyssinian cabbage), Brassica elongata (elongated mustard), Brassica fruticulosa (Mediterranean cabbage), Brassica hilarionis (St. Hilarion cabbage), Brassica juncea (Indian mustard, brown and leaf mustards, Sarepta mustard), Brassica napus (rapeseed, canola, rutabaga, Siberian kale), Brassica narinosa (broadbeaked mustard), Brassica nigra (black mustard), Brassica oleracea (kale, cabbage, collard greens, broccoli, cauliflower, kai-lan, brussels sprouts, kohlrabi), Brassica perviridis (tender green, mustard spinach), Brassica rapa (Chinese cabbage, turnip, rapini, komatsuna), Brassica rupestris, Brassica spinescens, or Brassica tournefortii (Asian mustard).

In some aspects, the plant, plant part, or plant cell is from the Thlaspi genus, such as Thlaspi alliaceum (roadside penny-cress), Thlaspi arcticum (arctic penny-cress), Thlaspi arvense (field penny-cress), Thlaspi caerulescens (alpine penny-cress), Thlaspi californicum (Kneeland Prairie penny-cress), Thlaspi cyprium (Cyprus penny-cress), Thlaspi fendleri (Fendler's penny-cress), Thlaspi idahoense (Idaho pennycress), Thlaspi jankae (Slovak penny-cress), Thlaspi montanum (alpine penny-cress), Thlaspi parviflorum (meadow penny-cress), Thlaspi perfoliatum (Cotswold penny-cress), Thlaspi praecox (early penny-cress), or Thlaspi rotundifolium (round-leaved penny-cress). In some aspects, the plant, plant part, or plant cell is from the Glycine genus, such as Glycine albicans, Glycine aphyonota, Glycine arenaria, Glycine argyria, Glycine canescens, Glycine clandestine, Glycine curvata, Glycine cyrtoloba, Glycine falcata, Glycine gracei, Glycine hirticaulis, Glycine hirticaulis subsp., Glycine lactovirens, Glycine latifolia, Glycine latrobeana, Glycine microphylla, Glycine montis- douglas, Glycine peratosa, Glycine pescadrensis, Glycine pindanica, Glycine pullenii, Glycine remota, Glycine rubiginosa, Glycine stenophita, Glycine syndetika, Glycine tabacina, Glycine tonientella, Glycine soja, or Glycine max.

VII. Transformed or Modified Plants

Provided are modified plants, including plant cells, plant parts, etc., generated by the methods disclosed herein.

In some aspects, the plants have increased tolerance to a herbicide. In some examples, the herbicide is an ALS inhibitor, such as sulfonylamino carbonyl triazolinones, such as propoxycarbazone-sodium (PROP) or a derivative or analog thereof. In some aspects, a transformed plant tissue is produced from a transformed plant cell. In some aspects, a transformed plantlet is produced from a transformed plant tissue and wherein the transformed plantlet has increased tolerance to a herbicide as compared to an untransformed control plantlet. In some aspects, a progeny of the transformed plantlet is produced and wherein the progeny has increased tolerance to a herbicide as compared to an untransformed control plantlet. In some aspects, the transformed plantlet or the progeny of the transformed plantlet is grown into a mature transformed plant, and wherein the mature transformed plant has increased tolerance to a herbicide as compared to a mature untransformed control plant. In some aspects, the mature transformed plant or clone of the mature transformed plant is used in a breeding method taught herein.

In some aspects, the transformed plants of the present disclosure has at least about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 200%, increase in tolerance to a herbicide as compared to an untransformed control plant. In some examples, the herbicide is an ALS inhibitor, such as sulfonylamino carbonyl triazolinones, such as propoxycarbazone-sodium (PROP) or a derivative or analog thereof.

In some aspects, transformed plants, plant parts, or plant cells include one or more modified endogenous target genes, wherein the one or more modifications result in an enhanced expression of one or more of the target genes, and/or enhanced activity of one or more proteins encoded by the target genes (the target proteins), compared to the expression/activity of a corresponding gene/protein in an unmodified plant, plant part, or plant cell. For example, in some examples, a modified plant, plant part, or plant cell demonstrates enhanced expression of a target gene, and/or activity of a target protein. In some examples, the expression of the gene or activity of the protein (such as CYP81A-5A, 5B, or 5D) in a modified plant, plant part, or plant cell is enhanced by at least 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400% or higher compared to the expression of a corresponding gene/protein in an unmodified plant, plant part, or plant cell.

In some examples, the modified endogenous protein demonstrates enhanced binding affinity to another protein expressed by the modified plant cell or by another cell; enhanced signaling capacity; enhanced enzymatic activity; enhanced DNA-binding activity with respect to a specific DNA sequence; or enhanced ability to function as a scaffolding protein.

In some examples, the modified plants, plant parts, or plant cells described herein comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or more modified endogenous target genes encoding 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or more modified endogenous proteins, such as a CYP81A gene or protein, or regulators thereof.

In some aspects, transformed plants, plant parts, or plant cells include one or more exogenous genes, optionally integrated into the genome. In some examples, the exogenous gene is one or more of CYP81A- 5A, 5B and 5D genes, which endow or increase tolerance to a herbicide to the transformed plants.

In some examples, a transformed plant, plant part, plant cell, or seed, comprising one or more gain- of-function mutations in one or more endogenous herbicide detoxification genes, or comprising one or more exogenous herbicide detoxification genes integrated into a genome of the transformed plant, plant part, plant cell, or seed, wherein the one or more herbicide detoxification genes comprise: any of SEQ ID NOs: 1-7 (CYP81A-5A) or a nucleic acid sequence that comprises at least 80% identity to any of SEQ ID NOs: 1 -7; any of SEQ ID NOs: 8-13 (CYP81A-5B) or a nucleic acid sequence that comprises at least 80% identity to any of SEQ ID NOs: 8-13; and/or any of SEQ ID NOs: 14-19 (CYP81A-5D) or a nucleic acid sequence that comprises at least 80% identity to any of SEQ ID NOs: 14-19; or wherein the one or more herbicide detoxification genes encode a protein comprising a sequence of any of SEQ ID NOs: 99-104, or a sequence that comprises at least 80% identify to any of SEQ ID NOs: 99-104.

In some examples, the gain-of-function mutations increase binding affinity of the promoter region to a transcription factor or decrease binding affinity to a repressor. In some examples, the gain-of-function mutation is a single G insertion about 256 bp upstream of TATA box. In some examples, the single G insertion is immediately after a position corresponding to position 137 of SEQ ID NO: 1.

In some examples, the transformed plant, plant part, plant cell, or seed comprises any of SEQ ID NOs: 20-22, or a nucleic acid sequence that comprises at least 90% identity to any of SEQ ID NOs: 20-22 and has a G at a position corresponding to position 138 of SEQ ID NO: 20.

In some examples, the transformed plant has tolerance to a herbicide increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 75%, at least 90%, at least 100%, at least 200%, or at least 400% as compared to a control plant. In some examples, the herbicide is an ALS inhibitor, such as sulfonylamino carbonyl triazolinones, such as propoxycarbazone-sodium (PROP) or a derivative or analog thereof. In some examples, the tolerance to a herbicide is tolerance to a herbicide in presence of a herbicide safener. In some examples, the herbicide safener is cloquintocet-mexyl (CM) or a derivative or analog thereof. In some examples, the transformed plant, plant part, plant cell, or seed does not comprise a transgene used to generate the one or more gain-of-function mutations. In some examples, the transformed plant, plant part, plant cell, or seed is transgene free. In some examples, the transformed plant, plant part, plant cell, or seed comprises one or more transgenes.

OVERVIEW OF SEVERAL ASPECTS

Aspect 1. A method for generating a plant with increased tolerance to a herbicide, comprising: increasing expression of one or more herbicide detoxification genes, and/or increasing activity of one or more proteins encoded by the one or more herbicide detoxification genes in a plant, thereby generating the plant with increased tolerance to a herbicide, wherein the one or more herbicide detoxification genes comprise: any of SEQ ID NOs: 1-7 (CYP81A-5A) or a nucleic acid sequence that comprises at least 80% identity to any of SEQ ID NOs: 1-7; any of SEQ ID NOs: 8-13 (CYP81A-5B) or a nucleic acid sequence that comprises at least 80% identity to any of SEQ ID NOs: 8-13; and/or any of SEQ ID NOs: 14-19 (CYP8/A-5D) or a nucleic acid sequence that comprises at least 80% identity to any of SEQ ID NOs: 14-19; or wherein the one or more herbicide detoxification genes encode a protein comprising a sequence of any of SEQ ID NOs: 99-104, or a sequence that comprises at least 80% identify to any of SEQ ID NOs: 99-104.

Aspect 2. A method for generating a plant with increased tolerance to a herbicide, comprising: increasing expression of one or more herbicide detoxification genes, and/or increasing activity of one or more proteins encoded by the one or more herbicide detoxification genes in a plant cell or plant part, and growing the plant cell or plant part into a plant, thereby generating the plant with increased tolerance to a herbicide, wherein the one or more herbicide detoxification genes comprise: any of SEQ ID NOs: 1-7 CYP81A-5A) or a nucleic acid sequence that comprises at least 80% identity to any of SEQ ID NOs: 1-7; any of SEQ ID NOs: 8-13 (CYP81A-5B) or a nucleic acid sequence that comprises at least 80% identity to any of SEQ ID NOs: 8-13; and/or any of SEQ ID NOs: 14-19 (CYP81A-5D) or a nucleic acid sequence that comprises at least 80% identity to any of SEQ ID NOs: 14-19; or wherein the one or more herbicide detoxification genes encode a protein comprising a sequence of any of SEQ ID NOs: 99-104, or a sequence that comprises at least 80% identify to any of SEQ ID NOs: 99-104.

Aspect 3. The method of aspect 1 or 2, wherein the increasing expression of one or more herbicide detoxification genes, and/or increasing activity of one or more proteins encoded by the one or more herbicide detoxification genes comprises: introducing one or more exogenous nucleic acid molecules into the plant, thereby generating a transformed plant, or into the plant part or plant cell, thereby generating a transformed plant part or plant cell, wherein the one or more exogenous nucleic acid molecules comprise the one or more herbicide detoxification genes, increase expression of the one or more herbicide detoxification genes, and/or increase activity of the one or more proteins. Aspect 4. The method of aspect 3, wherein the one or more exogenous nucleic acid molecules generates one or more gain-of-function mutations in the one or more herbicide detoxification genes, wherein the one or more herbicide detoxification genes are endogenous to the plant, plant part, or plant cell.

Aspect 5. The method of aspect 4, wherein the one or more gain-of-function mutations are in the promoter region of the one or more herbicide detoxification genes, and increase expression of the one or more herbicide detoxification genes in response to a herbicide safener or herbicide.

Aspect 6. The method of aspect 5, wherein the herbicide safener is cloquintocet-mexyl (CM) or a derivative or analog thereof.

Aspect 7. The method of aspect 6, wherein the gain-of-function mutation is a single G insertion about 256 bp upstream of TATA box.

Aspect 8. The method of aspect 7, wherein the single G insertion is immediately after a position corresponding to position 137 of SEQ ID NO: 1.

Aspect 9. The method of any one of aspects 3-8, wherein the one or more exogenous nucleic acid molecules generate a mutated herbicide detoxification gene that comprises any of SEQ ID NOs: 20-22, or a nucleic acid sequence that comprises at least 90% identity to any of SEQ ID NOs: 20-22 and has a G at a position corresponding to position 138 of SEQ ID NO: 20.

Aspect 10. The method of any one of aspects 3-9, wherein the one or more exogenous nucleic acid molecules comprise one or more guide nucleic acid molecules that are complementary to one or more regions of the one or more herbicide detoxification genes, wherein the one or more herbicide detoxification genes are endogenous to the plant, plant part, or plant cell.

Aspect 11. The method of any one of aspects 3-10, wherein the one or more exogenous nucleic acid molecules further comprise a nucleic acid molecule encoding a Cas protein, or the method further comprises introducing one or more Cas proteins into the plant, plant part, or plant cell.

Aspect 12. The method of any one of aspects 3-11, wherein the one or more exogenous nucleic acid molecules are operably linked to a heterologous promoter.

Aspect 13. The method of any one of aspects 3-12, wherein the transformed plant, plant cell, or plant part comprises one or more gain-of-function mutations in the one or more herbicide detoxification genes, wherein the one or more herbicide detoxification genes are endogenous to the plant, plant part, or plant cell.

Aspect 14. The method of any one of aspects 3-13, wherein the transformed plant, plant part, or plant cell comprises the one or more exogenous nucleic acid molecules comprising the one or more herbicide detoxification genes, wherein the one or more exogenous herbicide detoxification genes are integrated or not integrated into a genome of the transformed plant, plant part, or plant cell.

Aspect 15. The method of aspect 14, wherein the plant, plant part, or plant cell does not comprise the one or more herbicide detoxification genes before introduction of the one or more exogenous nucleic acid molecules. Aspect 16. The method of any one of aspects 1-14, wherein the plant, plant part, or plant cell is from the Poaceae family.

Aspect 17. The method of aspect 16, wherein the Poaceae is a cereal grass.

Aspect 18. The method of aspect 17, wherein the cereal grass is wheat.

Aspect 19. The method of aspect 15, wherein the plant is a dicot, or the plant part or plant cell is from a dicot.

Aspect 20. The method of any one of aspects 1-19, wherein the expression of the one or more herbicide detoxification genes, and/or activity of the one or more proteins is increased as compared to a control plant, plant part, or plant cell.

Aspect 21. The method of any one of aspects 1-20, wherein the expression of the one or more herbicide detoxification genes, and/or activity of the one or more proteins is increased by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 120%, 150%, or 200% as compared to a control plant, plant part, or plant cell.

Aspect 22. The method of any one of aspects 1-21, wherein the tolerance to a herbicide is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 75%, at least 90%, at least 100%, at least 200%, or at least 400% as compared to a control plant.

Aspect 23. The method of any one of aspects 3-22, further comprising producing a transformed plant tissue from the transformed plant cell.

Aspect 24. The method of any one of aspects 2-23, further comprising producing a transformed plantlet from the transformed plant part or plant cell, or from the transformed plant tissue, wherein the transformed plantlet has increased tolerance to a herbicide.

Aspect 25. The method of aspect 24, further comprising producing a transformed progeny from the transformed plantlet, wherein the transformed progeny has increased tolerance to a herbicide.

Aspect 26. The method of aspect 24 or 25, further comprising growing the transformed plantlet or the transformed progeny into a transformed plant, wherein the transformed plant has increased tolerance to a herbicide.

Aspect 27. The method of any one of aspects 3-26, further comprising using the transformed plant or a clone of the transformed plant in a breeding method.

Aspect 28. The method of aspect 27, wherein the breeding method comprises selfing or crossing the transformed plant or clone of the transformed plant.

Aspect 29. A transformed plant, transformed plant part, or transformed plant cell made by the method of any one of aspects 3-28, or a transformed plant tissue made by the method of aspect 23, or a transformed plantlet made by the method of aspect 24, or a transformed progeny made by the method of aspect 25.

Aspect 30. The method of any one of aspects 1-28, or the transformed plant, transformed plant part, transformed plant cell, transformed plant tissue, transformed plantlet, or transformed progeny of aspect 29, wherein the transformed plant, transformed plant part, transformed plant cell, transformed plant tissue, transformed plantlet, or transformed progeny further comprises one or more additional exogenous nucleic acids encoding one or more proteins that confer upon the transformed plant, transformed plant part, transformed plant cell, transformed plant tissue, transformed plantlet, or transformed progeny a desired trait, wherein the desired trait is one or more of drought tolerance, heat tolerance, low or high soil pH level tolerance, salt tolerance, resistance to an insect, resistance to a bacterial disease, resistance to a viral disease, resistance to a fungal disease, resistance to a nematode, resistance to a pest, male sterility, abiotic stress tolerance, modified phosphorus characteristics, modified antioxidant characteristics, modified essential seed amino acid characteristics, decreased phytate, modified fatty acid metabolism, and modified carbohydrate metabolism.

Aspect 31. A method of producing a commodity plant product, comprising collecting or producing the commodity plant product from the transformed plant, transformed plant part, transformed plant cell, transformed plant tissue, transformed plantlet, or transformed progeny of any one of aspects 3-30, optionally, wherein the commodity plant product comprises a non-native nucleic acid molecule or protein from the transformed plant, transformed plant part, transformed plant cell, transformed plant tissue, transformed plantlet, or transformed progeny; and optionally, wherein the commodity product comprises a protein concentrate, protein isolate, leaves, extract, oil, bean, and/or seed.

Aspect 32. A method of producing plant seed, comprising crossing the transformed plant, transformed plant part, transformed plant cell, transformed plant tissue, transformed plantlet, or transformed progeny of any one of aspects 3-30 with itself or a second plant.

Aspect 33. The method of any one of aspects 2-28 and 30-32, or the transformed plant part of aspect 25 or 26, wherein the plant part is a protoplast, leaf, stem, root, root tips, anther, pistil, stamen, seed, embryo, pollen, ovule, microspore, sporophyte, gametophyte, cotyledon, hypocotyl, flower, shoot, tissue, petiole, or meristematic cell.

Aspect 34. A method for breeding a plant with increased tolerance to a herbicide, comprising crossing the transformed plant of aspect 29 with a second plant; obtaining seed from the crossing; planting the seeds and growing the seeds to progeny plants; and selecting from the progeny plants those with increased tolerance to a herbicide.

Aspect 35. The method of aspect 34, further comprising producing clones of the progeny plants, wherein the clones are selected based on increased tolerance to a herbicide.

Aspect 36. A seed that produces or is produced by the transformed plant of aspect 29, wherein the seed comprises one or more gain-of-function mutations in the one or more endogenous herbicide detoxification genes, and/or comprises the one or more exogenous herbicide detoxification genes.

Aspect 37. A transformed plant, plant part, plant cell, or seed, comprising one or more gain-of- function mutations in one or more endogenous herbicide detoxification genes, or comprising one or more exogenous herbicide detoxification genes integrated into a genome of the transformed plant, plant part, plant cell, or seed, wherein the one or more herbicide detoxification genes comprise: any of SEQ ID NOs: 1-7 (CYP81A-5A) or a nucleic acid sequence that comprises at least 80% identity to any of SEQ ID NOs: 1-7; any of SEQ ID NOs: 8-13 (CYP81A-5B) or a nucleic acid sequence that comprises at least 80% identity to any of SEQ ID NOs: 8-13; and/or any of SEQ ID NOs: 14-19 (CYP81A-5D) or a nucleic acid sequence that comprises at least 80% identity to any of SEQ ID NOs: 14-19; or wherein the one or more herbicide detoxification genes encode a protein comprising a sequence of any of SEQ ID NOs: 99-104, or a sequence that comprises at least 80% identify to any of SEQ ID NOs: 99-104.

Aspect 38. The transformed plant, plant part, plant cell, or seed of aspect 37, which does not comprise a transgene used to generate the one or more gain-of-function mutations.

Aspect 39. The transformed plant, plant part, plant cell, or seed of aspect 37 or 38, wherein the gain-of-function mutation is a single G insertion about 256 bp upstream of TATA box.

Aspect 40. The transformed plant, plant part, plant cell, or seed of aspect 39, wherein the single G insertion is immediately after a position corresponding to position 137 of SEQ ID NO: 1.

Aspect 41. The transformed plant, plant part, plant cell, or seed of any one of aspects 37-40, wherein the transformed plant, plant part, plant cell, or seed comprises any of SEQ ID NOs: 20-22, or a nucleic acid sequence that comprises at least 90% identity to any of SEQ ID NOs: 20-22 and has a G at a position corresponding to position 138 of SEQ ID NO: 20.

Aspect 42. The transformed plant, plant part, plant cell, or seed of any one of aspects 37-41, which is transgene-free.

Aspect 43. The transformed plant, plant part, plant cell, or seed of any one of aspects 37-41, which comprises one or more transgenes.

Aspect 44. The transformed plant, plant part, plant cell, or seed of any one of aspects 37-43, wherein the plant is a monocot or dicot.

Aspect 45. The transformed plant of any one of aspects 37-44, which has tolerance to a herbicide increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 75%, at least 90%, at least 100%, at least 200%, or at least 400% as compared to a control plant.

Aspect 46. The transformed plant of aspect 45, wherein the tolerance to a herbicide is tolerance to a herbicide in presence of a herbicide safener.

Aspect 47. The transformed plant of aspect 46, wherein the herbicide safener is cloquintocet-mexyl (CM) or a derivative or analog thereof.

Aspect 48. The method or transformed plant of any one of aspects 1-46, wherein the herbicide is propoxycarbazone-sodium (PROP) or a derivative or analog thereof.

Aspect 49. A method of growing the transformed plant, plant part, plant cell, or seed of any one of aspects 33-47 in the presence of a herbicide.

Aspect 50. The method of aspect 49, wherein the herbicide is an acetolactate synthase (ALS) inhibitor.

Aspect 51. The method of aspect 50, wherein the ALS inhibitor is a sulfonylamino carbonyl triazolinone. Aspect 52. The method of aspect 51, wherein the sulfonylamino carbonyl triazolinone is propoxycarbazone-sodium (PROP) or an analog or derivative thereof.

Aspect 53. The method of any one of aspects 49-52, further comprising growing the transformed plant, plant part, plant cell, or seed in the presence of a herbicide safener.

Aspect 54. The method of aspect 53, wherein the herbicide safener is cloquintocet-mexyl (CM) or a derivative or analog thereof.

EXAMPLES

The following examples are provided to illustrate particular features of certain aspects of the disclosure, but the scope of the claims should not be limited to those features exemplified.

Previous research utilizing wheat alien substitution and nullisomic-tetrasomic (NT) lines showed that allohexaploid bread wheat (Triticum aestivum L.) lacking chromosome 5A exhibited increased sensitivity to halauxifen-methyl (HM), indicating genes necessary for HM tolerance are located on this chromosome. It is hypothesized that these lines displayed reduced tolerance because they lacked genes encoding enzymes capable of detoxifying HA , the biologically active form of HM, to non-phytotoxic metabolites.

The present application first identifies the genes encoding the enzymes that catalyze reactions in the HA detoxification pathway, and provides methods for increasing expression of these genes, and/or levels and/or activities of proteins encoded by these genes to increase herbicide tolerance in a plant. These genes can also be utilized in plant transformation to endow HM tolerance in plant species lacking natural HM tolerance, such as soybeans, cotton, and other dicots. The encoded enzymes could potentially detoxify multiple herbicides, making these genes even more valuable in breeding programs aimed at improving herbicide tolerance in crops. Additionally, these genes can be utilized for in vitro metabolism assays with E. coli or yeast cells (Abdollahi et al., 2021; Dimaano et al., 2020) to screen and predict whole-plant tolerance to numerous experimental herbicides prior to performing whole-plant experiments.

Liquid chromatography-mass spectrometry (LC-MS) experiments were conducted to measure HA levels in the alien substitution and NT lines that previously displayed variable HM tolerance in greenhouse experimentation. These results show that wheat lines lacking chromosome 5A have a reduced ability to detoxify HA, while lines possessing wheat chromosome 5A maintain the ability to detoxify HA. Genes potentially involved in HA detoxification pathways were identified via RNA-Seq and gene-specific RT- qPCR, including candidate CYP81A genes located on chromosome 5A, 5B and 5D (denoted as CYP81A-5A, CYP81A-5B, and CYP81A-5D), CYP71C genes, and UDP-dependent glucosyltransferase (UGT) genes. CYP81A-5A, CYP81A-5B, and CYP81A-5D were shown to be CM inducible but not HM inducible. CRISPR-editing was used to create a mutation in the promoter region of CYP81A-5A. This mutant allele was inherited in a Mendelian fashion and had no effect on several developmental traits. Progeny homozygous for the mutant allele displayed an increase in expression following CM treatment and increased tolerance to herbicide treatment relative to unedited wheat. EXAMPLE 1 METABOLISM OF HALAUXFEN-METHYL IS REGULATED BY GENES LOCATED ON GROUP 5 CHROMOSOMES OF HEXAPLOID BREAD WHEAT (TRITICVM AESTIVUM L.) Previous research utilizing allohexaploid wheat (Triticum aestivum L.) alien substitution lines and nullisomic-tetrasomic (NT) lines indicated plants lacking chromosome 5A are more sensitive to the synthetic auxin herbicide, halauxifen-methyl (HM). Wheat de-esterifies HM to the biologically active form, halauxifen acid (HA), but wheat is naturally tolerant via rapid detoxification of HA to non-toxic metabolites. It is hypothesized that genes encoding HA-detoxifying enzymes are located on chromosome 5A and, as a result, lines lacking this chromosome display increased sensitivity due to reduced detoxification of HA. To directly test this hypothesis, two excised leaf assays with unlabeled HM were performed. The first assay utilized group 5 alien substitution lines (denoted as 5A, 5B and 5D), the unaltered wheat variety, ‘Chinese Spring’ (CS), and Aegilops searsii (AS), an HM-sensitive, diploid wheat relative used to make the substitution lines. The second excised leaf assay utilized NT accessions (denoted as N5A-T5D and N5D- T5A) and CS.

Metabolites were extracted from leaf tissue with 90% methanol and analyzed via LC-MS to measure the abundance of HA at 2, 8, 12 and 24 hours after treatment (HAT). The highest levels of HA were detected in AS at 8, 12, 24 HAT, with 8.5-, 7.4-, and 5.6-fold greater HA levels compared to CS at each respective time point.

Among group 5 alien substitution accessions, only the 5A alien substitution had significantly higher levels of HA at 8, 12 and 24 HAT relative to CS, which were 3.8-, 3.2-, and 2.4-fold higher than CS, respectively. These results show that AS and the 5A alien substitution accession had significantly reduced rates of HA detoxification relative to CS, whereas CS, 5B, and 5D rapidly detoxified HA.

Results for NT accessions showed that higher levels of HA were detected in the accession lacking 5A (N5A-T5D) at 12 and 24 HAT (2.7-fold increase at each timepoint relative to CS), while levels of HA detected in the accession tetrasomic for 5A (N5D-T5A) did not significantly differ from CS at each timepoint. Since only accessions possessing endogenous 5A genes (CS, N5D-T5A, and alien substitution accessions for 5B and 5D) rapidly detoxified HA.

Previous HM phenotyping experimentation with wheat alien substitution and nullisomic-tetrasomic (NT) indicated genes encoding these enzymes are located on the group 5 chromosomes of wheat, especially chromosome 5A (Obenland & Riechers, 2020). In summary, increased sensitivity to HM was displayed by wheat lines lacking chromosome 5A relative to unaltered ‘Chinese Spring’ (CS) (Figures 2A-2C); however, HM tolerance in wheat lines possessing 5A was comparable CS (Figures 2A-2C). It is hypothesized that the increased HM sensitivity in these plants is due to reduced detoxification of HA, which was the result of the loss of necessary genes on chromosome 5A encoding HA-detoxifying enzymes. However, metabolism of HA was not measured in the previous experiment (Obenland & Riechers, 2020). To test this hypothesis, HA abundance in CS, AS, NT lines, and the alien substitution lines utilized in previous experiments was measured via excised leaf assays and subsequent LC-MS analysis.

Materials and Methods

Chemicals and Plant Materials:

Analytical grade standards of HM and HA (95.9% and 99.5% pure, respectively) were provided by Corteva Agriscience (Indianapolis, IN, USA). All other analytical grade reagents were purchased through Fisher Scientific (Thermo-Fisher, Hanover Park, Illinois) or Sigma Chemical (Millipore Sigma, St. Louis, Missouri, USA). Seed for CS, alien substitution lines (5A, 5B, and 5D), and NT lines were acquired from the Kansas State Wheat Genetics Resource Center, and Aegilops searsii (2n = 2x = 14; SS; PI 599163; AS) seed was acquired from the National Small Grains Collection of the US Department of Agriculture/ Agricultural Research Service.

Sowing of Plants, Excised Leaf Assay, Collection of Tissue and Data Analysis:

To promote uniform germination, seeds were subjected to a cold treatment by placing them on water-soaked filter paper in Petri dishes in a 5°C cold room for three days. Seedlings were cultivated in a Conviron Gen 1000 growth chamber until seedlings produced 2-3 leaves (Zadoks stage 12-13) with conditions of 28/22°C day/night and a 16:8 h photoperiod. The LED lights of the growth chamber provided 550 pmol m^-2 s^-1 photon flux at the plant canopy level.

The excised leaf assay and metabolite extraction protocols utilized for our experiments are based on a modified protocol from previous studies (Concepcion et al., 2021; Lygin et al., 2018; Ma et al., 2015). For each experiment, the two oldest leaves were cut at the collar, washed with deionized water, and transferred to 15 niL plastic tubes containing 0.1 M Tris-Cl (pH = 6.0) and incubated in the growth chamber for an hour. After incubation, plants designated for HM treatment were transferred to tubes containing 300 pM HM and untreated plants were transferred to tubes containing 0.1 M Tris-Cl (pH = 6.0). At 2 hours after treatment (HAT), leaves were rinsed with deionized water, fresh weights were recorded (approximately 200 mg of fresh weight per sample), leaves were frozen in liquid nitrogen, and samples stored at -80 °C until metabolite extraction. Samples designated for later timepoints (8, 12, and 24 HAT) were rinsed with deionized water and transferred to tubes containing * strength Murashige and Skoog salt solution. These same harvesting methods were also utilized at the later timepoints.

Frozen tissue (approximately 0.15 g) was ground in liquid nitrogen with a mortar and pestle and compounds were extracted in 1 mL of 90% (v/v) methanol. After the first extraction and centrifugation at 12,000 xg, the supernatant was removed, and a second extraction was performed with the remaining plant material by adding 1 mL of 90% (v/v) methanol. After another centrifugation, the pellet was discarded, and the second supernatant was combined with the first supernatant, resulting in a final volume of 2 mL. Samples were dried and concentrated under nitrogen gas and reconstituted with 250 pL of water : acetonitrile (1 : 1, v/v) containing 0.1% formic acid. Quality control (QC) samples were then prepared by combining aliquots of each sample for injection throughout each experimental run (Dunn et aL, 2011). Standards for HM and HA were also prepared for sample submission. All samples were stored at -80°C until further analysis.

Samples were submitted to the Roy J. Carver Metabolomics Facility at the University of Illinois at Urbana-Champaign for analysis using previously described methods (Elolimy et al., 2019). Prior to analysis, all samples were spiked with 4-chloro-DL-PHE to serve as an internal standard. Samples were then analyzed with a Q-Exactive MS system (Thermo, Bremen, Germany), and LC separation was conducted with a Dionex Ultimate 3000 series HPLC equipped with a Phenomenex C18 column (4.6 xlOO mm, 2.6pm). Mobile phases consisted of A [H2O with 0.1% formic acid (v/v)] and B [acetonitrile with 0.1% formic acid (v/v)]. The flow rate was set at 0.25 mL min^-1 with a linear gradient starting at 100% A for 3 min. The gradient then transitioned to 100% B (20-30 min) and returned to 100% A (31-36 min). 20 pL of each sample was injected and the autosampler temperature was set at 15°C. Mass spectra were then acquired under both positive (sheath gas flow rate: 45; aux gas flow rate: 11; sweep gas flow rate: 2; spray volt-age: 3.5 kV; capillary temp: 250 °C; Aux gas heater temp: 415 °C) and negative electrospray ionization (sheath gas flow rate: 45; aux gas flow rate: 11; sweep gas flow rate: 2; spray voltage: -2.5 kV; capillary temp: 250 °C; Aux gas heater temp: 415 °C). The full scan mass spectrum resolution was set to 70,000 with a scan range of m/z, 67 ~ m/z, 1000, and the AGC target was 1 E6 with a maximum injection time of 200 ms. The chromatographic analysis was conducted in a randomized sequence order including QC samples injected at the beginning of the analysis to equilibrate the analytical platform and after every 10 test samples to evaluate the stability of the experimental procedure (Dunn et al., 2011; Godzien et al., 2015; Sangster et al., 2006; Wehrens et al., 2016). Raw data files obtained in full-MS mode (samples, procedural blank and QC) and data obtained in full-MS followed by data-dependent MS2 were processed using MS-DIAL v.4.9221218 (with open source publicly available El spectra library) following the parameters previously described (Concepcion et al., 2021; Tsugawa et al., 2015). Peak area of each metabolite feature was normalized based on QC samples and amount of internal standard. Normalized peak areas for HA and HM were then exported into a .csv file for further statistical analysis.

The first experiment included CS, AS, and three alien substitution lines for 5A, 5B, and 5D, and the second experiment included CS, N5A-T5D and N5D-T5A. This first experiment utilized seven biological replicates per treatment per timepoint and the second experiment utilized five biological replicates per treatment per timepoint. CS served as a positive control in both experiments since it is capable of rapid HA detoxification, while AS served as a negative control due to its sensitivity to HM (Obenland & Riechers, 2020). Comparisons of the peak areas of HA among populations and timepoints were performed with the lme4 package (Bates et al., 2015) in R (version 4.2.0) using RStudio (Version 2023.03.0). For both experiments a mixed effects model was utilized where population, timepoint and their interactions were treated as fixed effects and replicates were random effects. Data was subjected to ANOVA and means were separated with Fisher’s LSD (a = 0.05). Results

LC-MS Analysis of CS, AS, and Alien Substitution Lines

When measuring HA abundance, CS and AS exhibited anticipated results: HA levels in CS remained relatively low throughout the entirety of the time course, while HA levels in AS increased over time and significantly differed from CS at 8, 12, and 24 HAT (Figure 3). At these time points, HA levels in AS were 8.5-, 7.4-, and 5.6-fold higher, respectively, than CS. Among the alien substitution lines, only 5A significantly accumulated HA relative to CS at 8, 12, and 24 HAT (Figure 3), with levels being 3.8-, 3.2-, and 2.4-fold higher than CS, respectively. In contrast, the HA levels in 5B and 5D were not significantly different from CS at any given time point.

LC-MS Analysis of CS and NT Lines

Similar to the previous experiment, CS exhibited anticipated results: HA levels remained relatively low throughout the entirety of the time course and did not significantly change throughout the time course (Figure 4). Only N5A-T5D displayed significantly higher levels of HA relative to CS at 12 and 24 HAT (approximately 3-fold), while HA levels in N5D-T5A did not significantly differ from CS throughout the time course.

Overall results showed that lines lacking wheat chromosome 5A (AS, N5A-T5D, and the 5A alien substitution line) have a reduced ability to detoxify HA, while lines possessing wheat chromosome 5 A (CS, N5D-T5A and the 5B and 5D alien substitution lines) maintain the ability to detoxify HA.

As mentioned previously, after HM is applied and absorbed through the leaf tissue, it is de-esterified to HA (Figure 1). HA is a phytotoxic, transient Phase I metabolite that is subsequently O-demethylated then conjugated with glucose (Phase II; Figure 1) that leads to HA detoxification (Dzikowski et al., 2016). The O-demethylation and glucose conjugation reactions are likely catalyzed by cytochrome P450s (CYPs) and UDP-dependent glucosyltransferases (UGTs), respectively, which commonly mediate synthetic auxin herbicide detoxification in grasses (Frear, 1995; Riechers et al., 2010; Sterling & Hall, 1997; Zhang & Yang, 2021). The presence of HA in all examined tissue (Figures 3 and 4) indicates all lines possess active esterases to convert HM to HA. However, the activity of esterases, CYPs, and UGTs could vary between lines, which may need to be considered when explaining the results of the experiment. For example, HA levels in the 5B alien substitution line could be low due to a loss of a more efficient esterase to convert HM to HA. Overall, future experimentation should be performed to measure the abundance of the O- demethylated and glucose conjugate of HA to further confirm altered detoxification in the alien substitution lines, which would require analytical standards. Since these metabolites could be present but in low abundance, especially the (9-demethylated metabolite, a whole-plant assay may be more desirable, especially if trying to detect and measure metabolites beyond 24 hours. It is hypothesized that the O-demethylated metabolite and glucose conjugate of HA will either not be detected or be in lower abundance in lines lacking 5A relative to unaltered CS, which should rapidly accumulate these metabolites. The results of these experiments provide evidence that HA-detoxifying genes are located on chromosome 5A in wheat. This information can be utilized in future experiments to identify potential candidate genes for further examination. Common experiments to identify candidate genes, such as RNA- Seq or genome wide association studies, often yield numerous potential candidate genes. This is especially true for wheat, which has a relatively large (approximately 16 Gb) and redundant genome (IWGSC, 2018), meaning that not only could a given gene be identified as significant but also its homoeologs.

Potential candidate genes could be CYPs and UGTs. CYPs perform irreversible oxidation reactions that predispose the molecule to glucose conjugation by UGTs (Frear, 1995; Riechers et aL, 2010; Sterling & Hall, 1997; Zhang & Yang, 2021). CYPs have been implicated in the detoxification of numerous herbicides targeting varying sites of action in members of the Poaceae family, which currently has the highest number of reported herbicide-detoxifying CYPs of any plant family (Dimaano & Iwakami, 2021; Han et al., 2020; Pan et al., 2022; Zheng et aL, 2022). Members of the CYP81A subfamily have by far been the most commonly identified (Table 1), but members of CYP72A and CYP71C have also been identified (Table 2). Some examples in wheat, include evidence of CYP involvement in diclofop-methyl (an acetyl-CoA carboxylase inhibitor) detoxification due to identification of ring hydroxylation and subsequent glucose conjugation (Tanaka et aL, 1990; Zimmerlin & Durst, 1990, 1992), and an in vitro yeast assay using wheat CYP71C6V1 demonstrated metabolism of several acetolactate synthase! ALS)-inhibiting herbicides, including chlorsulfuron, triasulfuron, metsulfuron-metyl, bensulfuron-metyl, and tribenuron-metyl (Xiang et aL, 2006).

Compared to CYPs, genes encoding herbicide-detoxifying UGTs are not as well characterized. However, their role in herbicide detoxification has been well documented (Frear, 1995; Riechers et aL, 2010; Sterling & Hall, 1997; Zhang & Yang, 2021) and their expression is often induced by herbicides and herbicide safeners (Baek et aL, 2019; Duhoux et aL, 2015; Edwards et aL, 2005; Gaines et aL, 2014). Some examples in wheat include detection of glucosylated metabolites of isoproturon, a Photosystem II (PSII) inhibitor, (Lu et al., 2015) and florasulam, an ALS inhibitor (DeBoer et aL, 2006). For other Poaceae crops, four genes in Oryza sativa are implicated in the detoxification 2,4-D and inhibitors of PSII, 4- hydroxyphenylpyruvate dioxygenase, and very-long-chain fatty acid elongase (Brazier-Hicks et aL, 2018; Liu et aL, 2019; Su et aL, 2019; Zhang et aL, 2017). For weedy Poaceae species, an Alopecurus myosuroides UGT was implicated in the resistance towards several herbicides (Brazier et aL, 2002).

Table 1: List of herbicide-detoxifying CYP81As in crop and weed species. Abbreviations: ALS = acetolactate synthase; ACCase = acetyl-CoA carboxylase; DOXPS = l-deoxy-d-xylulose-5-phosphate synthase; HPPD = 4-hydroxyphenylpyruvate dioxygenase; PDS = phytoene desaturase; PPO = protoporphyrinogen oxidase; PSII = photosystem II.

Table 2: List of herbicide-detoxifying CYPs identified in members of Poaceae outside of the CYP81 A subfamily. Abbreviations: ALS, acetolactate synthase; ACCase, acetyl-CoA carboxylase. | | ;

EXAMPLE 2

IDENTIFICATION OF CANDIDATE HERBICIDE-DETOXIFYING GENES IN HEXAPLOID BREAD WHEAT (TRITICUM AESTIVUM L.) VIA RNA-SEQ ANALYSIS OF UNTREATED AND

CLOQUINTOCET-MEXYL-TREATED LEAVES

Like other members of Poaceae, allohexaploid wheat (Triticum aestivum L.) displays natural tolerance to synthetic auxin herbicides due to rapid metabolic detoxification; however, genes encoding these detoxifying enzymes are often not identified nor characterized. Expression of some detoxification genes is increased by herbicide safeners, which are commonly applied with herbicides to enhance herbicide tolerance. The expression of genes induced by the safener indicates the encoded enzyme may play a role in herbicide metabolism. Cloquintocet-mexyl (CM) is a common herbicide safener applied in tank mixtures with the synthetic auxin herbicide, HM. Since it is possible that genes encoding HA-detoxifying enzymes are induced by CM, experiments were conducted to identify candidate herbicide -detoxifying genes via RNA-Seq by comparing untreated and CM-treated leaf tissues. Among several candidates, a member of the CYP81A subfamily of CYPs was identified, which is a noteworthy CYP subfamily due to its members responsible for detoxification of herbicides from various classes, including synthetic auxin herbicides. The candidate gene and its homoeologs are located on the group 5 chromosomes of wheat (denoted as CYP81A- 5A, CYP81A-5B, and CYP81A-5D), and it was hypothesized that the homoeologs varied in terms of basal expression and inductions from HM and CM. Expressions of CYP81A-5A, CYP81A-5B, and CYP81A-5D in untreated wheat leaf tissue, and leaf tissue treated with foliar applications of CM, HM and the combination of CM + HM were measured by TaqMan RT-qPCR over time. Overall, results demonstrate basal expression of these CYPs is relatively high compared to the reference gene (P-tubulin), expression between these CYPs varies over time, and these CYPs are CM inducible but not HM inducible.

CYPs were implicated in herbicide detoxification in some tolerant crops and resistant weeds (Dimaano & Iwakami, 2021; Gaines et al., 2020; Nandula et al., 2019). In related grass species, maize (Zea mays) CYP81A9 and rice (Oryza sativa) CYP81A6 encode CYPs governing tolerance to several herbicides, including synthetic auxins in the case of CYP81A9 (Nordby et al., 2008; Pan et al., 2006; Zhang et al., 2007). However, a specific gene encoding a synthetic auxin-detoxifying CYP has not been identified nor characterized in wheat.

Safeners are commonly applied with herbicides to large-seeded cereals to reduce herbicide injury, which is accomplished by inducing expression and activity of herbicide detoxification and transporter enzymes (Hatzios & Burgos, 2004; Kraehmer et al., 2014; Riechers & Green, 2017). Thus, induced expression by safeners, indicates the encoded enzymes may play a role in herbicide metabolism (Edwards et al., 2005; Hatzios & Burgos, 2004; Riechers et al. 2010), although further biochemical studies are required to functionally validate this theory. In the case of HM, CM is commonly utilized to prevent wheat injury. Published transcriptome analyses of safeners in cereal crops are rare (Baek et al., 2019; Brazier-Hicks et aL, 2020), and currently only one published paper has reported wheat transcriptomic data in response to a safener, mefenpyr-diethyl (Yuan et al., 2021).

Materials and Methods

Chemicals and Plant Materials:

Chemicals used in the following experiments include CM (formulated as a 25% active ingredient wettable powder) and the Elevore formulation of HM. Seed for the winter wheat variety ‘Kaskaskia’ (Kolb & Smith, 2001) was provided by Dr. Frederic Kolb at the University of Illinois at Urbana-Champaign.

Seed Sowing, Treatment Application, and Collection of Tissue:

For both RNA-Seq and RT-qPCR experiments, seeds were planted in 382 cm³ pots containing a 1 : 1 : 1 soil mixture of soil, peat, and sand. Pots were placed in a greenhouse room with a 14-hour day length and a constant 21 to 23°C temperature band. Natural light was supplemented with halide lamps delivering 800 pmol m^-2 s^-1 photon flux to the plant canopy. When seedlings produced 1-2 leaves (Zadoks stages 11- 12), treatments were applied using a compressed air research sprayer calibrated to deliver 187 L ha^-1 at 275 kPa with an even flat-fan nozzle.

For the RNA-Seq experiment untreated plants (UT) were sprayed with a 0.1% solution of nonionic surfactant (NIS), while safener-treated plants were sprayed with a solution containing 15 g a.i. ha^-1 of CM and 0.1% NIS (Taylor et al., 2013). After application of treatments, plants were returned to the greenhouse room until leaf tissue was harvested at 6 hours after treatment (HAT). At harvest, the first leaves were cut at the collar from five plants to achieve 500 mg of leaf tissues being collected, which were then frozen with liquid nitrogen, and stored in a -80°C freezer until RNA extraction.

For the RT-qPCR experiment, treatments from the RNA-Seq experiment were included with the addition of 5 g a.e. ha ¹ of HM and a combination of CM and HM treatments (CM+HM). All treatments included 0.1% NIS. Harvesting procedures are the same as previously mentioned but were performed at 3, 6, and 12 HAT.

RNA Extraction, Library Construction, and Transcriptomic Analysis:

Total RNA extraction was isolated using previously described methods (Obenland et al., 2019), and RNA concentration and purity were determined with a NanoDrop 1000 spectrophotometer (Thermo Scientific, USA). RNA samples with concentrations above 100 ng/ L, A260/A280 ratios above 1.8, and ratios between 2.0 and 2.3 were used in downstream processes. Each RNA sample (10 pg) was treated with TURBO™ DNase using the TURBO DNA-free™ Kit (Thermo Scientific, USA) using the manufacturer’s protocol in order to eliminate genomic DNA contamination. Concentration of DNase-treated RNA was determined with a Qubit 2.0 fluorometer (Invitrogen, USA) using the manufacturer’s protocol.

Six RNA samples (three untreated samples and three CM-treated samples) were submitted to Roy J. Carver Biotechnology Center for the construction of RNAseq libraries. An AATI Fragment Analyzer was used to evaluate integrity of the RNA samples, which indicated the 28S and 16S bands were very prominent and degradation was not detected. Libraries were quantitated by qPCR and sequenced on one lane for 151 cycles from each end of the fragments on a NovaSeq 6000, generating 150-bp paired end reads. RNA-Seq data quality was estimated by FastQC v0.12.0 (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/) with low quality sequences filtered with fastp v0.17.0 (Chen et al., 2018). None of the reads failed to pass the filter (Table 3). The ‘Chinese Spring’ reference genome (IWGSC RefSeq vl.l) and functional gene annotations were downloaded from URGI (wheat-urgi.versailles.inra.fr/). Salmon/ 1.10.1 was used to align clean reads to the reference genome and quantify reads (Patro et aL, 2017). All tests of significance for differential expression were analyzed with modules from the edgeR package (Robinson et al., 2010) in R (version 4.2.0) using RStudio (Version 2023.03.0), in which the TMM normalization was utilized to adjust expression values to a common scale. Gene expression levels were calculated using counts-per-million (CPM) and transformed to logi counts per million. Differentially-expressed genes (DEGs) were identified if fold changes in expression were >5 or <-5 and the false-discovery rate (FDR) was <0.1. Molecular functions of DEGs were assigned Gene Ontology (GO) annotations with agriGO v2.0, which were used to perform GO enrichment analysis with default settings (Du et al., 2010; Tian et al., 2017).

RNA Extraction, Primer Design, Thermal Cycling Conditions, and Data Analysis for TaqMan RT- qPCR:

RNA was prepared, evaluated for quality, and quantitated utilizing the same methods described in the previous section. However, instead of utilizing an AATI Fragment Analyzer to evaluate integrity, total RNA was examined for quality after denaturation at 55 °C in the presence of formamide and formaldehyde, then integrity of rRNA bands was visualized with ethidium bromide in a 1.2% agarose gel containing 0.4 M formaldehyde (Xu et al., 2002).

TaqMan primers and probes were designed with AllelelD 7 (PREMIER Biosoft, USA) for the candidate gene TraesCS5A02G394800.1 and its homoeologs TraesCS5B02G402800.1 and TraesCS5D02G407300.1 (denoted as CYP81A-5A, CYP81A-5B, and CYP81A-5D, respectively; Tables 4-7). Primers and probe were also designed for a -tubulin gene (ft-TUB TraesCS7D02G454200.1) to serve as a reference gene (Tables 4-7). This gene was chosen based on previous results indicating stable expression in numerous wheat leaf tissue (Zhang et al., 2013). PCR efficiencies of CYPs and ft-TUB primers were calculated in SDS 2.3 software (Applied Biosystems, USA) with six-point standard curves in a 10-fold dilution series of RNA (Tables 5-6).

RT-qPCR was conducted using a 7900 HT Sequence Detection System (Applied Biosystems, USA) and reactions were performed in 20 pL volumes following the manufacturer’s protocol (TaqMan™ RNA-to- Ct™ 1-Step Kit; Applied Biosystems, USA). The following program was used for RT-qPCR: 48 °C for 15 minutes, 95 °C for 10 minutes, followed by 40 cycles at 95 °C for 15 seconds and 60 °C for 1 minute. Each sample was analyzed in three technical replicates and mean cycle threshold (Ct) values were calculated. Reverse-transcription negative controls were included to verify genomic DNA contamination was not contributing to Ct values. CM- and HM-induced gene expression for each CYP gene was calculated relative to transcript levels in the nontreated control samples (per tissue and timepoint) and normalized using ft-TUB a reference gene with the 2^{- ACt} method.

Comparisons of the AACt and Ct values of genes were performed with the lme4 package (Bates et al., 2015) in R (version 4.2.0) using RStudio (Version 2023.03.0). A mixed effects model was utilized for both experiments where gene, treatment, and their interactions were treated as fixed effects and replicates were treated as random effects. Data was subjected to ANOVA and means were separated with Fisher’s protected LSD (a = 0.05). Results

RNA-Seq and AgriGO Categories of Upregulated Genes

Among all RNA-Seq libraries, 81% of reads mapped uniquely to the reference genome, 76.4% of reads were mapped to a gene, 10% of reads were not mapped to a gene, 3% of reads were unmapped, 11% of reads were multi-mapped, and only 0.1% of reads were ambiguous (Table 3). In terms of significant DEGs, 101 genes were induced, and two genes were repressed by CM (Figure 5). Based on their functional annotations, the majority of these DEGs encode proteins associated with Phase I, II and III metabolic detoxification reactions while the remaining DEGs are associated with stress/defense response, amino acid metabolism, heavy metal binding, or encode transcription factors (Figure 6). The two repressed genes are annotated as a peroxidase and a “negative regulator of resistance” (Figure 6), which indicate CM represses a few stress-related genes and likely triggers some level of oxidative stress to fine-tune safener-mediated transcriptional regulation.

The 103 significant DEGs were utilized for GO enrichment analysis with agriGO v2.0, and 99 (96.1%) DEGs were assigned GO terms. GO enrichment analysis show that a significant amount of GO terms were associated with transferase (both glutathione 5-transferases and UGTs) and oxidoreductase activity (Figures 7-9). Results of GO enrichment analysis corroborate the results of the tree map (Figure 6) and further elaborate on the function of the encoded proteins of the DEGs by indicating what substrates or molecular bonds are involved in the reactions catalyzed by the proteins. For example, the GO terms assigned to a specific gene may not only indicate it is a UGT, but also could catalyze glucose conjugations with abscisic acid or indole-3-acetate (Figure 7).

Of the 103 significant DEGs, five genes (three UGTs and two CYPs) are located on the group 5 chromosomes with fold inductions for these genes ranging from approximately 5 to 23 (Figure 10). Based on Phytozome BLAST results (https://phytozome-next.jgi.doe.gov/), TraesCS5A02G472300.1 and TraesCS5A02G397800.1 have high similarity to members of CYP71C and CYP81A (Table 7), respectively. Both CYPs were assigned G0:0003824 (catalytic activity), G0:0016491 (oxidoreductase activity), and G0:0046914 (transition metal ion binding). The G0:0016491 (oxidoreductase activity), would be especially typical of a CYP involved with herbicide detoxification. Based on sequence similarity, TraesCS5D02G404200.1 and TraesCS5A02G394800.1 are likely homoeologs (93.8% identity) and have high similarity to a member of UGT85A in rice. Both UGTs were assigned all the GO terms specifying the glucosyltransferase activities (far right of Figure 7) from abscisic acid to flavonol glucosyltransferase activities. TraesCS5B02G305600.1 shows high similarity to a salicylic acid glucosyltransferase 1 (SGT1) in rice, which catalyzes the formation of glucoside and glucose esters of salicylic acid (Li et aL, 2022). This UGT was assigned the same specific glucosyltransferase GO terms as the previous two UGTs, except for the terms associated with cytokinin, cis-zeatin, and hydroquinone activity (Figure 7). The described functions of SGT1 are corroborated by the assigned GO terms of G0:0052639 (salicylic acid glucosyltransferase (ester-forming) activity) and G0:0052640 (salicylic acid glucosyltransferase (glucoside-forming) activity). TraesCS5A02G397800.1 and its homoeologs, TraesCS5B02G402800.1 and TraesCS5D02G407300.1 (denoted as CYP81A-5A, CYP81A-5B, and CYP81A-5D, respectively) were selected for further analysis via gene-specific RT-qPCR. Due to the high sequence similarity between CYP81A-5A, CYP81A-5B, and CYP81A-5D (Table 8), TaqMan methods were chosen to achieve homoeolog discrimination.

Expression of CYP81A-5A, CYP81A-5B, and CYP81A-5D

Overall, expression levels for all genes and fold inductions for the CYPs fluctuated throughout the time-course, and results indicate all three CYPs are CM-inducible but not HM-inducible (Figures 11-12). The mean Ct for the HM treatment at each timepoint were not significantly different from untreated controls, resulting in small (approximately 2.5-fold or less) fold changes in expression (Figures 11-12). In contrast CM and CM+HM treatments significantly induced expression, especially at 3 HAT and 12 HAT. At each timepoint, a CYP was induced by CM was also induced by CM+HM and their fold inductions were not significantly different from each other (Figure 11). These results indicate no additive or synergistic effect from the HM treatment. Additionally, it is worth noting the expression of all CYPs in UT tissue was relatively high and often is not significantly different from [>-TUB. especially at 3 HAT where all CYPs are not significantly different from -TUB (Figures 12-13). CYP81 A-5A expression in UT tissue was not significantly different from fl-TUB at 3 and 6 HAT (Figure 13), but across all time points CM increases CYP81A-5A expression to either comparable levels or levels higher than [S-TUB (Figure 12). These results show that CYP expression is relatively high in UT tissue, suggesting that these CYPs are likely needed for one or more endogenous functions at this growth stage in wheat.

Within each timepoint fi-TU displayed stable, non-significantly different expression in response to all treatments (Figure 12), indicating its suitability as a reference gene for calculating fold changes for each CYP per timepoint. All three CYPs were significantly induced by CM at 3 HAT and 12 HAT. Expression at 3 HAT was approximately 7-fold greater than UT tissue for CYP81A-5A and CYP81A-5B and 21-fold greater than UT tissue for CYP81A-5D. Expression at 12 HAT was approximately 10-, 4-, and 30-fold greater than expression in the UT tissue for CYP81A-5A, CYP81A-5B, and CYP81A-5D, respectively (Figure 11). At 6 HAT, only the expression of CYP81A-5D is significantly induced by CM and CM+HM (Figure 12) with both treatments resulting in an approximate 7-fold induction in expression (Figure 11).

Results of the RT-qPCR experiment indicate the expression between the three homoeologs varies over time, and all three homoeologs are induced, though not equally, by CM (Figures 11-12)..

The fluctuations over time in basal expression and CM inductions in RT-qPCR results (Figures 11- 12) could indicate regulation by circadian clock. Plant biological activities show diurnal variation and the circadian clock coordinates plant activities in response to environmental cues, such as light and temperature (McClung, 2006). Additionally, nearly all the genes associated with stress signaling pathway are regulated by the circadian clock, which synchronizes them for improved fitness and optimized development (Covington et al., 2008; Green et al., 2002; Nozue & Maloof, 2006). To measure the effect of the circadian clock on target genes, reference genes lacking variation over a time course experiment would be required (Jain et al., 2018) or absolute quantification of transcripts could be performed by making standards to extrapolate CYP transcript abundance in samples. -TUB would not be a suitable reference gene for this objective because it displays variation between timepoints with Ct values ranging from 26.6 at 3 HAT to 24.4 at 12 HAT (Figure 12).

CYP81A-5A, -5B, and -51) can be subject to further characterization of ‘promiscuous’ herbicide detoxification. Such a finding would further illustrate the concept of non-specific substrate binding, and these genes would have great potential and value for genetic transformation of relevant crop genomes lacking natural herbicide tolerance. Additionally, successfully identified candidate genes could be utilized for in vitro metabolism assays with E. coli or yeast cells (Abdollahi et al., 2021; Dimaano et al., 2020) to screen and predict whole-plant tolerance to numerous experimental herbicides, especially potential analogs of HM or any other herbicide that the encoded enzyme detoxifies, prior to performing whole-plant pheno typing.

Other significant DEGs identified by RNA-Seq also warrant exploration in the future, especially since reactions catalyzed by CYPs result in metabolites with some phytotoxicity and still require further detoxification by UGTs (Dimaano & fwakami, 2021 ; Gaines et al., 2020). With that in mind, TraesCS5D02G404200.1 (UGT-5D), TraesCS5A02G394800.1 (UGT-5A), or TraesCS5B02G305600.1 (UGT-5B) could catalyze the conjugation reaction for the metabolite formed by CYP81A-5A. The CYP, TraesCS5A02G472300.1 (CYP71C-5A), resembling members of CYP71C is noteworthy given evidence showing CYP71C6V1 involvement in sulfonylurea herbicide detoxification in wheat (Xiang et al., 2006). Based on sequence similarity (55%), TraesCS5A02G472300.1 (CYP71C-5A) is not a homoeolog of CYP71C6V1 (located on chromosome 5D) due to it barely meeting the threshold to be in the same CYP sub-family (Dimaano & Iwakami, 2021; Nelson, 2009) and homoeologs usually have very high sequence similarity (Glover et al., 2016). Additionally, there is evidence illustrating a correlation of higher expression of CYP71C members in nicosulfuron-tolerant Zea mays cultivars (Liu et aL, 2018) and a cyhalofop-butyl- resistant Leptochloa chinensis population (Zhang et al., 2022) relative to sensitive populations.

Any selected candidate genes can be edited with CRISPR/Cas9 methods. It is therefore hypothesized that CRISPR/Cas9-mediated modifications in CYP81A-5A or other genes identified above that result in either altered expression or altered protein activity will have a commensurate effect on natural or CM-induced HM tolerance (and/or tolerance to other wheat-selective herbicides).

Table 3: RNA-Seq libraries prepared from untreated (UT) and cloquintocet-mexyl(CM)-treated wheat leaf tissue.

Table 4: Reference genes and target genes selected for TaqMan RT-qPCR experiment.

Table 5: Primer and probe sequences.

Table 6: Primer and probe details and efficiencies.

Table 7: Predicted amplified sequences of TaqMan primers and probes. The underlined sequences correspond to the binding sites of the forward and reverse primers, respectively. Underlined and bolded sequences correspond to the binding sites of the TaqMan probes.

Table 8: Percent identity matrix comparing CYP81As of wheat, rice and maize. The encoded protein for TraesCS5A02G397800.1 and its homoeologs, TraesCS5B02G402800.1 and TraesCS5D02G407300, are denoted as CYP81A-5A, CYP81A-5B, and CYP81A-5D, respectively. GenBank accession for CYP81A6 and CYP81A9 are ABC69856.1 and ACG28028.1, respectively.

EXAMPLE 3 CRISPR/CAS9 EDITING OF A CYP81A HOMOEOLOG IN HEXAPLOID BREAD WHEAT (TRITICVM AESTIVUM L.)

Members of the CYP81A family of CYPs catalyze the detoxification of several herbicide classes, including synthetic auxin herbicides. While the CYP81A family has been examined in several Poaceae crop and weed species, research is lacking for Triticum aestivum (allohexaploid bread wheat). Previous phenotypic and metabolic experiments in our lab indicated genes on wheat chromosome 5A significantly contribute to natural HM tolerance, a synthetic auxin herbicide. Following HM de-esterification, natural tolerance in wheat is achieved through rapid detoxification of the biologically active form, HA, and detoxification is enhanced by the herbicide safener, CM. Furthermore, RNA-Seq and RT-qPCR experiments indicated expression of a CYPS I A located on chromosome 5A (CYP81A-5A) was significantly induced by CM. Since CYPs are commonly involved with Phase I detoxification of various herbicides with differing sites of action, including synthetic auxins, it is hypothesized the CYP81A-5A encodes an HA- detoxifying enzyme capable of detoxifying other relevant wheat herbicides, such as florasulam and propoxycarbazone-sodium, and fenoxaprop-P-ethyl. To address this hypothesis, CRISPR/Cas9 technology was employed to edit CYP81A-5A with the goal of creating wheat lines with altered expression or function/activity of CYP81A-5A. The CRISPR plasmid was designed to deliver Cas9 and five guide RNAs targeting the coding region and the putative promoter of CYP81A-5A using the spring wheat cultivar, CB037.

These efforts resulted in three mutant, Cas9-free plants with an insertion in the putative promoter of CYP81A-5A. All three Cas9-free plants possessed the same mutant allele with a single nucleotide insertion within the promoter region. The plants varied in their zygosity for the mutant allele with two individuals being heterozygous and one being homozygous. Progeny segregating for the mutant allele were examined for various developmental traits. Results indicated that plants homozygous and heterozygous for the mutant allele did not differ in these traits from plants homozygous for the wild type alleles. However, plants homozygous for the mutant allele displayed higher inductions from CM treatment (1.6-fold) and increased tolerance to propoxycarbazone-sodium (1.4-fold) relative to unaltered wheat, showing that a single nucleotide insertion altered the transcriptional regulation of CYP81A-5A under CM treatments, and CYP81A- 5A plays a role in propoxycarbazone-sodium detoxification in wheat.

Based on the results obtained in the previous examples, it is hypothesized that CYP81A-5A encodes an HA-detoxifying enzyme, and other selective herbicides utilized in wheat, such as florasulam (FLOR), propoxycarbazone-sodium (PROP), and fenoxaprop-P-ethyl (FEN) may also be detoxified by CYP81A-5A. To address this hypothesis, CRISPR/Cas9 technology was used to introduce mutations in the putative promoter and/or coding regions of CYP81A-5A, with the goal of creating wheat lines with altered expression or function/activity of CYP81A-5A. Next, these wheat lines were characterized for alterations in CM induction and wheat-selective herbicide tolerance. As some CYP81As have been reported before as being involved in detoxifying herbicides with different sites of action (Iwakami et al., 2019; Li et al., 2013; Nordby et al., 2008; Pan et al., 2006; Zhang et al., 2002; Zheng et al., 2022), HM and other wheat-selective herbicides (where selectivity is based on oxidative metabolism) were examined, including two acetolactate synthase inhibitors (florasulam (FLOR) and propoxycarbazone-sodium (PROP)) and one acetyl-CoA carboxylase inhibitor (fenoxaprop-P-ethyl (FEN)).

Materials and Methods

Chemicals:

Chemicals used in the following experiments include CM (formulated as a 25% active ingredient wettable powder), the Elevore formulation of HM, the Defender formulation of FLOR, the Olympus formulation of PROP, and the Acclaim Extra formulation of FEN.

CR1SPR/Cas9 Editing, DNA Extraction, and Producing Cas9-free Progeny:

Genomic DNA was extracted using the DNeasy Plant Mini Kit (Qiagen, USA) and PCR was performed using Platinum™ SuperFi II PCR Master Mix (Invitrogen, USA). Cas9 was detected using Cas9- specific PCR primers (Table 9) with products visualized on an EtBr-stained 0.7% agarose gel. The wild type alleles of CYP81A-5A and its homoeologs on chromosomes 5B and 5D (denoted as CYP81A-5B and CYP81 A-5D. respectively) were sequenced in spring wheat variety ‘CB037’ (CB) via Sanger sequencing by the UIUC Core Sequencing Facility. PCR and Sanger sequencing primers are listed in Tables 9-10. Additionally, PCR parameters for each set of primers and the sequencing results of the genes in CB are listed in Tables 11-12. The specificity of CYP primers was tested by utilizing DNA from CB (positive control) and appropriate nullisomic-tetrasomic (NT) lines (negative control), and specificity of Cas9 primers was tested by utilizing DNA from CB (negative for Cas9) and a T i mutant (positive for Cas9). PCR products were visualized on an EtBr-stained 0.7% agarose gel.

The wheat line, CB, was utilized for CRISPR-editing techniques (Clemente and Mitra, 2004). The CRISPR plasmid encoded five single guide RNAs (sgRNAs) targeting the coding regions and promoter of CYP81A-5A (Figure 14). To progeny were self-fertilized to produce Ti progeny, which were all positive for Cas9. To create Cas9-free progeny, Ti plants with mutations in CYP81A-5A were crossed with CB037 to produce Fi progeny. Fi plants were then self-fertilized to produce F2 progeny. When three mutant, Cas9- free F2 progeny were identified with Sanger sequencing (Table 13), PCR subcloning was performed to confirm whether these plants were homozygous or heterozygous for the mutant allele.

Purified amplicons were ligated into pCR 4 Blunt-TOPO cloning vector from the Invitrogen™ Zero Blunt™ TOPO™ PCR Cloning Kit for Sequencing (Invitrogen, USA), and plasmids were transformed into competent Escherichia coli cells. Cells were cultured on ampicillin (100 pg/mL) agar plates, and single colonies were selected for liquid culture inoculation. Recombinant plasmids were purified with an LBlue Mini Plasmid Kit (IBI Scientific, USA). The presence of ~3.0 kb inserts was verified by digesting plasmids with EcoRI and visualizing the insert with a 0.7% agarose gel stained with ethidium bromide. Plasmid samples were submitted for Sanger sequencing at the UIUC Core Sequencing Facility using primers listed in Table 10. The homoeologs, CYP81A-5B and CYP81A-5D, were also sequenced to check for possible off- target editing (Tables 12-13). These F2 progeny were self-fertilized to produce F3 and F4 progeny utilized for subsequent experiments.

Treatments, RNA Extraction, TaqMan RT-qPCR and Statistical Analysis:

F4 progeny (CBE) descended from F2-3 (Table 13) and CB were planted in 382 cm³ pots containing a 1 : 1 : 1 soil mixture of soil, peat, and sand. Pots were placed in a greenhouse room with a 14-hour day length and a constant 21 to 23 °C temperature band. Natural light was supplemented with halide lamps delivering 800 pmol m^-2 s^-1 photon flux to the plant canopy. When seedlings produced 1-2 leaves (Zadoks stages 11-12), treatments were applied using a compressed air research sprayer calibrated to deliver 187 L ha ¹ at 275 kPa with an even flat-fan nozzle. Untreated plants (UT) were sprayed with a 0.1% solution of nonionic surfactant (N1S), while CM-treated plants were sprayed with a solution containing 15 g a.i. ha^-1 of CM and 0.1% NIS (Taylor et al., 2013). After application of treatments, plants were returned to the greenhouse until tissue harvest at 3 hours after treatment (HAT). At harvest, 500 mg of leaf tissue was collected, frozen with liquid nitrogen, and stored in a -80°C freezer until RNA extraction.

Total RNA was isolated using previously described methods (Obenland et al., 2019) for three biological replicates per treatment, and RNA concentration and purity were determined with a NanoDrop 1000 spectrophotometer (Thermo Scientific, USA). Samples with concentrations above 100 ng/pL, A260/A280 ratios above 1.8, and A260/A230 ratios between 2.0 and 2.3 were used in downstream processes. Each sample (10 pg) was treated with TURBO™ DNase using the TURBO DNA-free™ Kit (Thermo Scientific, USA) using the manufacturer’s protocol in order to eliminate genomic DNA contamination. Concentration of DNase-treated RNA was determined with a Qubit 2.0 fluorometer (Invitrogen, USA) using the manufacturer’s protocol. Total RNA was examined for integrity after denaturation at 55 °C in the presence of formamide and formaldehyde, and rRNA bands was visualized with ethidium bromide in a 1.2% agarose gel containing 0.4 M formaldehyde (Xu et al., 2002).

Due to the high sequence similarity between CYP81A-5A, CYP81A-5B, and CYP81A-5D (Table 8), TaqMan methods were chosen to achieve homoeolog discrimination. TaqMan primers and probes were designed with AllelelD 7 (PREMIER Biosoft, USA) for the candidate gene TraesCS5A02G394800.1 (CYP81A-5A) and [!- tubulin ([-TUB'. TraesCS7D02G454200.1) to serve as a reference gene (Tables 14-17), and [i-TUB was chosen based on previous results indicating stable expression in wheat leaf tissue (Zhang et al., 2013). PCR efficiencies of CYP81A-5A and [i-TUB primers were calculated in SDS 2.3 software (Applied Biosystems, USA) with six-point standard curves in a 10-fold dilution series of RNA (Tables 15- 16).

RT-qPCR was conducted using a 7900 HT Sequence Detection System (Applied Biosystems, USA) and reactions were performed in 20 pL volumes following the manufacturer’s protocol (TaqMan™ RNA-to- Ct™ 1-Step Kit; Applied Biosystems, USA). The following program was used for RT-qPCR: 48°C for 15 minutes, 95 °C for 10 minutes, followed by 40 cycles at 95 °C for 15 seconds and 60 °C for 1 minute. Each sample was analyzed in three technical replicates and mean cycle threshold (Ct) values were calculated. Reverse-transcription negative controls were included to verify genomic DNA contamination was not contributing to Ct values. CM-induced gene expression for CYP81A-5A gene was calculated relative to transcript levels in the nontreated control samples and normalized using fi-TUR as a reference gene with the 2’^AACt method.

Comparisons of the AACt and Ct values of genes were performed with the lme4 package (Bates et al., 2015) in R (version 4.2.0) using RStudio (Version 2023.03.0). A mixed effects model was utilized where population, treatment, and their interactions were treated as fixed effects and replicates were treated as random effects. Data was subjected to ANOVA and means were separated with Fisher’s protected LSD (a = 0.01).

Genotyping, Developmental Trait Phenotyping, and Statistical Analysis:

Seeds of F3 progeny descended from F2-I (Table 13) were planted in 9,464 cm³ pots containing a 1: 1: 1 soil mixture of soil, peat, and sand. Pots were arranged in a completely randomized design in a greenhouse room with a 15-hour day length and a constant 24 to 27 °C temperature band. Natural light was supplemented with halide lamps delivering 800 pmol m^-2 s^-1 photon flux to the plant canopy. Genotyping of 41 plants was performed by extracting genomic DNA from leaf tissue using the DNeasy Plant Mini Kit (Qiagen, USA) and sequencing CYP81A-5A using methods described in Example 3 (Tables 9-11).

Plants were phenotyped for the following traits: days to flowering (DTF), days to maturity (DTM), number of productive tillers (NPT), seeds per spikes (SPS), spike length (SL), yield, and dry biomass. DTF and DTM dates were recorded when half the spikes had emerged or browned, respectively. Three spikes from each plant were utilized for recording SL and SPS with SL being measured from the base of rachis to the topmost spikelet. When all the spikes of the productive tillers browned, they were harvested, dried, and the seeds were weighed for yield. The remaining aboveground biomass was harvested at the soil line, dried, and the dry biomass was recorded.

All tests of significance were performed in R (version 4.2.0) using RStudio (Version 2023.03.0). The Least-Square (LS) means for each trait were calculated using the emmeans package (version 1.8.5), the Pearson's X² test was performed with the R stats package (version 3.6.2), and comparisons of the LS means performed with the lme4 package (Bates et aL, 2015). A mixed effects model was utilized where genotype, agronomic trait, and their interactions were treated as fixed effects and replicates were treated as random effects. Data was subjected to ANOVA and means were separated with Fisher’s LSD (a = 0.01).

Herbicide Phenotyping and Statistical Analysis:

Seeds of CBE, CB, and Aegilops searsii (AS; a halauxifen-sensitive diploid wheat relative; Obenland & Riechers, 2020) were planted in 382 cm³ pots containing a 1: 1: 1 soil mixture of soil, peat, and sand. Pots were placed in a greenhouse room with a 14-hour day length and a constant 21 to 23 °C temperature band. Natural light was supplemented with halide lamps delivering 800 pmol m“² s^-1 photon flux to the plant canopy. When seedlings produced 1-2 leaves (Zadoks stages 11-12), treatments were applied using a compressed air research sprayer calibrated to deliver 187 L ha^-1 at 275 kPa with an even flatfan nozzle. UT plants were treated with a solution of 1% solution of methylated seed oil (MSO) and 2.5% ammonium sulfate (AMS), HM-treated plants were treated with 40 g a.e. ha ¹, FLOR-treated plants were treated with 10 g a.i. ha ¹, PROP-treated plants were treated with 5.5 g a.i. ha ¹, and FEN-treated plants were treated with 46.2 g a.i. ha ¹. All herbicide treatments included 1% MSO and 2.5% AMS. CB served as a positive control that displays tolerance to these herbicides under field conditions and AS served as a negative control that is sensitive to these herbicides. These herbicide rates were chosen because they caused 30-40% biomass reductions in CB and >50% biomass reductions in AS. After application of treatments, plants were returned to the greenhouse and were arranged in a completely randomized design. At 21 days after treatment, above ground biomass was harvested, dried in a 65°C oven, and dry biomass was recorded. Five biological replicates were utilized per treatment and the experiment was performed twice.

All tests of significance were performed in R (version 4.2.0) using RStudio (Version 2023.03.0) and comparison of means were performed with the R stats package (version 3.6.2). A fixed effects model was utilized where population, treatment, and their interactions, were treated as fixed effects. There was no significant replicate effect. Data was subjected to ANOVA and means were separated with Fisher’s protected LSD (a = 0.01).

Results

Production of Cas9-Free Progeny

Of the 49 Fi progeny, 14 mutant plants were identified (28.5%), which were all positive for Cas9. Among 20 F progeny, three plants were Cas9-free and possessed mutations in the putative promoter of CYP81A-5A (6.1%). One F₂ plant is homozygous and the remaining two plants are heterozygous for the mutant allele (Table 13). The homoeologs, CYP81A-5B and CYP81A-5D, in these three F₂ progeny have not been edited due to off-target editing by CRISPR/Cas9 (Tabic 13). All plants had the same single G insertion in the promotor region of CYP81A-5A, which was targeted by sgRNA 1 (Table 12 and Figure 14). Progeny from these F plants were utilized for subsequent experiments.

Expression of CYP81A-5A in CB and CBE

TaqMan RT-qPCR indicated CM induction of CYP81A-5A is approximately 1.6-fold higher in CBE relative to expression in CB (Figure 15). The Ct values among treated or untreated CM or CBE for fi-TUB were not statistically significant (Figure 16). For CYP81A-5A, the Ct values for UT tissue in CB and CBE was comparable to the results of fi-TUB, showing that the expression of these two genes is comparable, and the expression of CYP81A-5A in UT tissue for both CB and CBE were not significantly different (Figure 16). In contrast, Ct values for CYP81A-5A in CM-treated tissue for both populations are significantly lower than fi-TUB (Figure 16). Overall, these results indicate the insertion in the promoter of CYP81A-5A resulted in increased induction from CM treatment. Phenotyping of Growth and Development Traits and Herbicide Tolerance

Among all traits, no significant difference between the three genotypes was identified (Table 18), indicating the insertion in the promoter of CYP81A-5A has no effect on these traits. The non-significant result of the Pearson's X² test indicates any differences in the number of observed genotypes from expected ratio of genotypes (1:2:1) is due to random chance and the mutant allele exhibits stable Mendelian inheritance (Table 18).

In the herbicide phenotyping experiment, mean biomasses for CB and CBE (both UT) were not significantly different (1.85 g and 1.88 g, respectively). CB and AS responded as expected with biomass reductions ranging from 30% to 40% in CB and >50% biomass reductions in AS for each treatment. Interestingly, when evaluating changes in herbicide tolerance between CB and CBE, the only significant change occurred with PROP, where the biomass in CBE is approximately 1.4-fold higher than the biomass of CB (Figure 17-18). Furthermore, the CBE and CB plants treated with PROP visibly differ in size, with CBE being larger and more comparable in size to the respective UT plant (Figure 18). For all other herbicides, the results for CBE are not statistically significant from results of CB (Figure 17). Results indicate the insertion in the promoter of CYP81A-5A resulted in increased tolerance towards PROP, likely by increasing expression of CYP81A-5A.

While a relatively low number of mutant, Cas9-free plants were identified (Table 13), the editing efficiency is consistent with previous findings for CRISPR/Cas9 editing in wheat (Howells et al., 2018; Xu et al., 2022). Additionally, the mutant allele displayed stable, Mendelian inheritance. Due to the high similarity of homoeolog coding sequences (Table 8), it is often difficult to design homoeolog-specific sgRNAs for CRISPR/Cas9 editing in wheat. However, the only identified edit occurring in the promoter region where there is more disparity between homoeologs may partly explain why it was identified in this region rather than the coding sequence. Generally, sgRNA design greatly affects editing efficiency (Xu et al., 2022; Zischewski et aL, 2017) and sgRNAs targeting all three homoeologs have lower editing efficiency than sgRNAs specifically targeting one or two homoeologs (Howells et al., 2018).

Additionally, it is possible mutations in the coding sequence of CYP81A-5A results in lethality. Given that endogenous substrates of CYP81As are unknown (Dimaano & Iwakami, 2021) and CYPs serve roles in biosynthesis of primary and secondary metabolites, the possibility of a deleterious mutation resulting in the repression of some essential biosynthesis pathway cannot be ruled out. Base editing is possible with Cas9 base editors that consist of a dead Cas9 domain fused to a cytidine deaminase enzyme and a sgRNA capable of converting G to A and C to T (Komor et aL, 2016). The same base conversions can also be achieved with a Cas9 fused with a transfer RNA adenosine deaminase (Gaudelli et aL, 2017). Progeny with a deleterious mutation, such as a frameshift or a premature stop codon, in the coding sequence of CYP81A- 5A can be used to determine whether tolerance to treated herbicides (i.e. HM, FEN, and FLOR) is altered by mutant CYP81A-5A alleles. With such progeny, it is hypothesized that if CYP81A-5A detoxifies these herbicides, then the plants will be more sensitive than unaltered wheat, or if CYP81A-5A does not detoxify these herbicides, then the plant will display no change in tolerance relative to unaltered wheat.

The above results showed that the insertion in the CYP81A-5A promoter increased the rate of transcription of CYP81A-5A in response to CM treatments, and increased PROP tolerance of the wheat, suggesting that CYP81A-5A likely serves a role in PROP detoxification in wheat.

Furthermore, the lack of altered response from HM, FEN, and FLOR in CBE relative to CB does not indicate a lack metabolism of these herbicides (Figure 17). In fact, it might indicate the detoxification enzymes between CB and CBE are performing with equal efficiency.

Given that transcription factors (TFs) are often identified in RNA-Seq experiments as significantly induced or repressed by both herbicides and herbicide safeners (Baek et al., 2019; Brazier-Hicks et al., 2020; Chen et al., 2023; Wang et al., 2021; Yuan et al., 2021; Zhang et al., 2022b), it is plausible for TFs to affect the expression of genes encoding herbicide-detoxifying enzymes. It is hypothesized that the insertion in the CYP81A-5A promoter is either improving the binding of a TF that enhances transcription or inhibiting the binding of a TF that represses transcription, resulting in increased expression of CYP81A-5A after treatments of CM and/or PROP. Examination of the edited and unedited CYP81A-5A promoters with the Plant Cisacting Regulatory DNA Elements (PLACE) database (Higo et al., 1999) indicates there is no change in putative promoter-TF binding motifs. However, the insertion in the mutant allele is adjacent to the binding site for a Dof protein (the binding sequence is AAAG) and about 11-bp from the binding site for a GATA protein (the binding sequence is GATA). Assays that can be used to directly examine differences in TF binding between the wild type and mutant alleles include yeast 1 hybrid assays, chromatin immunoprecipitation (ChIP) assays, and ChlP-Seq assays (Li et al., 2020; Nie et al., 2009; Park, 2009; Reece-Hoyes & Walhout, 2012; Tock et al., 2021; Zhang et al., 2022b).

Table 9: PCR primers for CYP81A-5A, CYP81A-5B, and CYP81A-5D.

Table 10: Internal primers utilized for Sanger sequencing for CYP81A-5A, CYP81A-5B, and CYP81A-5D. The first eight primer sets were used for sequencing CYP81A-5A and the remaining six primer sets were used for sequencing CYP81A-5B and CYP81A-5D.

Table 11: PCR parameters of target genes for primers in Table 9

Table 12: Sequences of CYP81A-5A, CYP81A-5B, CYP81A-5D and Cas9 in wheat varieties CB037 and ‘Chinese Spring’. Regions that are italicized correspond to binding sites of amplification primers. The putative TATA box is both bolded and italicized. For CYP81A-5A the binding sites for the guide RNAs are listed in bold and underlined and the protospacer adjacent motif (PAM) sequences are italicized. The insertion in the CYP81A-5A mutant allele is shown in a larger font.

GG7’/lC/\C/AG77'C7GC777bAC'V TTTCACCACTTGTATACTCTGCAAGTAAACGAAG

CCGCTCACAAAGTCAGTGAGTGGACAACTACGTCAGAGCTTTTCCCTATTTTAA

TTCATCAGAGCTTCTTGTTTCGGAAGGAAAGGTCGCAAGATACAGAACCCCA

ATTCGAGTTGGCCTTTTGCATTCGGAAGGAAGGGGTCCGGATCCAAGCTCCGC

GCGGGGAGCAAGACATGCCCACCTCCCACGCAGTACGAACCAAATAATCAAT

AACTCTGTCAGACTGTCATGGGCGCTAACCTCCATCTCCACGGACGTCAGGCAT

GGCGTATTACGCAGCTTAAGCTGCCCCGCAAGGCAAGCCAAGCCAAGCCATG

GGGACCCGGCCCGGCGCGTGATACAG7A7A7AGGCCCGCCATGCATCAAGACA

GACACATCAGAGCATCCATCACTTCCCCACAGACCACGGACGGTCAGCCATGG

ATAAGGCATACATTGCCGTCCTCTCCTTCGCCTTCCTCTTCCTGCTCCACTACAT

TCTGGGCAAGAAGAGCAATGGCAGCAAGGGCGCCGCGCACCTCCCGCCGAGCC

CCCCGGCCGTCCCGTTCCTCGGCCACCTCCACCTCGTGGAGAAGCCGCTGCACG

CCGCGCTGTGCCGCCTCGGGGCGCGCTACGGCTCGGTCTTCTCGCTGCGGCTCG

GCGCGCGCAACGCCGTGGTGGTCTCCTCGCCGGCGTGCGCCAGGGAGTGCTTC

ACGGACCACGACGTGGCCTTCGCCAATCGGCCCCAGTTCCCCTCGCAGATGCTC

GTCTCCTACGGCGGCACCTCGCTCGTCAGCTCCAGCTACGGCCCGCACTGGC

GCAACCTCCGCCGCGTCGCCGCCGTGCGGCTGCTCTCCGCGCACCGCGTCGCCG

GCATGTCGGGCGTCATCGCCGCCGAGGTGCGCGCCATGGCGCGCCGCCTGTGC

CGCGCCGCCGCGGCGTGCCCCGCCCGGGrGGAGCTCAAGCGGAGCCTCTTCG

AGCTCTCCCTCAGCGTGCTCATGGAGACCATCGCGCGGACCAAGGGGACCCGG

TCGGAGGCGGACGGCGACACGGACATGTCCCTGGAGGCGCAGGAGTTCAAGCA

GGTGGTGGACGAGATCATCCCGCTCATCGGCGCCGCCAACGTGTGGGACTACC

TGCCGGTGATGCGGTGGCTCGACGTGTCCGGCGTGAGGAGCCGGATCCTGGCC

A portion ACGGTGAGCAGGAGGGACGCCTTCCTCCATCGGCTCATCGACGCCGAGCGGCG of SEQ ID GAGGATGGAGGAGGGCGGCGACGAGGGCGAGAAGAAGAGCATGATCGCCGTG NO: 1 CTCCTCACTCTGCAGAAGACAGAGCCGGAGCTGTACACTGATCAGATGATCATT

GCTCTGTGTGCGGTAAGTCCCTCTTCCACTTTGGTTGCTTCTCACATACCCGAGT (from

ACTTCAACTGTCGGTTTCTTCATTAAGGACATTGGCAATTTGGCATCATCAGTA promoter GATTTTTGACTCGTTTAAGTCATCAGATAGCAACAACTCGACCAGAGTCATTATo end of 3’ GTTAGTCCAGTACTCCACTTTTTATCTATCTTATCATATATCTAATCAGATAGTG UTR) GTCACTCAACGTAGGCGAGTAACACGGCCGTGTCACTTAGCTGGATGAATCCC

ACTTCCCACATGTTTATTTCTAGTAAATTTTGGCCTTTTCCCACTCGAGTACTTG

ACGTTAAGGCTCTCTTTGCCTTCGTAGGATTTGTGTAGTATTCTACTAAATTTTT

AATTTTTTTTTACCCTAATAGGTCTCGAACGTTCGGAATTTAAGCATGTCATCCT

GAATGTTAACCCATGACAACTTTGTTCCAGTTCATTTTTTATCAGAAAATTGCCA

TGTTTGTCAACCTTATCTTAACTAAAGTTCGGATTGCCATGCTTAAAATTTAAAT

Table 13: Sequencing results of CYP81A-5A, CYP81A-5B, CYP81A-5D in F₂ Progeny.

Table 14: Reference gene and target gene selected for RT-qPCR experiment.

Table 15: Primer and probe sequences.

Table 16: Primer details and PCR efficiencies.

Table 17: Predicted amplified sequences of TaqMan primers and probes. The underlined sequences correspond to the binding sites of the forward and reverse primers, respectively. Underlined and bolded sequences correspond to the binding sites of the TaqMan probe.

Table 18: Results of growth and development phenotyping and X² test of F3 plants. The Least Square (LS) means are reported in the table for each trait, and the standard error of the mean is reported in parentheses below the LS mean.

Exemplary guide RNA sequences (5’-3’):

Target Sequence 1 (including PAM): GCTTCTTGTTTCGGAAGGAAAGG (SEQ ID NO: 84)

Guide Sequence 1: GCUUCUUGUUUCGGAAGGAA (SEQ ID NO: 85)

Exemplary sgRNA Sequence 1 :

GCUUCUUGUUUCGGAAGGAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCC

GUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU (SEQ ID NO: 86)

Target Sequence 2 (including PAM): CTTGCTCCCCGCGCGGAGCT7GG (SEQ ID NO: 87)

Guide Sequence 2: CUUGCUCCCCGCGCGGAGCU (SEQ ID NO: 88)

Exemplary sgRNA Sequence 2:

CUUGCUCCCCGCGCGGAGCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCC

GUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU (SEQ ID NO: 89) Target Sequence 3 (including PAM): GCAGCTTAAGCTGCCCCGCAAGG (SEQ ID NO: 90)

Guide Sequence 3: GCAGCUUAAGCUGCCCCGCA (SEQ ID NO: 91)

Exemplary sgRNA Sequence 3:

GCAGCUUAAGCUGCCCCGCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCC

GUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU (SEQ ID NO: 92)

Target Sequence 4 (including PAM): TCGCTCGTCAGCTCCAGCTACGG (SEQ ID NO: 93)

Guide Sequence 4: UCGCUCGUCAGCUCCAGCUA (SEQ ID NO: 94)

Exemplary sgRNA Sequence 4:

UCGCUCGUCAGCUCCAGCUAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCC

GUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU (SEQ ID NO: 95)

Target Sequence 5 (including PAM): CGCGGCGTGCCCCGCCCGGGTGG (SEQ ID NO: 96)

Guide Sequence 5: CGCGGCGUGCCCCGCCCGGG (SEQ ID NO: 97)

Exemplary sgRNA Sequence 5 :

CGCGGCGUGCCCCGCCCGGGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCC

GUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU (SEQ ID NO: 98)

Exemplary Protein Sequences:

When the edit (e.g., generated by a CRISPR/Cas system) is in a regulatory region (such as a promoter), the protein sequence generated from the edited gene is the same as the one generated from the gene before the edit (such as a wild type gene).

CB037 CYP81A-5A wild type protein sequence

MDKAYIAVLSFAFLFLLHYILGKKSNGSKGAAHLPPSPPAVPFLGHLHLVEKPLHAALCRLGARYG

SVFSLRLGARNAVVVSSPACARECFTDHDVAFANRPQFPSQMLVSYGGTSLVSSSYGPHWRNLRR

VAAVRLLSAHRVAGMSGVIAAEVRAMARRLCRAAAACPARVELKRSLFELSLSVLMETIARTKGT

RSEADGDTDMSLEAQEFKQVVDEIIPLIGAANVWDYLPVMRWLDVSGVRSRILATVSRRDAFLHRL

IDAERRRMEEGGDEGEKKSMIAVLLTLQKTEPELYTDQMIIALCANMFVAGTETTSSTIEWAMSLLL

NHPAALNKAQAEMDASIGTSRMVTADDVPRLSYLQCIISETLRLYPAAPLLLPHESSADCKVGGYD

VPSGTMLIVNAYAIHRDPAVWEDPTAFRPERFEDGMGDGLLLMPFGMGRRRCPGEALALQTVGVV

LGTLVQCFDWERVDGVEVDMTEGVGITMPKAVALEAVCRPRAAMRDVLQKL (SEQ ID NO: 99)

‘Chinese Spring’ CYP81A-5A wild type protein sequence

MDKAYIAVLSFAFLFLLHYILGKKSNGSKGAAHLPPSPPAVPFLGHLHLVEKPLHAALCRLGARYG

SVFSLRLGARNAVVVSSPACARECFTDHDVAFANRPQFPSQMLVSYGGTSLVSSSYGPHWRNLRR

VAAVRLLSAHRVAGMSGVIAAEVRAMARRLCRAAAACPARVELKRSLFELSLSVLMETIARTKGT

RSEADGDTDMSLEAQEFKQVVDEIIPLIGAANVWDYLPVMRWLDVSGVRSRILATVSRRDAFLHRL

IDAERRRMEEGGDEGEKKSMIAVLLTLQKTEPELYTDQMIIALCANMFVAGTETTSSTIEWAMSLLL

NHPAALNKAQAEMDASIGTSRMVTADDVPRLSYLQCIISETLRLYPAAPLLLPHESSADCKVGGYD

VPSGTMLIVNAYAIHRDPAVWEDPTAFRPERFEDGMGDGLLLMPFGMGRRRCPGEALALQTVGVV

LGTLVQCFDWERVDRVEVDMTEGVGITMPKAVALEAVCRPRAAMRDVLQKL (SEQ ID NO: 100)

CB037 CYP81A-5B wild type protein sequence MDKAYIAVLSFAFLFLLHYILGKKSNGSKGAVQLPPSPPAIPFFGHLHLVEKPLHAALCRLGARYGP VFSLRLGARNAVVVSSPACARECFTEHDVAFANRPQFPSQMLVSYGGTSLVSSSYGPHWRNLRRV AAVRLLSAHRVAGMSGVIAAEVRAMARRLCRAAAASTGGGAARVELKRSLFELSLSVLMETIAQT KGTRPEADGDTDMSLEAQEFKQVVDEIIPLIGAANVWDYLPVMRWFDVSGVRSRILATVSRRDAFL

HRLIDAERRRMDEGGAGDEGEKKSMIAVLLTLQKTEPELYTDQMIIALCANLFVAGTETTSTTIEW

AMSLLLNHPAALKKAQAEMDASIGASRMVAADDVPRLSYFQCIINETLRLYPAAPLLLPHESSADC KVGGYDVPSGTMLIVNAYAIHRDPAVWEDPAAFRPERFEDGKADGLLLMPFGMGRRRCPGETLAL QTVGVVLGTLVQCFDWDRVDGAEVDMTEGVGITMPKAVALEAVCRPRAAMGDVLQKL (SEQ ID NO: 101)

‘Chinese Spring’ CYP81A-5B wild type protein sequence

MDKAYIAVLSFAFLFLLHYILGKKSNGSKGAVQLPPSPPAIPFFGHLHLVEKPLHAALCRLGARYGP

VFSLRLGARNAVVVSSPACARECFTEHDVAFANRPQFPSQMLVSYGGTSLVSSSYGPHWRNLRRV

AAVRLLSAHRVAGMSGVIAAEVRAMARRLCRAAAASTGGGAARVELKRSLFELSLSVLMETIAQT

KGTRPEADGDTDMSLEAQEFKQVVDEIIPLIGAANVWDYLPVMRWFDVSGVRSRILATVSRRDAFL

HRLIDAERRRMDEGGAGDEGEKKSMIAVLLTLQKTEPELYTDQMIIALCANLFVAGTETTSTTIEW

AMSLLLNHPAALKKAQAEMDASIGASRMVAADDVPRLSYLQCIINETLRLYPAAPLLLPHESSADC KVGGYDVPSGTMLIVNAYAIHRDPAVWEDPAAFRPERFEDGKADGLLLMPFGMGRRRCPGETLAL QTVGVVLGTLVQCFDWDRVDGAEVDMTEGVGITMPKAVALEAVCRPRAAMGDVLQKL (SEQ ID NO: 102)

CB037 and ‘Chinese Spring’ CYP81A-5D wild type protein sequence (allele 1)

MDKAYIAVLSFAFLFLLHYILGRKSNGSKGAVHLPPSPPAVPFFGHLHLVEKPLHAALCRLGARLGP

VFSLRLGARNAVVVSSPACARECFTDHDVAFANRPQFPSQMLVSYGGTSLVSSSYGPHWRNLRRV

AAVRLLSAHRVTGMSGVGAARVELKRSLFELSLSVLMETIARTKGTRSEADGDTDMSLEAQEFKQ VVDEIIPLIGAANVWDYLPVMRWFDVSGVRSRILATVSRRDAFLHRLIDAERRRMEEGGDDGEKKS

MIAVLLTLQKTEPELYTDQMIIALCANMFVAGTETTSSTIEWAMSLLLNHPAALKKAQAEMDASIG TSRMVTADDVPRLSYLQCIINETLRLYPAAPLLLPHESSADCKVGGYDVPSGTMLIVNAYAIHRDPA VWEDPTAFRPERFEDGKGDGLLLMPFGMGRRRCPGETLALQTVGVVLGTLVQCFDWERVDGLEV DMTEGVG1TMPKSVALEAVCRPRAAMRDVLQKL (SEQ ID NO: 103)

CB037 and ‘Chinese Spring’ CYP81A-5D wild type protein sequence (allele 2)

MDKAYIAVLSFAFLFLLHYILGRKSNGSKGAVHLPPSPPAVPFFGHLHLVEKPLHAALCRLGARLGP

VFSLRLGARNAVVVSSPACARECFTDHDVAFANRPQFPSQMLVSYGGTSLVSSSYGPHWRNLRRV AAVRLLSAHRVTGMSGVIAAEVRAMARRLCRAAAASPGGDGAARVELKRSLFELSLSVLMETIAR TKGTRSEADGDTDMSLEAQEFKQVVDEIIPLIGAANVWDYLPVMRWFDVSGVRSRILATVSRRDAF

LHRLIDAERRRMEEGGDDGEKKSMIAVLLTLQKTEPELYTDQMIIALCANMFVAGTETTSSTIEWA MSLLLNHPAALKKAQAEMDASIGTSRMVTADDVPRLSYLQCIINETLRLYPAAPLLLPHESSADCK VGGYDVPSGTMLIVNAYAIHRDPAVWEDPTAFRPERFEDGKGDGLLLMPFGMGRRRCPGETLALQ TVGVVLGTLVQCFDWERVDGLEVDMTEGVGITMPKSVALEAVCRPRAAMRDVLQKL (SEQ ID NO: 104)

It will be apparent that the precise details of the methods or compositions described may be varied or modified without departing from the spirit of the described aspects of the disclosure. We claim all such modifications and variations that fall within the scope and spirit of the claims below. REFERENCES

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Claims

We claim:

1. A method for generating a plant with increased tolerance to a herbicide, comprising: increasing expression of one or more herbicide detoxification genes, and/or increasing activity of one or more proteins encoded by the one or more herbicide detoxification genes in a plant, thereby generating the plant with increased tolerance to a herbicide, wherein the one or more herbicide detoxification genes comprise: any of SEQ ID NOs: 1-7 (CYP81A-5A) or a nucleic acid sequence that comprises at least 80% identity to any of SEQ ID NOs: 1-7; any of SEQ ID NOs: 8-13 (CYP81A-5B) or a nucleic acid sequence that comprises at least 80% identity to any of SEQ ID NOs: 8-13; and/or any of SEQ ID NOs: 14-19 (CYP81A-5D) or a nucleic acid sequence that comprises at least 80% identity to any of SEQ ID NOs: 14-19; or wherein the one or more herbicide detoxification genes encode a protein comprising a sequence of any of SEQ ID NOs: 99-104, or a sequence that comprises at least 80% identify to any of SEQ ID NOs: 99- 104.

2. A method for generating a plant with increased tolerance to a herbicide, comprising: increasing expression of one or more herbicide detoxification genes, and/or increasing activity of one or more proteins encoded by the one or more herbicide detoxification genes in a plant cell or plant part, and growing the plant cell or plant part into a plant, thereby generating the plant with increased tolerance to a herbicide, wherein the one or more herbicide detoxification genes comprise: any of SEQ ID NOs: 1-7 (CYP81A-5A) or a nucleic acid sequence that comprises at least 80% identity to any of SEQ ID NOs: 1-7; any of SEQ ID NOs: 8-13 (CYP81A-5B) or a nucleic acid sequence that comprises at least 80% identity to any of SEQ ID NOs: 8-13; and/or any of SEQ ID NOs: 14-19 (CYP81A-5D) or a nucleic acid sequence that comprises at least 80% identity to any of SEQ ID NOs: 14-19; or wherein the one or more herbicide detoxification genes encode a protein comprising a sequence of any of SEQ ID NOs: 99-104, or a sequence that comprises at least 80% identify to any of SEQ ID NOs: 99- 104.

3. The method of claim 1, wherein the increasing expression of one or more herbicide detoxification genes, and/or increasing activity of one or more proteins encoded by the one or more herbicide detoxification genes comprises: introducing one or more exogenous nucleic acid molecules into the plant, thereby generating a transformed plant, or into the plant part or plant cell, thereby generating a transformed plant part or plant cell, wherein the one or more exogenous nucleic acid molecules comprise the one or more herbicide detoxification genes, increase expression of the one or more herbicide detoxification genes, and/or increase activity of the one or more proteins.

4. The method of claim 3, wherein the one or more exogenous nucleic acid molecules generates one or more gain-of-function mutations in the one or more herbicide detoxification genes, wherein the one or more herbicide detoxification genes are endogenous to the plant, plant part, or plant cell.

5. The method of claim 4, wherein the one or more gain-of-function mutations are in the promoter region of the one or more herbicide detoxification genes, and increase expression of the one or more herbicide detoxification genes in response to a herbicide safener or herbicide.

6. The method of claim 5, wherein the herbicide safener is cloquintocet-mexyl (CM) or a derivative or analog thereof.

7. The method of claim 6, wherein the gain-of-function mutation is a single G insertion about 256 bp upstream of TATA box.

8. The method of claim 7, wherein the single G insertion is immediately after a position corresponding to position 137 of SEQ ID NO: 1.

9. The method of claim 3, wherein the one or more exogenous nucleic acid molecules generate a mutated herbicide detoxification gene that comprises any of SEQ ID NOs: 20-22, or a nucleic acid sequence that comprises at least 90% identity to any of SEQ ID NOs: 20-22 and has a G at a position corresponding to position 138 of SEQ ID NO: 20.

10. The method of claim 3, wherein the one or more exogenous nucleic acid molecules comprise one or more guide nucleic acid molecules that are complementary to one or more regions of the one or more herbicide detoxification genes, wherein the one or more herbicide detoxification genes are endogenous to the plant, plant part, or plant cell.

11. The method of claim 3, wherein the one or more exogenous nucleic acid molecules further comprise a nucleic acid molecule encoding a Cas protein, or the method further comprises introducing one or more Cas proteins into the plant, plant part, or plant cell.

12. The method of claim 3, wherein the one or more exogenous nucleic acid molecules are operably linked to a heterologous promoter.

13. The method of claim 3, wherein the transformed plant, plant cell, or plant part comprises one or more gain-of-function mutations in the one or more herbicide detoxification genes, wherein the one or more herbicide detoxification genes are endogenous to the plant, plant part, or plant cell.

14. The method of claim 3, wherein the transformed plant, plant part, or plant cell comprises the one or more exogenous nucleic acid molecules comprising the one or more herbicide detoxification genes, wherein the one or more exogenous herbicide detoxification genes are integrated or not integrated into a genome of the transformed plant, plant part, or plant cell.

15. The method of claim 14, wherein the plant, plant part, or plant cell does not comprise the one or more herbicide detoxification genes before introduction of the one or more exogenous nucleic acid molecules.

16. The method of claim 1, wherein the plant, plant part, or plant cell is from the Poaceae family.

17. The method of claim 16, wherein the Poaceae is a cereal grass.

18. The method of claim 17, wherein the cereal grass is wheat.

19. The method of claim 15, wherein the plant is a dicot, or the plant part or plant cell is from a dicot.

20. The method of claim 1, wherein the expression of the one or more herbicide detoxification genes, and/or activity of the one or more proteins is increased as compared to a control plant, plant part, or plant cell.

21. The method of claim 1, wherein the expression of the one or more herbicide detoxification genes, and/or activity of the one or more proteins is increased by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 120%, 150%, or 200% as compared to a control plant, plant part, or plant cell.

22. The method of claim 1, wherein the tolerance to a herbicide is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 75%, at least 90%, at least 100%, at least 200%, or at least 400% as compared to a control plant.

23. The method of claim 3, further comprising producing a transformed plant tissue from the transformed plant cell.

24. The method of claim 2, further comprising producing a transformed plantlet from the transformed plant part or plant cell, or from the transformed plant tissue, wherein the transformed plantlet has increased tolerance to a herbicide.

25. The method of claim 24, further comprising producing a transformed progeny from the transformed plantlet, wherein the transformed progeny has increased tolerance to a herbicide.

26. The method of claim 25, further comprising growing the transformed plantlet or the transformed progeny into a transformed plant, wherein the transformed plant has increased tolerance to a herbicide.

27. The method of claim 3, further comprising using the transformed plant or a clone of the transformed plant in a breeding method.

28. The method of claim 27, wherein the breeding method comprises selfing or crossing the transformed plant or clone of the transformed plant.

29. A transformed plant, transformed plant part, or transformed plant cell made by the method of claim 3.

30. The method of claim 1, or the transformed plant, transformed plant part, transformed plant cell, transformed plant tissue, transformed plantlet, or transformed progeny of claim 29, wherein the transformed plant, transformed plant part, transformed plant cell, transformed plant tissue, transformed plantlet, or transformed progeny further comprises one or more additional exogenous nucleic acids encoding one or more proteins that confer upon the transformed plant, transformed plant part, transformed plant cell, transformed plant tissue, transformed plantlet, or transformed progeny a desired trait, wherein the desired trait is one or more of drought tolerance, heat tolerance, low or high soil pH level tolerance, salt tolerance, resistance to an insect, resistance to a bacterial disease, resistance to a viral disease, resistance to a fungal disease, resistance to a nematode, resistance to a pest, male sterility, abiotic stress tolerance, modified phosphorus characteristics, modified antioxidant characteristics, modified essential seed amino acid characteristics, decreased phytate, modified fatty acid metabolism, and modified carbohydrate metabolism.

31. A method of producing a commodity plant product, comprising collecting or producing the commodity plant product from the transformed plant, transformed plant part, transformed plant cell, transformed plant tissue, transformed plantlet, or transformed progeny of claim 29, optionally, wherein the commodity plant product comprises a non-native nucleic acid molecule or protein from the transformed plant, transformed plant part, transformed plant cell, transformed plant tissue, transformed plantlet, or transformed progeny; and optionally, wherein the commodity product comprises a protein concentrate, protein isolate, leaves, extract, oil, bean, and/or seed.

32. A method of producing plant seed, comprising crossing the transformed plant, transformed plant part, transformed plant cell, transformed plant tissue, transformed plantlet, or transformed progeny of claim 29 with itself or a second plant.

33. The method of claim 1, wherein the plant part is a protoplast, leaf, stem, root, root tips, anther, pistil, stamen, seed, embryo, pollen, ovule, microspore, sporophyte, gametophyte, cotyledon, hypocotyl, flower, shoot, tissue, petiole, or meristematic cell.

34. A method for breeding a plant with increased tolerance to a herbicide, comprising crossing the transformed plant of claim 29 with a second plant; obtaining seed from the crossing; planting the seeds and growing the seeds to progeny plants; and selecting from the progeny plants those with increased tolerance to a herbicide.

35. The method of claim 34, further comprising producing clones of the progeny plants, wherein the clones are selected based on increased tolerance to a herbicide.

36. A seed that produces or is produced by the transformed plant of claim 29, wherein the seed comprises one or more gain-of-function mutations in the one or more endogenous herbicide detoxification genes, and/or comprises the one or more exogenous herbicide detoxification genes.

37. A transformed plant, plant part, plant cell, or seed, comprising one or more gain-of-function mutations in one or more endogenous herbicide detoxification genes, or comprising one or more exogenous herbicide detoxification genes integrated into a genome of the transformed plant, plant part, plant cell, or seed, wherein the one or more herbicide detoxification genes comprise: any of SEQ ID NOs: 1-7 (CYP81A-5A) or a nucleic acid sequence that comprises at least 80% identity to any of SEQ ID NOs: 1-7; any of SEQ ID NOs: 8-13 (CYP81A-5B) or a nucleic acid sequence that comprises at least 80% identity to any of SEQ ID NOs: 8-13; and/or any of SEQ ID NOs: 14-19 (CYP81A-5D) or a nucleic acid sequence that comprises at least 80% identity to any of SEQ ID NOs: 14-19; or wherein the one or more herbicide detoxification genes encode a protein comprising a sequence of any of SEQ ID NOs: 99-104, or a sequence that comprises at least 80% identify to any of SEQ ID NOs: 99- 104.

38. The transformed plant, plant part, plant cell, or seed of claim 37, which does not comprise a transgene used to generate the one or more gain-of-function mutations.

39. The transformed plant, plant part, plant cell, or seed of claim 37, wherein the gain-of-function mutation is a single G insertion about 256 bp upstream of TATA box.

40. The transformed plant, plant part, plant cell, or seed of claim 39, wherein the single G insertion is immediately after a position corresponding to position 137 of SEQ ID NO: 1.

41. The transformed plant, plant part, plant cell, or seed of claim 37, wherein the transformed plant, plant part, plant cell, or seed comprises any of SEQ ID NOs: 20-22, or a nucleic acid sequence that comprises at least 90% identity to any of SEQ ID NOs: 20-22 and has a G at a position corresponding to position 138 of SEQ ID NO: 20.

42. The transformed plant, plant part, plant cell, or seed of claim 37, which is transgene-free.

43. The transformed plant, plant part, plant cell, or seed of claim 37, which comprises one or more transgenes.

44. The transformed plant, plant part, plant cell, or seed of claim 37, wherein the plant is a monocot or dicot.

45. The transformed plant of claim 37, which has tolerance to a herbicide increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 75%, at least 90%, at least 100%, at least 200%, or at least 400% as compared to a control plant.

46. The transformed plant of claim 45, wherein the tolerance to a herbicide is tolerance to a herbicide in presence of a herbicide safener.

47. The transformed plant of claim 46, wherein the herbicide safener is cloquintocet-mexyl (CM) or a derivative or analog thereof.

48. The method of claim 1, wherein the herbicide is propoxycarbazone-sodium (PROP) or a derivative or analog thereof.

49. A method of growing the transformed plant, plant part, plant cell, or seed of claim 37 in the presence of a herbicide.

50. The method of claim 49, wherein the herbicide is an acetolactate synthase (ALS) inhibitor.

51. The method of claim 50, wherein the ALS inhibitor is a sulfonylamino carbonyl triazolinone.

52. The method of claim 51 , wherein the sulfonylamino carbonyl triazolinone is propoxycarbazone-sodium (PROP) or an analog or derivative thereof.

53. The method of claim 49, further comprising growing the transformed plant, plant part, plant cell, or seed in the presence of a herbicide safener.

54. The method of claim 53, wherein the herbicide safener is cloquintocet-mexyl (CM) or a derivative or analog thereof.