Compositions and methods for enhanced translation in plants

Abstract

Disclosed are expression cassettes for enhancing translation of a gene comprising at least a portion of the 5' UTR of MYB28 operably linked to: (a) a gene to be expressed downstream of the 5' UTR of MYB28; (b) a 3' UTR downstream of the gene to be expressed; and (c) a promoter upstream of the 5' UTR of MYB28. Also disclosed are nucleic acid vectors comprising a disclosed expression cassette; recombinant plant cells capable of exhibiting enhanced translation of a gene, wherein said cell comprises a construct comprising at least a portion of the 5' UTR of MYB28 gene upstream of the gene to be expressed; and plants comprising a disclosed recombinant plant cell.

Description

COMPOSITIONS AND METHODS FOR ENHANCED TRANSLATION IN PLANTS CROSS-REFERENCE TO RELATED APPLICATION[S]

[0001] This application claims priority to, and the benefit of, U.S. Provisional Patent Application Serial No. 63 / 680,004 filed on August 6, 2024, the entire contents of which are incorporated herein by reference as if set forth in its entirety. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under Grant No. 2142898, awarded by the National Science Foundation. The government has certain rights in the invention. SEQUENCE LISTING

[0003] The instant application contains a Sequence Listing which has been submitted electronically in .XML format and is hereby incorporated by reference in its entirety. Said .XML copy, created on August 5, 2025, is named “T19283WO001(222112_2500) sequence listing.xml” and is 86,016 bytes in size. SUMMARY

[0004] In accordance with the purpose(s) of the disclosure, as embodied and broadly described herein, the disclosure, in one aspect, relates to methods and compositions for enhanced expression of a gene by enhanced translation of the gene, e.g., by operably linking a disclosed 5’ UTR to the gene whose expression is to be enhanced. In a further aspect, the enhanced expression relates to enhanced expression MYB28, and thereby, enhancing the expression of genes regulated by MYB28.

[0005] Disclosed are expression cassettes for enhancing translation of a gene comprising at least a portion of the 5’ UTR of MYB28 operably linked to: (a) a gene to be expressed downstream of the 5’ UTR of MYB28; (b) a 3’ UTR downstream of the gene to be expressed; and (c) a promoter upstream of the 5’ UTR of MYB28.

[0006] Also disclosed are nucleic acid vectors comprising a disclosed expression cassette.

[0007] Also disclosed herein are methods of enhancing the translation of a gene to be expressed in a plant comprising introducing a construct comprising at least a portion of the 5’ UTR of MYB28 into the cells of said plant upstream of the gene to be expressed.

[0008] Also disclosed herein are recombinant plant cells capable of exhibiting enhanced translation of a gene, wherein said cell comprises a construct comprising at least a portion of the 5’ UTR of MYB28 gene upstream of the gene to be expressed.

[0009] Also disclosed herein are a plant or plants comprising a disclosed recombinant plant cell.

[0010] While aspects of the present disclosure can be described and claimed in a particular statutory class, such as the system statutory class, this is for convenience only and one of skill in the art will understand that each aspect of the present disclosure can be described and claimed in any statutory class. Unless otherwise expressly stated, it is in no way intended that any method or aspect set forth herein be construed as requiring that its steps be performed in a specific order. Accordingly, where a method claim does not specifically state in the claims or descriptions that the steps are to be limited to a specific order, it is no way intended that an order be inferred, in any respect. This holds for any possible non-express basis for interpretation, including matters of logic with respect to arrangement of steps or operational flow, plain meaning derived from grammatical organization or punctuation, or the number or type of aspects described in the specification. BRIEF DESCRIPTION OF THE FIGURES

[0011] The accompanying figures, which are incorporated in and constitute a part of this specification, illustrate several aspects and together with the description serve to explain the principles of the disclosure.

[0012] FIGs. 1A-1C show representative data that single locus dominant mutations are responsible for the restored high-auxin morphological phenotype in ref5 suppressors: 19-6 and 30-3. FIG.1A shows a representative schematic of the IAOx-mediated metabolic network in Arabidopsis, as well as the major route for IAA biosynthesis. FIG.1A shows a representative data for 3-week-old 19-6 (rhax1-1D) and 30-3 (rhax1-2D) compared to wild type (WT) and ref5. FIG.1A shows representative data of number of plants in the F1 and F2 generations showing either rhax-looking or ref5-looking phenotype of ref5 x 19-6, ref5 x 30-3, and 19-6 x 30-3 crosses.

[0013] FIGs.2A-2B show representative BSA analysis of SNP occurrence ratios in rhax- looking and ref5-looking pools of F2 populations from ref5 crossed with rhax1-1D (19-6). FIG. 2A shows representative BSA results showing the SNP occurrence ratios in ref5-looking versus rhax-looking Arabidopsis mutant populations across chromosomes 1 to 5. Each scatter plot is dedicated to a chromosome and plots the rhax-looking SNP occurrence ratio againstthe ref5-looking SNP occurrence ratio. Vertical dashed lines indicate the targeted occurrence ratio thresholds of 0.6 and 0.7 in the rhax-looking pool, while the horizontal dashed line marks the zero SNP occurrence ratio threshold in the ref5-looking pool. SNPs that are unique to the rhax-looking pool and fall within the 0.6 to 0.7 occurrence ratio range are highlighted with red dots. FIG.2B shows a representative summary list of the highlighted SNPs from BSA analysis. This table specifically points out SNPs that are uniquely present in the rhax-looking pool with an occurrence ratio in the specified range (red dots on scatter plot), which are of particular interest for further genetic analysis.

[0014] FIGs. 3A-3D show representative data pertaining to expression profiles and co- expression analysis of candidate genes identified by BSA. FIG.3A shows a representative heat map showing the expression levels of 12 candidate genes identified through BSA in WT, ref5, rhax1-1D. These genes are listed along the vertical axis with different Arabidopsis genotypes arrayed along the horizontal axis. The color gradient represents Z-score of the expression level in Transcripts Per Million (TPM), with yellow indicating high expression and blue indicating low expression across different genotypes. FIG.3B shows a table summarizing representative data pertaining to the fold changes and associated P-values (in parentheses) for the RNA-seq data of these 12 genes across all genotype combinations. FIG.3C shows representative Venn diagrams illustrating the overlap between genes upregulated in rhax1-1D compared to ref5 (a total of 993 genes) and the top 50 genes co-expressed with each candidate gene, as retrieved from the ATTED database. Each diagram corresponds to one of the candidate genes. FIG.3D shows a list of 30 genes that are up-regulated in rhax1-1D and co-expressed with MYB28 shown in FIG.3C.

[0015] FIGs. 4A-4D show representative data pertaining to rhax1-1D and rhax1-2D both have a C-to-T mutation in the 5′ UTR of MYB28 and show increased aliphatic glucosinolates. FIG.4A shows a diagram of MYB28 isoforms 1 and 2 with the identified C-to-T mutation in rhax1-1D and rhax1-2D. Grey box indicates 5′ UTR, white box indicates exons, and the intervening line denotes an intron. The rhax1-1D and rhax1-2D mutations occur 152 and 165 bp, respectively, upstream of the ATG translation start site of MYB28 isoform 2. FIG.4B shows representative data relating to MYB28 expression level as Transcripts Per Million (TPM) from RNA-seq data in rhax1-1D compared to WT, ref5 (n = 3). Data represent means ± SD. FIG. 4A shows a schematic of the aliphatic glucosinolate biosynthesis pathway with heatmaps of the fold changes in genes encoding for enzymes and activators involved in the aliphatic glucosinolate biosynthesis pathway in Arabidopsis from RNA-seq. Gene names highlighted in red represent those enzymes that were upregulated in rhax1-1D when compared to ref5. Other enzymes in the pathway are labeled in blue for differentiation. FIG. 4A shows 3MSOP,4MSOB, and 8MSOO glucosinolate content in 3-week-old whole aerial parts of WT, ref5, rhax1-1D, rhax1-2D (n = 4). Data represent means ± SD. with a schematic of aliphatic glucosinate biosynthesis pathway.

[0016] FIGs.5A-5B show representative data pertaining to the observation that rhax1-1D and rhax1-2D mutations are sufficient to increase production of aliphatic glucosinolates. FIG. 5A shows representative 3-week-old rhax1-1D / WT and rhax1-2D / WT with WT. FIG.5B shows 4MSOB and 8MSOO glucosinolate content in 3-week-old whole aerial parts of rhax1-1D / WT and rhax1-2D / WT with WT (n = 3). Data represent mean ± SD.

[0017] FIGs.6A-6C show representative data pertaining to the observation that a single C- to-T gain-of-function mutation in 5′ UTR of MYB28 in rhax1-1D is responsible for the increase of aliphatic glucosinolates. FIG. 6A shows diagrams of the genetic alterations in MYB28CR / rhax1-1D-3 and MYB28CR / rhax1-1D-4. Yellow highlight represents the 20 bp target site for the CRISPR construct. Green highlight represents the PAM recognition site. Red highlight represents the genetic alterations of the MYB28 CRISPR line. FIG. 6A shows representative 3-week-old rhax1-1D and MYB28 CRISPR lines in rhax1-1D, MYB28CR / rhax1- 1D-3 and MYB28CR / rhax1-1D-4, compared to WT and ref5. (c) 4MSOB and 8MSOO glucosinolate content in 3-week-old whole aerial parts of MYB28CR / rhax1-1D-3 and MYB28CR / rhax1-1D-4 compared to WT, ref5, rhax1-1D, and rhax1-2D (n = 4). Data represent means ± SD; and “n.d.” refers to not detected.

[0018] FIG.7 shows a restriction map of the disclosed 5’ UTR (corresponding to SEQ ID NO:1).

[0019] FIG. 8 shows a plasmid map of a disclosed plasmid expression construct, pCHF- MYB28 (rhax1-1D)-MYB28 CDS (corresponding to SEQ ID NO: 4).

[0020] FIG.9 shows a plasmid map of a disclosed plasmid expression construct, pCHF3- MYB28(Wild type)-MYB28_CDS (corresponding to SEQ ID NO: 5).

[0021] FIG.10 shows a plasmid map of a disclosed plasmid expression construct, p35S- 5UTR-wt-GFP. The wild-type 5’UTR adjacent to the coding sequence for wild-type GFP is transcriptionally driven by a 35S promoter.

[0022] FIG.11 shows a plasmid map of a disclosed plasmid expression construct, p35S- 5UTR-mut-GFP. The mutated 5’UTR adjacent to the coding sequence for wild-type GFP is transcriptionally driven by a 35S promoter.

[0023] FIG.12 shows alignment of 5’UTR sequences of MYB28 aligned with the indicatedBrassica crops showing that the ‘C’ sequences of both rhax1-1 and rhax1-2 positions are conserved. In the figure the red circle highlights TTCTC: Rhax1-2D position (C to ‘T’ mutation in rhax1-2D mutant); and the blue circle highlights TCTG: Rhax1-1D position (C to ‘T’ mutation in rhax1-1D mutant). The query sequence is based on SEQ ID NO:1 and SEQ ID NO:2.

[0024] FIG.13 shows alignment of 5’UTR sequences of MYB28 aligned with the indicated Brassica crops showing that the ‘C’ sequences of both rhax1-1 and rhax1-2 positions are conserved. In the figure the red circle highlights TTCTC: Rhax1-2D position (C to ‘T’ mutation in rhax1-2D mutant); and the blue circle highlights TCTG: Rhax1-1D position (C to ‘T’ mutation in rhax1-1D mutant). The query sequence is based on SEQ ID NO:1 and SEQ ID NO:2.

[0025] FIG.14 shows alignment of 5’UTR sequences of MYB28 aligned with the indicated Brassica crops showing that the ‘C’ sequences of both rhax1-1 and rhax1-2 positions are conserved. In the figure the red circle highlights TTCTC: Rhax1-2D position (C to ‘T’ mutation in rhax1-2D mutant); and the blue circle highlights TCTG: Rhax1-1D position (C to ‘T’ mutation in rhax1-1D mutant). The query sequence is based on SEQ ID NO:1 and SEQ ID NO:2.

[0026] FIGs.15A-15D show a single nucleotide change increases the translation of MYB28, leading to increased glucosinolate production in Arabidopsis. (FIG. 15A) Schematic representation of the MYB28 genomic DNA bound to HA-tag driven by a 2-kilobase sequence of MYB28 promoter, illustrating MYB28:HA expression under the control of either the native (p2KbC::MYB28:HA) or modified (p2KbT::MYB28:HA) 5’UTR. The yellow line in promoter indicates the site of mutation (152bp upstream of transcription start site). (FIG.15B) Aliphatic glucosinolate content (3MSOP, 4MSOB, and 8MSOO) in mature siliques from 7-week-old T1 transgenic myb28 myb29 plants expressing HA-tagged MYB28 under the native promoter carrying the native 5’UTR (p2KbC::MYB28:HA) and modified 5’UTR containing a T substitution 152 bp upstream of the transcription start site (p2KbT::MYB28:HA). Asterisks (*) represent not detected. (FIG.15C) RT-PCR analysis of MYB28:HA transcript levels in 3-week-old rosettes of T1 transgenic lines. ACTIN2 was used as loading control. (FIG.15D) Western blot analysis of MYB28-HA protein levels in 3-week-old rosettes of T1 transgenic lines. Ponceau staining shows equal loading.

[0027] FIGs. 16A-16C show the 5′UTR carrying the rhax1-1D mutation activates the translation of eGFP. (FIG. 16A) Schematic representation of the MYB285′UTR, with GFP expression driven by the 35S promoter, illustrating GFP expression under the control of either the native (p35::5′UTRC:GFP) or modified (p35::5′UTRT:GFP) 5′UTR. The yellow line in 5′UTR indicates the site of mutation (152bp upstream of transcription start site). (FIG.16B) RT-PCR analysis of GFP transcript levels in 3-week-old rosettes from T1 transgenic lines expressingGFP under the control of either the native or modified 5′UTR. The primer binding positions, labeled P1 and P2, are highlighted to indicate the regions used for RT-PCR amplification. ACTIN2 was used as loading control. (FIG.16C) Western blot analysis of GFP protein levels in corresponding T1 transgenic lines. Ponceau staining shows equal loading.

[0028] FIGs.17A-17B show myb28uORF-Δ3bpa CRISPR-mediated transgenic line carrying a 3bp deletion mutation in the 5′UTR increases aliphatic glucosinolate production in Arabidopsis. (FIG. 17A) Schematic diagram of myb28uORF-Δ3bpshowing 3bp deletion. Green arrow represents 117bp µORF of MYB28. Asterisks represent the ref5 rhax1-1D and ref5 rhax1-2D mutations. Highlighted elements: yellow (20bp target site), green (PAM sequence), and red (induced mutations). (FIG.17B) 3MSOP, 4MSOB, and 8MSOO content in 3-week-old rosette of myb28uORF-Δ3bpcompared to WT (n=3). Data represent means ± SD.

[0029] FIG.18 shows conserved 5′UTR Sequences in Brassica Crops. Alignment of 5′UTR sequences from several Brassica species reveals conserved regions, including the rhax1-1D and rhax1-2D elements, a three-base-pair deletion mutation (highlighted in red lines), and upstreamORF (uORF)(purple line). Sequences shown correspond to Arabidopsis thaliana MYB28 and its homologs in Brassica juncea (Braju.18G182400), Brassica oleracea (LOC106327854; NCBI accession: XM_013766140), Brassica rapa (Brapa.I00661), and Camelina sativa (CsCN113611.18G182300).

[0030] Additional advantages of the disclosure will be set forth in part in the description which follows, and in part will be obvious from the description, or can be learned by practice of the disclosure. The advantages of the disclosure will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the disclosure, as claimed. DETAILED DESCRIPTION

[0031] The present disclosure can be understood more readily by reference to the following detailed description of the disclosure and the Examples included therein. A. DEFINITIONS

[0032] Throughout the present disclosure, a number of terms are used. In order to provide a clear and consistent understanding of the specification and claims, including the scope to be given such terms, the following definitions are provided.

[0033] As used herein, “comprising” is to be interpreted as specifying the presence of thestated features, integers, steps, or components as referred to, but does not preclude the presence or addition of one or more features, integers, steps, or components, or groups thereof. Moreover, each of the terms “by”, “comprising,” “comprises”, “comprised of,” “including,” “includes,” “included,” “involving,” “involves,” “involved,” and “such as” are used in their open, non-limiting sense and may be used interchangeably. Further, the term “comprising” is intended to include examples and aspects encompassed by the terms “consisting essentially of” and “consisting of.” Similarly, the term “consisting essentially of” is intended to include examples encompassed by the term “consisting of.

[0034] As used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a gene,” “a 5’ UTR,” or “a promoter” includes mixtures of two or more such genes, 5’ UTRs, or promoters, and the like.

[0035] Ranges can be expressed herein as from “about” one particular value, and / or to “about” another particular value. When such a range is expressed, a further aspect includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms a further aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0036] References in the specification and concluding claims to parts by weight of a particular element or component in a composition denotes the weight relationship between the element or component and any other elements or components in the composition or article for which a part by weight is expressed. Thus, in a compound containing 2 parts by weight of component X and 5 parts by weight component Y, X and Y are present at a weight ratio of 2:5, and are present in such ratio regardless of whether additional components are contained in the compound.

[0037] A weight percent (wt. %) of a component, unless specifically stated to the contrary, is based on the total weight of the formulation or composition in which the component is included.

[0038] The term “about” as used herein means greater or lesser than the value or range ofvalues stated by 10 percent, but is not intended to designate any value or range of values to only this broader definition. Each value or range of values preceded by the term “about” is also intended to encompass the embodiment of the stated absolute value or range of values.

[0039] As used herein, the terms “optional” or “optionally” means that the subsequently described event or circumstance can or can not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

[0040] The term “introduced” in the context of inserting a nucleic acid into a cell, means “transfection” or “transformation” or “transduction” and includes reference to the incorporation of a nucleic acid into a eukaryotic or prokaryotic cell where the nucleic acid may be incorporated into the genome of the cell (e.g., chromosome, plasmid, plastid or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed.

[0041] As used herein “recombinant” includes reference to a cell or vector, that has been modified by the introduction of a heterologous nucleic acid or that the cell is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found in identical form within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under-expressed or not expressed at all as a result of deliberate human intervention. The term “recombinant” as used herein does not encompass the alteration of the cell or vector by naturally occurring events (e.g., spontaneous mutation, natural transformation / transduction / transposition) such as those occurring without deliberate human intervention.

[0042] As used herein, the term “intron” refers to any nucleic acid sequence comprised in a gene (or expressed polynucleotide sequence of interest) that is transcribed but not translated. Introns include untranslated nucleic acid sequence within an expressed sequence of DNA, as well as the corresponding sequence in RNA molecules transcribed therefrom. A construct described herein can also contain sequences that enhance translation and / or mRNA stability such as introns. An example of one such intron is the first intron of gene II of the histone H3 variant of Arabidopsis thaliana or any other commonly known intron sequence. Introns can be used in combination with a promoter sequence to enhance translation and / or mRNA stability.

[0043] The term “isolated”, as used herein means having been removed from its natural environment, or removed from other compounds present when the compound is first formed. The term “isolated” embraces materials isolated from natural sources as well as materials (e.g., nucleic acids and proteins) recovered after preparation by recombinant expression in a host cell, or chemically-synthesized compounds such as nucleic acid molecules, proteins, andpeptides.

[0044] The term “purified”, as used herein relates to the isolation of a molecule or compound in a form that is substantially free of contaminants normally associated with the molecule or compound in a native or natural environment, or substantially enriched in concentration relative to other compounds present when the compound is first formed, and means having been increased in purity as a result of being separated from other components of the original composition. The term “purified nucleic acid” is used herein to describe a nucleic acid sequence which has been separated, produced apart from, or purified away from other biological compounds including, but not limited to polypeptides, lipids and carbohydrates, while effecting a chemical or functional change in the component (e.g., a nucleic acid may be purified from a chromosome by removing protein contaminants and breaking chemical bonds connecting the nucleic acid to the remaining DNA in the chromosome).

[0045] The term “synthetic”, as used herein refers to a polynucleotide (i.e., a DNA or RNA) molecule that was created via chemical synthesis as an in vitro process. For example, a synthetic DNA may be created during a reaction within an Eppendorf™ tube, such that the synthetic DNA is enzymatically produced from a native strand of DNA or RNA. Other laboratory methods may be utilized to synthesize a polynucleotide sequence. Oligonucleotides may be chemically synthesized on an oligo synthesizer via solid-phase synthesis using phosphoramidites. The synthesized oligonucleotides may be annealed to one another as a complex, thereby producing a “synthetic” polynucleotide. Other methods for chemically synthesizing a polynucleotide are known in the art, and can be readily implemented for use in the present disclosure.

[0046] For the purposes of the present disclosure, a “gene,” includes a DNA region encoding a gene product (see infra), as well as all DNA regions which regulate the production of the gene product, whether or not such regulatory sequences are adjacent to coding and / or transcribed sequences. Accordingly, a gene includes, but is not necessarily limited to, promoter sequences, terminators, translational regulatory sequences such as ribosome binding sites and internal ribosome entry sites, enhancers, silencers, insulators, boundary elements, replication origins, matrix attachment sites and locus control regions.

[0047] As used herein the terms “native” or “natural” define a condition found in nature. A “native DNA sequence” is a DNA sequence present in nature that was produced by natural means or traditional breeding techniques but not generated by genetic engineering (e.g., using molecular biology / transformation techniques).

[0048] As used herein a “transgene” is defined to be a nucleic acid sequence that encodes a gene product, including for example, but not limited to, an mRNA. In one embodiment the transgene is an exogenous nucleic acid, where the transgene sequence has been introduced into a host cell by genetic engineering (or the progeny thereof) where the transgene is not normally found. In one example, a transgene encodes an industrially or pharmaceutically useful compound, or a gene encoding a desirable agricultural trait (e.g., an herbicide- resistance gene). In yet another example, a transgene is an antisense nucleic acid sequence, wherein expression of the antisense nucleic acid sequence inhibits expression of a target nucleic acid sequence. In one embodiment the transgene is an endogenous nucleic acid, wherein additional genomic copies of the endogenous nucleic acid are desired, or a nucleic acid that is in the antisense orientation with respect to the sequence of a target nucleic acid in a host organism.

[0049] A “gene product” as defined herein is any product produced by the gene. For example, the gene product can be the direct transcriptional product of a gene (e.g., mRNA, tRNA, rRNA, antisense RNA, interfering RNA, ribozyme, structural RNA or any other type of RNA) or a protein produced by translation of a mRNA. Gene products also include RNAs which are modified, by processes such as capping, polyadenylation, methylation, and editing, and proteins modified by, for example, methylation, acetylation, phosphorylation, ubiquitination, ADP-ribosylation, myristilation, and glycosylation. Gene expression can be influenced by external signals, for example, exposure of a cell, tissue, or organism to an agent that increases or decreases gene expression. Expression of a gene can also be regulated anywhere in the pathway from DNA to RNA to protein. Regulation of gene expression occurs, for example, through controls acting on transcription, translation, RNA transport and processing, degradation of intermediary molecules such as mRNA, or through activation, inactivation, compartmentalization, or degradation of specific protein molecules after they have been made, or by combinations thereof. Gene expression can be measured at the RNA level or the protein level by any method known in the art, including, without limitation, Northern blot, RT-PCR, Western blot, or in vitro, in situ, or in vivo protein activity assay(s).

[0050] As used herein the term “gene expression” relates to the process by which the coded information of a nucleic acid transcriptional unit (including, e.g., genomic DNA) is converted into an operational, non-operational, or structural part of a cell, often including the synthesis of a protein. Gene expression can be influenced by external signals; for example, exposure of a cell, tissue, or organism to an agent that increases or decreases gene expression. Expression of a gene can also be regulated anywhere in the pathway from DNA to RNA to protein. Regulation of gene expression occurs, for example, through controls acting ontranscription, translation, RNA transport and processing, degradation of intermediary molecules such as mRNA, or through activation, inactivation, compartmentalization, or degradation of specific protein molecules after they have been made, or by combinations thereof. Gene expression can be measured at the RNA level or the protein level by any method known in the art, including, without limitation, Northern blot, RT-PCR, Western blot, or in vitro, in situ, or in vivo protein activity assay(s).

[0051] As used herein, the term “nucleic acid molecule” (or “nucleic acid” or “polynucleotide”) may refer to a polymeric form of nucleotides, which may include both sense and anti-sense strands of RNA, cDNA, genomic DNA, and synthetic forms and mixed polymers of the above. A nucleotide may refer to a ribonucleotide, deoxyribonucleotide, or a modified form of either type of nucleotide. A “nucleic acid molecule” as used herein is synonymous with “nucleic acid” and “polynucleotide”. A nucleic acid molecule is usually at least 10 bases in length, unless otherwise specified. The term may refer to a molecule of RNA or DNA of indeterminate length. The term includes single- and double-stranded forms of DNA. A nucleic acid molecule may include either or both naturally-occurring and modified nucleotides linked together by naturally occurring and / or non-naturally occurring nucleotide linkages.

[0052] Nucleic acid molecules may be modified chemically or biochemically, or may contain non-natural or derivatized nucleotide bases, as will be readily appreciated by those of skill in the art. Such modifications include, for example, labels, methylation, substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications (e.g., uncharged linkages: for example, methyl phosphonates, phosphotriesters, phosphoramidites, carbamates, etc.; charged linkages: for example, phosphorothioates, phosphorodithioates, etc.; pendent moieties: for example, peptides; intercalators: for example, acridine, psoralen, etc.; chelators; alkylators; and modified linkages: for example, alpha anomeric nucleic acids, etc.). The term “nucleic acid molecule” also includes any topological conformation, including single-stranded, double-stranded, partially duplexed, triplexed, hairpinned, circular, and padlocked conformations.

[0053] Transcription proceeds in a 5′ to 3′ manner along a DNA strand. This means that RNA is made by the sequential addition of ribonucleotide-5′-triphosphates to the 3′ terminus of the growing chain (with a requisite elimination of the pyrophosphate). In either a linear or circular nucleic acid molecule, discrete elements (e.g., particular nucleotide sequences) may be referred to as being “upstream” or “5′” relative to a further element if they are bonded or would be bonded to the same nucleic acid in the 5′ direction from that element. Similarly, discrete elements may be “downstream” or “3′” relative to a further element if they are or would be bonded to the same nucleic acid in the 3′ direction from that element.

[0054] Oligonucleotide: An oligonucleotide is a short nucleic acid polymer. Oligonucleotides may be formed by cleavage of longer nucleic acid segments, or by polymerizing individual nucleotide precursors. Automated synthesizers allow the synthesis of oligonucleotides up to several hundred base pairs in length. Because oligonucleotides may bind to a complementary nucleotide sequence, they may be used as probes for detecting DNA or RNA. Oligonucleotides composed of DNA (oligodeoxyribonucleotides) may be used in PCR, a technique for the amplification of small DNA sequences. In PCR, the oligonucleotide is typically referred to as a “primer”, which allows a DNA polymerase to extend the oligonucleotide and replicate the complementary strand.

[0055] As used herein, the term “sequence identity” or “identity”, as used herein in the context of two nucleic acid or polypeptide sequences, may refer to the residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window.

[0056] As used herein, the term “percentage of sequence identity” may refer to the value determined by comparing two optimally aligned sequences (e.g., nucleic acid sequences, and amino acid sequences) over a comparison window, wherein the portion of the sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleotide or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to yield the percentage of sequence identity.

[0057] Methods for aligning sequences for comparison are well-known in the art. Various programs and alignment algorithms are described in, for example: Smith and Waterman (1981) Adv. Appl. Math.2:482; Needleman and Wunsch (1970) J. Mol. Biol.48:443; Pearson and Lipman (1988) Proc. Natl. Acad. Sci. U.S.A. 85:2444; Higgins and Sharp (1988) Gene 73:237-44; Higgins and Sharp (1989) CABIOS 5:151-3; Corpet et al. (1988) Nucleic Acids Res.16:10881-90; Huang et al. (1992) Comp. Appl. Biosci.8:155-65; Pearson et al. (1994) Methods Mol. Biol. 24:307-31; Tatiana et al. (1999) FEMS Microbiol. Lett. 174:247-50. A detailed consideration of sequence alignment methods and homology calculations can be found in, e.g., Altschul et al. (1990) J. Mol. Biol.215:403-10.

[0058] The National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST™; Altschul et al. (1990)) is available from several sources, including theNational Center for Biotechnology Information (Bethesda, Md.), and on the internet, for use in connection with several sequence analysis programs. A description of how to determine sequence identity using this program is available on the internet under the “help” section for BLAST™. For comparisons of nucleic acid sequences, the “Blast 2 sequences” function of the BLAST™ (Blastn) program may be employed using the default parameters. Nucleic acid sequences with even greater similarity to the reference sequences will show increasing percentage identity when assessed by this method.

[0059] As used herein the term “operably linked” relates to a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked with a coding sequence when the promoter affects the transcription or expression of the coding sequence. When recombinantly produced, operably linked nucleic acid sequences are generally contiguous and, where necessary to join two protein-coding regions, in the same reading frame. However, elements need not be contiguous to be operably linked.

[0060] As used herein, the term “promoter” refers to a region of DNA that generally is located upstream (towards the 5′ region of a gene) of a gene and is needed to initiate and drive transcription of the gene. A promoter may permit proper activation or repression of a gene that it controls. A promoter may contain specific sequences that are recognized by transcription factors. These factors may bind to a promoter DNA sequence, which results in the recruitment of RNA polymerase, an enzyme that synthesizes RNA from the coding region of the gene. The promoter generally refers to all gene regulatory elements located upstream of the gene, including, upstream promoters, 5′ UTR, introns, and leader sequences.

[0061] As used herein, the term “upstream-promoter” refers to a contiguous polynucleotide sequence that is sufficient to direct initiation of transcription. As used herein, an upstream- promoter encompasses the site of initiation of transcription with several sequence motifs, which include TATA Box, initiator sequence, TFIIB recognition elements and other promoter motifs (Jennifer, E. F. et al., (2002) Genes & Dev., 16: 2583-2592). The upstream promoter provides the site of action to RNA polymerase II which is a multi-subunit enzyme with the basal or general transcription factors like, TFIIA, B, D, E, F and H. These factors assemble into a transcription pre initiation complex that catalyzes the synthesis of RNA from DNA template.

[0062] The activation of the upstream-promoter is done by the additional sequence of regulatory DNA sequence elements to which various proteins bind and subsequently interact with the transcription initiation complex to activate gene expression. These gene regulatoryelements sequences interact with specific DNA-binding factors. These sequence motifs may sometimes be referred to as cis-elements. Such cis-elements, to which tissue-specific or development-specific transcription factors bind, individually or in combination, may determine the spatiotemporal expression pattern of a promoter at the transcriptional level. These cis- elements vary widely in the type of control they exert on operably linked genes. Some elements act to increase the transcription of operably-linked genes in response to environmental responses (e.g., temperature, moisture, and wounding). Other cis-elements may respond to developmental cues (e.g., germination, seed maturation, and flowering) or to spatial information (e.g., tissue specificity). See, for example, Langridge et al., (1989) Proc. Natl. Acad. Sci. USA 86:3219-23. These cis-elements are located at a varying distance from transcription start point, some cis-elements (called proximal elements) are adjacent to a minimal core promoter region while other elements can be positioned several kilobases upstream or downstream of the promoter (enhancers).

[0063] As used herein, the terms “5′ untranslated region” or “5′UTR” is defined as the untranslated segment in the 5′ terminus of pre-mRNAs or mature mRNAs. For example, on mature mRNAs, a 5′UTR typically harbors on its 5′ end a 7-methylguanosine cap and is involved in many processes such as splicing, polyadenylation, mRNA export towards the cytoplasm, identification of the 5′ end of the mRNA by the translational machinery, and protection of the mRNAs against degradation.

[0064] As used herein, the terms “transcription terminator” is defined as the transcribed segment in the 3′ terminus of pre-mRNAs or mature mRNAs. For example, longer stretches of DNA beyond “polyadenylation signal” site is transcribed as a pre-mRNA. This DNA sequence usually contains transcription termination signal for the proper processing of the pre-mRNA into mature mRNA.

[0065] As used herein, the term “3′ untranslated region” or “3′ UTR” is defined as the untranslated segment in a 3′ terminus of the pre-mRNAs or mature mRNAs. For example, on mature mRNAs this region harbors the poly-(A) tail and is known to have many roles in mRNA stability, translation initiation, and mRNA export. In addition, the 3′ UTR is considered to include the polyadenylation signal and transcription terminator.

[0066] As used herein, the term “polyadenylation signal” designates a nucleic acid sequence present in mRNA transcripts that allows for transcripts, when in the presence of a poly-(A) polymerase, to be polyadenylated on the polyadenylation site, for example, located 10 to 30 bases downstream of the poly-(A) signal. Many polyadenylation signals are known in the art and are useful for the present invention. An exemplary sequence includes AAUAAA andvariants thereof, as described in Loke J., et al., (2005) Plant Physiology 138(3); 1457-1468.

[0067] A “DNA binding transgene” is a polynucleotide coding sequence that encodes a DNA binding protein. The DNA binding protein is subsequently able to bind to another molecule. A binding protein can bind to, for example, a DNA molecule (a DNA-binding protein), a RNA molecule (an RNA-binding protein), and / or a protein molecule (a protein-binding protein). In the case of a protein-binding protein, it can bind to itself (to form homodimers, homotrimers, etc.) and / or it can bind to one or more molecules of a different protein or proteins. A binding protein can have more than one type of binding activity. For example, zinc finger proteins have DNA-binding, RNA-binding, and protein-binding activity.

[0068] Examples of DNA binding proteins include, for example; meganucleases, zinc fingers, CRISPRs, and TALE binding domains that can be “engineered” to bind to a predetermined nucleotide sequence. Typically, the engineered DNA binding proteins (e.g., zinc fingers, CRISPRs, or TALEs) are proteins that are non-naturally occurring. Non-limiting examples of methods for engineering DNA-binding proteins are design and selection. A designed DNA binding protein is a protein not occurring in nature whose design / composition results principally from rational criteria. Rational criteria for design include application of substitution rules and computerized algorithms for processing information in a database storing information of existing ZFP, CRISPR, and / or TALE designs and binding data. See, for example, U.S. Pat. Nos.6,140,081; 6,453,242; and 6,534,261; see also WO 98 / 53058; WO 98 / 53059; WO 98 / 53060; WO 02 / 016536 and WO 03 / 016496 and U.S. Publication Nos. 20110301073, 20110239315 and 20119145940.

[0069] A “zinc finger DNA binding protein” (or binding domain) is a protein, or a domain within a larger protein, that binds DNA in a sequence-specific manner through one or more zinc fingers, which are regions of amino acid sequence within the binding domain whose structure is stabilized through coordination of a zinc ion. The term zinc finger DNA binding protein is often abbreviated as zinc finger protein or ZFP. Zinc finger binding domains can be “engineered” to bind to a predetermined nucleotide sequence. Non-limiting examples of methods for engineering zinc finger proteins are design and selection. A designed zinc finger protein is a protein not occurring in nature whose design / composition results principally from rational criteria. Rational criteria for design include application of substitution rules and computerized algorithms for processing information in a database storing information of existing ZFP designs and binding data. See, for example, U.S. Pat. Nos. 6,140,081; 6,453,242; 6,534,261 and 6,794,136; see also WO 98 / 53058; WO 98 / 53059; WO 98 / 53060; WO 02 / 016536 and WO 03 / 016496.

[0070] In other examples, the DNA-binding domain of one or more of the nucleases comprises a naturally occurring or engineered (non-naturally occurring) TAL effector DNA binding domain. See, e.g., U.S. Patent Publication No. 20110301073, incorporated by reference in its entirety herein. The plant pathogenic bacteria of the genus Xanthomonas are known to cause many diseases in important crop plants. Pathogenicity of Xanthomonas depends on a conserved type III secretion (T3S) system which injects more than different effector proteins into the plant cell. Among these injected proteins are transcription activator- like (TALEN) effectors which mimic plant transcriptional activators and manipulate the plant transcriptome (see Kay et al., (2007) Science 318:648-651). These proteins contain a DNA binding domain and a transcriptional activation domain. One of the most well characterized TAL-effectors is AvrBs3 from Xanthomonas campestgris pv. Vesicatoria (see Bonas et al., (1989) Mol Gen Genet 218: 127-136 and WO2010079430). TAL-effectors contain a centralized domain of tandem repeats, each repeat containing approximately 34 amino acids, which are key to the DNA binding specificity of these proteins. In addition, they contain a nuclear localization sequence and an acidic transcriptional activation domain (for a review see Schornack S, et al., (2006) J Plant Physiol 163(3): 256-272). In addition, in the phytopathogenic bacteria Ralstonia solanacearum two genes, designated brg11 and hpx17 have been found that are homologous to the AvrBs3 family of Xanthomonas in the R. solanacearum biovar strain GMI1000 and in the biovar 4 strain RS 1000 (See Heuer et al., (2007) Appl and Enviro Micro 73(13): 4379-4384). These genes are 98.9% identical in nucleotide sequence to each other but differ by a deletion of 1,575 bp in the repeat domain of hpx17. However, both gene products have less than 40% sequence identity with AvrBs3 family proteins of Xanthomonas. See, e.g., U.S. Patent Publication No.20110301073, incorporated by reference in its entirety.

[0071] Specificity of these TAL effectors depends on the sequences found in the tandem repeats. The repeated sequence comprises approximately 102 bp and the repeats are typically 91-100% homologous with each other (Bonas et al., ibid). Polymorphism of the repeats is usually located at positions 12 and 13 and there appears to be a one-to-one correspondence between the identity of the hypervariable diresidues at positions 12 and 13 with the identity of the contiguous nucleotides in the TAL-effector's target sequence (see Moscou and Bogdanove, (2009) Science 326:1501 and Boch et al., (2009) Science 326:1509- 1512). Experimentally, the natural code for DNA recognition of these TAL-effectors has been determined such that an HD sequence at positions 12 and 13 leads to a binding to cytosine (C), NG binds to T, NI to A, C, G or T, NN binds to A or G, and ING binds to T. These DNA binding repeats have been assembled into proteins with new combinations and numbers of repeats, to make artificial transcription factors that are able to interact with new sequencesand activate the expression of a non-endogenous reporter gene in plant cells (Boch et al., ibid). Engineered TAL proteins have been linked to a FokI cleavage half domain to yield a TAL effector domain nuclease fusion (TALEN) exhibiting activity in a yeast reporter assay (plasmid- based target).

[0072] The CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) / Cas (CRISPR Associated) nuclease system is a recently engineered nuclease system based on a bacterial system that can be used for genome engineering. It is based on part of the adaptive immune response of many bacteria and Archaea. When a virus or plasmid invades a bacterium, segments of the invader's DNA are converted into CRISPR RNAs (crRNA) by the ‘immune’ response. This crRNA then associates, through a region of partial complementarity, with another type of RNA called tracrRNA to guide the Cas9 nuclease to a region homologous to the crRNA in the target DNA called a “protospacer.” Cas9 cleaves the DNA to generate blunt ends at the double-stranded break (DSB) at sites specified by a 20-nucleotide guide sequence contained within the crRNA transcript. Cas9 requires both the crRNA and the tracrRNA for site specific DNA recognition and cleavage. This system has now been engineered such that the crRNA and tracrRNA can be combined into one molecule (the “single guide RNA”), and the crRNA equivalent portion of the single guide RNA can be engineered to guide the Cas9 nuclease to target any desired sequence (see Jinek et al., (2012) Science 337, pp.816-821, Jinek et al., (2013), eLife 2:e00471, and David Segal, (2013) eLife 2:e00563). Thus, the CRISPR / Cas system can be engineered to create a DSB at a desired target in a genome, and repair of the DSB can be influenced by the use of repair inhibitors to cause an increase in error prone repair.

[0073] In other examples, the DNA binding transgene is a site-specific nuclease that comprises an engineered (non-naturally occurring) Meganuclease (also described as a homing endonuclease). The recognition sequences of homing endonucleases or meganucleases such as I-SceI, I-CeuI, PI-PspI, PI-Sce, I-SceIV, I-CsmI, I-PanI, I-SceII, I-PpoI, I-SceIII, I-CreI, I-TevI, I-TevII and I-TevIII are known. See also U.S. Pat. No.5,420,032; U.S. Pat. No.6,833,252; Belfort et al., (1997) Nucleic Acids Res.25:3379-303388; Dujon et al., (1989) Gene 82:115-118; Perler et al., (1994) Nucleic Acids Res. 22, 11127; Jasin (1996) Trends Genet. 12:224-228; Gimble et al., (1996) J. Mol. Biol. 263:163-180; Argast et al., (1998) J. Mol. Biol. 280:345-353 and the New England Biolabs catalogue. In addition, the DNA-binding specificity of homing endonucleases and meganucleases can be engineered to bind non-natural target sites. See, for example, Chevalier et al., (2002) Molec. Cell 10:895- 905; Epinat et al., (2003) Nucleic Acids Res.531:2952-2962; Ashworth et al., (2006) Nature 441:656-659; Paques et al., (2007) Current Gene Therapy 7:49-66; U.S. Patent PublicationNo. 20070117128. The DNA-binding domains of the homing endonucleases and meganucleases may be altered in the context of the nuclease as a whole (i.e., such that the nuclease includes the cognate cleavage domain) or may be fused to a heterologous cleavage domain.

[0074] As used herein, the term “transformation” encompasses all techniques that a nucleic acid molecule can be introduced into such a cell. Examples include, but are not limited to: transfection with viral vectors; transformation with plasmid vectors; electroporation; lipofection; microinjection (Mueller et al., (1978) Cell 15:579-85); Agrobacterium-mediated transfer; direct DNA uptake; WHISKERS™-mediated transformation; and microprojectile bombardment. These techniques may be used for both stable transformation and transient transformation of a plant cell. “Stable transformation” refers to the introduction of a nucleic acid fragment into a genome of a host organism resulting in genetically stable inheritance. Once stably transformed, the nucleic acid fragment is stably integrated in the genome of the host organism and any subsequent generation. Host organisms containing the transformed nucleic acid fragments are referred to as “transgenic” organisms. “Transient transformation” refers to the introduction of a nucleic acid fragment into the nucleus, or DNA-containing organelle, of a host organism resulting in gene expression without genetically stable inheritance.

[0075] An exogenous nucleic acid sequence. In one example, a transgene is a gene sequence (e.g., an herbicide-resistance gene), a gene encoding an industrially or pharmaceutically useful compound, or a gene encoding a desirable agricultural trait. In yet another example, the transgene is an antisense nucleic acid sequence, wherein expression of the antisense nucleic acid sequence inhibits expression of a target nucleic acid sequence. A transgene may contain regulatory sequences operably linked to the transgene (e.g., a promoter). In some embodiments, a polynucleotide sequence of interest is a transgene. However, in other embodiments, a polynucleotide sequence of interest is an endogenous nucleic acid sequence, wherein additional genomic copies of the endogenous nucleic acid sequence are desired, or a nucleic acid sequence that is in the antisense orientation with respect to the sequence of a target nucleic acid molecule in the host organism.

[0076] As used herein, the term a transgenic “event” is produced by transformation of plant cells with heterologous DNA, i.e., a nucleic acid construct that includes a transgene of interest, regeneration of a population of plants resulting from the insertion of the transgene into the genome of the plant, and selection of a particular plant characterized by insertion into a particular genome location. The term “event” refers to the original transformant and progeny of the transformant that include the heterologous DNA. The term “event” also refers to progeny produced by a sexual outcross between the transformant and another variety that includes thegenomic / transgene DNA. Even after repeated back-crossing to a recurrent parent, the inserted transgene DNA and flanking genomic DNA (genomic / transgene DNA) from the transformed parent is present in the progeny of the cross at the same chromosomal location. The term “event” also refers to DNA from the original transformant and progeny thereof comprising the inserted DNA and flanking genomic sequence immediately adjacent to the inserted DNA that would be expected to be transferred to a progeny that receives inserted DNA including the transgene of interest as the result of a sexual cross of one parental line that includes the inserted DNA (e.g., the original transformant and progeny resulting from selfing) and a parental line that does not contain the inserted DNA.

[0077] As used herein, the terms “Polymerase Chain Reaction” or “PCR” define a procedure or technique in which minute amounts of nucleic acid, RNA and / or DNA, are amplified as described in U.S. Pat. No.4,683,195 issued Jul.28, 1987. Generally, sequence information from the ends of the region of interest or beyond needs to be available, such that oligonucleotide primers can be designed; these primers will be identical or similar in sequence to opposite strands of the template to be amplified. The 5′ terminal nucleotides of the two primers may coincide with the ends of the amplified material. PCR can be used to amplify specific RNA sequences, specific DNA sequences from total genomic DNA, and cDNA transcribed from total cellular RNA, bacteriophage or plasmid sequences, etc. See generally Mullis et al., Cold Spring Harbor Symp. Quant. Biol., 51:263 (1987); Erlich, ed., PCR Technology, (Stockton Press, N Y, 1989).

[0078] As used herein, the term “primer” refers to an oligonucleotide capable of acting as a point of initiation of synthesis along a complementary strand when conditions are suitable for synthesis of a primer extension product. The synthesizing conditions include the presence of four different deoxyribonucleotide triphosphates and at least one polymerization-inducing agent such as reverse transcriptase or DNA polymerase. These are present in a suitable buffer, which may include constituents which are co-factors or which affect conditions such as pH and the like at various suitable temperatures. A primer is preferably a single strand sequence, such that amplification efficiency is optimized, but double stranded sequences can be utilized.

[0079] As used herein, the term “vector” is used interchangeably with the terms “construct”, “cloning vector” and “expression vector” and means the vehicle by which a DNA or RNA sequence (e.g. a foreign gene) can be introduced into a host cell, so as to transform the host and promote expression (e.g. transcription and translation) of the introduced sequence. A “non-viral vector” is intended to mean any vector that does not comprise a virus or retrovirus. In some embodiments a “vector” is a sequence of DNA comprising at least one origin of DNAreplication and at least one selectable marker gene. Examples include, but are not limited to, a plasmid, cosmid, bacteriophage, bacterial artificial chromosome (BAC), or virus that carries exogenous DNA into a cell. A vector can also include one or more genes, antisense molecules, and / or selectable marker genes and other genetic elements known in the art. A vector may transduce, transform, or infect a cell, thereby causing the cell to express the nucleic acid molecules and / or proteins encoded by the vector. The term “plasmid” defines a circular strand of nucleic acid capable of autosomal replication in either a prokaryotic or a eukaryotic host cell. The term includes nucleic acid which may be either DNA or RNA and may be single- or double-stranded. The plasmid of the definition may also include the sequences which correspond to a bacterial origin of replication.

[0080] As used herein, the term “selectable marker gene” as used herein defines a gene or other expression cassette which encodes a protein which facilitates identification of cells into which the selectable marker gene is inserted. For example, a “selectable marker gene” encompasses reporter genes as well as genes used in plant transformation to, for example, protect plant cells from a selective agent or provide resistance / tolerance to a selective agent. In one embodiment only those cells or plants that receive a functional selectable marker are capable of dividing or growing under conditions having a selective agent. Examples of selective agents can include, for example, antibiotics, including spectinomycin, neomycin, kanamycin, paromomycin, gentamicin, and hygromycin. These selectable markers include neomycin phosphotransferase (npt II), which expresses an enzyme conferring resistance to the antibiotic kanamycin, and genes for the related antibiotics neomycin, paromomycin, gentamicin, and G418, or the gene for hygromycin phosphotransferase (hpt), which expresses an enzyme conferring resistance to hygromycin. Other selectable marker genes can include genes encoding herbicide resistance including bar or pat (resistance against glufosinate ammonium or phosphinothricin), acetolactate synthase (ALS, resistance against inhibitors such as sulfonylureas (SUs), imidazolinones (IMIs), triazolopyrimidines (TPs), pyrimidinyl oxybenzoates (POB s), and sulfonylamino carbonyl triazolinones that prevent the first step in the synthesis of the branched-chain amino acids), glyphosate, 2,4-D, and metal resistance or sensitivity. Examples of “reporter genes” that can be used as a selectable marker gene include the visual observation of expressed reporter gene proteins such as proteins encoding β- glucuronidase (GUS), luciferase, green fluorescent protein (GFP), yellow fluorescent protein (YFP), DsRed, β-galactosidase, chloramphenicol acetyltransferase (CAT), alkaline phosphatase, and the like. The phrase “marker-positive” refers to plants that have been transformed to include a selectable marker gene.

[0081] As used herein, the term “detectable marker” refers to a label capable of detection,such as, for example, a radioisotope, fluorescent compound, bioluminescent compound, a chemiluminescent compound, metal chelator, or enzyme. Examples of detectable markers include, but are not limited to, the following: fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors), enzymatic labels (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), chemiluminescent, biotinyl groups, predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags). In an embodiment, a detectable marker can be attached by spacer arms of various lengths to reduce potential steric hindrance.

[0082] As used herein, the terms “cassette”, “expression cassette” and “gene expression cassette” refer to a segment of DNA that can be inserted into a nucleic acid or polynucleotide at specific restriction sites or by homologous recombination. As used herein the segment of DNA comprises a polynucleotide that encodes a polypeptide of interest, and the cassette and restriction sites are designed to ensure insertion of the cassette in the proper reading frame for transcription and translation. In an embodiment, an expression cassette can include a polynucleotide that encodes a polypeptide of interest and having elements in addition to the polynucleotide that facilitate transformation of a particular host cell. In an embodiment, a gene expression cassette may also include elements that allow for enhanced expression of a polynucleotide encoding a polypeptide of interest in a host cell. These elements may include, but are not limited to: a promoter, a minimal promoter, an enhancer, a response element, a terminator sequence, a polyadenylation sequence, and the like.

[0083] As used herein a “linker” or “spacer” is a bond, molecule or group of molecules that binds two separate entities to one another. Linkers and spacers may provide for optimal spacing of the two entities or may further supply a labile linkage that allows the two entities to be separated from each other. Labile linkages include photocleavable groups, acid-labile moieties, base-labile moieties and enzyme-cleavable groups. The terms “polylinker” or “multiple cloning site” as used herein defines a cluster of three or more Type-2 restriction enzyme sites located within 10 nucleotides of one another on a nucleic acid sequence. In other instances, the term “polylinker” as used herein refers to a stretch of nucleotides that are targeted for joining two sequences via any known seamless cloning method (i.e., Gibson Assembly®, NEBuilder HiFiDNA Assembly®, Golden Gate Assembly, BioBrick® Assembly, etc.). Constructs comprising a polylinker are utilized for the insertion and / or excision of nucleic acid sequences such as the coding region of a gene.

[0084] As used herein, the term “control” refers to a sample used in an analytical procedure for comparison purposes. A control can be “positive” or “negative”. For example, where thepurpose of an analytical procedure is to detect a differentially expressed transcript or polypeptide in cells or tissue, it is generally preferable to include a positive control, such as a sample from a known plant exhibiting the desired expression, and a negative control, such as a sample from a known plant lacking the desired expression.

[0085] As used herein, the term “plant” includes a whole plant and any descendant, cell, tissue, or part of a plant. A class of plant that can be used in the present invention is generally as broad as the class of higher and lower plants amenable to mutagenesis including angiosperms (monocotyledonous and dicotyledonous plants), gymnosperms, ferns and multicellular algae. Thus, “plant” includes dicot and monocot plants. The term “plant parts” include any part(s) of a plant, including, for example and without limitation: seed (including mature seed and immature seed); a plant cutting; a plant cell; a plant cell culture; a plant organ (e.g., pollen, embryos, flowers, fruits, shoots, leaves, roots, stems, and explants). A plant tissue or plant organ may be a seed, protoplast, callus, or any other group of plant cells that is organized into a structural or functional unit. A plant cell or tissue culture may be capable of regenerating a plant having the physiological and morphological characteristics of the plant from which the cell or tissue was obtained, and of regenerating a plant having substantially the same genotype as the plant. In contrast, some plant cells are not capable of being regenerated to produce plants. Regenerable cells in a plant cell or tissue culture may be embryos, protoplasts, meristematic cells, callus, pollen, leaves, anthers, roots, root tips, silk, flowers, kernels, ears, cobs, husks, or stalks.

[0086] Plant parts include harvestable parts and parts useful for propagation of progeny plants. Plant parts useful for propagation include, for example and without limitation: seed; fruit; a cutting; a seedling; a tuber; and a rootstock. A harvestable part of a plant may be any useful part of a plant, including, for example and without limitation: flower; pollen; seedling; tuber; leaf; stem; fruit; seed; and root.

[0087] A plant cell is the structural and physiological unit of the plant, comprising a protoplast and a cell wall. A plant cell may be in the form of an isolated single cell, or an aggregate of cells (e.g., a friable callus and a cultured cell), and may be part of a higher organized unit (e.g., a plant tissue, plant organ, and plant). Thus, a plant cell may be a protoplast, a gamete producing cell, or a cell or collection of cells that can regenerate into a whole plant. As such, a seed, which comprises multiple plant cells and is capable of regenerating into a whole plant, is considered a “plant cell” in embodiments herein.

[0088] Unless otherwise specifically explained, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to whichthis disclosure belongs. Definitions of common terms in molecular biology can be found in, for example: Lewin, Genes V, Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, Blackwell Science Ltd., 1994 (ISBN 0-632- 02182-9); and Meyers (ed.), Molecular Biology and Biotechnology: A Comprehensive Desk Reference, VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8).

[0089] A base “position”, as used herein, refers to the location of a given base or nucleotide residue within a designated nucleic acid. The designated nucleic acid may be defined by alignment (see below) with a reference nucleic acid.

[0090] Unless otherwise expressly stated, it is in no way intended that any method set forth herein be construed as requiring that its steps be performed in a specific order. Accordingly, where a method claim does not actually recite an order to be followed by its steps or it is not otherwise specifically stated in the claims or descriptions that the steps are to be limited to a specific order, it is no way intended that an order be inferred, in any respect. This holds for any possible non-express basis for interpretation, including: matters of logic with respect to arrangement of steps or operational flow; plain meaning derived from grammatical organization or punctuation; and the number or type of aspects described in the specification. B. INTRODUCTION

[0091] The present disclosure pertains to compositions and methods a 5′ UTR that enhances translation in plants, e.g., enhances translation in specialized metabolism. As discussed herein below, and with data in the Examples, the present disclosure relates to mechanisms governing the metabolic interplay of tryptophan-derived metabolites demonstrated using a genetic screening using mutagenized Arabidopsis mutant cyp83b1 (ref5). ref5 was observed to be deficient of indole glucosinolates while it concurrently enhances auxin production derived from indole-3-acetaldoxime (IAOx), a precursor of indole glucosinolates and a substrate of CYP83B1. Two ref5 suppressors are disclosed herein that show restored high-auxin-related morphology indicative of reduced auxin contents or impaired auxin signaling. Genetic analyses discussed herein below show that single locus dominant mutations in the mutants are associated with the phenotype, and they are likely alleles of the same gene, leading to our designation of of these two dominant mutants as rhax1-1D (restored high auxin1-1D) and rhax1-2D.

[0092] Herein below, e.g., in the Examples, bulk-segregation analysis was used to precisely mapped the rhax1-1D mutation to the 5′ UTR of MYB28, a transcriptional activator for the biosynthesis of aliphatic glucosinolates derived from methionine. rhax1-2D carries a mutation13 bp upstream of the rhax1-1D mutation in the MYB285′ UTR. It was observed that although MYB28 transcript levels remained unaffected in the mutants, the expression of an array of genes regulated by MYB28 substantially increased. Without wishing to be bound by a particular theory, it is believed that these data are consistent with translational activation by the mutations in the 5′ UTR. Without wishing to be bound by a particular theory, it is believed that the mutants in the 5′ UTR are linked to the observation that rhax1 mutants accumulate higher levels of aliphatic glucosinolates. Notably, removing the original ref5 mutation from rhax1 mutants sustained the elevated glucosinolate levels, indicating that the 5′ UTR mutation alone is sufficient to activate MYB28 translation. The present disclosure relates to methods and compositions utilizing a 5′ UTR, i.e., a 5′ UTR of MYB28 comprising the rhax1- 1D mutation (SEQ ID NO: 1) or a 5’ UTR comprising the rhax1-2D (SEQ ID NO: 2). The disclosed compositions and methods are useful in regulating MYB28 translation and modulating metabolic networks involving specialized metabolites, including glucosinolates and auxin in plants.

[0093] The central dogma of life, the transmission of genetic information from DNA through RNA to proteins, governs all biological processes in living organisms. Specifically, DNA is transcribed to RNA, which is then translated into proteins. Although the 5′ UTR is not translated to amino acids, the 5′ UTR is known to play a role in the regulation of translation. However, the mechanism(s) of 5′ UTR-mediated translation regulation in plants remains poorly understood.

[0094] 5′ UTR is a part of mRNA that is not translated to protein. Yet, it is necessary for proper translation by protecting mRNA from degradation, transporting mRNA to the cytosol where translation occurs, and recruiting translational machineries. Recent studies suggest that the 5′ UTR is also involved in the regulation of translation (Ref. No.28: Yang et al., 2021; Ref. No.26: Xiang et al., 2023). Presence of an open reading frame (ORF) in the 5′ UTR, referred to as upstream ORF (uORF), can repress translation (Ref. No. 25: Wiese et al., 2004; Ref. No.22: Tanaka et al., 2016; Ref. No.27: Xu et al., 2017; Ref. No.26: Xiang et al., 2023). In a 2023 study, the presence of RNA hairpin structure within the transcribed uORF impedes translation at the main ORF of the mRNA (Ref. No.26: Xiang et al., 2023). The uORF has been shown to be associated with the repression of translation in a condition-dependent manner (Ref. No.25: Wiese et al., 2004; Ref. No.15: Pajerowska-Mukhtar et al., 2012; Ref. No.22: Tanaka et al., 2016). However, besides the uORF, additional elements in the 5′ UTR function in regulating translation (Ref. No.18: Pickering & Willis, 2005; Ref. No.12: Leppek et al., 2018). In mammalians, a structural 5′ UTR element known as the iron responsive element is a target for binding by iron-regulatory proteins, binding of these proteins prevents translationinitiation complex from binding to the mRNA (Ref. No.10: Leibold & Guo, 1992; Ref. No.5: Gray & Hentze, 1994; Ref. No.6: Guo et al., 1995; Ref. No.14: Muckenthaler et al., 1998). Yet, the mechanism(s) of these elements in the 5′ UTR remain poorly understood in plants. While investigating metabolic interplay between auxin biosynthesis and indole glucosinolate production, we identified a crucial role of the 5′ UTR in translation regulation in plants.

[0095] Auxins are plant growth hormones, and plants produce a couple of compounds having auxin activities. Among them, IAA (indole-3-acetic acid) is the most potent auxin that plays an essential role in growth and development. The de novo biosynthesis of IAA directly impacts localized IAA concentration in plants (Ref. No.30: Zhao, 2018). Biosynthesis of IAA occurs primarily through a two-step pathway called the YUCCA pathway (Ref. No.13: Mashiguchi et al., 2011). However, evidence suggests that there are alternative pathways to produce IAA in plants (Ref. No.30: Zhao, 2018; Ref. No.13: Mashiguchi et al., 2011). One proposed route of IAA production is the IAOx-derived IAA biosynthesis pathway converts IAOx (indole-3- acetaldoxime) to IAA, which is present in both dicots and monocots such as maize and sorghum (Ref. No.16: Perez et al., 2021; Ref. No.17: Perez et al., 2023). Although the IAOx- derived IAA pathway has been proposed decades ago, how IAA is produced from IAOx remains unknown. In addition to IAA production, IAOx metabolism affects phenylpropanoid production (Ref. No. 8: Kim et al., 2015). The accumulation of IAOx and / or its derivatives represses the phenylpropanoid pathway and one mechanism includes accelerated degradation of phenylalanine ammonia-lyase (PAL) (Ref. No. 9: Kim et al., 2020). PAL is ubiquitinated by specific F-box proteins and polyubiquitinated PAL is targeted by 26S proteasome, which results in its degradation (Ref. No.9: Kim et al., 2020). Arabidopsis plants with increased IAOx have enhanced expression of the PAL-interacting F-box proteins and reduced PAL activity and phenylpropanoids (Ref. No.9: Kim et al., 2020). Given that IAOx metabolism is linked with multiple metabolic pathways; plant growth hormone auxin, defense compounds glucosinolates, and phenylpropanoids, alteration of IAOx metabolism may affect plant growth and development and stress response substantially. With the aim to understand the IAOx-derived IAA production, we utilized an Arabidopsis mutant called ref5. In Arabidopsis, IAOx is a precursor for the defense compound indole glucosinolates in addition to a precursor of IAA (Ref. No. 31: Zhao et al., 2002; see also FIG. 1A). REF5 encodes CYP83B1 that converts IAOx to indole-3-acetonitrile oxide in the indole glucosinolate biosynthesis pathway (Ref. No.1: Bak et al., 2001). ref5 has a missense mutation of REF5 showing increased IAOx and IAA, leading to characteristics of high-auxin morphological phenotype such as narrow downward-curled leaves and elongated hypocotyls (Ref. No. 8: Kim et al., 2015). The accumulation of IAA in ref5 and its characteristic high auxin phenotype results from the increased flux toward the IAOx-derived IAA biosynthesis pathway from the accumulated IAOx(Ref. No.8: Kim et al., 2015). As discussed below, e.g., Examples, genes functioning in the metabolic interplay between the IAOx-derived IAA biosynthesis and the IAOx-derived indole glucosinolates were identified using ref5 suppressor screening and the characterization of two ref5 suppressors demonstrates a pivotal role of the 5′ UTR in translation in plants.

[0096] In various further aspects, the present disclosure pertains to enhance expression of MYB28 regulated genes by introducing into a plant or plant cells a MYB28 expression construct comprising a disclosed 5’ UTR or by specific modification of a MYB285’ UTR to have a disclosed 5’ UTR sequence. C. 5’ UNTRANSLATED REGION

[0097] In various aspects, the present disclosure pertains to a 5’ UTR comprising at least a portion of the disclosed 5’ UTR of MYB28 that can be used to prepare expression constructs disclosed herein, wherein said constructs can be used to transform a plant cell. In a further aspect, the disclosed 5’ UTR comprises a sequence having at least 80%, 85%, 90%, 92.5%, 95%, 97.5%, 99%, or 99.9% homology to at least a portion of the 5’ UTR of MYB28. In a further aspect, the disclosed 5’ UTR comprises a sequence having at least 80%, 85%, 90%, 92.5%, 95%, 97.5%, 99%, or 99.9% identity to at least a portion of the 5’ UTR of MYB28

[0098] In a further aspect, the disclosed 5’ UTR comprises a sequence having at least 90% sequence homology to SEQ ID NO:1 or SEQ ID NO: 2. In a still further aspect, the disclosed 5’ UTR comprises a sequence having at least 95% sequence homology to SEQ ID NO:1 or SEQ ID NO: 2. In a yet further aspect, the disclosed 5’ UTR comprises a sequence having at least 97% sequence homology to SEQ ID NO:1 or SEQ ID NO: 2. In an even further aspect, the disclosed 5’ UTR comprises a sequence having at least 98% sequence homology to SEQ ID NO:1 or SEQ ID NO: 2. In a still further aspect, the disclosed 5’ UTR comprises a sequence having at least 99% sequence homology to SEQ ID NO:1 or SEQ ID NO: 2.

[0099] In a further aspect, the disclosed 5’ UTR comprises a sequence having at least 90% sequence identity to SEQ ID NO:1 or SEQ ID NO: 2. In a still further aspect, the disclosed 5’ UTR comprises a sequence having at least 95% sequence identity to SEQ ID NO:1 or SEQ ID NO: 2. In a yet further aspect, the disclosed 5’ UTR comprises a sequence having at least 97% sequence identity to SEQ ID NO:1 or SEQ ID NO: 2. In an even further aspect, the disclosed 5’ UTR comprises a sequence having at least 98% sequence identity to SEQ ID NO:1 or SEQ ID NO: 2. In a still further aspect, the disclosed 5’ UTR comprises a sequence having at least 99% sequence identity to SEQ ID NO:1 or SEQ ID NO: 2.

[0100] In a further aspect, the disclosed 5’ UTR has SEQ ID NO:1 or SEQ ID NO: 2. In aneven further aspect, the disclosed 5’ UTR consists essentially of SEQ ID NO:1 or SEQ ID NO: 2. D. 3’ UNTRANSLATED REGION

[0101] In various aspects, the present disclosure pertains to a 3’ UTR suitable for being operably linked to a gene to be expressed in a plant cell or can be used to prepare expression constructs disclosed herein, wherein said constructs can be used to transform a plant cell.

[0102] In a further aspect, the suitable 3’ UTR comprises at least a portion of the 3’ UTR of MYB28. In a still further aspect, the 3’ UTR of MYB28 having at least 80%, 85%, 90%, 92.5%, 95%, 97.5%, 99%, or 99.9% sequence homology to MYB28. In a still further aspect, the 3’ UTR of MYB28 having at least 80%, 85%, 90%, 92.5%, 95%, 97.5%, 99%, or 99.9% sequence identity to MYB28. In an even further aspect, the 3’ UTR of MYB28 has the 3’ UTR sequenced designated in SEQ ID NO: 9 or SEQ ID NO:10.

[0103] In a further aspect, the 3' UTR comprises a sequence having at least 90% sequence homology to the 3’ UTR sequenced designated in SEQ ID NO: 9 or SEQ ID NO:10. In a further aspect, the 3' UTR comprises a sequence at least 95% sequence homology to the 3’ UTR sequenced designated in SEQ ID NO: 9 or SEQ ID NO:10. In a further aspect, the 3' UTR comprises a sequence having at least 97% sequence homology to the 3’ UTR sequenced designated in SEQ ID NO: 9 or SEQ ID NO:10. In a further aspect, the 3' UTR comprises a sequence having at least 98% sequence homology to the 3’ UTR sequenced designated in SEQ ID NO: 9 or SEQ ID NO:10. In a further aspect, the 3' UTR comprises having a sequence at least 99% sequence homology to the 3’ UTR sequenced designated in SEQ ID NO: 9 or SEQ ID NO:10. E. EXPRESSION CONSTRUCTS

[0104] In various aspects, the present disclosure pertains to a nucleic acid expression construct for enhancing translation of a gene comprising at least a portion of the disclosed 5’ UTR of MYB28 operably linked to: (a) a gene to be expressed downstream of the disclosed 5’ UTR of MYB28; and (b) a 3' UTR downstream of the gene to be expressed. In a further aspect, the nucleic acid expression construct further comprises a promoter upstream of the disclosed 5’ UTR of MYB28. In a further aspect, the the disclosed 5’ UTR of MYB28 is a 5' UTR of MYB28 as disclosed herein above.

[0105] In a further aspect, the gene to be expressed is at least 80%, 85%, 90%, 92.5%, 95%, 97.5%, 99%, or 99.9% sequence homology to MYB28. In a still further aspect, the gene to beexpressed is at least at least 90% sequence homology to MYB28. In a yet further aspect, the gene to be expressed is at least at least 95% sequence homology to MYB28. In an even further aspect, the gene to be expressed is at least at least 97% sequence homology to MYB28. In a still further aspect, the gene to be expressed is at least at least 98% sequence homology to MYB28. In a yet further aspect, the gene to be expressed is at least at least 99% sequence homology to MYB28. In an even further aspect, the MYB28 has SEQ ID NO as disclosed herein below.

[0106] In a further aspect, the gene to be expressed is at least 80%, 85%, 90%, 92.5%, 95%, 97.5%, 99%, or 99.9% sequence identity to MYB28. In a still further aspect, the gene to be expressed is at least at least 90% sequence identity to MYB28. In a yet further aspect, the gene to be expressed is at least at least 95% sequence identity to MYB28. In an even further aspect, the gene to be expressed is at least at least 97% sequence identity to MYB28. In a still further aspect, the gene to be expressed is at least at least 98% sequence identity to MYB28. In a yet further aspect, the gene to be expressed is at least at least 99% sequence identity to MYB28. In an even further aspect, the MYB28 has SEQ ID NO as disclosed herein below.

[0107] Other genes to be expressed, i.e., a transgener, can be used in the disclosed expression contructs or plasmid vectors, e.g., reporter genes. Transgenes of interest that are suitable for use in the present disclosed constructs include, but are not limited to, coding sequences that confer (1) resistance to pests or disease, (2) tolerance to herbicides, (3) value added agronomic traits, such as; yield improvement, nitrogen use efficiency, water use efficiency, and nutritional quality, (4) binding of a protein to DNA in a site specific manner, (5) expression of small RNA, and (6) selectable markers. In accordance with one embodiment, the transgene encodes a selectable marker or a gene product conferring insecticidal resistance, herbicide tolerance, small RNA expression, nitrogen use efficiency, water use efficiency, or nutritional quality.

[0108] In a further aspect, the gene to be expressed is a selectable marker gene. Various selectable markers also described as reporter genes can be operably linked to the linked to a disclosed 5’ UTR. The operably linked sequences can then be incorporated into a chosen vector to allow for identification and selectable of transformed plants (“transformants”). Many methods are available to confirm expression of selectable markers in transformed plants, including for example DNA sequencing and PCR (polymerase chain reaction), Southern blotting, RNA blotting, immunological methods for detection of a protein expressed from the vector. But, usually the reporter genes are observed through visual observation of proteins that when expressed produce a colored product. Exemplary reporter genes are known in theart and encode β-glucuronidase (GUS), luciferase, green fluorescent protein (GFP), yellow fluorescent protein (YFP, Phi-YFP), red fluorescent protein (DsRFP, RFP, etc.), β- galactosidase, and the like (See Sambrook, et al., Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Press, N.Y., 2001, the content of which is incorporated herein by reference in its entirety).

[0109] Selectable marker genes are utilized for selection of transformed cells or tissues. Selectable marker genes include genes encoding antibiotic resistance, such as those encoding neomycin phosphotransferase II (NEO), spectinomycin / streptinomycin resistance (AAD), and hygromycin phosphotransferase (HPT or HGR) as well as genes conferring resistance to herbicidal compounds. Herbicide resistance genes generally code for a modified target protein insensitive to the herbicide or for an enzyme that degrades or detoxifies the herbicide in the plant before it can act. For example, resistance to glyphosate has been obtained by using genes coding for mutant target enzymes, 5-enolpyruvylshikimate-3- phosphate synthase (EPSPS). Genes and mutants for EPSPS are well known, and further described below. Resistance to glufosinate ammonium, bromoxynil, and 2,4- dichlorophenoxyacetate (2,4-D) have been obtained by using bacterial genes encoding PAT or DSM-2, a nitrilase, an AAD-1, or an AAD-12, each of which are examples of proteins that detoxify their respective herbicides.

[0110] In an embodiment, selectable marker genes include, but are not limited to genes encoding: 2,4-D; neomycin phosphotransferase II; cyanamide hydratase; aspartate kinase; dihydrodipicolinate synthase; tryptophan decarboxylase; dihydrodipicolinate synthase and desensitized aspartate kinase; bar gene; tryptophan decarboxylase; neomycin phosphotransferase (NEO); hygromycin phosphotransferase (HPT or HYG); dihydrofolate reductase (DHFR); phosphinothricin acetyltransferase; 2,2-dichloropropionic acid dehalogenase; acetohydroxyacid synthase; 5-enolpyruvyl-shikimate-phosphate synthase (aroA); haloarylnitrilase; acetyl-coenzyme A carboxylase; dihydropteroate synthase (sul I); and 32 kD photosystem II polypeptide (psbA). An embodiment also includes selectable marker genes encoding resistance to: chloramphenicol; methotrexate; hygromycin; spectinomycin; bromoxynil; glyphosate; and phosphinothricin. The above list of selectable marker genes is not meant to be limiting. Any reporter or selectable marker gene are encompassed by the present disclosure.

[0111] In a further aspect, the promoter is a promoter for constitutive expression in a plant.

[0112] In a further aspect, the promoter is a promoter for tissue-specific expression in a plant.

[0113] The nucleic acid vector of any one of claims 1-25, wherein the promoter is a promoter for tissue-specific expression in a plant.

[0114] In some aspects, the expression cassette or construct is operably linked to an Agrobacterium T-DNA border. In accordance with one embodiment the recombinant gene cassette further comprises a first and second T-DNA border, wherein the first T-DNA border is operably linked to one end of the gene construct, and the second T-DNA border is operably linked to the other end of the gene construct. The first and second Agrobacterium T-DNA borders can be independently selected from T-DNA border sequences originating from bacterial strains selected from the group consisting of a nopaline synthesizing Agrobacterium T-DNA border, an ocotopine synthesizing Agrobacterium T-DNA border, a mannopine synthesizing Agrobacterium T-DNA border, a succinamopine synthesizing Agrobacterium T- DNA border, or any combination thereof. In one embodiment an Agrobacterium strain selected from the group consisting of a nopaline synthesizing strain, a mannopine synthesizing strain, a succinamopine synthesizing strain, or an octopine synthesizing strain is provided, wherein said strain comprises a plasmid wherein the plasmid comprises a transgene operably linked to a sequence F. TRANSFORMATION

[0115] Suitable methods for transformation of plants include any method by which DNA can be introduced into a cell, for example and without limitation: electroporation (see, e.g., U.S. Pat. No. 5,384,253); micro-projectile bombardment (see, e.g., U.S. Pat. Nos. 5,015,580, 5,550,318, 5,538,880, 6,160,208, 6,399,861, and 6,403,865); Agrobacterium-mediated transformation (see, e.g., U.S. Pat. Nos. 5,635,055, 5,824,877, 5,591,616; 5,981,840, and 6,384,301); and protoplast transformation (see, e.g., U.S. Pat. No.5,508,184).

[0116] A DNA construct may be introduced directly into the genomic DNA of the plant cell using techniques such as agitation with silicon carbide fibers (see, e.g., U.S. Pat. Nos. 5,302,523 and 5,464,765), or the DNA constructs can be introduced directly to plant tissue using biolistic methods, such as DNA particle bombardment (see, e.g., Klein et al. (1987) Nature 327:70-73). Alternatively, the DNA construct can be introduced into the plant cell via nanoparticle transformation (see, e.g., US Patent Publication No. 20090104700, which is incorporated herein by reference in its entirety).

[0117] In addition, gene transfer may be achieved using non-Agrobacterium bacteria or viruses such as Rhizobium sp. NGR234, Sinorhizoboium meliloti, Mesorhizobium loti, potato virus X, cauliflower mosaic virus and cassava vein mosaic virus and / or tobacco mosaic virus,see, e.g., Chung et al. (2006) Trends Plant Sci.11(1):1-4.

[0118] Through the application of transformation techniques, cells of virtually any plant species may be stably transformed, and these cells may be developed into transgenic plants by well-known techniques. For example, techniques that may be particularly useful in the context of cotton transformation are described in U.S. Pat. Nos. 5,846,797, 5,159,135, 5,004,863, and 6,624,344; techniques for transforming Brassica plants in particular are described, for example, in U.S. Pat. No.5,750,871; techniques for transforming soy bean are described, for example, in U.S. Pat. No.6,384,301; and techniques for transforming maize are described, for example, in U.S. Pat. Nos. 7,060,876 and 5,591,616, and International PCT Publication WO 95 / 06722.

[0119] After effecting delivery of an exogenous nucleic acid to a recipient cell, a transformed cell is generally identified for further culturing and plant regeneration. In order to improve the ability to identify transformants, one may desire to employ a selectable marker gene with the transformation vector used to generate the transformant. In an illustrative embodiment, a transformed cell population can be assayed by exposing the cells to a selective agent or agents, or the cells can be screened for the desired marker gene trait.

[0120] Cells that survive exposure to a selective agent, or cells that have been scored positive in a screening assay, may be cultured in media that supports regeneration of plants. In an embodiment, any suitable plant tissue culture media may be modified by including further substances, such as growth regulators. Tissue may be maintained on a basic media with growth regulators until sufficient tissue is available to begin plant regeneration efforts, or following repeated rounds of manual selection, until the morphology of the tissue is suitable for regeneration (e.g., at least 2 weeks), then transferred to media conducive to shoot formation. Cultures are transferred periodically until sufficient shoot formation has occurred. Once shoots are formed, they are transferred to media conducive to root formation. Once sufficient roots are formed, plants can be transferred to soil for further growth and maturity.

[0121] MOLECULAR CONFIRMATION

[0122] A transformed plant cell, callus, tissue or plant may be identified and isolated by selecting or screening the engineered plant material for traits encoded by the marker genes present on the transforming DNA. For instance, selection can be performed by growing the engineered plant material on media containing an inhibitory amount of the antibiotic or herbicide to which the transforming gene construct confers resistance. Further, transformed plants and plant cells can also be identified by screening for the activities of any visible markergenes (e.g., the β-glucuronidase, luciferase, or gfp genes) that may be present on the recombinant nucleic acid constructs. Such selection and screening methodologies are well known to those skilled in the art. Molecular confirmation methods that can be used to identify transgenic plants are known to those with skill in the art. Several exemplary methods are further described below.

[0123] Molecular Beacons have been described for use in sequence detection. Briefly, a FRET oligonucleotide probe is designed that overlaps the flanking genomic and insert DNA junction. The unique structure of the FRET probe results in it containing a secondary structure that keeps the fluorescent and quenching moieties in close proximity. The FRET probe and PCR primers (one primer in the insert DNA sequence and one in the flanking genomic sequence) are cycled in the presence of a thermostable polymerase and dNTPs. Following successful PCR amplification, hybridization of the FRET probe(s) to the target sequence results in the removal of the probe secondary structure and spatial separation of the fluorescent and quenching moieties. A fluorescent signal indicates the presence of the flanking genomic / transgene insert sequence due to successful amplification and hybridization. Such a molecular beacon assay for detection of as an amplification reaction is an embodiment of the subject disclosure.

[0124] Hydrolysis probe assay, otherwise known as TAQMAN® (Life Technologies, Foster City, Calif.), is a method of detecting and quantifying the presence of a DNA sequence. Briefly, a FRET oligonucleotide probe is designed with one oligo within the transgene and one in the flanking genomic sequence for event-specific detection. The FRET probe and PCR primers (one primer in the insert DNA sequence and one in the flanking genomic sequence) are cycled in the presence of a thermostable polymerase and dNTPs. Hybridization of the FRET probe results in cleavage and release of the fluorescent moiety away from the quenching moiety on the FRET probe. A fluorescent signal indicates the presence of the flanking / transgene insert sequence due to successful amplification and hybridization. Such a hydrolysis probe assay for detection of as an amplification reaction is an embodiment of the subject disclosure.

[0125] KASPar® assays are a method of detecting and quantifying the presence of a DNA sequence. Briefly, the genomic DNA sample comprising the integrated gene expression cassette polynucleotide is screened using a polymerase chain reaction (PCR) based assay known as a KASPar® assay system. The KASPar® assay used in the practice of the subject disclosure can utilize a KASPar® PCR assay mixture which contains multiple primers. The primers used in the PCR assay mixture can comprise at least one forward primer and at least one reverse primer. The forward primer contains a sequence corresponding to a specific region of the DNA polynucleotide, and the reverse primer contains a sequence correspondingto a specific region of the genomic sequence. In addition, the primers used in the PCR assay mixture can comprise at least one forward primer and at least one reverse primer. For example, the KASPar® PCR assay mixture can use two forward primers corresponding to two different alleles and one reverse primer. One of the forward primers contains a sequence corresponding to specific region of the endogenous genomic sequence. The second forward primer contains a sequence corresponding to a specific region of the DNA polynucleotide. The reverse primer contains a sequence corresponding to a specific region of the genomic sequence. Such a KASPar® assay for detection of an amplification reaction is an embodiment of the subject disclosure.

[0126] In some embodiments the fluorescent signal or fluorescent dye is selected from the group consisting of a HEX fluorescent dye, a FAM fluorescent dye, a JOE fluorescent dye, a TET fluorescent dye, a Cy 3 fluorescent dye, a Cy 3.5 fluorescent dye, a Cy 5 fluorescent dye, a Cy 5.5 fluorescent dye, a Cy 7 fluorescent dye, and a ROX fluorescent dye.

[0127] In other embodiments the amplification reaction is run using suitable second fluorescent DNA dyes that are capable of staining cellular DNA at a concentration range detectable by flow cytometry, and have a fluorescent emission spectrum which is detectable by a real time thermocycler. It should be appreciated by those of ordinary skill in the art that other nucleic acid dyes are known and are continually being identified. Any suitable nucleic acid dye with appropriate excitation and emission spectra can be employed, such as YO-PRO- 1®, SYTOX Green®, SYBR Green I®, SYTO11®, SYTO12®, SYTO13®, BOBO®, YOYO®, and TOTO®. In one embodiment, a second fluorescent DNA dye is SYTO13® used at less than 10 μM, less than 4 μM, or less than 2.7 μM.

[0128] In further embodiments, Next Generation Sequencing (NGS) can be used for detection. As described by Brautigma et al., 2010, DNA sequence analysis can be used to determine the nucleotide sequence of the isolated and amplified fragment. The amplified fragments can be isolated and sub-cloned into a vector and sequenced using chain-terminator method (also referred to as Sanger sequencing) or Dye-terminator sequencing. In addition, the amplicon can be sequenced with Next Generation Sequencing. NGS technologies do not require the sub-cloning step, and multiple sequencing reads can be completed in a single reaction. Three NGS platforms are commercially available, the Genome Sequencer FLX™ from 454 Life Sciences / Roche, the Illumina Genome Analyser™ from Solexa and Applied Biosystems' SOLiD™ (acronym for: ‘Sequencing by Oligo Ligation and Detection’). In addition, there are two single molecule sequencing methods that are currently being developed. These include the true Single Molecule Sequencing (tSMS) from Helicos Bioscience™ and the Single Molecule Real Time™ sequencing (SMRT) from Pacific Biosciences.

[0129] The Genome Sequencher FLX™ which is marketed by 454 Life Sciences / Roche is a long read NGS, which uses emulsion PCR and pyrosequencing to generate sequencing reads. DNA fragments of 300-800 bp or libraries containing fragments of 3-20 kb can be used. The reactions can produce over a million reads of about 250 to 400 bases per run for a total yield of 250 to 400 megabases. This technology produces the longest reads but the total sequence output per run is low compared to other NGS technologies.

[0130] The Illumina Genome Analyser™ which is marketed by Solexa™ is a short read NGS which uses sequencing by synthesis approach with fluorescent dye-labeled reversible terminator nucleotides and is based on solid-phase bridge PCR. Construction of paired end sequencing libraries containing DNA fragments of up to 10 kb can be used. The reactions produce over 100 million short reads that are 35-76 bases in length. This data can produce from 3-6 gigabases per run.

[0131] The Sequencing by Oligo Ligation and Detection (SOLiD) system marketed by Applied Biosystems™ is a short read technology. This NGS technology uses fragmented double stranded DNA that are up to 10 kb in length. The system uses sequencing by ligation of dye-labelled oligonucleotide primers and emulsion PCR to generate one billion short reads that result in a total sequence output of up to 30 gigabases per run.

[0132] tSMS of Helicos Bioscience™ and SMRT of Pacific Biosciences™ apply a different approach which uses single DNA molecules for the sequence reactions. The tSMS Helicos™ system produces up to 800 million short reads that result in 21 gigabases per run. These reactions are completed using fluorescent dye-labelled virtual terminator nucleotides that is described as a ‘sequencing by synthesis’ approach.

[0133] The SMRT Next Generation Sequencing system marketed by Pacific Biosciences™ uses a real time sequencing by synthesis. This technology can produce reads of up to 1,000 bp in length as a result of not being limited by reversible terminators. Raw read throughput that is equivalent to one-fold coverage of a diploid human genome can be produced per day using this technology.

[0134] In another embodiment, the detection can be completed using blotting assays, including Western blots, Northern blots, and Southern blots. Such blotting assays are commonly used techniques in biological research for the identification and quantification of biological samples. These assays include first separating the sample components in gels by electrophoresis, followed by transfer of the electrophoretically separated components from the gels to transfer membranes that are made of materials such as nitrocellulose, polyvinylidenefluoride (PVDF), or Nylon. Analytes can also be directly spotted on these supports or directed to specific regions on the supports by applying vacuum, capillary action, or pressure, without prior separation. The transfer membranes are then commonly subjected to a post-transfer treatment to enhance the ability of the analytes to be distinguished from each other and detected, either visually or by automated readers.

[0135] In a further embodiment the detection can be completed using an ELISA assay, which uses a solid-phase enzyme immunoassay to detect the presence of a substance, usually an antigen, in a liquid sample or wet sample. Antigens from the sample are attached to a surface of a plate. Then, a further specific antibody is applied over the surface so it can bind to the antigen. This antibody is linked to an enzyme, and, in the final step, a substance containing the enzyme's substrate is added. The subsequent reaction produces a detectable signal, most commonly a color change in the substrate. G. TRANSGENIC PLANTS

[0136] In various aspects, the present disclosure pertains to a plant, plant tissue, or plant cell comprising a disclosed 5’ UTR. In a further aspect, the plant, plant tissue, or plant cell can further comprise a gene to be expressed operably linked downstream of the disclosed 5’ UTR, such the gene to be expressed is operatively linked upstream of a disclosed 3’ UTR.

[0137] In a further aspect, the present disclosure pertains to a plant, plant tissue, or plant cell comprising a nucleic acid vector for enhancing the translation of a gene comprising at least a portion of the disclosed 5’ UTR of MYB28 operably linked to (a) a gene to be expressed downstream of the disclosed 5’ UTR of MYB28; (b) a 3’ UTR downstream of the gene to be expressed; and (c) a promoter upstream of the disclosed 5’ UTR of MYB28 as disclosed herein above.

[0138] In some aspects, the plant is either a monocotyledonous or a dicotyledonous plant. Specific examples of plants include maize, wheat, rice, sorghum, oats, rye, bananas, sugar cane, soybean, cotton, Arabidopsis, tobacco, sunflower, and canola. In aspects, such plants may be transformed, wherein the transgene is inserted into the genome of said plant.

[0139] In a further aspect, the present disclosure relates to a method for producing a transgenic plant cell. Such a method utilizes transforming a plant cell with a gene expression cassette nucleic acid vector for enhancing the translation of a gene comprising at least a portion of the disclosed 5’ UTR of MYB28 operably linked to (a) a gene to be expressed downstream of the disclosed 5’ UTR of MYB28; and (b) a 3’ UTR downstream of the gene to be expressed.

[0140] In a further aspect, the disclosed method further comprises isolating the transformed plant cell comprising the gene expression cassette. Further, the method considers producing a transgenic plant cell comprising at least a portion of the disclosed 5’ UTR of MYB28 operably linked to (a) a gene to be expressed downstream of the disclosed 5’ UTR of MYB28; and (b) a 3’ UTR downstream of the gene to be expressed.

[0141] In a further aspect, the disclosed methods include regenerating the transgenic plant cell into a transgenic plant. In addition, the disclosed methods include obtaining the transgenic plant, wherein the transgenic plant comprises the gene expression cassette comprising the at least a portion of the disclosed 5’ UTR of MYB28 operably linked to (a) a gene to be expressed downstream of the disclosed 5’ UTR of MYB28; and (b) a 3’ UTR downstream of the gene to be expressed. In such an aspect, the method of transforming a plant cell is performed with a plant transformation method. In other aspect, the method of transforming a plant cell results in a polynucleotide sequence of interest that is stably integrated into the genome of the transgenic plant cell.

[0142] In an embodiment, a plant, plant tissue, or plant cell according to the methods disclosed herein can be a dicotyledonous plant. The dicotyledonous plant, plant tissue, or plant cell can be, but not limited, to alfalfa, rapeseed, canola, Indian mustard, Ethiopian mustard, soybean, sunflower, cotton, beans, broccoli, cabbage, cauliflower, celery, cucumber, eggplant, lettuce; melon, pea, pepper, peanut, potato, pumpkin, radish, spinach, sugarbeet, sunflower, tobacco, tomato, and watermelon.

[0143] In an embodiment, a plant, plant tissue, or plant cell according to the methods disclosed herein can be a monocotyledonous plant. The monocotyledonous plant, plant tissue, or plant cell can be, but not limited, is selected from an Arabidopsis plant, a tobacco plant, a soybean plant, a canola plant, and a cotton plant.

[0144] One of skill in the art will recognize that after the exogenous sequence is stably incorporated in transgenic plants and confirmed to be operable, it can be introduced into other plants by sexual crossing. Any of a number of standard breeding techniques can be used, depending upon the species to be crossed.

[0145] The present disclosure also encompasses seeds of the transgenic plants described above, wherein the seed has the transgene or gene construct containing the gene regulatory elements of the subject disclosure. The present disclosure further encompasses the progeny, clones, cell lines or cells of the transgenic plants described above wherein said progeny, clone, cell line or cell has the transgene or gene construct containing the gene regulatory elementsof the subject disclosure.

[0146] The present disclosure also encompasses the cultivation of transgenic plants described above, wherein the transgenic plant has the transgene or gene construct containing the gene regulatory elements of the subject disclosure. Accordingly, such transgenic plants may be engineered to, inter alia, have one or more desired traits or transgenic events containing the gene regulatory elements of the subject disclosure, by being transformed with nucleic acid molecules according to the invention, and may be cropped or cultivated by any method known to those of skill in the art. H. GENETIC TRANSFORMATION METHODS AND PLANT REGENERATION

[0147] Gene transfer and genetic transformation methods for introducing engineered gRNA- Cas9 constructs into plant cells include, but are not limited to, protoplast transformation through calcium-, polyethylene glycol (PEG)- or electroporation-mediated uptake of naked DNA (see Paszkowski et al. (1984) EMBO J3:2717-2722; Potrykus et al. (1985) Molec. Gen. Genet. 199:169-177; Fromm et al. (1985) Proc. Nat. Acad. Sci. USA 82:5824-5828; and Shimamoto (1989) Nature 338:274-276) and electroporation of plant tissues (D'Halluin et al. (1992) Plant Cell 4:1495-1505). Additional methods for plant cell transformation include microinjection, silicon carbide mediated DNA uptake (Kaeppler et al. (1990) Plant Cell Reporter 9:415-418), and microprojectile bombardment (see Klein et al. (1988) Proc. Nat. Acad. Sci. USA 85:4305-4309; and Gordon-Kamm et al. (1990) Plant Cell 2:603-618). According to certain embodiments, gene constructs carrying gRNA-Cas9 nuclease can be introduced into plant cells by various methods, which include but are not limited to PEG- or electroporation-mediated protoplast transformation, tissue culture or plant tissue transformation by biolistic bombardment, or the Agrobacterium-mediated transient and stable transformation.

[0148] Target gene sequences for genome editing and genetic modification can be selected using methods known in the art, and as described elsewhere in this application. In a preferred embodiment, target sequences are identified that include or are proximal to protospacer adjacent motif (PAM). Once identified, the specific sequence can be targeted by synthesizing a pair of target-specific DNA oligonucleotides with appropriate cloning linkers, and phosphorylating, annealing, and ligating the oligonucleotides into a digested plasmid vector, as described herein. The plasmid vector comprising the target-specific oligonucleotides can then be used for transformation of a plant. In specific embodiments, the target gene sequences comprise a disease susceptibility gene.

[0149] Transformed plant cells which are produced by any of the above transformation techniques can be cultured to regenerate a whole plant which possesses the transformed genotype and thus the desired phenotype. Such regeneration techniques rely on manipulation of certain phytohormones in a tissue culture growth medium, typically relying on a biocide and / or herbicide marker or reporter marker genes (such as GFP or GUS gene) which has been introduced together with the desired nucleotide sequences. Plant regeneration from cultured protoplasts is described in Evans, et al., “Protoplasts Isolation and Culture” in Handbook of Plant Cell Culture, pp. 124-176, Macmillian Publishing Company, New York, 1983; and Binding, Regeneration of Plants, Plant Protoplasts, pp. 21-73, CRC Press, Boca Raton, 1985. Regeneration can also be obtained from plant callus, explants, organs, pollens, embryos or parts thereof. Such regeneration techniques are described generally in Klee et al (1987) Ann. Rev. of Plant Phys.38:467-486.

[0150] The methods for non-transgenic plant cell transfection do not depend on a particular method for introducing RNP into the cell. The RNP is provided to the cells and taken up into the cell interior. Introduction of the RNP may be accomplished by any method known, which permits the successful introduction of the RNP into the cells. Methods include but are not limited to such methods as transfection, microinjection, electroporation, nucleofection and lipofection. Preferably, a PEG transfection is used, as further detailed herein below.

[0151] CRISPR-Cas RNPs can be produced fast and delivered directly to the cells as completely functional complexes. Indeed, they are instantaneously active after transfection, and rapidly breakdown inside the cell. This rapid breakdown kinetics permits CRISPR-Cas RNPs to modify the target genes with lower off-target effects. However, a successful editing using CRISPR-Cas RNPs always rely on an efficient CRISPR RNA (crRNA) and a solid delivery method which can avoid the influence of cell wall as a major barrier. In this regard, plant protoplasts generated by the removal of cell wall using enzymatic digestion provides a promising strategy to improve the efficiency of CRISPR-Cas RNP systems, since the lack of the cell wall makes it possible to employ transfection or electroporation for RNPs and / or nucleic acid deliveries. Moreover, the protoplast can be used to analyze target site mutagenesis efficiency and can be regenerated into plant.

[0152] Among the CRISPR systems, Cpf1 as the effector of the CRISPR locus is identified as a class two CRISPR which recognizes the target DNA region via protospacer adjacent motif (PAM) scanning (PAM in Cpf1 is highly specific to the 5′-TTTV-3′). CRISPR-Cpf1 systems create 5′ staggered ends, which potentially can facilitate precise gene replacement using non- homologous end joining (NHEJ), moreover it cleaves DNA at sites distal to the PAM. Such distal cleavage allows previously mutated sequences to be severed repeatedly, promotinghomology-dependent repair (HDR) (Safari, F., Zare, K., Negandaripour, M., Barekati- Mowahed, M., and Ghasemi, Y. (2019) CRISPR Cpf1 proteins: structure, function and implications for genome editing. Cell Biosci., 9, 36.). To date three homologues of Cpfl, including Francisella novicida (FnCpf1), Acidaminococcus sp (AsCpf1), and Lachnospiraceae bacterium (LbCpf1) have been applied to genome engineering of different organisms. I. NON-TRANSGENIC PLANT CELL TRANSFECTION OF RIBONUCLEOPROTEIN (RNPS) COMPLEXES

[0153] The methods for non-transgenic plant cell transfection do not depend on a particular method for introducing RNP into the cell. The RNP is provided to the cells and taken up into the cell interior. Introduction of the RNP may be accomplished by any method known, which permits the successful introduction of the RNP into the cells. Methods include but are not limited to such methods as transfection, microinjection, electroporation, nucleofection and lipofection. Preferably, a PEG transfection is used, as further detailed herein below.

[0154] CRISPR-Cas RNPs can be produced fast and delivered directly to the cells as completely functional complexes. Indeed, they are instantaneously active after transfection, and rapidly breakdown inside the cell. This rapid breakdown kinetics permits CRISPR-Cas RNPs to modify the target genes with lower off-target effects. However, a successful editing using CRISPR-Cas RNPs always rely on an efficient CRISPR RNA (crRNA) and a solid delivery method which can avoid the influence of cell wall as a major barrier. In this regard, plant protoplasts generated by the removal of cell wall using enzymatic digestion provides a promising strategy to improve the efficiency of CRISPR-Cas RNP systems, since the lack of the cell wall makes it possible to employ transfection or electroporation for RNPs and / or nucleic acid deliveries. Moreover, the protoplast can be used to analyze target site mutagenesis efficiency and can be regenerated into plant.

[0155] Among the CRISPR systems, Cpf1 as the effector of the CRISPR locus is identified as a class two CRISPR which recognizes the target DNA region via protospacer adjacent motif (PAM) scanning (PAM in Cpf1 is highly specific to the 5′-TTTV-3′). CRISPR-Cpf1 systems create 5′ staggered ends, which potentially can facilitate precise gene replacement using non- homologous end joining (NHEJ), moreover it cleaves DNA at sites distal to the PAM. Such distal cleavage allows previously mutated sequences to be severed repeatedly, promoting homology-dependent repair (HDR) (Safari, F., Zare, K., Negandaripour, M., Barekati- Mowahed, M., and Ghasemi, Y. (2019) CRISPR Cpf1 proteins: structure, function and implications for genome editing. Cell Biosci., 9, 36.). To date three homologues of Cpfl, including Francisella novicida (FnCpf1), Acidaminococcus sp (AsCpf1), and Lachnospiraceaebacterium (LbCpf1) have been applied to genome engineering of different organisms. J. REFERENCES

[0156] References are cited herein throughout using the format of reference number(s) enclosed by parentheses corresponding to one or more of the following numbered references. For example, citation of references numbers 1 and 2 immediately herein below would be indicated in the disclosure as (Refs.1 and 2).

[0157] Ref.1: Bak, S., Tax, F. E., Feldmann, K. A., Galbraith, D. W., & Feyereisen, R. (2001). CYP83B1, a Cytochrome P450 at the Metabolic Branch Point in Auxin and Indole Glucosinolate Biosynthesis in Arabidopsis. The Plant Cell, 13(1), 101–111.

[0158] Ref.2: Brown, P. D., Tokuhisa, J. G., Reichelt, M., & Gershenzon, J. (2003). Variation of glucosinolate accumulation among different organs and developmental stages of Arabidopsis thaliana. Phytochemistry, 62(3), 471–481.

[0159] Ref.3: Gigolashvili, T., Engqvist, M., Yatusevich, R., Müller, C., & Flügge, U. (2008). HAG2 / MYB76 and HAG3 / MYB29 exert a specific and coordinated control on the regulation of aliphatic glucosinolate biosynthesis in Arabidopsis thaliana. New Phytologist, 177(3), 627– 642.

[0160] Ref. 4: Gigolashvili, T., Yatusevich, R., Berger, B., Müller, C., & Flügge, U. (2007). The R2R3-MYB transcription factor HAG1 / MYB28 is a regulator of methionine-derived glucosinolate biosynthesis in Arabidopsis thaliana. The Plant Journal, 51(2), 247–261.

[0161] Ref.5: Gray, N. K., & Hentze, M. W. (1994). Iron regulatory protein prevents binding of the 43S translation pre-initiation complex to ferritin and eALAS mRNAs. The EMBO Journal, 13(16), 3882–3891.

[0162] Ref. 6: Guo, B., Phillips, J. D., Yu, Y., & Leibold, E. A. (1995). Iron Regulates the Intracellular Degradation of Iron Regulatory Protein 2 by the Proteasome. Journal of Biological Chemistry, 270(37), 21645–21651.

[0163] Ref.7: Harun, S., Abdullah-Zawawi, M.-R., Goh, H.-H., & Mohamed-Hussein, Z.-A. (2020). A Comprehensive Gene Inventory for Glucosinolate Biosynthetic Pathway in Arabidopsis thaliana. Journal of Agricultural and Food Chemistry, 68(28), 7281–7297.

[0164] Ref. 8: Kim, J. I., Dolan, W. L., Anderson, N. A., & Chapple, C. (2015). Indole Glucosinolate Biosynthesis Limits Phenylpropanoid Accumulation in Arabidopsis thaliana. The Plant Cell, 27(5), 1529–1546.

[0165] Ref. 9: Kim, J. I., Zhang, X., Pascuzzi, P. E., Liu, C., & Chapple, C. (2020). Glucosinolate and phenylpropanoid biosynthesis are linked by proteasome-dependent degradation of PAL. New Phytologist, 225(1), 154–168.

[0166] Ref.10: Leibold, E. A., & Guo, B. (1992). Iron-Dependent Regulation of Ferritin and Transferrin Receptor Expression by the Iron-Responsive Element Binding Protein. Annual Review of Nutrition, 12(1), 345–368.

[0167] Ref.12: Leppek, K., Das, R., & Barna, M. (2018). Functional 5′ UTR mRNA structures in eukaryotic translation regulation and how to find them. Nature Reviews Molecular Cell Biology, 19(3), 158–174.

[0168] Ref.13: Mashiguchi, K., Tanaka, K., Sakai, T., Sugawara, S., Kawaide, H., Natsume, M., Hanada, A., Yaeno, T., Shirasu, K., Yao, H., McSteen, P., Zhao, Y., Hayashi, K., Kamiya, Y., & Kasahara, H. (2011). The main auxin biosynthesis pathway in Arabidopsis. Proceedings of the National Academy of Sciences, 108(45), Article 45.

[0169] Ref. 14: Muckenthaler, M., Gray, N. K., & Hentze, M. W. (1998). IRP-1 Binding to Ferritin mRNA Prevents the Recruitment of the Small Ribosomal Subunit by the Cap-Binding Complex eIF4F. Molecular Cell, 2(3), 383–388.

[0170] Ref. 15: Pajerowska-Mukhtar, K. M., Wang, W., Tada, Y., Oka, N., Tucker, C. L., Fonseca, J. P., & Dong, X. (2012). The HSF-like Transcription Factor TBF1 Is a Major Molecular Switch for Plant Growth-to-Defense Transition. Current Biology, 22(2), 103–112.

[0171] Ref.16: Perez, V. C., Dai, R., Bai, B., Tomiczek, B., Askey, B. C., Zhang, Y., Rubin, G. M., Ding, Y., Grenning, A., Block, A. K., & Kim, J. (2021). Aldoximes are precursors of auxins in Arabidopsis and maize. New Phytologist, 231(4), 1449–1461.

[0172] Ref.17: Perez, V. C., Dai, R., Tomiczek, B., Mendoza, J., Wolf, E. S. A., Grenning, A., Vermerris, W., Block, A. K., & Kim, J. (2023). Metabolic link between auxin production and specialized metabolites in Sorghum bicolor. Journal of Experimental Botany, 74(1), 364–376.

[0173] Ref. 18: Pickering, B. M., & Willis, A. E. (2005). The implications of structured 5′ untranslated regions on translation and disease. Seminars in Cell & Developmental Biology, 16(1), 39–47.

[0174] Ref.19: Shin, D., Perez, V. C., Dickinson, G. K., Zhao, H., Dai, R., Tomiczek, B., Cho, K. H., Zhu, N., Koh, J., Grenning, A., & Kim, J. (2023). Altered methionine metabolism impacts phenylpropanoid production and plant development in Arabidopsis thaliana. The PlantJournal, 116(1), 187–200.

[0175] Ref.20: Sønderby, I. E., Burow, M., Rowe, H. C., Kliebenstein, D. J., & Halkier, B. A. (2010). A Complex Interplay of Three R2R3 MYB Transcription Factors Determines the Profile of Aliphatic Glucosinolates in Arabidopsis. Plant Physiology, 153(1), 348–363.

[0176] Ref.21: Sønderby, I. E., Hansen, B. G., Bjarnholt, N., Ticconi, C., Halkier, B. A., & Kliebenstein, D. J. (2007). A Systems Biology Approach Identifies a R2R3 MYB Gene Subfamily with Distinct and Overlapping Functions in Regulation of Aliphatic Glucosinolates. PLoS ONE, 2(12), e1322.

[0177] Ref.22: Tanaka, M., Sotta, N., Yamazumi, Y., Yamashita, Y., Miwa, K., Murota, K., Chiba, Y., Hirai, M. Y., Akiyama, T., Onouchi, H., Naito, S., & Fujiwara, T. (2016). The Minimum Open Reading Frame, AUG-Stop, Induces Boron-Dependent Ribosome Stalling and mRNA Degradation. The Plant Cell, 28(11), 2830–2849.

[0178] Ref. 23: Till, B. J., Reynolds, S. H., Greene, E. A., Codomo, C. A., Enns, L. C., Johnson, J. E., Burtner, C., Odden, A. R., Young, K., Taylor, N. E., Henikoff, J. G., Comai, L., & Henikoff, S. (2003). Large-Scale Discovery of Induced Point Mutations With High- Throughput TILLING. Genome Research, 13(3), 524–530.

[0179] Ref.24: Wachsman, G., Modliszewski, J. L., Valdes, M., & Benfey, P. N. (2017). A SIMPLE Pipeline for Mapping Point Mutations. Plant Physiology, 174(3), 1307–1313.

[0180] Ref.25: Wiese, A., Elzinga, N., Wobbes, B., & Smeekens, S. (2004). A Conserved Upstream Open Reading Frame Mediates Sucrose-Induced Repression of Translation[W]. The Plant Cell, 16(7), 1717–1729.

[0181] Ref.26: Xiang, Y., Huang, W., Tan, L., Chen, T., He, Y., Irving, P. S., Weeks, K. M., Zhang, Q. C., & Dong, X. (2023). Pervasive downstream RNA hairpins dynamically dictate start-codon selection. Nature, 621(7978), 423–430.

[0182] Ref. 27: Xu, G., Yuan, M., Ai, C., Liu, L., Zhuang, E., Karapetyan, S., Wang, S., & Dong, X. (2017). uORF-mediated translation allows engineered plant disease resistance without fitness costs. Nature, 545(7655), 491–494.

[0183] Ref.28: Yang, X., Yu, H., Sun, W., Ding, L., Li, J., Cheema, J., Ramirez-Gonzalez, R., Zhao, X., Martín, A. C., Lu, F., Liu, B., Uauy, C., Ding, Y., & Zhang, H. (2021). Wheat in vivo RNA structure landscape reveals a prevalent role of RNA structure in modulating translational subgenome expression asymmetry. Genome Biology, 22(1), 326.

[0184] Ref. 29: Zhang, D., Song, Y. H., Dai, R., Lee, T. G., & Kim, J. (2020). Aldoxime Metabolism Is Linked to Phenylpropanoid Production in Camelina sativa. Frontiers in Plant Science, 11.

[0185] Ref. 30: Zhao, Y. (2018). Essential Roles of Local Auxin Biosynthesis in Plant Development and in Adaptation to Environmental Changes. Annual Review of Plant Biology, 69(1), 417–435.

[0186] Ref. 31: Zhao, Y., Hull, A. K., Gupta, N. R., Goss, K. A., Alonso, J., Ecker, J. R., Normanly, J., Chory, J., & Celenza, J. L. (2002). Trp-dependent auxin biosynthesis in Arabidopsis: Involvement of cytochrome P450s CYP79B2 and CYP79B3. Genes & Development, 16(23), Article 23.

[0187] From the foregoing, it will be seen that aspects herein are well adapted to attain all the ends and objects hereinabove set forth together with other advantages which are obvious and which are inherent to the structure.

[0188] While specific elements and steps are discussed in connection to one another, it is understood that any element and / or steps provided herein is contemplated as being combinable with any other elements and / or steps regardless of explicit provision of the same while still being within the scope provided herein.

[0189] It will be understood that certain features and subcombinations are of utility and may be employed without reference to other features and subcombinations. This is contemplated by and is within the scope of the claims.

[0190] Since many possible aspects may be made without departing from the scope thereof, it is to be understood that all matter herein set forth or shown in the accompanying drawings and detailed description is to be interpreted as illustrative and not in a limiting sense.

[0191] It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only, and is not intended to be limiting. The skilled artisan will recognize many variants and adaptations of the aspects described herein. These variants and adaptations are intended to be included in the teachings of this disclosure and to be encompassed by the claims herein.

[0192] Now having described the aspects of the present disclosure, in general, the following Examples describe some additional aspects of the present disclosure. While aspects of thepresent disclosure are described in connection with the following examples and the corresponding text and figures, there is no intent to limit aspects of the present disclosure to this description. On the contrary, the intent is to cover all alternatives, modifications, and equivalents included within the spirit and scope of the present disclosure. K. EXAMPLES

[0193] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the compounds, compositions, articles, devices and / or methods claimed herein are made and evaluated, and are intended to be purely exemplary of the disclosure and are not intended to limit the scope of what the inventors regard as their disclosure. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in ^C or is at ambient temperature, and pressure is at or near atmospheric. 1. MATERIALS AND METHODS

[0194] PLANT MATERIALS AND GROWTH CONDITIONS: Arabidopsis thaliana Col-0 was used as wild type plants. Plants were soil-grown in conditions as stated in Shin et al., 2023 at 22°C ± 1°C under a 16-h : 8-h, light : dark photoperiod with fluorescent lighting intensity of 140 µE m-2sec-1. Seeds were directly planted on soil after 3 d of cold treatment at 4°C. The ref5 is ref5- 1 and ref5-1 mutation was confirmed with PCR and enzyme digestion following the genotype information available in Kim et al., 2015 (Ref. No.8).

[0195] HYPOCOTYL LENGTH MEASUREMENT: Following the procedure in Perez et al., 2021 (Ref. No.16), 9-day-old plants were photographed and hypocotyl length were measured with ImageJ software (v.1.53).

[0196] GENOMIC DNA PREPARATION AND BSA ANALYSIS: Two groups of plants were collected from F2 segregation populations of ref5 crossed with 19-6 (rhax1-1D).73 ref5-looking plants and 235 rhax-looking plants were collected in two separate tubes and frozen immediately in liquid nitrogen. The frozen samples were ground, and genomic DNA was extracted with CTAB method. Briefly, extracted DNA was incubated with RNase A (10 mg / mL), then mixed with equal parts of chloroform:isoamyl alcohol solution (24:1) and incubated further with 0.7 volume of isopropanol before washing with 70% ethanol and centrifuged, dried, and resuspended in Tris-EDTA buffer. Whole genome sequencing was conducted at Novogene Co. Mutations in the BSA sequences were analyzed using the SIMPLE pipeline (Wachsman et al., 2017). Arabidopsis TAIR10 was used for the analysis of mutations. The ratio of each mutation sitewas calculated by the number of reads containing mutation per number of total reads mapped to the mutation site (Ref. No.24: Wachsman et al., 2017).

[0197] RNA PREPARATION AND RNA-SEQ: Total RNA was extracted from whole aerial parts of 2-week-old wild type, ref5, and rhax1 in triplicate using the TRIzol method as per the manufacturer′ s protocol (15596018; Thermo Fisher Scientific). Briefly, frozen samples were ground and centrifuged with 1 mL of TRIzol. Supernatant was transferred and mixed with 200 µL of chloroform before centrifuging and transferring the upper aqueous layer. Then, RNA was precipitated with 500 µL of isopropanol before centrifuging to pellet, washed with 75% ethanol, dried, resuspended in DEPC treated water. RNA-seq analysis was performed following the method described in Perez et al., 2021. Briefly, total RNA sample were sequenced using Illumina NovaSeq 6000 system. Raw reads were trimmed and filtered to align to Arabidopsis TAIR10 for differential expression analysis.

[0198] GENERATION OF CRISPR LINES: To generate the Arabidopsis MYB28 CRISPR construct, the target sequence GGCTTCTAGTTCCAACCCTA in the conserved exon of MYB28 was cloned into the binary vector pHSE401 using BsaI enzyme digestion and NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs, Ipswich, MA, USA), generating U6-26P:MYB28gRNA construct. The U6-26P:MYB28gRNA construct was confirmed by sequencing and introduced into Agrobacterium tumefaciens (GV3101) following a method described in Zhang et al., 2020. Agrobacteria containing U6-26P:MYB28gRNA were used to transform rhax1-1D via a floral dipping method (Zhang et al., 2020). Open flowers of mature plants were dipped into a 5% sucrose and 0.01% (v / v) Silwet L-77 (PhytoTech, S7777) solution containing suspended Agrobacteria harboring the construct. More than 6 T1 plants were screened by selection on Murashige and Skoog (MS) media containing 0.5% sucrose, 0.22% MS basal salts, 0.025% MES hydrate and 0.8% agar with 50 mg / L Kanamycin.

[0199] GLUCOSINOLATE ANALYSIS: Glucosinolates were extracted following the method described in Perez et al., 2021. Soluble metabolite extracts came from whole aerial parts of soil-grown 3-week-old Arabidopsis plants using 50% methanol (v / v) incubated at 65°C for 1^h. Samples were centrifuged at 10^000 x^g for 10^min, and the supernatant was collected. The High-performance liquid chromatography (HPLC) analysis of metabolites was performed using an UltiMate 3000 HPLC system (Thermo Fisher Scientific, Waltham, MA, USA). The system was equipped with an autosampler that was cooled to 10°C and a diode array detector (DAD). For desulfoglucosinolate quantification, samples were extracted with 50% methanol containing 250^μM sinigrin (internal standard). One hundred fifty microliters of the extract were incubated with 200^μL of QAE Sephadex solution (Sigma-Aldrich) for 5^min at room temperature. Then, the beads were washed twice with 50% methanol and twice withautoclaved MilliQ water. After the final wash, 100^μL of MilliQ water containing sulfatase (Sigma-Aldrich) was added to the samples, which were then incubated at 37°C for 6^h. Ten microliters were analyzed using the HPLC equipped with an AcclaimTM120 C18 column (150^mm^×^4.6^mm, 5^μm) (Thermo Fisher Scientific). Metabolites were separated by utilizing a mobile phase composed of solvent A (water) and solvent B (100% acetonitrile), and a linear gradient program of solvent B 2–12% over 10^min, 12–15% over 15^min, 15–95% over 30 sec, and 95% for over 1^min. The flow rate was set at 0.75^mL^min−1, and the column temperature was maintained at 40°C. The content of desulfo-glucosinolates was quantified using the peak area at 220^nm and response factors (Brown et al., 2003). Glucosinolate peaks were validated with authentic standards.

[0200] GENERATION OF ARABIDOPSIS TRANSGENIC LINES EXPRESSING GFP DRIVEN BY THE 35S PROMOTER: Two GFP expression binary vectors (p35S::5’UTRWT:GFP and p35S::5’UTRmut:GFP) were generated. To generate GFP expression binary vectors, eGFP reporter constructs were introduced into the pCHF3 vector (Company, Location). The 236bp insert of the 5′UTR of MYB28 was produced from genomic DNA. The coding sequence of the eGFP gene was amplified and fused to the 236bp 5′UTR insert using NEBuilder® HiFi DNA Assembly (New England Biolabs, Ipswich, MA) to generate p35S::5’UTRWT:GFP and p35S::5’UTRmut:GFP. p35S::5’UTRWT:GFP and p35S::5’UTRmut:GFP were introduced into Agrobacterium tumefaciens (GV3101) following a method described in Zhang et al., 2020. Agrobacteria containing p35S::5’UTRWT:GFP and p35S::5’UTRmut:GFP were used to transform wild type via a floral dipping method (Zhang et al., 2020). Open flowers of mature plants were dipped into a 5% sucrose and 0.01% (v / v) Silwet L-77 (PhytoTech, S7777) solution containing suspended Agrobacteria harboring the constructs. More than 4 T1 plants were screened by selection on Murashige and Skoog (MS) media containing 0.5% sucrose, 0.22% MS basal salts, 0.025% MES hydrate and 0.8% agar with 50 mg / L Spectinomycin.

[0201] WESTERN BLOTTING WITH GFP: Crude plant protein for western-blot analysis was extracted from 2-week-old seedlings of transgenic lines containing p35S::5’UTRWT:GFP and p35S::5’UTRmut:GFP. Protein concentration was determined by Bradford assay (Bradford, 1976). Thirty micrograms of total protein were loaded into 12% acrylamide gel per well for SDS-PAGE. The proteins were electrophoretically transferred to PVDF membranes (Millipore Sigma, Burlington, MA) using Mini PROTEAN® Tetra Cell (Bio-Rad, Hercules, CA). Anti-GFP antibodies from mouse and anti-mouse antibodies (source) were used for western-blot analysis.

[0202] GENERATION OF ARABIDOPSIS TRANSGENIC LINES EXPRESSING MYB28 DRIVEN BY A MYB28 PROMOTER: To generate MYB28 complementation lines, the promoter and codingsequence of MYB28 was introduced into the pCHF3 vector (Company, Location). MYB28 insert fragments containing 2392bp of promoter and the coding sequence of MYB28 was produced from genomic DNA. Insert fragments were then introduced into the pCHF3 vector using NEBuilder® HiFi DNA Assembly (NEB #E5520S; New England Biolabs, Ipswich, MA) to generate 35S::2kb(WT):MYB28 and 35S::2kb(rhax1-1D ref5):MYB28. 35S::2kb(WT):MYB28 and 35S::2kb(rhax1-1D ref5):MYB28 were introduced into Agrobacterium tumefaciens (GV3101) following a method described in Zhang et al., 2020. Agrobacteria containing 35S::2kb(WT):MYB28 and 35S::2kb(rhax1-1D ref5):MYB28 were used to transform myb28 myb29 via a floral dipping method (Zhang et al., 2020). Open flowers of mature plants were dipped into a 5% sucrose and 0.01% (v / v) Silwet L-77 (PhytoTech, S7777) solution containing suspended Agrobacteria harboring the constructs. More than 2 T1 plants were screened by selection on Murashige and Skoog (MS) media containing 0.5% sucrose, 0.22% MS basal salts, 0.025% MES hydrate and 0.8% agar with 50 mg / L Spectinomycin. 2. RESULTS

[0203] ISOLATION OF RHAX1 MUTANTS: To elucidate mechanisms underlying the biosynthesis of IAA derived from IAOx, (FIG.1A), we screened the ref5 suppressor pool which was a bulk seed collection pooled from 48 flats of ethylmethanesulfonate (EMS)-treated ref5 M2 plants (Kim et al., 2015). From the screening, two independent lines of ref5 suppressors, 19-6 from flat #19 and 30-3 from flat #30, were identified as they displayed relieved high-auxin morphological phenotypes typical of ref5, such as narrow downward-curled leaves (FIG.1B) and elongated hypocotyls (FIG.1C).

[0204] As they were isolated from different flats and displayed restored high auxin phenotypes, they are presumably independent suppressors having alterations of the IAOx- derived IAA metabolism or auxin signaling. To understand genetic characteristics of these mutations, they were crossed with ref5. All F1 plants (19-6 X ref5 and 30-3 X ref5) displayed restored high auxin-looking phenotype (or rhax-looking phenotype) and F2 progenies showed rhax-looking phenotype or ref5-looking phenotype with 3 to 1 segregation ratio, suggesting that the mutations responsible for the phenotype in both suppressors result from single locus dominant mutations (FIG.1D). Notably, all F1 plants of 19-6 crossed with 30-3 (19-6 X 30-3) displayed rhax-looking phenotype and there was no segregation of morphological phenotypes in the F2 population of 19-6 crossed with 30-3. It is likely that the responsible mutations in both suppressors are physically very close, or they are alleles. Thus, we renamed these two dominant mutants restored high auxin phenotype 1-1D (rhax1-1D) and rhax1-2D.

[0205] IDENTIFICATION OF RHAX1 MUTATIONS: To identify the gene responsible for the restored morphological phenotype of rhax1-1D, we conducted bulk segregation analysis (BSA). Genomic DNA was extracted from a segregation population of F2 progeny of rhax1-1D heterozygous plants (FIG. 1D). A total of 235 rhax-looking plants (flat rosettes and short hypocotyls) and 73 ref5-looking plants (epinasty rosettes and elongated hypocotyls) were pooled for genomic DNA extraction. Genomic DNA samples from the rhax-looking pool and the ref5-looking pool were sequenced using Illumina NovaSeq, generating 4.94Gb and 6.5Gb of sequences for each pool, respectively. A total of 18,766 mutation sites were identified in the BSA sequences. As EMS primarily induces G-to-A or C-to-T changes (Ref. No.23: Till et al., 2003), 4,860 mutations were identified as EMS-induced single nucleotide polymorphisms (SNPs) (FIG.2A). Considering a dominant mutant causing the rhax-looking phenotype, the SNP occurrence ratio at the target site was expected to be 0.66 in the rhax-looking pool and zero in the ref5-looking pool. Out of the 4860 SNPs, 13 SNPs exhibited a zero SNP occurrence ratio in the ref5-looking pool with a 0.6~0.7 ratio in rhax-looking pool (FIG.2A). Out of the 13 SNPs showing the expected occurrence ratio, 12 SNPs were clustered across in chromosome 5 (FIG.2B).

[0206] To determine global expression changes in rhax1-1D, RNA-sequencing (RNA-seq) was conducted with wild type (WT), ref5, and rhax1-1D. In rhax1-1D, 993 genes were up- regulated, while 885 genes were down-regulated compared to ref5. Of the 12 candidate genes clustered in chromosome 5, none of them showed a significant expression alteration derived from the rhax1-1D mutation (FIGs.3A-3B). The expression of HAI1 is up-regulated in the ref5 background regardless of the presence of the rhax1-1D mutation (FIG. 3B). We then conducted co-expression analysis, comparing the mis-regulated genes in rhax1-1D with the top 50 co-expressed genes of each candidate gene (FIG.3C). Interestingly, 60% of the top 50 co-expressed genes of MYB28 were upregulated in rhax1-1D (FIG.3C). The expression of these 30 co-expressed genes of MYB28 was induced in rhax1-1D compared to both wild type and ref5 (FIG.3D), However, there was no significant increase in expression in ref5 compared to wild type. This suggests the relevance of the rhax1-1D mutation in the increased expression of MYB28 co-expression genes (FIG.3D). The BSA of rhax1-1D identified a C-to-T SNP in the 5′ UTR of MYB28, located 152 bp upstream of the ATG translation start site of MYB28 isoform 2 (FIG.2B). Given our genetic study indicating a possibility of rhax1-2D as an allele of rhax1-1D, we sequenced the 5′ UTR region of MYB28 in rhax1-2D and identified a C-to-T SNP, which was located 13 bp upstream of the rhax1-1D mutation (FIG.4A).

[0207] THE GAIN-OF-FUNCTION MUTATIONS IN RHAX1-1D AND RHAX1-2D INCREASE MYB28 ACTIVITY: RNA-seq analysis revealed that the expression of MYB28 in rhax1-1D remainsunchanged FIG.4B). However, the majority of MYB28 co-expressed genes were upregulated due to rhax1-1D mutation FIG.3D). MYB28 is a master regulator of the aliphatic glucosinolate biosynthesis (Ref. No.21: Sønderby et al., 2007, 2010). As an activator, MYB28 activates the expression of an array of aliphatic glucosinolate biosynthesis genes (Ref. No.3: Gigolashvili et al., 2007; Ref. No.4: Gigolashvili et al., 2008). Our RNA-seq analysis indicated that most of aliphatic glucosinolate biosynthesis genes were upregulated in rhax1-1D (FIG.4C) (Harun et al., 2020). Consistently, contents of 4MSOB (4-Methylsulfinylbutylglucosinolate) and 8MSOO (8-Methylsulfinyloctylglucosinolate), two major aliphatic glucosinolates in leaf samples, increased in rhax1-1D and rhax1-2D (FIG.4D). The presented data suggest that the SNPs in rhax1-1D and rhax1-2D increased MYB28 activities without affecting the expression of MYB28, likely through increasing its translation.

[0208] As rhax1-1D is a suppressor of ref5, it contains a defect in the REF5 that functions in tryptophan-derived indole glucosinolate biosynthesis. To examine if the increased aliphatic glucosinolate production in rhax1-1D requires the ref5 mutation, we generated this gain-of- function mutation in the wild-type background by crossing rhax1-1D with wild type. rhax1- 1D / WT has a functional REF5 but contains the rhax1-1D homozygous mutation. As seen in FIG.5A, rhax1-1D / WT is indistinguishable from wild type. However, the levels of aliphatic glucosinolates in rhax1-1D / WT increased substantially compared to wild type (FIG. 5B), demonstrating that the SNP in the 5′ UTR of MYB28 in rhax1-1D is sufficient to increase glucosinolate production.

[0209] To gain more insight on the effects of the 5′ UTR SNP in MYB28, we removed functional MYB28 from rhax1-1D using a CRISPR construct targeting the conserved exon of both isoforms of MYB28. Two MYB28 CRISPR lines were generated in the rhax1-1D (MYB28CR / rhax1-1D) background. MYB28CR / rhax1-1D-3 and -4 lines contain a single bp deletion and a single bp insertion mutation, respectively, resulting in an early translation termination site (FIG. 6A). Interestingly, MYB28CR / rhax1-1D-3 and -4 displays high-auxin morphological phenotype, concluding that the mutation in 5′ UTR of MYB28 in rhax1-1D is responsible for its restored high-auxin phenotype (FIG. 6B). MYB28CR / rhax1-1D-3 and -4 showed significantly reduced concentrations of the aliphatic glucosinolates (FIG.6C). This demonstrates the 5′ UTR SNP of MYB28 in rhax1-1D is responsible for the increased expression of glucosinolate biosynthesis genes and consequently the accumulation of aliphatic glucosinolates, likely due to increased MYB28 activity.

[0210] A SNP MUTATION OF THE 5’UTR INCREASES GFP PROTEIN CONTENT: To assess the impact of the SNP mutation of the 5’UTR on translation, two expression vectors (p35S::5’UTRWT:GFP and p35S::5’UTRmut:GFP) were generated (FIGs.10-11). Both havethe same promoter and 5’UTR, but one has ‘C’ at the rhax1-1D mutation position while the other has ‘T’ at the same position. Then, we introduced these binary vectors to Arabidopsis wild type and identified two single-insertion homozygous lines for each construct. Then, we analyzed GFP protein contents in these four individual plants. As shown in Fig 7b, both #3 and #4 lines carrying the mutated ‘T’ at the rhax1-1D position produced GFP more than lines #1 and #2 carrying the wild-type 5’UTR, suggesting that the single nucleotide change in the 5’UTR is sufficient enough to activate translation.

[0211] FIGs.15A-15D show a single nucleotide change increases the translation of MYB28, leading to increased glucosinolate production in Arabidopsis. (FIG. 15A) Schematic representation of the MYB28 genomic DNA carrying a HA-tag driven by a 2-kilobase sequence of MYB28 promoter, illustrating MYB28:HA expression under the control of the native promoter carrying either the native 5’UTR (p2KbC::MYB28:HA) or the modified 5’UTR (p2KbT::MYB28:HA). The yellow line in promoter indicates the site of mutation (152bp upstream of transcription start site). (FIG. 15B) Aliphatic glucosinolate content (3MSOP, 4MSOB, and 8MSOO) in mature siliques from 7-week-old T1 transgenic myb28 myb29 plants expressing HA-tagged MYB28 with the native 5’UTR (p2KbC::MYB28:HA) and the modified 5’UTR containing a T substitution 152 bp upstream of the transcription start site (p2KbT::MYB28:HA). Asterisks (*) represent not detected. (FIG. 15C) RT-PCR analysis of MYB28:HA transcript levels in 3-week-old rosettes of T1 transgenic lines. ACTIN2 was used as loading control. (FIG.15D) Western blot analysis of MYB28-HA protein levels in 3-week- old rosettes of T1 transgenic lines. Ponceau staining shows equal loading.

[0212] FIGs. 16A-16C show the 5′UTR carrying the rhax1-1D mutation activates the translation of eGFP. (FIG. 16A) Schematic representation of the MYB285′UTR, with GFP expression driven by the 35S promoter, illustrating GFP expression under the control of either the native (p35::5′UTRC:GFP) or modified (p35::5′UTRT:GFP) 5′UTR. The yellow line in 5′UTR indicates the site of mutation (152bp upstream of transcription start site). (FIG.16B) RT-PCR analysis of GFP transcript levels in 3-week-old rosettes from T1 transgenic lines expressing GFP under the control of either the native or modified 5′UTR. The primer binding positions, labeled P1 and P2, are highlighted to indicate the regions used for RT-PCR amplification. ACTIN2 was used as loading control. (FIG.16C) Western blot analysis of GFP protein levels in corresponding T1 transgenic lines. Ponceau staining shows equal loading.

[0213] FIGs. 17A-17B show a 3bp deletion mutation in the 5′UTR increases aliphatic glucosinolate production in Arabidopsis. (FIG. 17A) Schematic diagram and sequencing chromatograms of myb28uORF-Δ3bpcompared to wild type show 3bp deletion mutation in myb28uORF-Δ3bp. Green arrow represents 117bp uORF of MYB28. Asterisks represent the ref5rhax1-1D and ref5 rhax1-2D mutations. Highlighted elements: yellow (20bp target site), green (PAM sequence), and red (induced mutations). (FIG. 17B) 3MSOP, 4MSOB, and 8MSOO content in 3-week-old rosette of myb28uORF-Δ3bpcompared to WT (n=3). Data represent means ± SD.

[0214] FIG.18 shows conserved 5′UTR Sequences in Brassica Crops. Alignment of 5′UTR sequences from several Brassica species reveals conserved regions, including the rhax1-1D and rhax1-2D elements, a three-base-pair deletion mutation (highlighted in red lines), and upstreamORF (uORF)(purple line). Sequences shown correspond to Arabidopsis thaliana MYB28 and its homologs in Brassica juncea (Braju.18G182400), Brassica oleracea (LOC106327854; NCBI accession: XM_013766140), Brassica rapa (Brapa.I00661), and Camelina sativa (CsCN113611.18G182300).

[0215] It will be apparent to those skilled in the art that various modifications and variations can be made in the present disclosure without departing from the scope or spirit of the disclosure. Other aspects of the disclosure will be apparent to those skilled in the art from consideration of the specification and practice of the disclosure disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the disclosure being indicated by the following claims. L. DISCLOSED SEQUENCES WITH SEQ ID NOS

[0216] The following sequences are useful in the compositions and methods of the present disclosure. The sequences are also found in SEQ ID NO: 1 (Arabidopsis thaliana 5′ untranslated region (5′ UTR) rhax1-1D (restored high auxin 1-1D); for translational enhancement; 300 bp) TCTTTTCCATAAGCTTATAACATATATTTTTTTTAAAATCTACTCTGCGTTAAAAAAATGAA AACACGTAGCAGCAGTGTGGGTAAGATCAAAGGGTGTTTCTCGATCAGTTTCATATTCAGAT GTATCAGAGTTCTCATTAACAGATTTGTTTCTTTTTCCTTATCTGATTAAACAATTTCCTTC AGAATTTTACTTTTTTGAACATATATAGTTTTTCTCTGTTCCTATATCTTGAGTTTTGTGAG AGGTTAATTATATGAAATTTTACGCATTATTGTTCATCTATATCGAAAAACA SEQ ID NO: 2 (Arabidopsis thaliana 5′ untranslated region (5′ UTR) rhax1-2D (restored high auxin 1-1D); for translational enhancement; 300 bp) TCTTTTCCATAAGCTTATAACATATATTTTTTTTAAAATCTACTCTGCGTTAAAAAAATGAA AACACGTAGCAGCAGTGTGGGTAAGATCAAAGGGTGTTTCTCGATCAGTTTCATATTCAGAT GTATCAGAGTTTTCATTAACAGATCTGTTTCTTTTTCCTTATCTGATTAAACAATTTCCTTC AGAATTTTACTTTTTTGAACATATATAGTTTTTCTCTGTTCCTATATCTTGAGTTTTGTGAG AGGTTAATTATATGAAATTTTACGCATTATTGTTCATCTATATCGAAAAACASEQ ID NO: 3 (Arabidopsis thaliana 5′untranslated region (5′ UTR); wild type; 300 bp) TCTTTTCCATAAGCTTATAACATATATTTTTTTTAAAATCTACTCTGCGTTAAAAAAATGAA AACACGTAGCAGCAGTGTGGGTAAGATCAAAGGGTGTTTCTCGATCAGTTTCATATTCAGAT GTATCAGAGTTCTCATTAACAGATCTGTTTCTTTTTCCTTATCTGATTAAACAATTTCCTTC AGAATTTTACTTTTTTGAACATATATAGTTTTTCTCTGTTCCTATATCTTGAGTTTTGTGAG AGGTTAATTATATGAAATTTTACGCATTATTGTTCATCTATATCGAAAAACA SEQ ID NO: 4 (plasmid construct comprising the MYB28 coding DNA sequence; 12,737 bp) TTGATCCCGAGGGGAACCCTGTGGTTGGCATGCACATACAAATGGACGAACGGATAAACCTT TTCACGCCCTTTTAAATATCCGTTATTCTAATAAACGCTCTTTTCTCTTAGGTTTACCCGCC AATATATCCTGTCAAACACTGATAGTTTAAACTGAAGGCGGGAAACGACAATCTGATCCAAG CTCAAGCTGCTCTAGCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATG CAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGA GTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGT GGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGATAATCA ATACTTATTGACTAAAATTTTCCCAAAAGAAAGAAGAATCAAATGATTACTCTATGTAGTAA CCCAAACTGATCCTAACAAAATTGTAGAAATGCAGATGGTTTAAATATGTGGCGCTCTCATA AAACTCCTACTTCAGGTAATCTTTTTACACAGTTTGGAGCTATCGTAGCTCTTAACATTTTC ACTCCAGCAATGACTAGAACCAACAGAACAATGAGAGATTGGCTTCTATCCATAGAAAGCTT CAACACGAAAACCGACCAAAACGAAATGTTAAACCCAAGCCTTCTTCAAGCATAGCTGTATC ATATTCTATCTTCCTTGTAAGAGTTCCTTTTGTTAAAAACTAAATACTAAATCCGACTTAAA GAATAATAATCAAGAACTTCAAAATAGCAAAGTAAAATATACACACGCACAAATTGATAAGA GTTCACTTAGCTTGCAGTACGAGAACTAGGCAGGGGCAGACCTAGCTTAAGAGTGTAGGTGT GGCAGGTGTTTAATTATATAGAATTTACTTTGTGGCACTAACATATTTTTGTTTTATAATGC AAAATAAGATGTTAAATTTGATTAAATTTATATACAATACAAGTTTGTGTTCTATGTAAAAT ATTTTTCTAGATCAAACAAGGTCTAGTTTTAAACGATCCATGGGAGTATAAATTTTATCTTT TTCACTCTACCTTGAAAAATGCGCATGAGATAAAATCATAGGTACATATACATACGTGAAGA ATAGCATCAGAAAATATTGTTCTAACTATTCCGATAACTAACAAAACCTTAGGAAACCTCAT CAAGACTAGTTCGAATTAAATCATAAGGTTTAGGTTGAGAGAGTCAAAGAGGGAATGATATA AATAGAAGAATATTTTTTGTTTAAGAATGATTTTTAGACAATGGAAAGAAGAATATGTTAAG GTGGTATAGACGACGAGCAATAATACAACAGCCACAAAAGTGGCAAACAAAAAGGACCTACG CTGGAAAAAAAACACGTGATGTTACAATCACCCTTTCATTCTCAATGATGAACAATAATGTT TATTATTGATAAGAAAAACAATAATGTAAATTTATACTTTCTCGTTAACCAGATTTGTTTTT TCATTATGCGTTTGCAGTATAAAAATAGTAAAATACGTTTTAAGTATTAAACTGTTTGATAG TTTTTTTTTATATATATACCTAACAGAAACCAACTATTTAAACAATACAAAAATATCTGCAA AGATATATATATAATACAATCGAATTCTTAAAAGTTATATATATTTGCAAACGTCCCTTTAG TTATTCCCCTCCAACTCTCCATGTTGGATCAATCATTCAATTTTTTTTTAATAACCAAAAGT TAAATGTACAAATATGCAAGAACCTACAGGTACGTTTACGTGATATATAAATTAAAATATTG CATCTCGTACCGAAGCGCATTACCGTATTTAAAATACCTGAAAGTAGGAAAATATAGTACTA TACAAACACCACTTTTCGGACATTATTTTCATAGAAAAGTTACGAATTATCCTTTTTAACTA TTGATCTATTTAAATAATTTACTAACCATAACTATCTTGTTACGTTTTCACAAAAAAAAAAA AAAAAAAATCTCATTACGTACGTGTATATATATGGAATAGCTCATAACCTCACCACTACCAC AGAAATCATGCCTCTTGGTTCTTTTCCATAAGCTTATAACATATATTTTTTTTAAAATCTAC TCTGCGTTAAAAAAATGAAAACACGTAGCAGCAGTGTGGGTAAGATCAAAGGGTGTTTCTCG ATCAGTTTCATATTCAGATGTATCAGAGTTCTCATTAACAGATTTGTTTCTTTTTCCTTATC TGATTAAACAATTTCCTTCAGAATTTTACTTTTTTGAACATATATAGTTTTTCTCTGTTCCT ATATCTTGAGTTTTGTGAGAGGTTAATTATATGAAATTTTACGCATTATTGTTCATCTATAT CGAAAAACAATGTCAAGAAAGCCATGTTGCGTCGGAGAAGGCTTGAAGAAAGGAGCATGGAC CACCGAGGAGGACAAGAAACTCATCTCTTACATCCACGACCACGGCGAGGGAGGCTGGCGCGACATTCCCCAAAAAGCTGGGTTGAAACGGTGTGGAAAGAGTTGTAGACTGCGATGGACCAAC TACCTTAAACCTGAGATCAAAAGAGGCGAGTTTAGTTCAGAGGAAGAGCAGATTATCATCAT GCTTCATGCTTCTCGTGGCAACAAGTGGTCGGTCATAGCGAGACATTTACCTAGAAGAACAG ACAACGAGATCAAGAACTACTGGAACACGCATCTCAAAAAACGTTTGATGGAACAGGGTATT GATCCCGTGACTCACAAGCCACTGGCTTCTAGTTCCAACCCTACGGTCGATGAGAATTTGAA TTCCCCAAATGCCTCTAGTTCCGACAAGCAATACTCCCGATCGAGCTCAATGCCTTTTCTGT CTCGTCCTCCTCCATCCAGTTGCAACATGGTTTCCAAGGTCTCCGAGCTTAGCAGCAATGAT GGGACACCGATTCAAGGCAGTTCCTTGAGTTGCAAGAAACGTTTCAAGAAATCAAGTTCTAC ATCAAGGCTCTTGAACAAAGTTGCGGCTAAGGCCACTTCCATCAAAGATATATTGTCGGCTT CCATGGAAGGTAGCTTGAGTGCTACTACAATATCACATGCAAGCTTTTTTAATGGCTTCACT GAGCAGATTCGCAATGAAGAGGATAGTTCTAACACATCCCTGACAAATACTCTTGCTGAATT TGATCCCTTCTCCCCATCATCGTTGTACCCCGAACATGAGATCAATGCTACTTCTGATCTCA ACATGGACCAAGATTACGATTTTTCACAATTTTTCGAAAAATTCGGAGGAGATAACCACAAT GAGGAGAACAGTATGAATGATCTCCTTATGTCCGATGTTTCCCAAGAAGTCTCATCAACTAG CGTTGATGATCAAGACAATATGGTAGGAAACTTCGAGGGATGGTCAAATTATCTTCTTGACC ATACCAATTTTATGTATGACACCGACTCAGACTCGCTTGAAAAGCATTTCATATATCCATAC GATGTTCCAGATTATGCTTAGGAGCTTTCGTTCGTATCATCGGTTTCGACAACGTTCGTCAA GTTCAATGCATCAGTTTCATTGCGCACACACCAGAATCCTACTGAGTTTGAGTATTATGGCA TTGGGAAAACTGTTTTTCTTGTACCATTTGTTGTGCTTGTAATTTACTGTGTTTTTTATTCG GTTTTCGCTATCGAACTGTGAAATGGAAATGGATGGAGAAGAGTTAATGAATGATATGGTCC TTTTGTTCATTCTCAAATTAATATTATTTGTTTTTTCTCTTATTTGTTGTGTGTTGAATTTG AAAATATAAGAGATATGCAAACATTTTGTTTTGAGTAAAAATGTGTCAAATCGTGGCCTCTA ATGACCGAAGTTAATATGAGGAGTAAAACACTTGTAGTTGTACCATTATGCTTATTCACTAG GCAACAAATATATTTTCAGACCTAGAAAAGCTGCAAATGTTACTGAATACAAGTATGTCCTC TTGTGTTTTAGACATTTATGAACTTTCCTTTATGTAATTTTCCAGAATCCTTGTCAGATTCT AATCATTGCTTTATAATTATAGTTATACTCATGGATTTGTAGTTGAGTATGAAAATATTTTT TAATGCATTTTATGACTTGCCAATTGATTGACAACATGCATCAATCGAAGCTTGGCACTGGC CGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAG CACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAA CAGTTGCGCAGCCTGAATGGCGAATGCTAGAGCAGCTTGCCAACATGGTGGAGCACGACACT CTCGTCTACTCCAAGAATATCAAAGATACAGTCTCAGAAGACCAAAGGGCTATTGAGACTTT TCAACAAAGGGTAATATCGGGAAACCTCCTCGGATTCCATTGCCCAGCTATCTGTCACTTCA TCAAAAGGACAGTAGAAAAGGAAGGTGGCACCTACAAATGCCATCATTGCGATAAAGGAAAG GCTATCGTTCAAGATGCCTCTGCCGACAGTGGTCCCAAAGATGGACCCCCACCCACGAGGAG CATCGTGGAAAAAGAAGACGTTCCAACCACGTCTTCAAAGCAAGTGGATTGATGTGATAACA TGGTGGAGCACGACACTCTCGTCTACTCCAAGAATATCAAAGATACAGTCTCAGAAGACCAA AGGGCTATTGAGACTTTTCAACAAAGGGTAATATCGGGAAACCTCCTCGGATTCCATTGCCC AGCTATCTGTCACTTCATCAAAAGGACAGTAGAAAAGGAAGGTGGCACCTACAAATGCCATC ATTGCGATAAAGGAAAGGCTATCGTTCAAGATGCCTCTGCCGACAGTGGTCCCAAAGATGGA CCCCCACCCACGAGGAGCATCGTGGAAAAAGAAGACGTTCCAACCACGTCTTCAAAGCAAGT GGATTGATGTGATATCTCCACTGACGTAAGGGATGACGCACAATCCCACTATCCTTCGCAAG ACCCTTCCTCTATATAAGGAAGTTCATTTCATTTGGAGAGGACACGCTGAAATCACCAGTCT CTCTCTACAAATCTATCTCTCTCGATTCGCAGATCTGTCGATCGACCATGGGGATTGAACAA GATGGATTGCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACTGGGC ACAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGCGCAGGGGCGCCCGG TTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTCCAGGACGAGGCAGCGCGG CTATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGTGCTCGACGTTGTCACTGAAGC GGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACCTTG CTCCTGCCGAGAAAGTATCCATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATCCG GCTACCTGCCCATTCGACCACCAAGCGAAACATCGCATCGAGCGAGCACGTACTCGGATGGA AGCCGGTCTTGTCGATCAGGATGATCTGGACGAAGAGCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGCGCATGCCCGACGGCGAGGATCTCGTCGTGACACATGGCGAT GCCTGCTTGCCGAATATCATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCG GCTGGGTGTGGCGGACCGCTATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGC TTGGCGGCGAATGGGCTGACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGCAG CGCATCGCCTTCTATCGCCTTCTTGACGAGTTCTTCTGAGCGGGACTCTGGGGTTCGGATCG ATCCTCTAGCTAGAGTCGATCGACATCGAGTTTCTCCATAATAATGTGTGAGTAGTTCCCAG ATAAGGGAATTAGGGTTCTTATAGGGTTTCGCTCACGTGTTGAGCATATAAGAAACCCTTAG TATGTATTTGTATTTGTAAAATACTTCTATCAATAAAATTTCTAATTCCTAAAACCAAAATC CAGTACTAAAATCCAGATCACCTAAAGTCCCTATAGATCCCCCGAATTAATTCGGCGTTAAT TCAGTACATTAAAAACGTCCGCAATGTGTTATTAAGTTGTCTAAGCGTCAATTTGTTTACAC CACAATATATCCTGCCACCAGCCAGCCAACAGCTCCCCGACCGGCAGCTCGGCACAAAATCA CCACTCGATACAGGCAGCCCATCAGTCCGGGACGGCGTCAGCGGGAGAGCCGTTGTAAGGCG GCAGACTTTGCTCATGTTACCGATGCTATTCGGAAGAACGGCAACTAAGCTGCCGGGTTTGA AACACGGATGATCTCGCGGAGGGTAGCATGTTGATTGTAACGATGACAGAGCGTTGCTGCCT GTGATCAATTCGGGCACGAACCCAGTGGACATAAGCCTCGTTCGGTTCGTAAGCTGTAATGC AAGTAGCGTAACTGCCGTCACGCAACTGGTCCAGAACCTTGACCGAACGCAGCGGTGGTAAC GGCGCAGTGGCGGTTTTCATGGCTTCTTGTTATGACATGTTTTTTTGGGGTACAGTCTATGC CTCGGGCATCCAAGCAGCAAGCGCGTTACGCCGTGGGTCGATGTTTGATGTTATGGAGCAGC AACGATGTTACGCAGCAGGGCAGTCGCCCTAAAACAAAGTTAAACATCATGGGGGAAGCGGT GATCGCCGAAGTATCGACTCAACTATCAGAGGTAGTTGGCGTCATCGAGCGCCATCTCGAAC CGACGTTGCTGGCCGTACATTTGTACGGCTCCGCAGTGGATGGCGGCCTGAAGCCACACAGT GATATTGATTTGCTGGTTACGGTGACCGTAAGGCTTGATGAAACAACGCGGCGAGCTTTGAT CAACGACCTTTTGGAAACTTCGGCTTCCCCTGGAGAGAGCGAGATTCTCCGCGCTGTAGAAG TCACCATTGTTGTGCACGACGACATCATTCCGTGGCGTTATCCAGCTAAGCGCGAACTGCAA TTTGGAGAATGGCAGCGCAATGACATTCTTGCAGGTATCTTCGAGCCAGCCACGATCGACAT TGATCTGGCTATCTTGCTGACAAAAGCAAGAGAACATAGCGTTGCCTTGGTAGGTCCAGCGG CGGAGGAACTCTTTGATCCGGTTCCTGAACAGGATCTATTTGAGGCGCTAAATGAAACCTTA ACGCTATGGAACTCGCCGCCCGACTGGGCTGGCGATGAGCGAAATGTAGTGCTTACGTTGTC CCGCATTTGGTACAGCGCAGTAACCGGCAAAATCGCGCCGAAGGATGTCGCTGCCGACTGGG CAATGGAGCGCCTGCCGGCCCAGTATCAGCCCGTCATACTTGAAGCTAGACAGGCTTATCTT GGACAAGAAGAAGATCGCTTGGCCTCGCGCGCAGATCAGTTGGAAGAATTTGTCCACTACGT GAAAGGCGAGATCACCAAGGTAGTCGGCAAATAATGTCTAGCTAGAAATTCGTTCAAGCCGA CGCCGCTTCGCCGGCGTTAACTCAAGCGATTAGATGCACTAAGCACATAATTGCTCACAGCC AAACTATCAGGTCAAGTCTGCTTTTATTATTTTTAAGCGTGCATAATAAGCCCTACACAAAT TGGGAGATATATCATGCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCA GACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTG CTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAA CTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTG TAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCT AATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAA GACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCC AGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGC CACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAG AGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGC CACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAA CGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCT TTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACC GCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCT GATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATGGTGCACTCTCA GTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATACACTCCGCTATCGCTACGTGACT GGGTCATGGCTGCGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTT TTCACCGTCATCACCGAAACGCGCGAGGCAGGGTGCCTTGATGTGGGCGCCGGCGGTCGAGT GGCGACGGCGCGGCTTGTCCGCGCCCTGGTAGATTGCCTGGCCGTAGGCCAGCCATTTTTGA GCGGCCAGCGGCCGCGATAGGCCGACGCGAAGCGGCGGGGCGTAGGGAGCGCAGCGACCGAA GGGTAGGCGCTTTTTGCAGCTCTTCGGCTGTGCGCTGGCCAGACAGTTATGCACAGGCCAGG CGGGTTTTAAGAGTTTTAATAAGTTTTAAAGAGTTTTAGGCGGAAAAATCGCCTTTTTTCTC TTTTATATCAGTCACTTACATGTGTGACCGGTTCCCAATGTACGGCTTTGGGTTCCCAATGT ACGGGTTCCGGTTCCCAATGTACGGCTTTGGGTTCCCAATGTACGTGCTATCCACAGGAAAG AGACCTTTTCGACCTTTTTCCCCTGCTAGGGCAATTTGCCCTAGCATCTGCTCCGTACATTA GGAACCGGCGGATGCTTCGCCCTCGATCAGGTTGCGGTAGCGCATGACTAGGATCGGGCCAG CCTGCCCCGCCTCCTCCTTCAAATCGTACTCCGGCAGGTCATTTGACCCGATCAGCTTGCGC ACGGTGAAACAGAACTTCTTGAACTCTCCGGCGCTGCCACTGCGTTCGTAGATCGTCTTGAA CAACCATCTGGCTTCTGCCTTGCCTGCGGCGCGGCGTGCCAGGCGGTAGAGAAAACGGCCGA TGCCGGGATCGATCAAAAAGTAATCGGGGTGAACCGTCAGCACGTCCGGGTTCTTGCCTTCT GTGATCTCGCGGTACATCCAATCAGCTAGCTCGATCTCGATGTACTCCGGCCGCCCGGTTTC GCTCTTTACGATCTTGTAGCGGCTAATCAAGGCTTCACCCTCGGATACCGTCACCAGGCGGC CGTTCTTGGCCTTCTTCGTACGCTGCATGGCAACGTGCGTGGTGTTTAACCGAATGCAGGTT TCTACCAGGTCGTCTTTCTGCTTTCCGCCATCGGCTCGCCGGCAGAACTTGAGTACGTCCGC AACGTGTGGACGGAACACGCGGCCGGGCTTGTCTCCCTTCCCTTCCCGGTATCGGTTCATGG ATTCGGTTAGATGGGAAACCGCCATCAGTACCAGGTCGTAATCCCACACACTGGCCATGCCG GCCGGCCCTGCGGAAACCTCTACGTGCCCGTCTGGAAGCTCGTAGCGGATCACCTCGCCAGC TCGTCGGTCACGCTTCGACAGACGGAAAACGGCCACGTCCATGATGCTGCGACTATCGCGGG TGCCCACGTCATAGAGCATCGGAACGAAAAAATCTGGTTGCTCGTCGCCCTTGGGCGGCTTC CTAATCGACGGCGCACCGGCTGCCGGCGGTTGCCGGGATTCTTTGCGGATTCGATCAGCGGC CGCTTGCCACGATTCACCGGGGCGTGCTTCTGCCTCGATGCGTTGCCGCTGGGCGGCCTGCG CGGCCTTCAACTTCTCCACCAGGTCATCACCCAGCGCCGCGCCGATTTGTACCGGGCCGGAT GGTTTGCGACCGTCACGCCGATTCCTCGGGCTTGGGGGTTCCAGTGCCATTGCAGGGCCGGC AGACAACCCAGCCGCTTACGCCTGGCCAACCGCCCGTTCCTCCACACATGGGGCATTCCACG GCGTCGGTGCCTGGTTGTTCTTGATTTTCCATGCCGCCTCCTTTAGCCGCTAAAATTCATCT ACTCATTTATTCATTTGCTCATTTACTCTGGTAGCTGCGCGATGTATTCAGATAGCAGCTCG GTAATGGTCTTGCCTTGGCGTACCGCGTACATCTTCAGCTTGGTGTGATCCTCCGCCGGCAA CTGAAAGTTGACCCGCTTCATGGCTGGCGTGTCTGCCAGGCTGGCCAACGTTGCAGCCTTGC TGCTGCGTGCGCTCGGACGGCCGGCACTTAGCGTGTTTGTGCTTTTGCTCATTTTCTCTTTA CCTCATTAACTCAAATGAGTTTTGATTTAATTTCAGCGGCCAGCGCCTGGACCTCGCGGGCA GCGTCGCCCTCGGGTTCTGATTCAAGAACGGTTGTGCCGGCGGCGGCAGTGCCTGGGTAGCT CACGCGCTGCGTGATACGGGACTCAAGAATGGGCAGCTCGTACCCGGCCAGCGCCTCGGCAA CCTCACCGCCGATGCGCGTGCCTTTGATCGCCCGCGACACGACAAAGGCCGCTTGTAGCCTT CCATCCGTGACCTCAATGCGCTGCTTAACCAGCTCCACCAGGTCGGCGGTGGCCCATATGTC GTAAGGGCTTGGCTGCACCGGAATCAGCACGAAGTCGGCTGCCTTGATCGCGGACACAGCCA AGTCCGCCGCCTGGGGCGCTCCGTCGATCACTACGAAGTCGCGCCGGCCGATGGCCTTCACG TCGCGGTCAATCGTCGGGCGGTCGATGCCGACAACGGTTAGCGGTTGATCTTCCCGCACGGC CGCCCAATCGCGGGCACTGCCCTGGGGATCGGAATCGACTAACAGAACATCGGCCCCGGCGA GTTGCAGGGCGCGGGCTAGATGGGTTGCGATGGTCGTCTTGCCTGACCCGCCTTTCTGGTTA AGTACAGCGATAACCTTCATGCGTTCCCCTTGCGTATTTGTTTATTTACTCATCGCATCATA TACGCAGCGACCGCATGACGCAAGCTGTTTTACTCAAATACACATCACCTTTTTAGACGGCG GCGCTCGGTTTCTTCAGCGGCCAAGCTGGCCGGCCAGGCCGCCAGCTTGGCATCAGACAAAC CGGCCAGGATTTCATGCAGCCGCACGGTTGAGACGTGCGCGGGCGGCTCGAACACGTACCCG GCCGCGATCATCTCCGCCTCGATCTCTTCGGTAATGAAAAACGGTTCGTCCTGGCCGTCCTG GTGCGGTTTCATGCTTGTTCCTCTTGGCGTTCATTCTCGGCGGCCGCCAGGGCGTCGGCCTC GGTCAATGCGTCCTCACGGAAGGCACCGCGCCGCCTGGCCTCGGTGGGCGTCACTTCCTCGC TGCGCTCAAGTGCGCGGTACAGGGTCGAGCGATGCACGCCAAGCAGTGCAGCCGCCTCTTTCACGGTGCGGCCTTCCTGGTCGATCAGCTCGCGGGCGTGCGCGATCTGTGCCGGGGTGAGGGT AGGGCGGGGGCCAAACTTCACGCCTCGGGCCTTGGCGGCCTCGCGCCCGCTCCGGGTGCGGT CGATGATTAGGGAACGCTCGAACTCGGCAATGCCGGCGAACACGGTCAACACCATGCGGCCG GCCGGCGTGGTGGTGTCGGCCCACGGCTCTGCCAGGCTACGCAGGCCCGCGCCGGCCTCCTG GATGCGCTCGGCAATGTCCAGTAGGTCGCGGGTGCTGCGGGCCAGGCGGTCTAGCCTGGTCA CTGTCACAACGTCGCCAGGGCGTAGGTGGTCAAGCATCCTGGCCAGCTCCGGGCGGTCGCGC CTGGTGCCGGTGATCTTCTCGGAAAACAGCTTGGTGCAGCCGGCCGCGTGCAGTTCGGCCCG TTGGTTGGTCAAGTCCTGGTCGTCGGTGCTGACGCGGGCATAGCCCAGCAGGCCAGCGGCGG CGCTCTTGTTCATGGCGTAATGTCTCCGGTTCTAGTCGCAAGTATTCTACTTTATGCGACTA AAACACGCGACAAGAAAACGCCAGGAAAAGGGCAGGGCGGCAGCCTGTCGCGTAACTTAGGA CTTGTGCGACATGTCGTTTTCAGAAGACGGCTGCACTGAACGTCAGAAGCCGACTGCACTAT AGCAGCGGAGGGGTTGGATCAAAGTAC SEQ ID NO: 5 (plasmid construct comprising the MYB28 coding DNA sequence; 12,737 bp) TTGATCCCGAGGGGAACCCTGTGGTTGGCATGCACATACAAATGGACGAACGGATAAACCTT TTCACGCCCTTTTAAATATCCGTTATTCTAATAAACGCTCTTTTCTCTTAGGTTTACCCGCC AATATATCCTGTCAAACACTGATAGTTTAAACTGAAGGCGGGAAACGACAATCTGATCCAAG CTCAAGCTGCTCTAGCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATG CAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGA GTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGT GGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGATAATCA ATACTTATTGACTAAAATTTTCCCAAAAGAAAGAAGAATCAAATGATTACTCTATGTAGTAA CCCAAACTGATCCTAACAAAATTGTAGAAATGCAGATGGTTTAAATATGTGGCGCTCTCATA AAACTCCTACTTCAGGTAATCTTTTTACACAGTTTGGAGCTATCGTAGCTCTTAACATTTTC ACTCCAGCAATGACTAGAACCAACAGAACAATGAGAGATTGGCTTCTATCCATAGAAAGCTT CAACACGAAAACCGACCAAAACGAAATGTTAAACCCAAGCCTTCTTCAAGCATAGCTGTATC ATATTCTATCTTCCTTGTAAGAGTTCCTTTTGTTAAAAACTAAATACTAAATCCGACTTAAA GAATAATAATCAAGAACTTCAAAATAGCAAAGTAAAATATACACACGCACAAATTGATAAGA GTTCACTTAGCTTGCAGTACGAGAACTAGGCAGGGGCAGACCTAGCTTAAGAGTGTAGGTGT GGCAGGTGTTTAATTATATAGAATTTACTTTGTGGCACTAACATATTTTTGTTTTATAATGC AAAATAAGATGTTAAATTTGATTAAATTTATATACAATACAAGTTTGTGTTCTATGTAAAAT ATTTTTCTAGATCAAACAAGGTCTAGTTTTAAACGATCCATGGGAGTATAAATTTTATCTTT TTCACTCTACCTTGAAAAATGCGCATGAGATAAAATCATAGGTACATATACATACGTGAAGA ATAGCATCAGAAAATATTGTTCTAACTATTCCGATAACTAACAAAACCTTAGGAAACCTCAT CAAGACTAGTTCGAATTAAATCATAAGGTTTAGGTTGAGAGAGTCAAAGAGGGAATGATATA AATAGAAGAATATTTTTTGTTTAAGAATGATTTTTAGACAATGGAAAGAAGAATATGTTAAG GTGGTATAGACGACGAGCAATAATACAACAGCCACAAAAGTGGCAAACAAAAAGGACCTACG CTGGAAAAAAAACACGTGATGTTACAATCACCCTTTCATTCTCAATGATGAACAATAATGTT TATTATTGATAAGAAAAACAATAATGTAAATTTATACTTTCTCGTTAACCAGATTTGTTTTT TCATTATGCGTTTGCAGTATAAAAATAGTAAAATACGTTTTAAGTATTAAACTGTTTGATAG TTTTTTTTTATATATATACCTAACAGAAACCAACTATTTAAACAATACAAAAATATCTGCAA AGATATATATATAATACAATCGAATTCTTAAAAGTTATATATATTTGCAAACGTCCCTTTAG TTATTCCCCTCCAACTCTCCATGTTGGATCAATCATTCAATTTTTTTTTAATAACCAAAAGT TAAATGTACAAATATGCAAGAACCTACAGGTACGTTTACGTGATATATAAATTAAAATATTG CATCTCGTACCGAAGCGCATTACCGTATTTAAAATACCTGAAAGTAGGAAAATATAGTACTA TACAAACACCACTTTTCGGACATTATTTTCATAGAAAAGTTACGAATTATCCTTTTTAACTA TTGATCTATTTAAATAATTTACTAACCATAACTATCTTGTTACGTTTTCACAAAAAAAAAAA AAAAAAAATCTCATTACGTACGTGTATATATATGGAATAGCTCATAACCTCACCACTACCAC AGAAATCATGCCTCTTGGTTCTTTTCCATAAGCTTATAACATATATTTTTTTTAAAATCTAC TCTGCGTTAAAAAAATGAAAACACGTAGCAGCAGTGTGGGTAAGATCAAAGGGTGTTTCTCGATCAGTTTCATATTCAGATGTATCAGAGTTCTCATTAACAGATCTGTTTCTTTTTCCTTATC TGATTAAACAATTTCCTTCAGAATTTTACTTTTTTGAACATATATAGTTTTTCTCTGTTCCT ATATCTTGAGTTTTGTGAGAGGTTAATTATATGAAATTTTACGCATTATTGTTCATCTATAT CGAAAAACAATGTCAAGAAAGCCATGTTGCGTCGGAGAAGGCTTGAAGAAAGGAGCATGGAC CACCGAGGAGGACAAGAAACTCATCTCTTACATCCACGACCACGGCGAGGGAGGCTGGCGCG ACATTCCCCAAAAAGCTGGGTTGAAACGGTGTGGAAAGAGTTGTAGACTGCGATGGACCAAC TACCTTAAACCTGAGATCAAAAGAGGCGAGTTTAGTTCAGAGGAAGAGCAGATTATCATCAT GCTTCATGCTTCTCGTGGCAACAAGTGGTCGGTCATAGCGAGACATTTACCTAGAAGAACAG ACAACGAGATCAAGAACTACTGGAACACGCATCTCAAAAAACGTTTGATGGAACAGGGTATT GATCCCGTGACTCACAAGCCACTGGCTTCTAGTTCCAACCCTACGGTCGATGAGAATTTGAA TTCCCCAAATGCCTCTAGTTCCGACAAGCAATACTCCCGATCGAGCTCAATGCCTTTTCTGT CTCGTCCTCCTCCATCCAGTTGCAACATGGTTTCCAAGGTCTCCGAGCTTAGCAGCAATGAT GGGACACCGATTCAAGGCAGTTCCTTGAGTTGCAAGAAACGTTTCAAGAAATCAAGTTCTAC ATCAAGGCTCTTGAACAAAGTTGCGGCTAAGGCCACTTCCATCAAAGATATATTGTCGGCTT CCATGGAAGGTAGCTTGAGTGCTACTACAATATCACATGCAAGCTTTTTTAATGGCTTCACT GAGCAGATTCGCAATGAAGAGGATAGTTCTAACACATCCCTGACAAATACTCTTGCTGAATT TGATCCCTTCTCCCCATCATCGTTGTACCCCGAACATGAGATCAATGCTACTTCTGATCTCA ACATGGACCAAGATTACGATTTTTCACAATTTTTCGAAAAATTCGGAGGAGATAACCACAAT GAGGAGAACAGTATGAATGATCTCCTTATGTCCGATGTTTCCCAAGAAGTCTCATCAACTAG CGTTGATGATCAAGACAATATGGTAGGAAACTTCGAGGGATGGTCAAATTATCTTCTTGACC ATACCAATTTTATGTATGACACCGACTCAGACTCGCTTGAAAAGCATTTCATATATCCATAC GATGTTCCAGATTATGCTTAGGAGCTTTCGTTCGTATCATCGGTTTCGACAACGTTCGTCAA GTTCAATGCATCAGTTTCATTGCGCACACACCAGAATCCTACTGAGTTTGAGTATTATGGCA TTGGGAAAACTGTTTTTCTTGTACCATTTGTTGTGCTTGTAATTTACTGTGTTTTTTATTCG GTTTTCGCTATCGAACTGTGAAATGGAAATGGATGGAGAAGAGTTAATGAATGATATGGTCC TTTTGTTCATTCTCAAATTAATATTATTTGTTTTTTCTCTTATTTGTTGTGTGTTGAATTTG AAAATATAAGAGATATGCAAACATTTTGTTTTGAGTAAAAATGTGTCAAATCGTGGCCTCTA ATGACCGAAGTTAATATGAGGAGTAAAACACTTGTAGTTGTACCATTATGCTTATTCACTAG GCAACAAATATATTTTCAGACCTAGAAAAGCTGCAAATGTTACTGAATACAAGTATGTCCTC TTGTGTTTTAGACATTTATGAACTTTCCTTTATGTAATTTTCCAGAATCCTTGTCAGATTCT AATCATTGCTTTATAATTATAGTTATACTCATGGATTTGTAGTTGAGTATGAAAATATTTTT TAATGCATTTTATGACTTGCCAATTGATTGACAACATGCATCAATCGAAGCTTGGCACTGGC CGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAG CACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAA CAGTTGCGCAGCCTGAATGGCGAATGCTAGAGCAGCTTGCCAACATGGTGGAGCACGACACT CTCGTCTACTCCAAGAATATCAAAGATACAGTCTCAGAAGACCAAAGGGCTATTGAGACTTT TCAACAAAGGGTAATATCGGGAAACCTCCTCGGATTCCATTGCCCAGCTATCTGTCACTTCA TCAAAAGGACAGTAGAAAAGGAAGGTGGCACCTACAAATGCCATCATTGCGATAAAGGAAAG GCTATCGTTCAAGATGCCTCTGCCGACAGTGGTCCCAAAGATGGACCCCCACCCACGAGGAG CATCGTGGAAAAAGAAGACGTTCCAACCACGTCTTCAAAGCAAGTGGATTGATGTGATAACA TGGTGGAGCACGACACTCTCGTCTACTCCAAGAATATCAAAGATACAGTCTCAGAAGACCAA AGGGCTATTGAGACTTTTCAACAAAGGGTAATATCGGGAAACCTCCTCGGATTCCATTGCCC AGCTATCTGTCACTTCATCAAAAGGACAGTAGAAAAGGAAGGTGGCACCTACAAATGCCATC ATTGCGATAAAGGAAAGGCTATCGTTCAAGATGCCTCTGCCGACAGTGGTCCCAAAGATGGA CCCCCACCCACGAGGAGCATCGTGGAAAAAGAAGACGTTCCAACCACGTCTTCAAAGCAAGT GGATTGATGTGATATCTCCACTGACGTAAGGGATGACGCACAATCCCACTATCCTTCGCAAG ACCCTTCCTCTATATAAGGAAGTTCATTTCATTTGGAGAGGACACGCTGAAATCACCAGTCT CTCTCTACAAATCTATCTCTCTCGATTCGCAGATCTGTCGATCGACCATGGGGATTGAACAA GATGGATTGCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACTGGGC ACAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGCGCAGGGGCGCCCGG TTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTCCAGGACGAGGCAGCGCGG ĵķCTATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGTGCTCGACGTTGTCACTGAAGC GGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACCTTG CTCCTGCCGAGAAAGTATCCATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATCCG GCTACCTGCCCATTCGACCACCAAGCGAAACATCGCATCGAGCGAGCACGTACTCGGATGGA AGCCGGTCTTGTCGATCAGGATGATCTGGACGAAGAGCATCAGGGGCTCGCGCCAGCCGAAC TGTTCGCCAGGCTCAAGGCGCGCATGCCCGACGGCGAGGATCTCGTCGTGACACATGGCGAT GCCTGCTTGCCGAATATCATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCG GCTGGGTGTGGCGGACCGCTATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGC TTGGCGGCGAATGGGCTGACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGCAG CGCATCGCCTTCTATCGCCTTCTTGACGAGTTCTTCTGAGCGGGACTCTGGGGTTCGGATCG ATCCTCTAGCTAGAGTCGATCGACATCGAGTTTCTCCATAATAATGTGTGAGTAGTTCCCAG ATAAGGGAATTAGGGTTCTTATAGGGTTTCGCTCACGTGTTGAGCATATAAGAAACCCTTAG TATGTATTTGTATTTGTAAAATACTTCTATCAATAAAATTTCTAATTCCTAAAACCAAAATC CAGTACTAAAATCCAGATCACCTAAAGTCCCTATAGATCCCCCGAATTAATTCGGCGTTAAT TCAGTACATTAAAAACGTCCGCAATGTGTTATTAAGTTGTCTAAGCGTCAATTTGTTTACAC CACAATATATCCTGCCACCAGCCAGCCAACAGCTCCCCGACCGGCAGCTCGGCACAAAATCA CCACTCGATACAGGCAGCCCATCAGTCCGGGACGGCGTCAGCGGGAGAGCCGTTGTAAGGCG GCAGACTTTGCTCATGTTACCGATGCTATTCGGAAGAACGGCAACTAAGCTGCCGGGTTTGA AACACGGATGATCTCGCGGAGGGTAGCATGTTGATTGTAACGATGACAGAGCGTTGCTGCCT GTGATCAATTCGGGCACGAACCCAGTGGACATAAGCCTCGTTCGGTTCGTAAGCTGTAATGC AAGTAGCGTAACTGCCGTCACGCAACTGGTCCAGAACCTTGACCGAACGCAGCGGTGGTAAC GGCGCAGTGGCGGTTTTCATGGCTTCTTGTTATGACATGTTTTTTTGGGGTACAGTCTATGC CTCGGGCATCCAAGCAGCAAGCGCGTTACGCCGTGGGTCGATGTTTGATGTTATGGAGCAGC AACGATGTTACGCAGCAGGGCAGTCGCCCTAAAACAAAGTTAAACATCATGGGGGAAGCGGT GATCGCCGAAGTATCGACTCAACTATCAGAGGTAGTTGGCGTCATCGAGCGCCATCTCGAAC CGACGTTGCTGGCCGTACATTTGTACGGCTCCGCAGTGGATGGCGGCCTGAAGCCACACAGT GATATTGATTTGCTGGTTACGGTGACCGTAAGGCTTGATGAAACAACGCGGCGAGCTTTGAT CAACGACCTTTTGGAAACTTCGGCTTCCCCTGGAGAGAGCGAGATTCTCCGCGCTGTAGAAG TCACCATTGTTGTGCACGACGACATCATTCCGTGGCGTTATCCAGCTAAGCGCGAACTGCAA TTTGGAGAATGGCAGCGCAATGACATTCTTGCAGGTATCTTCGAGCCAGCCACGATCGACAT TGATCTGGCTATCTTGCTGACAAAAGCAAGAGAACATAGCGTTGCCTTGGTAGGTCCAGCGG CGGAGGAACTCTTTGATCCGGTTCCTGAACAGGATCTATTTGAGGCGCTAAATGAAACCTTA ACGCTATGGAACTCGCCGCCCGACTGGGCTGGCGATGAGCGAAATGTAGTGCTTACGTTGTC CCGCATTTGGTACAGCGCAGTAACCGGCAAAATCGCGCCGAAGGATGTCGCTGCCGACTGGG CAATGGAGCGCCTGCCGGCCCAGTATCAGCCCGTCATACTTGAAGCTAGACAGGCTTATCTT GGACAAGAAGAAGATCGCTTGGCCTCGCGCGCAGATCAGTTGGAAGAATTTGTCCACTACGT GAAAGGCGAGATCACCAAGGTAGTCGGCAAATAATGTCTAGCTAGAAATTCGTTCAAGCCGA CGCCGCTTCGCCGGCGTTAACTCAAGCGATTAGATGCACTAAGCACATAATTGCTCACAGCC AAACTATCAGGTCAAGTCTGCTTTTATTATTTTTAAGCGTGCATAATAAGCCCTACACAAAT TGGGAGATATATCATGCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCA GACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTG CTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAA CTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTG TAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCT AATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAA GACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCC AGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGC CACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAG AGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGC CACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAA CGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCT ĵĸTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACC GCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCT GATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATGGTGCACTCTCA GTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATACACTCCGCTATCGCTACGTGACT GGGTCATGGCTGCGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTG CTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTT TTCACCGTCATCACCGAAACGCGCGAGGCAGGGTGCCTTGATGTGGGCGCCGGCGGTCGAGT GGCGACGGCGCGGCTTGTCCGCGCCCTGGTAGATTGCCTGGCCGTAGGCCAGCCATTTTTGA GCGGCCAGCGGCCGCGATAGGCCGACGCGAAGCGGCGGGGCGTAGGGAGCGCAGCGACCGAA GGGTAGGCGCTTTTTGCAGCTCTTCGGCTGTGCGCTGGCCAGACAGTTATGCACAGGCCAGG CGGGTTTTAAGAGTTTTAATAAGTTTTAAAGAGTTTTAGGCGGAAAAATCGCCTTTTTTCTC TTTTATATCAGTCACTTACATGTGTGACCGGTTCCCAATGTACGGCTTTGGGTTCCCAATGT ACGGGTTCCGGTTCCCAATGTACGGCTTTGGGTTCCCAATGTACGTGCTATCCACAGGAAAG AGACCTTTTCGACCTTTTTCCCCTGCTAGGGCAATTTGCCCTAGCATCTGCTCCGTACATTA GGAACCGGCGGATGCTTCGCCCTCGATCAGGTTGCGGTAGCGCATGACTAGGATCGGGCCAG CCTGCCCCGCCTCCTCCTTCAAATCGTACTCCGGCAGGTCATTTGACCCGATCAGCTTGCGC ACGGTGAAACAGAACTTCTTGAACTCTCCGGCGCTGCCACTGCGTTCGTAGATCGTCTTGAA CAACCATCTGGCTTCTGCCTTGCCTGCGGCGCGGCGTGCCAGGCGGTAGAGAAAACGGCCGA TGCCGGGATCGATCAAAAAGTAATCGGGGTGAACCGTCAGCACGTCCGGGTTCTTGCCTTCT GTGATCTCGCGGTACATCCAATCAGCTAGCTCGATCTCGATGTACTCCGGCCGCCCGGTTTC GCTCTTTACGATCTTGTAGCGGCTAATCAAGGCTTCACCCTCGGATACCGTCACCAGGCGGC CGTTCTTGGCCTTCTTCGTACGCTGCATGGCAACGTGCGTGGTGTTTAACCGAATGCAGGTT TCTACCAGGTCGTCTTTCTGCTTTCCGCCATCGGCTCGCCGGCAGAACTTGAGTACGTCCGC AACGTGTGGACGGAACACGCGGCCGGGCTTGTCTCCCTTCCCTTCCCGGTATCGGTTCATGG ATTCGGTTAGATGGGAAACCGCCATCAGTACCAGGTCGTAATCCCACACACTGGCCATGCCG GCCGGCCCTGCGGAAACCTCTACGTGCCCGTCTGGAAGCTCGTAGCGGATCACCTCGCCAGC TCGTCGGTCACGCTTCGACAGACGGAAAACGGCCACGTCCATGATGCTGCGACTATCGCGGG TGCCCACGTCATAGAGCATCGGAACGAAAAAATCTGGTTGCTCGTCGCCCTTGGGCGGCTTC CTAATCGACGGCGCACCGGCTGCCGGCGGTTGCCGGGATTCTTTGCGGATTCGATCAGCGGC CGCTTGCCACGATTCACCGGGGCGTGCTTCTGCCTCGATGCGTTGCCGCTGGGCGGCCTGCG CGGCCTTCAACTTCTCCACCAGGTCATCACCCAGCGCCGCGCCGATTTGTACCGGGCCGGAT GGTTTGCGACCGTCACGCCGATTCCTCGGGCTTGGGGGTTCCAGTGCCATTGCAGGGCCGGC AGACAACCCAGCCGCTTACGCCTGGCCAACCGCCCGTTCCTCCACACATGGGGCATTCCACG GCGTCGGTGCCTGGTTGTTCTTGATTTTCCATGCCGCCTCCTTTAGCCGCTAAAATTCATCT ACTCATTTATTCATTTGCTCATTTACTCTGGTAGCTGCGCGATGTATTCAGATAGCAGCTCG GTAATGGTCTTGCCTTGGCGTACCGCGTACATCTTCAGCTTGGTGTGATCCTCCGCCGGCAA CTGAAAGTTGACCCGCTTCATGGCTGGCGTGTCTGCCAGGCTGGCCAACGTTGCAGCCTTGC TGCTGCGTGCGCTCGGACGGCCGGCACTTAGCGTGTTTGTGCTTTTGCTCATTTTCTCTTTA CCTCATTAACTCAAATGAGTTTTGATTTAATTTCAGCGGCCAGCGCCTGGACCTCGCGGGCA GCGTCGCCCTCGGGTTCTGATTCAAGAACGGTTGTGCCGGCGGCGGCAGTGCCTGGGTAGCT CACGCGCTGCGTGATACGGGACTCAAGAATGGGCAGCTCGTACCCGGCCAGCGCCTCGGCAA CCTCACCGCCGATGCGCGTGCCTTTGATCGCCCGCGACACGACAAAGGCCGCTTGTAGCCTT CCATCCGTGACCTCAATGCGCTGCTTAACCAGCTCCACCAGGTCGGCGGTGGCCCATATGTC GTAAGGGCTTGGCTGCACCGGAATCAGCACGAAGTCGGCTGCCTTGATCGCGGACACAGCCA AGTCCGCCGCCTGGGGCGCTCCGTCGATCACTACGAAGTCGCGCCGGCCGATGGCCTTCACG TCGCGGTCAATCGTCGGGCGGTCGATGCCGACAACGGTTAGCGGTTGATCTTCCCGCACGGC CGCCCAATCGCGGGCACTGCCCTGGGGATCGGAATCGACTAACAGAACATCGGCCCCGGCGA GTTGCAGGGCGCGGGCTAGATGGGTTGCGATGGTCGTCTTGCCTGACCCGCCTTTCTGGTTA AGTACAGCGATAACCTTCATGCGTTCCCCTTGCGTATTTGTTTATTTACTCATCGCATCATA TACGCAGCGACCGCATGACGCAAGCTGTTTTACTCAAATACACATCACCTTTTTAGACGGCG GCGCTCGGTTTCTTCAGCGGCCAAGCTGGCCGGCCAGGCCGCCAGCTTGGCATCAGACAAAC ĵĹCGGCCAGGATTTCATGCAGCCGCACGGTTGAGACGTGCGCGGGCGGCTCGAACACGTACCCG GCCGCGATCATCTCCGCCTCGATCTCTTCGGTAATGAAAAACGGTTCGTCCTGGCCGTCCTG GTGCGGTTTCATGCTTGTTCCTCTTGGCGTTCATTCTCGGCGGCCGCCAGGGCGTCGGCCTC GGTCAATGCGTCCTCACGGAAGGCACCGCGCCGCCTGGCCTCGGTGGGCGTCACTTCCTCGC TGCGCTCAAGTGCGCGGTACAGGGTCGAGCGATGCACGCCAAGCAGTGCAGCCGCCTCTTTC ACGGTGCGGCCTTCCTGGTCGATCAGCTCGCGGGCGTGCGCGATCTGTGCCGGGGTGAGGGT AGGGCGGGGGCCAAACTTCACGCCTCGGGCCTTGGCGGCCTCGCGCCCGCTCCGGGTGCGGT CGATGATTAGGGAACGCTCGAACTCGGCAATGCCGGCGAACACGGTCAACACCATGCGGCCG GCCGGCGTGGTGGTGTCGGCCCACGGCTCTGCCAGGCTACGCAGGCCCGCGCCGGCCTCCTG GATGCGCTCGGCAATGTCCAGTAGGTCGCGGGTGCTGCGGGCCAGGCGGTCTAGCCTGGTCA CTGTCACAACGTCGCCAGGGCGTAGGTGGTCAAGCATCCTGGCCAGCTCCGGGCGGTCGCGC CTGGTGCCGGTGATCTTCTCGGAAAACAGCTTGGTGCAGCCGGCCGCGTGCAGTTCGGCCCG TTGGTTGGTCAAGTCCTGGTCGTCGGTGCTGACGCGGGCATAGCCCAGCAGGCCAGCGGCGG CGCTCTTGTTCATGGCGTAATGTCTCCGGTTCTAGTCGCAAGTATTCTACTTTATGCGACTA AAACACGCGACAAGAAAACGCCAGGAAAAGGGCAGGGCGGCAGCCTGTCGCGTAACTTAGGA CTTGTGCGACATGTCGTTTTCAGAAGACGGCTGCACTGAACGTCAGAAGCCGACTGCACTAT AGCAGCGGAGGGGTTGGATCAAAGTAC SEQ ID NO: 6 (Synthetic DNA; Oligonucleotide CRISPR target sequence, 20 bp). GGCTTCTAGTTCCAACCCTA SEQ ID NO: 7 (35S-5UTR-WT-GFP, 11,419 bp). Bold text indicates the 35S promoter region; underlined text indicates the 256 bp region of the 5’ UTR; italicized text indicates the EGFP coding region; and underlined with italicized text indicates the RBCS E9 terminator region. TTGATCCCGAGGGGAACCCTGTGGTTGGCATGCACATACAAATGGACGAACGGATAAACCTTTTCACGCCC TTTTAAATATCCGTTATTCTAATAAACGCTCTTTTCTCTTAGGTTTACCCGCCAATATATCCTGTCAAACA CTGATAGTTTAAACTGAAGGCGGGAAACGACAATCTGATCCAAGCTCAAGCTGCTCTAGCCAATACGCAAA CCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGG CAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTC CGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTAC GAATTCGGTCCCCAGATTAGCCTTTTCAATTTCAGAAAGAATGCTAACCCACAGATGGTTAGAGAGGCTTA CGCAGCAGGTCTCATCAAGACGATCTACCCGAGCAATAATCTCCAGGAAATCAAATACCTTCCCAAGAAGG TTAAAGATGCAGTCAAAAGATTCAGGACTAACTGCATCAAGAACACAGAGAAAGATATATTTCTCAAGATC AGAAGTACTATTCCAGTATGGACGATTCAAGGCTTGCTTCACAAACCAAGGCAAGTAATAGAGATTGGAGT CTCTAAAAAGGTAGTTCCCACTGAATCAAAGGCCATGGAGTCAAAGATTCAAATAGAGGACCTAACAGAAC TCGCCGTAAAGACTGGCGAACAGTTCATACAGAGTCTCTTACGACTCAATGACAAGAAGAAAATCTTCGTC AACATGGTGGAGCACGACACACTTGTCTACTCCAAAAATATCAAAGATACAGTCTCAGAAGACCAAAGGGC AATTGAGACTTTTCAACAAAGGGTAATATCCGGAAACCTCCTCGGATTCCATTGCCCAGCTATCTGTCACT TTATTGTGAAGATAGTGGAAAAGGAAGGTGGCTCCTACAAATGCCATCATTGCGATAAAGGAAAGGCCATC GTTGAAGATGCCTCTGCCGACAGTGGTCCCAAAGATGGACCCCCACCCACGAGGAGCATCGTGGAAAAAGA AGACGTTCCAACCACGTCTTCAAAGCAAGTGGATTGATGTGATATCTCCACTGACGTAAGGGATGACGCAC AATCCCACTATCCTTCGCAAGACCCTTCCTCTATATAAGGAAGTTCATTTCATTTGGAGAGAACACGGGGC ACGTAGCAGCAGTGTGGGTAAGATCAAAGGGTGTTTCTCGATCAGTTTCATATTCAGATGTATCAGAGTTC TCATTAACAGATCTGTTTCTTTTTCCTTATCTGATTAAACAATTTCCTTCAGAATTTTACTTTTTTGAACA TATATAGTTTTTCTCTGTTCCTATATCTTGAGTTTTGTGAGAGGTTAATTATATGAAATTTTACGCATTAT TGTTCATCTATATCGAAAAACAATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGG TCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTAC GGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCAC CCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCG CCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGG CAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGA AGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCAC TACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTC CGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGA TCACTCTCGGCATGGACGAGCTGTACAAGTAATGCAGAGCTTTCGTTCGTATCATCGGTTTCGACAACGTT CGTCAAGTTCAATGCATCAGTTTCATTGCGCACACACCAGAATCCTACTGAGTTTGAGTATTATGGCATTG GGAAAACTGTTTTTCTTGTACCATTTGTTGTGCTTGTAATTTACTGTGTTTTTTATTCGGTTTTCGCTATC GAACTGTGAAATGGAAATGGATGGAGAAGAGTTAATGAATGATATGGTCCTTTTGTTCATTCTCAAATTAA TATTATTTGTTTTTTCTCTTATTTGTTGTGTGTTGAATTTGAAAATATAAGAGATATGCAAACATTTTGTT TTGAGTAAAAATGTGTCAAATCGTGGCCTCTAATGACCGAAGTTAATATGAGGAGTAAAACACTTGTAGTT GTACCATTATGCTTATTCACTAGGCAACAAATATATTTTCAGACCTAGAAAAGCTGCAAATGTTACTGAAT ACAAGTATGTCCTCTTGTGTTTTAGACATTTATGAACTTTCCTTTATGTAATTTTCCAGAATCCTTGTCAG ATTCTAATCATTGCTTTATAATTATAGTTATACTCATGGATTTGTAGTTGAGTATGAAAATATTTTTTAAT GCATTTTATGACTTGCCAATTGATTGACAACATGCATCAATCGAAGCTTGGCACTGGCCGTCGTTTTACAA CGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTG GCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGCTAGA GCAGCTTGCCAACATGGTGGAGCACGACACTCTCGTCTACTCCAAGAATATCAAAGATACAGTCTCAGAAG ACCAAAGGGCTATTGAGACTTTTCAACAAAGGGTAATATCGGGAAACCTCCTCGGATTCCATTGCCCAGCT ATCTGTCACTTCATCAAAAGGACAGTAGAAAAGGAAGGTGGCACCTACAAATGCCATCATTGCGATAAAGG AAAGGCTATCGTTCAAGATGCCTCTGCCGACAGTGGTCCCAAAGATGGACCCCCACCCACGAGGAGCATCG TGGAAAAAGAAGACGTTCCAACCACGTCTTCAAAGCAAGTGGATTGATGTGATAACATGGTGGAGCACGAC ACTCTCGTCTACTCCAAGAATATCAAAGATACAGTCTCAGAAGACCAAAGGGCTATTGAGACTTTTCAACA AAGGGTAATATCGGGAAACCTCCTCGGATTCCATTGCCCAGCTATCTGTCACTTCATCAAAAGGACAGTAG AAAAGGAAGGTGGCACCTACAAATGCCATCATTGCGATAAAGGAAAGGCTATCGTTCAAGATGCCTCTGCC GACAGTGGTCCCAAAGATGGACCCCCACCCACGAGGAGCATCGTGGAAAAAGAAGACGTTCCAACCACGTC TTCAAAGCAAGTGGATTGATGTGATATCTCCACTGACGTAAGGGATGACGCACAATCCCACTATCCTTCGC AAGACCCTTCCTCTATATAAGGAAGTTCATTTCATTTGGAGAGGACACGCTGAAATCACCAGTCTCTCTCT ACAAATCTATCTCTCTCGATTCGCAGATCTGTCGATCGACCATGGGGATTGAACAAGATGGATTGCACGCA GGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCACAACAGACAATCGGCTGCTCTGA TGCCGCCGTGTTCCGGCTGTCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCC TGAATGAACTCCAGGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGTG CTCGACGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCAGGATCTCCTGTC ATCTCACCTTGCTCCTGCCGAGAAAGTATCCATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATC CGGCTACCTGCCCATTCGACCACCAAGCGAAACATCGCATCGAGCGAGCACGTACTCGGATGGAAGCCGGT CTTGTCGATCAGGATGATCTGGACGAAGAGCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAA GGCGCGCATGCCCGACGGCGAGGATCTCGTCGTGACACATGGCGATGCCTGCTTGCCGAATATCATGGTGG AAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGACCGCTATCAGGACATAGCG TTGGCTACCCGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGCTGACCGCTTCCTCGTGCTTTACGGTAT CGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATCGCCTTCTTGACGAGTTCTTCTGAGCGGGACTCTGGG GTTCGGATCGATCCTCTAGCTAGAGTCGATCGACATCGAGTTTCTCCATAATAATGTGTGAGTAGTTCCCA GATAAGGGAATTAGGGTTCTTATAGGGTTTCGCTCACGTGTTGAGCATATAAGAAACCCTTAGTATGTATT TGTATTTGTAAAATACTTCTATCAATAAAATTTCTAATTCCTAAAACCAAAATCCAGTACTAAAATCCAGA TCACCTAAAGTCCCTATAGATCCCCCGAATTAATTCGGCGTTAATTCAGTACATTAAAAACGTCCGCAATG TGTTATTAAGTTGTCTAAGCGTCAATTTGTTTACACCACAATATATCCTGCCACCAGCCAGCCAACAGCTC CCCGACCGGCAGCTCGGCACAAAATCACCACTCGATACAGGCAGCCCATCAGTCCGGGACGGCGTCAGCGG GAGAGCCGTTGTAAGGCGGCAGACTTTGCTCATGTTACCGATGCTATTCGGAAGAACGGCAACTAAGCTGC CGGGTTTGAAACACGGATGATCTCGCGGAGGGTAGCATGTTGATTGTAACGATGACAGAGCGTTGCTGCCT GTGATCAATTCGGGCACGAACCCAGTGGACATAAGCCTCGTTCGGTTCGTAAGCTGTAATGCAAGTAGCGT AACTGCCGTCACGCAACTGGTCCAGAACCTTGACCGAACGCAGCGGTGGTAACGGCGCAGTGGCGGTTTTC ATGGCTTCTTGTTATGACATGTTTTTTTGGGGTACAGTCTATGCCTCGGGCATCCAAGCAGCAAGCGCGTT ACGCCGTGGGTCGATGTTTGATGTTATGGAGCAGCAACGATGTTACGCAGCAGGGCAGTCGCCCTAAAACA AAGTTAAACATCATGGGGGAAGCGGTGATCGCCGAAGTATCGACTCAACTATCAGAGGTAGTTGGCGTCAT CGAGCGCCATCTCGAACCGACGTTGCTGGCCGTACATTTGTACGGCTCCGCAGTGGATGGCGGCCTGAAGC CACACAGTGATATTGATTTGCTGGTTACGGTGACCGTAAGGCTTGATGAAACAACGCGGCGAGCTTTGATC AACGACCTTTTGGAAACTTCGGCTTCCCCTGGAGAGAGCGAGATTCTCCGCGCTGTAGAAGTCACCATTGT TGTGCACGACGACATCATTCCGTGGCGTTATCCAGCTAAGCGCGAACTGCAATTTGGAGAATGGCAGCGCA ATGACATTCTTGCAGGTATCTTCGAGCCAGCCACGATCGACATTGATCTGGCTATCTTGCTGACAAAAGCAAGAGAACATAGCGTTGCCTTGGTAGGTCCAGCGGCGGAGGAACTCTTTGATCCGGTTCCTGAACAGGATCT ATTTGAGGCGCTAAATGAAACCTTAACGCTATGGAACTCGCCGCCCGACTGGGCTGGCGATGAGCGAAATG TAGTGCTTACGTTGTCCCGCATTTGGTACAGCGCAGTAACCGGCAAAATCGCGCCGAAGGATGTCGCTGCC GACTGGGCAATGGAGCGCCTGCCGGCCCAGTATCAGCCCGTCATACTTGAAGCTAGACAGGCTTATCTTGG ACAAGAAGAAGATCGCTTGGCCTCGCGCGCAGATCAGTTGGAAGAATTTGTCCACTACGTGAAAGGCGAGA TCACCAAGGTAGTCGGCAAATAATGTCTAGCTAGAAATTCGTTCAAGCCGACGCCGCTTCGCCGGCGTTAA CTCAAGCGATTAGATGCACTAAGCACATAATTGCTCACAGCCAAACTATCAGGTCAAGTCTGCTTTTATTA TTTTTAAGCGTGCATAATAAGCCCTACACAAATTGGGAGATATATCATGCATGACCAAAATCCCTTAACGT GAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCT GCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGC TACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAG CCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACC AGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGG CGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTG AGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGT AAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTC CTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGG AAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCC TGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCC GAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACG CATCTGTGCGGTATTTCACACCGCATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAG CCAGTATACACTCCGCTATCGCTACGTGACTGGGTCATGGCTGCGCCCCGACACCCGCCAACACCCGCTGA CGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCA TGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGGCAGGGTGCCTTGATGTGGGCGCCGGCGGTC GAGTGGCGACGGCGCGGCTTGTCCGCGCCCTGGTAGATTGCCTGGCCGTAGGCCAGCCATTTTTGAGCGGC CAGCGGCCGCGATAGGCCGACGCGAAGCGGCGGGGCGTAGGGAGCGCAGCGACCGAAGGGTAGGCGCTTTT TGCAGCTCTTCGGCTGTGCGCTGGCCAGACAGTTATGCACAGGCCAGGCGGGTTTTAAGAGTTTTAATAAG TTTTAAAGAGTTTTAGGCGGAAAAATCGCCTTTTTTCTCTTTTATATCAGTCACTTACATGTGTGACCGGT TCCCAATGTACGGCTTTGGGTTCCCAATGTACGGGTTCCGGTTCCCAATGTACGGCTTTGGGTTCCCAATG TACGTGCTATCCACAGGAAAGAGACCTTTTCGACCTTTTTCCCCTGCTAGGGCAATTTGCCCTAGCATCTG CTCCGTACATTAGGAACCGGCGGATGCTTCGCCCTCGATCAGGTTGCGGTAGCGCATGACTAGGATCGGGC CAGCCTGCCCCGCCTCCTCCTTCAAATCGTACTCCGGCAGGTCATTTGACCCGATCAGCTTGCGCACGGTG AAACAGAACTTCTTGAACTCTCCGGCGCTGCCACTGCGTTCGTAGATCGTCTTGAACAACCATCTGGCTTC TGCCTTGCCTGCGGCGCGGCGTGCCAGGCGGTAGAGAAAACGGCCGATGCCGGGATCGATCAAAAAGTAAT CGGGGTGAACCGTCAGCACGTCCGGGTTCTTGCCTTCTGTGATCTCGCGGTACATCCAATCAGCTAGCTCG ATCTCGATGTACTCCGGCCGCCCGGTTTCGCTCTTTACGATCTTGTAGCGGCTAATCAAGGCTTCACCCTC GGATACCGTCACCAGGCGGCCGTTCTTGGCCTTCTTCGTACGCTGCATGGCAACGTGCGTGGTGTTTAACC GAATGCAGGTTTCTACCAGGTCGTCTTTCTGCTTTCCGCCATCGGCTCGCCGGCAGAACTTGAGTACGTCC GCAACGTGTGGACGGAACACGCGGCCGGGCTTGTCTCCCTTCCCTTCCCGGTATCGGTTCATGGATTCGGT TAGATGGGAAACCGCCATCAGTACCAGGTCGTAATCCCACACACTGGCCATGCCGGCCGGCCCTGCGGAAA CCTCTACGTGCCCGTCTGGAAGCTCGTAGCGGATCACCTCGCCAGCTCGTCGGTCACGCTTCGACAGACGG AAAACGGCCACGTCCATGATGCTGCGACTATCGCGGGTGCCCACGTCATAGAGCATCGGAACGAAAAAATC TGGTTGCTCGTCGCCCTTGGGCGGCTTCCTAATCGACGGCGCACCGGCTGCCGGCGGTTGCCGGGATTCTT TGCGGATTCGATCAGCGGCCGCTTGCCACGATTCACCGGGGCGTGCTTCTGCCTCGATGCGTTGCCGCTGG GCGGCCTGCGCGGCCTTCAACTTCTCCACCAGGTCATCACCCAGCGCCGCGCCGATTTGTACCGGGCCGGA TGGTTTGCGACCGTCACGCCGATTCCTCGGGCTTGGGGGTTCCAGTGCCATTGCAGGGCCGGCAGACAACC CAGCCGCTTACGCCTGGCCAACCGCCCGTTCCTCCACACATGGGGCATTCCACGGCGTCGGTGCCTGGTTG TTCTTGATTTTCCATGCCGCCTCCTTTAGCCGCTAAAATTCATCTACTCATTTATTCATTTGCTCATTTAC TCTGGTAGCTGCGCGATGTATTCAGATAGCAGCTCGGTAATGGTCTTGCCTTGGCGTACCGCGTACATCTT CAGCTTGGTGTGATCCTCCGCCGGCAACTGAAAGTTGACCCGCTTCATGGCTGGCGTGTCTGCCAGGCTGG CCAACGTTGCAGCCTTGCTGCTGCGTGCGCTCGGACGGCCGGCACTTAGCGTGTTTGTGCTTTTGCTCATT TTCTCTTTACCTCATTAACTCAAATGAGTTTTGATTTAATTTCAGCGGCCAGCGCCTGGACCTCGCGGGCA GCGTCGCCCTCGGGTTCTGATTCAAGAACGGTTGTGCCGGCGGCGGCAGTGCCTGGGTAGCTCACGCGCTG CGTGATACGGGACTCAAGAATGGGCAGCTCGTACCCGGCCAGCGCCTCGGCAACCTCACCGCCGATGCGCG TGCCTTTGATCGCCCGCGACACGACAAAGGCCGCTTGTAGCCTTCCATCCGTGACCTCAATGCGCTGCTTA ACCAGCTCCACCAGGTCGGCGGTGGCCCATATGTCGTAAGGGCTTGGCTGCACCGGAATCAGCACGAAGTC GGCTGCCTTGATCGCGGACACAGCCAAGTCCGCCGCCTGGGGCGCTCCGTCGATCACTACGAAGTCGCGCC GGCCGATGGCCTTCACGTCGCGGTCAATCGTCGGGCGGTCGATGCCGACAACGGTTAGCGGTTGATCTTCCCGCACGGCCGCCCAATCGCGGGCACTGCCCTGGGGATCGGAATCGACTAACAGAACATCGGCCCCGGCGAG TTGCAGGGCGCGGGCTAGATGGGTTGCGATGGTCGTCTTGCCTGACCCGCCTTTCTGGTTAAGTACAGCGA TAACCTTCATGCGTTCCCCTTGCGTATTTGTTTATTTACTCATCGCATCATATACGCAGCGACCGCATGAC GCAAGCTGTTTTACTCAAATACACATCACCTTTTTAGACGGCGGCGCTCGGTTTCTTCAGCGGCCAAGCTG GCCGGCCAGGCCGCCAGCTTGGCATCAGACAAACCGGCCAGGATTTCATGCAGCCGCACGGTTGAGACGTG CGCGGGCGGCTCGAACACGTACCCGGCCGCGATCATCTCCGCCTCGATCTCTTCGGTAATGAAAAACGGTT CGTCCTGGCCGTCCTGGTGCGGTTTCATGCTTGTTCCTCTTGGCGTTCATTCTCGGCGGCCGCCAGGGCGT CGGCCTCGGTCAATGCGTCCTCACGGAAGGCACCGCGCCGCCTGGCCTCGGTGGGCGTCACTTCCTCGCTG CGCTCAAGTGCGCGGTACAGGGTCGAGCGATGCACGCCAAGCAGTGCAGCCGCCTCTTTCACGGTGCGGCC TTCCTGGTCGATCAGCTCGCGGGCGTGCGCGATCTGTGCCGGGGTGAGGGTAGGGCGGGGGCCAAACTTCA CGCCTCGGGCCTTGGCGGCCTCGCGCCCGCTCCGGGTGCGGTCGATGATTAGGGAACGCTCGAACTCGGCA ATGCCGGCGAACACGGTCAACACCATGCGGCCGGCCGGCGTGGTGGTGTCGGCCCACGGCTCTGCCAGGCT ACGCAGGCCCGCGCCGGCCTCCTGGATGCGCTCGGCAATGTCCAGTAGGTCGCGGGTGCTGCGGGCCAGGC GGTCTAGCCTGGTCACTGTCACAACGTCGCCAGGGCGTAGGTGGTCAAGCATCCTGGCCAGCTCCGGGCGG TCGCGCCTGGTGCCGGTGATCTTCTCGGAAAACAGCTTGGTGCAGCCGGCCGCGTGCAGTTCGGCCCGTTG GTTGGTCAAGTCCTGGTCGTCGGTGCTGACGCGGGCATAGCCCAGCAGGCCAGCGGCGGCGCTCTTGTTCA TGGCGTAATGTCTCCGGTTCTAGTCGCAAGTATTCTACTTTATGCGACTAAAACACGCGACAAGAAAACGC CAGGAAAAGGGCAGGGCGGCAGCCTGTCGCGTAACTTAGGACTTGTGCGACATGTCGTTTTCAGAAGACGG CTGCACTGAACGTCAGAAGCCGACTGCACTATAGCAGCGGAGGGGTTGGATCAAAGTAC SEQ ID NO: 8 (35S-5UTR-MUT-GFP, comprising a mutated GFP, 11,419 bp). Bold text indicates the 35S promoter region; underlined text indicates the 256 bp region of the 5’ UTR; italicized text indicates the EGFP coding region; and underlined with italicized text indicates the RBCS E9 terminator region. TTGATCCCGAGGGGAACCCTGTGGTTGGCATGCACATACAAATGGACGAACGGATAAACCTTTTCACG CCCTTTTAAATATCCGTTATTCTAATAAACGCTCTTTTCTCTTAGGTTTACCCGCCAATATATCCTGT CAAACACTGATAGTTTAAACTGAAGGCGGGAAACGACAATCTGATCCAAGCTCAAGCTGCTCTAGCCA ATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGA CTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTT TACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAAC AGCTATGACCATGATTACGAATTCGGTCCCCAGATTAGCCTTTTCAATTTCAGAAAGAATGCTAACCC ACAGATGGTTAGAGAGGCTTACGCAGCAGGTCTCATCAAGACGATCTACCCGAGCAATAATCTCCAGG AAATCAAATACCTTCCCAAGAAGGTTAAAGATGCAGTCAAAAGATTCAGGACTAACTGCATCAAGAAC ACAGAGAAAGATATATTTCTCAAGATCAGAAGTACTATTCCAGTATGGACGATTCAAGGCTTGCTTCA CAAACCAAGGCAAGTAATAGAGATTGGAGTCTCTAAAAAGGTAGTTCCCACTGAATCAAAGGCCATGG AGTCAAAGATTCAAATAGAGGACCTAACAGAACTCGCCGTAAAGACTGGCGAACAGTTCATACAGAGT CTCTTACGACTCAATGACAAGAAGAAAATCTTCGTCAACATGGTGGAGCACGACACACTTGTCTACTC CAAAAATATCAAAGATACAGTCTCAGAAGACCAAAGGGCAATTGAGACTTTTCAACAAAGGGTAATAT CCGGAAACCTCCTCGGATTCCATTGCCCAGCTATCTGTCACTTTATTGTGAAGATAGTGGAAAAGGAA GGTGGCTCCTACAAATGCCATCATTGCGATAAAGGAAAGGCCATCGTTGAAGATGCCTCTGCCGACAG TGGTCCCAAAGATGGACCCCCACCCACGAGGAGCATCGTGGAAAAAGAAGACGTTCCAACCACGTCTTCAAAGCAAGTGGATTGATGTGATATCTCCACTGACGTAAGGGATGACGCACAATCCCACTATCCTTCG CAAGACCCTTCCTCTATATAAGGAAGTTCATTTCATTTGGAGAGAACACGGGGCACGTAGCAGCAGTG TGGGTAAGATCAAAGGGTGTTTCTCGATCAGTTTCATATTCAGATGTATCAGAGTTCTCATTAACAGA TTTGTTTCTTTTTCCTTATCTGATTAAACAATTTCCTTCAGAATTTTACTTTTTTGAACATATATAGT TTTTCTCTGTTCCTATATCTTGAGTTTTGTGAGAGGTTAATTATATGAAATTTTACGCATTATTGTTC ATCTATATCGAAAAACAATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTC GAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTA CGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGA CCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTC AAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAA GACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACT TCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATC ATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAG CGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACA ACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTG CTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAATGCAGAGCTTT CGTTCGTATCATCGGTTTCGACAACGTTCGTCAAGTTCAATGCATCAGTTTCATTGCGCACACACCAG AATCCTACTGAGTTTGAGTATTATGGCATTGGGAAAACTGTTTTTCTTGTACCATTTGTTGTGCTTGT AATTTACTGTGTTTTTTATTCGGTTTTCGCTATCGAACTGTGAAATGGAAATGGATGGAGAAGAGTTA ATGAATGATATGGTCCTTTTGTTCATTCTCAAATTAATATTATTTGTTTTTTCTCTTATTTGTTGTGT GTTGAATTTGAAAATATAAGAGATATGCAAACATTTTGTTTTGAGTAAAAATGTGTCAAATCGTGGCC TCTAATGACCGAAGTTAATATGAGGAGTAAAACACTTGTAGTTGTACCATTATGCTTATTCACTAGGC AACAAATATATTTTCAGACCTAGAAAAGCTGCAAATGTTACTGAATACAAGTATGTCCTCTTGTGTTT TAGACATTTATGAACTTTCCTTTATGTAATTTTCCAGAATCCTTGTCAGATTCTAATCATTGCTTTAT AATTATAGTTATACTCATGGATTTGTAGTTGAGTATGAAAATATTTTTTAATGCATTTTATGACTTGC CAATTGATTGACAACATGCATCAATCGAAGCTTGGCACTGGCCGTCGTTTTACAACGTCGTGACTGGG AAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGC GAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGCTAGAGCAGCTT GCCAACATGGTGGAGCACGACACTCTCGTCTACTCCAAGAATATCAAAGATACAGTCTCAGAAGACCA AAGGGCTATTGAGACTTTTCAACAAAGGGTAATATCGGGAAACCTCCTCGGATTCCATTGCCCAGCTA TCTGTCACTTCATCAAAAGGACAGTAGAAAAGGAAGGTGGCACCTACAAATGCCATCATTGCGATAAA GGAAAGGCTATCGTTCAAGATGCCTCTGCCGACAGTGGTCCCAAAGATGGACCCCCACCCACGAGGAG CATCGTGGAAAAAGAAGACGTTCCAACCACGTCTTCAAAGCAAGTGGATTGATGTGATAACATGGTGG AGCACGACACTCTCGTCTACTCCAAGAATATCAAAGATACAGTCTCAGAAGACCAAAGGGCTATTGAG ACTTTTCAACAAAGGGTAATATCGGGAAACCTCCTCGGATTCCATTGCCCAGCTATCTGTCACTTCAT CAAAAGGACAGTAGAAAAGGAAGGTGGCACCTACAAATGCCATCATTGCGATAAAGGAAAGGCTATCG TTCAAGATGCCTCTGCCGACAGTGGTCCCAAAGATGGACCCCCACCCACGAGGAGCATCGTGGAAAAAGAAGACGTTCCAACCACGTCTTCAAAGCAAGTGGATTGATGTGATATCTCCACTGACGTAAGGGATGA CGCACAATCCCACTATCCTTCGCAAGACCCTTCCTCTATATAAGGAAGTTCATTTCATTTGGAGAGGA CACGCTGAAATCACCAGTCTCTCTCTACAAATCTATCTCTCTCGATTCGCAGATCTGTCGATCGACCA TGGGGATTGAACAAGATGGATTGCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTAT GACTGGGCACAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGCGCAGGGGCGCCC GGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTCCAGGACGAGGCAGCGCGGCTAT CGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGTGCTCGACGTTGTCACTGAAGCGGGAAGGGAC TGGCTGCTATTGGGCGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACCTTGCTCCTGCCGAGAAAGT ATCCATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATCCGGCTACCTGCCCATTCGACCACC AAGCGAAACATCGCATCGAGCGAGCACGTACTCGGATGGAAGCCGGTCTTGTCGATCAGGATGATCTG GACGAAGAGCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGCGCATGCCCGACGG CGAGGATCTCGTCGTGACACATGGCGATGCCTGCTTGCCGAATATCATGGTGGAAAATGGCCGCTTTT CTGGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGACCGCTATCAGGACATAGCGTTGGCTACCCGT GATATTGCTGAAGAGCTTGGCGGCGAATGGGCTGACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCC CGATTCGCAGCGCATCGCCTTCTATCGCCTTCTTGACGAGTTCTTCTGAGCGGGACTCTGGGGTTCGG ATCGATCCTCTAGCTAGAGTCGATCGACATCGAGTTTCTCCATAATAATGTGTGAGTAGTTCCCAGAT AAGGGAATTAGGGTTCTTATAGGGTTTCGCTCACGTGTTGAGCATATAAGAAACCCTTAGTATGTATT TGTATTTGTAAAATACTTCTATCAATAAAATTTCTAATTCCTAAAACCAAAATCCAGTACTAAAATCC AGATCACCTAAAGTCCCTATAGATCCCCCGAATTAATTCGGCGTTAATTCAGTACATTAAAAACGTCC GCAATGTGTTATTAAGTTGTCTAAGCGTCAATTTGTTTACACCACAATATATCCTGCCACCAGCCAGC CAACAGCTCCCCGACCGGCAGCTCGGCACAAAATCACCACTCGATACAGGCAGCCCATCAGTCCGGGA CGGCGTCAGCGGGAGAGCCGTTGTAAGGCGGCAGACTTTGCTCATGTTACCGATGCTATTCGGAAGAA CGGCAACTAAGCTGCCGGGTTTGAAACACGGATGATCTCGCGGAGGGTAGCATGTTGATTGTAACGAT GACAGAGCGTTGCTGCCTGTGATCAATTCGGGCACGAACCCAGTGGACATAAGCCTCGTTCGGTTCGT AAGCTGTAATGCAAGTAGCGTAACTGCCGTCACGCAACTGGTCCAGAACCTTGACCGAACGCAGCGGT GGTAACGGCGCAGTGGCGGTTTTCATGGCTTCTTGTTATGACATGTTTTTTTGGGGTACAGTCTATGC CTCGGGCATCCAAGCAGCAAGCGCGTTACGCCGTGGGTCGATGTTTGATGTTATGGAGCAGCAACGAT GTTACGCAGCAGGGCAGTCGCCCTAAAACAAAGTTAAACATCATGGGGGAAGCGGTGATCGCCGAAGT ATCGACTCAACTATCAGAGGTAGTTGGCGTCATCGAGCGCCATCTCGAACCGACGTTGCTGGCCGTAC ATTTGTACGGCTCCGCAGTGGATGGCGGCCTGAAGCCACACAGTGATATTGATTTGCTGGTTACGGTG ACCGTAAGGCTTGATGAAACAACGCGGCGAGCTTTGATCAACGACCTTTTGGAAACTTCGGCTTCCCC TGGAGAGAGCGAGATTCTCCGCGCTGTAGAAGTCACCATTGTTGTGCACGACGACATCATTCCGTGGC GTTATCCAGCTAAGCGCGAACTGCAATTTGGAGAATGGCAGCGCAATGACATTCTTGCAGGTATCTTC GAGCCAGCCACGATCGACATTGATCTGGCTATCTTGCTGACAAAAGCAAGAGAACATAGCGTTGCCTT GGTAGGTCCAGCGGCGGAGGAACTCTTTGATCCGGTTCCTGAACAGGATCTATTTGAGGCGCTAAATG AAACCTTAACGCTATGGAACTCGCCGCCCGACTGGGCTGGCGATGAGCGAAATGTAGTGCTTACGTTG TCCCGCATTTGGTACAGCGCAGTAACCGGCAAAATCGCGCCGAAGGATGTCGCTGCCGACTGGGCAATGGAGCGCCTGCCGGCCCAGTATCAGCCCGTCATACTTGAAGCTAGACAGGCTTATCTTGGACAAGAAG AAGATCGCTTGGCCTCGCGCGCAGATCAGTTGGAAGAATTTGTCCACTACGTGAAAGGCGAGATCACC AAGGTAGTCGGCAAATAATGTCTAGCTAGAAATTCGTTCAAGCCGACGCCGCTTCGCCGGCGTTAACT CAAGCGATTAGATGCACTAAGCACATAATTGCTCACAGCCAAACTATCAGGTCAAGTCTGCTTTTATT ATTTTTAAGCGTGCATAATAAGCCCTACACAAATTGGGAGATATATCATGCATGACCAAAATCCCTTA ACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTT TTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCG GATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGT CCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTC TGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGA CGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGA GCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAG GGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCA GGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTT GTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGG CCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATT ACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGA GGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATAT GGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATACACTCCGCTATCGCTAC GTGACTGGGTCATGGCTGCGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTG CTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACC GTCATCACCGAAACGCGCGAGGCAGGGTGCCTTGATGTGGGCGCCGGCGGTCGAGTGGCGACGGCGCG GCTTGTCCGCGCCCTGGTAGATTGCCTGGCCGTAGGCCAGCCATTTTTGAGCGGCCAGCGGCCGCGAT AGGCCGACGCGAAGCGGCGGGGCGTAGGGAGCGCAGCGACCGAAGGGTAGGCGCTTTTTGCAGCTCTT CGGCTGTGCGCTGGCCAGACAGTTATGCACAGGCCAGGCGGGTTTTAAGAGTTTTAATAAGTTTTAAA GAGTTTTAGGCGGAAAAATCGCCTTTTTTCTCTTTTATATCAGTCACTTACATGTGTGACCGGTTCCC AATGTACGGCTTTGGGTTCCCAATGTACGGGTTCCGGTTCCCAATGTACGGCTTTGGGTTCCCAATGT ACGTGCTATCCACAGGAAAGAGACCTTTTCGACCTTTTTCCCCTGCTAGGGCAATTTGCCCTAGCATC TGCTCCGTACATTAGGAACCGGCGGATGCTTCGCCCTCGATCAGGTTGCGGTAGCGCATGACTAGGAT CGGGCCAGCCTGCCCCGCCTCCTCCTTCAAATCGTACTCCGGCAGGTCATTTGACCCGATCAGCTTGC GCACGGTGAAACAGAACTTCTTGAACTCTCCGGCGCTGCCACTGCGTTCGTAGATCGTCTTGAACAAC CATCTGGCTTCTGCCTTGCCTGCGGCGCGGCGTGCCAGGCGGTAGAGAAAACGGCCGATGCCGGGATC GATCAAAAAGTAATCGGGGTGAACCGTCAGCACGTCCGGGTTCTTGCCTTCTGTGATCTCGCGGTACA TCCAATCAGCTAGCTCGATCTCGATGTACTCCGGCCGCCCGGTTTCGCTCTTTACGATCTTGTAGCGG CTAATCAAGGCTTCACCCTCGGATACCGTCACCAGGCGGCCGTTCTTGGCCTTCTTCGTACGCTGCAT GGCAACGTGCGTGGTGTTTAACCGAATGCAGGTTTCTACCAGGTCGTCTTTCTGCTTTCCGCCATCGG CTCGCCGGCAGAACTTGAGTACGTCCGCAACGTGTGGACGGAACACGCGGCCGGGCTTGTCTCCCTTCCCTTCCCGGTATCGGTTCATGGATTCGGTTAGATGGGAAACCGCCATCAGTACCAGGTCGTAATCCCA CACACTGGCCATGCCGGCCGGCCCTGCGGAAACCTCTACGTGCCCGTCTGGAAGCTCGTAGCGGATCA CCTCGCCAGCTCGTCGGTCACGCTTCGACAGACGGAAAACGGCCACGTCCATGATGCTGCGACTATCG CGGGTGCCCACGTCATAGAGCATCGGAACGAAAAAATCTGGTTGCTCGTCGCCCTTGGGCGGCTTCCT AATCGACGGCGCACCGGCTGCCGGCGGTTGCCGGGATTCTTTGCGGATTCGATCAGCGGCCGCTTGCC ACGATTCACCGGGGCGTGCTTCTGCCTCGATGCGTTGCCGCTGGGCGGCCTGCGCGGCCTTCAACTTC TCCACCAGGTCATCACCCAGCGCCGCGCCGATTTGTACCGGGCCGGATGGTTTGCGACCGTCACGCCG ATTCCTCGGGCTTGGGGGTTCCAGTGCCATTGCAGGGCCGGCAGACAACCCAGCCGCTTACGCCTGGC CAACCGCCCGTTCCTCCACACATGGGGCATTCCACGGCGTCGGTGCCTGGTTGTTCTTGATTTTCCAT GCCGCCTCCTTTAGCCGCTAAAATTCATCTACTCATTTATTCATTTGCTCATTTACTCTGGTAGCTGC GCGATGTATTCAGATAGCAGCTCGGTAATGGTCTTGCCTTGGCGTACCGCGTACATCTTCAGCTTGGT GTGATCCTCCGCCGGCAACTGAAAGTTGACCCGCTTCATGGCTGGCGTGTCTGCCAGGCTGGCCAACG TTGCAGCCTTGCTGCTGCGTGCGCTCGGACGGCCGGCACTTAGCGTGTTTGTGCTTTTGCTCATTTTC TCTTTACCTCATTAACTCAAATGAGTTTTGATTTAATTTCAGCGGCCAGCGCCTGGACCTCGCGGGCA GCGTCGCCCTCGGGTTCTGATTCAAGAACGGTTGTGCCGGCGGCGGCAGTGCCTGGGTAGCTCACGCG CTGCGTGATACGGGACTCAAGAATGGGCAGCTCGTACCCGGCCAGCGCCTCGGCAACCTCACCGCCGA TGCGCGTGCCTTTGATCGCCCGCGACACGACAAAGGCCGCTTGTAGCCTTCCATCCGTGACCTCAATG CGCTGCTTAACCAGCTCCACCAGGTCGGCGGTGGCCCATATGTCGTAAGGGCTTGGCTGCACCGGAAT CAGCACGAAGTCGGCTGCCTTGATCGCGGACACAGCCAAGTCCGCCGCCTGGGGCGCTCCGTCGATCA CTACGAAGTCGCGCCGGCCGATGGCCTTCACGTCGCGGTCAATCGTCGGGCGGTCGATGCCGACAACG GTTAGCGGTTGATCTTCCCGCACGGCCGCCCAATCGCGGGCACTGCCCTGGGGATCGGAATCGACTAA CAGAACATCGGCCCCGGCGAGTTGCAGGGCGCGGGCTAGATGGGTTGCGATGGTCGTCTTGCCTGACC CGCCTTTCTGGTTAAGTACAGCGATAACCTTCATGCGTTCCCCTTGCGTATTTGTTTATTTACTCATC GCATCATATACGCAGCGACCGCATGACGCAAGCTGTTTTACTCAAATACACATCACCTTTTTAGACGG CGGCGCTCGGTTTCTTCAGCGGCCAAGCTGGCCGGCCAGGCCGCCAGCTTGGCATCAGACAAACCGGC CAGGATTTCATGCAGCCGCACGGTTGAGACGTGCGCGGGCGGCTCGAACACGTACCCGGCCGCGATCA TCTCCGCCTCGATCTCTTCGGTAATGAAAAACGGTTCGTCCTGGCCGTCCTGGTGCGGTTTCATGCTT GTTCCTCTTGGCGTTCATTCTCGGCGGCCGCCAGGGCGTCGGCCTCGGTCAATGCGTCCTCACGGAAG GCACCGCGCCGCCTGGCCTCGGTGGGCGTCACTTCCTCGCTGCGCTCAAGTGCGCGGTACAGGGTCGA GCGATGCACGCCAAGCAGTGCAGCCGCCTCTTTCACGGTGCGGCCTTCCTGGTCGATCAGCTCGCGGG CGTGCGCGATCTGTGCCGGGGTGAGGGTAGGGCGGGGGCCAAACTTCACGCCTCGGGCCTTGGCGGCC TCGCGCCCGCTCCGGGTGCGGTCGATGATTAGGGAACGCTCGAACTCGGCAATGCCGGCGAACACGGT CAACACCATGCGGCCGGCCGGCGTGGTGGTGTCGGCCCACGGCTCTGCCAGGCTACGCAGGCCCGCGC CGGCCTCCTGGATGCGCTCGGCAATGTCCAGTAGGTCGCGGGTGCTGCGGGCCAGGCGGTCTAGCCTG GTCACTGTCACAACGTCGCCAGGGCGTAGGTGGTCAAGCATCCTGGCCAGCTCCGGGCGGTCGCGCCT GGTGCCGGTGATCTTCTCGGAAAACAGCTTGGTGCAGCCGGCCGCGTGCAGTTCGGCCCGTTGGTTGG TCAAGTCCTGGTCGTCGGTGCTGACGCGGGCATAGCCCAGCAGGCCAGCGGCGGCGCTCTTGTTCATG ĶķGCGTAATGTCTCCGGTTCTAGTCGCAAGTATTCTACTTTATGCGACTAAAACACGCGACAAGAAAACG CCAGGAAAAGGGCAGGGCGGCAGCCTGTCGCGTAACTTAGGACTTGTGCGACATGTCGTTTTCAGAAG ACGGCTGCACTGAACGTCAGAAGCCGACTGCACTATAGCAGCGGAGGGGTTGGATCAAAGTAC SEQ ID NO: 9 (Isoform 1 of MYB28; AT5G61420.1; 1952 bp). Bold text indicates the 5’ UTR region; underlined text indicates the coding region; and italicized text indicates the 3’ UTR region. TCTTTTCCATAAGCTTATAACATATATTTTTTTTAAAATCTACTCTGCGTTAAAAAAATGAAAACACG TAGCAGCAGTGTGGGTAAGATCAAAGGGTGTTTCTCGATCAGTTTCATATTCAGATGTATCAGAGTTC TCATTAACAGATCTGTTTCTTTTTCCTTATCTGATTAAACAATTTCCTTCAGAATTTTACTTTTTTGA ACATATATAGTTTTTCTCTGTTCCTATATCTTGAGTTTTGTGAGAGGTTAATTATATGAAATTTTACG CATTATTGTTCATCTATATCGAAAAACAATGTCAAGAAAGCCATGTTGCGTCGGAGAAGGCTTGAAGA AAGGAGCATGGACCACCGAGGAGGACAAGAAACTCATCTCTTACATCCACGACCACGGCGAGGGAGGC TGGCGCGACATTCCCCAAAAAGCTGGTTTATACAAATCTATACATACACTCATTTTTGTACTTGTTGT AGAAAATTGTTCTGATAAACATATTGTGTCTGATTAGGGTTGAAACGGTGTGGAAAGAGTTGTAGACT GCGATGGACCAACTACCTTAAACCTGAGATCAAAAGAGGCGAGTTTAGTTCAGAGGAAGAGCAGATTA TCATCATGCTTCATGCTTCTCGTGGCAACAAGTGGTCGGTCATAGCGAGACATTTACCTAGAAGAACA GACAACGAGATCAAGAACTACTGGAACACGCATCTCAAAAAACGTTTGATGGAACAGGGTATTGATCC CGTGACTCACAAGCCACTGGCTTCTAGTTCCAACCCTACGGTCGATGAGAATTTGAATTCCCCAAATG CCTCTAGTTCCGACAAGCAATACTCCCGATCGAGCTCAATGCCTTTTCTGTCTCGTCCTCCTCCATCC AGTTGCAACATGGTTTCCAAGGTCTCCGAGCTTAGCAGCAATGATGGGACACCGATTCAAGGCAGTTC CTTGAGTTGCAAGAAACGTTTCAAGAAATCAAGTTCTACATCAAGGCTCTTGAACAAAGTTGCGGCTA AGGCCACTTCCATCAAAGATATATTGTCGGCTTCCATGGAAGGTAGCTTGAGTGCTACTACAATATCA CATGCAAGCTTTTTTAATGGCTTCACTGAGCAGATTCGCAATGAAGAGGATAGTTCTAACACATCCCT GACAAATACTCTTGCTGAATTTGATCCCTTCTCCCCATCATCGTTGTACCCCGAACATGAGATCAATG CTACTTCTGATCTCAACATGGACCAAGATTACGATTTTTCACAATTTTTCGAAAAATTCGGAGGAGAT AACCACAATGAGGAGAACAGTATGAATGATCTCCTTATGTCCGATGTTTCCCAAGAAGTCTCATCAAC TAGCGTTGATGATCAAGACAATATGGTAGGAAACTTCGAGGGATGGTCAAATTATCTTCTTGACCATA CCAATTTTATGTATGACACCGACTCAGACTCGCTTGAAAAGCATTTCATATGAGTCTTCATATCCAAA CAGAAAGGTTTCAAACTATTCGACGACTTAAAATAATGGTTCTGTACCCAAGGTTAGTCGATTACTAA CTCGCTCGAACGAGATATTGTGTATGTATTAATTAGTATTTGGGTTGTTTACTATATGTCCAAGGCGT GTTTATTACGATGTTAAACAAGGGTTAATCTTAACACTTAAGTTTCCCCAAGAATAAATAAAATAGGG TTTGAGTTAGGGTTTCTCTTACATTGAGAACCATGCATGTAACCTCGCGAATCAATTGGTAATTGATT TGTGCGGGCCACGATGTTTATACTAATATTTCTTTCTAAAGCTT SEQ ID NO: 10 (Isoform 2 of MYB28; AT5G61420.2; 2119 bp). Bold text indicates the 5’ UTR region; underlined text indicates the coding region; and italicized text indicates the 3’ UTR region. AAAAAAAAAAAAAAAAAAATCTCATTACGTACGTGTATATATATGGAATAGCTCATAACCTCACCACTACC ACAGAAATCATGCCTCTTGGTTCTTTTCCATAAGCTTATAACATATATTTTTTTTAAAATCTACTCTGCGT TAAAAAAATGAAAACACGTAGCAGCAGTGTGGGTAAGATCAAAGGGTGTTTCTCGATCAGTTTCATATTCA GATGTATCAGAGTTCTCATTAACAGATCTGTTTCTTTTTCCTTATCTGATTAAACAATTTCCTTCAGAATT TTACTTTTTTGAACATATATAGTTTTTCTCTGTTCCTATATCTTGAGTTTTGTGAGAGGTTAATTATATGA AATTTTACGCATTATTGTTCATCTATATCGAAAAACAATGTCAAGAAAGCCATGTTGCGTCGGAGAAGGCT TGAAGAAAGGAGCATGGACCACCGAGGAGGACAAGAAACTCATCTCTTACATCCACGACCACGGCGAGGGA GGCTGGCGCGACATTCCCCAAAAAGCTGGGTTGAAACGGTGTGGAAAGAGTTGTAGACTGCGATGGACCAA CTACCTTAAACCTGAGATCAAAAGAGGCGAGTTTAGTTCAGAGGAAGAGCAGATTATCATCATGCTTCATG CTTCTCGTGGCAACAAGTGGTCGGTCATAGCGAGACATTTACCTAGAAGAACAGACAACGAGATCAAGAAC TACTGGAACACGCATCTCAAAAAACGTTTGATGGAACAGGGTATTGATCCCGTGACTCACAAGCCACTGGCTTCTAGTTCCAACCCTACGGTCGATGAGAATTTGAATTCCCCAAATGCCTCTAGTTCCGACAAGCAATACT CCCGATCGAGCTCAATGCCTTTTCTGTCTCGTCCTCCTCCATCCAGTTGCAACATGGTTTCCAAGGTCTCC GAGCTTAGCAGCAATGATGGGACACCGATTCAAGGCAGTTCCTTGAGTTGCAAGAAACGTTTCAAGAAATC AAGTTCTACATCAAGGCTCTTGAACAAAGTTGCGGCTAAGGCCACTTCCATCAAAGATATATTGTCGGCTT CCATGGAAGGTAGCTTGAGTGCTACTACAATATCACATGCAAGCTTTTTTAATGGCTTCACTGAGCAGATT CGCAATGAAGAGGATAGTTCTAACACATCCCTGACAAATACTCTTGCTGAATTTGATCCCTTCTCCCCATC ATCGTTGTACCCCGAACATGAGATCAATGCTACTTCTGATCTCAACATGGACCAAGATTACGATTTTTCAC AATTTTTCGAAAAATTCGGAGGAGATAACCACAATGAGGAGAACAGTATGAATGATCTCCTTATGTCCGAT GTTTCCCAAGAAGTCTCATCAACTAGCGTTGATGATCAAGACAATATGGTAGGAAACTTCGAGGGATGGTC AAATTATCTTCTTGACCATACCAATTTTATGTATGACACCGACTCAGACTCGCTTGAAAAGCATTTCATAT GAGTCTTCATATCCAAACAGAAAGGTTTCAAACTATTCGACGACTTAAAATAATGGTTCTGTACCCAAGGT TAGTCGATTACTAACTCGCTCGAACGAGATATTGTGTATGTATTAATTAGTATTTGGGTTGTTTACTATAT GTCCAAGGCGTGTTTATTACGATGTTAAACAAGGGTTAATCTTAACACTTAAGTTTCCCCAAGAATAAATA AAATAGGGTTTGAGTTAGGGTTTCTCTTACATTGAGAACCATGCATGTAACCTCGCGAATCAATTGGTAAT TGATTTGTGCGGGCCACGATGTTTATACTAATATTTCTTTCTAAAGCTTGTTTTATTTATCTTATTTCGTA GTAGTACTTCCCATTATAATCATCAGTGCCCATAATAGTCATCAATTATGCTC SEQ ID NO: 11 (AtMYB28.2-5’UTR-uORF sequence; 117 bp). ATGAAAACACGTAGCAGCAGTGTGGGTAAGATCAAAGGGTGTTTCTCGATCAGTTTCATATT CAGATGTATCAGAGTTCTCATTAACAGATCTGTTTCTTTTTCCTTATCTGATTAA SEQ ID NO: 12 (myb28uORF-3bp: AtMYB28.2-5’UTR-uORF with 3bp deletion mutation sequence; 114 bp). ATGAAAACACGTAGCAGCAGTGTGGGTAACAAAGGGTGTTTCTCGATCAGTTTCATATTCAG ATGTATCAGAGTTCTCATTAACAGATCTGTTTCTTTTTCCTTATCTGATTAA SEQ ID NO: 13 (AtMYB28.2-5’UTR sequence; 373 bp). TCTCATTACGTACGTGTATATATATGGAATAGCTCATAACCTCACCACTACCACAGAAATCA TGCCTCTTGGTTCTTTTCCATAAGCTTATAACATATATTTTTTTTAAAATCTACTCTGCGTT AAAAAAATGAAAACACGTAGCAGCAGTGTGGGTAAGATCAAAGGGTGTTTCTCGATCAGTTT CATATTCAGATGTATCAGAGTTCTCATTAACAGATCTGTTTCTTTTTCCTTATCTGATTAAA CAATTTCCTTCAGAATTTTACTTTTTTGAACATATATAGTTTTTCTCTGTTCCTATATCTTG AGTTTTGTGAGAGGTTAATTATATGAAATTTTACGCATTATTGTTCATCTATATCGAAAAAC A, 5’UTR sequences; 351 bp). ACCACCATCACAACTCACGCCTCTTACTCCTTCATGAGCTTCTTTATTCTGATCTAAGTATT ATCATTTGCAAATACTTATAGAAAGCAAAATTTGGAATCTACGAGAAAAACATGAAAACACC TAACAGCTCTGTGGGTGAAACCCAAGAGCGTTTCTCGATTAGTCTTATATACAGATGCATCA GAGTTCTCATCAACCGATCTATTTCTTTCTTATCTTGTTAGAAAAAAAAAAACTACCTATCA AATTTGGTCAAGTATATTTTTCTCTACATTTTTATTTTCTTGTGAGTTGTGTGAGAGGTTAT ATGAGAGTTTACCCATTAGTGTTCATATATATCGGAAAAAA SEQ ID NO: 15 (Brassica oleracea (LOC106327854; NCBI accession: XM_013766140), 5’UTR sequence; 356 bp). CACCACCATCACACAATTCATTGCTCTTCTTCACAAGTTTCTGTAGTCTGATCTTATAGTGT TATAAAAATACATATAATATTTCAAGGATATATAGAAGGCAAAAACTCAAAGTCTACTTGAA AAACATGAAAACACCTAGCAGCTCTGTGGGTAAGACCCAAGAGCGCTTCTCGATTAGTCTCA TATTCAGATGTATCAGAGTTCTCATTAACAGATCTATTTCTTTCTTACCTTTTTAGAAAATT TCCTTTCTGATTTTAGTTTCCTCAAGTATATTTTTCTCAATATAGTATTTCCTTTGGTATTG TTTGAACCTTTTACACATTAGTGTTCATCTATATATATCGGAGAAASEQ ID NO: 16 (Brassica rapa (Brara.I00661), 5’UTR sequence; 323 bp). ACAATTCACGCGCTCTTACTCCATGAACTTCTCTATTCTCATCCTAGTGTTATAATCTTGCA AAATTTGGAGTGTACGAGAAAAACATGAAAACGCCTAGTAGCTCTGTGGGTGAGACCCAAGA GCGTTTCTCGATTAGTTTCATATACAGATGCATCAGAGTTCTCATCAACCGATCTATTTCTT TCTTATCTTATTAGAAGAAAAAAAATCTTATCAAATTTTACTTTCCTGCAAGTATATTTTTC TTTACATTTTCATTTTCTTGAGTGTTATTTGAGTGAAGTTATATTAAATTGTAATAGAGTTC ATATATATCGAAA SEQ ID NO: 17 (Camelina sativa (CsCN113611.18G182300), 5’UTR sequences; 312 bp). CCACTATCACACAAGTCGTTTAGTCATGCCTCTTGGTTCATCTTCACAAGCTTCTTGATAGC TCTAATTAGTGTTTTGCTATTAAAAAAAAAAAAAAGAAGATTGTACGTAAAAAAAATGAAAA CACGTAGCAGCTGTGTGGGTAGGATCCAACAACGTTTCTCGATCTGTTTAATATTCCGATGC ATCAGAGTTCTCATTAACAGATCTATTTCTTTCTTATCTAGTTGAACAATATCCTCTGAAAT TTTAATTTTCAAAATATTTATATATTTCTCTATTTTTAAATATTGAGTGTTTGTGAGAGATT AT SEQ ID NO: 18 (A part of 5’UTR sequences shown in Fig. 12 for Camelina sativa; 58 bp). TCTCAATCTGTTTAATATTCCGATGCATCAGAGTTCTCATTAACAGATCTGTTTCTTT SEQ ID NO: 19 (A part of 5’UTR sequences shown in Fig. 12 for Brassica juncea; 66 bp). TCTCGATTAGTCTCATATTCAGATGTATCAGAGTTCTCATTAACAGATCTATTTCTTTCTTA CCTT SEQ ID NO: 20 (A part of 5’UTR sequences shown in Fig. 13 for Brassica napus; 66 bp). TCTCGATTAGTCTCATATTCAGATGTATCAGAGTTCTCATTAACAGATCTATTTCTTTCTTA CCTT SEQ ID NO: 21 (A part of 5’UTR sequences shown in Fig. 13 for Brassica oleracea; 58 bp). TCTCGATTAGTCTCATATTCAGATGTATCAGAGTTCTCATTAACAGATCTATTTCTTT SEQ ID NO: 22 (A part of 5’UTR sequences shown in Fig. 14 for Brassica rapa; 66 bp). TCTCGATTAGTCTCATATTCAGATGTATCAGAGTTCTCATTAACAGATCTATTTCTTTCTTA CCTT

Claims

CLAIMS What is claimed is:

1. An expression casette for enhancing translation of a gene comprising at least a portion of the 5’ UTR of MYB28 operably linked to: a) a gene to be expressed downstream of the 5’ UTR of MYB28; b) a 3’ UTR downstream of the gene to be expressed; and optionally c) a promoter upstream of the 5’ UTR of MYB28.

2. The expression casette of claim 1, wherein the gene has at least 90% sequence homology to MYB28.

3. The expression casette of claim 2, wherein the gene has at least 95% sequence homology to MYB28.

4. The expression casette of claim 3, wherein the gene has at least 97% sequence homology to MYB28.

5. The expression casette of claim 4, wherein the gene has at least 98% sequence homology to MYB28.

6. The expression casette of claim 5, wherein the gene has at least 99% sequence homology to MYB28.

7. The expression casette of claim 6, wherein the gene is MYB28.

8. The expression casette of claim 7, wherein the gene is MYB28 having SEQ ID NO: as disclosed herein.

9. The expression casette of claim 1, wherein the gene is a reporter gene.

10. The expression casette of claim any one of claims 1-9, wherein the 5’ UTR comprises a sequence at least 90% sequence homology to SEQ ID NO:1 or SEQ ID NO:

2.

11. The expression casette of claim 10, wherein the 5’ UTR comprises a sequence at least 95% sequence homology to SEQ ID NO:1 or SEQ ID NO:

2.

12. The expression casette of claim 10, wherein the 5’ UTR comprises a sequence at least 97% sequence homology to SEQ ID NO:1 or SEQ ID NO: 2.

13. The expression casette of claim 10, wherein the 5’ UTR comprises a sequence at least 98% sequence homology to SEQ ID NO:1 or SEQ ID NO:

2.

14. The expression casette of claim 10, wherein the 5’ UTR comprises a sequence at least 99% sequence homology to SEQ ID NO:1 or SEQ ID NO:

2.

15. The expression casette of claim 45, wherein the 5’ UTR comprises a sequence sequence identity to SEQ ID NO:1 or SEQ ID NO:

2.

16. The expression casette of claim 10, wherein the 5’ UTR has SEQ ID NO:1 or SEQ ID NO:

2.

17. The expression casette of claim 10, wherein the 5’ UTR consists essentially of SEQ ID NO:1 or SEQ ID NO:

2.

18. The expression casette of any one of claims 1-17, wherein the 3’ UTR comprises at least a portion the 3’ UTR of MYB28.

19. The expression casette of claim 18, wherein the 3’ UTR comprises a sequence at least 90% sequence homology to the designated 3’ UTR of SEQ ID NO:9 or SEQ ID NO:

10.

20. The expression casette of claim 18, wherein the 3’ UTR comprises a sequence at least 95% sequence homology to the designated 3’ UTR of SEQ ID NO:9 or SEQ ID NO:

10.

21. The expression casette of claim 18, wherein the 3’ UTR comprises a sequence at least 97% sequence homology to the designated 3’ UTR of SEQ ID NO:9 or SEQ ID NO:

10.

22. The expression casette of claim 18, wherein the 3’ UTR comprises a sequence at least 98% sequence homology to SEQ ID NO:1 or SEQ ID NO:

2.

23. The expression casette of claim 18, wherein the 3’ UTR comprises a sequence at least 99% sequence homology to the designated 3’ UTR of SEQ ID NO:9 or SEQ ID NO:

10.

24. The expression casette of claim 18, wherein the 3’ UTR has the designated 3’ UTR of SEQ ID NO:9 or SEQ ID NO:

10.

25. The expression casette of any one of claims 1-24, wherein the promoter is apromoter for constitutive expression in a plant.

26. The expression casette of any one of claims 1-25, wherein the promoter is a promoter for tissue-specific expression in a plant.

27. The expression casette of any one of claims 1-26, further comprising a transcriptional terminator.

28. A nucleic acid vector comprising the expression cassette of any one of claims 1-27.

29. The nucleic acid vector of claim 28, further comprising a selectable marker.

30. A method of enhancing the translation of a gene to be expressed in a plant comprising introducing a construct comprising at least a portion of the 5’ UTR of MYB28 into the cells of said plant upstream of the gene to be expressed.

31. The method of claim 30, wherein the gene has at least 90% sequence homology to MYB28 as disclosed herein.

32. The method of claim 31, wherein the gene has at least 95% sequence homology to MYB28 as disclosed herein.

33. The method of claim 32, wherein the gene has at least 97% sequence homology to MYB28 as disclosed herein.

34. The method of claim 33, wherein the gene has at least 98% sequence homology to MYB28 as disclosed herein.

35. The method of claim 34, wherein the gene has at least 99% sequence homology to MYB28 as disclosed herein.

36. The method of claim 35, wherein the gene is MYB28 as disclosed herein.

37. The method of claim 36, wherein the gene is MYB28 having SEQ ID NO as disclosed herein.

38. The method of claim 30, wherein the gene is a reporter gene.

39. The method of any one of claims 30-38, wherein the plant is a monocotyledonous plant.

40. The method of claim 39, wherein the monocotyledonous plant is selected from anArabidopsis plant, a tobacco plant, a soybean plant, a canola plant, and a cotton plant.

41. The method of claim 39, wherein the plant is a plant of the order Brassicale.

42. The method of claim 41, wherein the plant is a plant of the family Brassicaceae.

43. The method of claim 42, wherein the plant is a plant of the genus Arabidopsis.

44. The method of claim 43, wherein the plant is Arabidopsis thaliana.

45. The method of claim any one of claims 30-43, wherein the 5’ UTR comprises a sequence at least 90% sequence homology to SEQ ID NO:1 or SEQ ID NO:

2.

46. The method of claim 45, wherein the 5’ UTR comprises a sequence at least 95% sequence homology to SEQ ID NO:1 or SEQ ID NO:

2.

47. The method of claim 45, wherein the 5’ UTR comprises a sequence at least 97% sequence homology to SEQ ID NO:1 or SEQ ID NO:

2.

48. The method of claim 45, wherein the 5’ UTR comprises a sequence at least 98% sequence homology to SEQ ID NO:1 or SEQ ID NO:

2.

49. The method of claim 45, wherein the 5’ UTR comprises a sequence at least 99% sequence homology to SEQ ID NO:1 or SEQ ID NO:

2.

50. The method of claim 45, wherein the 5’ UTR comprises a sequence sequence identity to SEQ ID NO:1 or SEQ ID NO:

2.

51. The method of claim 45, wherein the 5’ UTR has SEQ ID NO:1 or SEQ ID NO:

2.

52. The method of claim 45, wherein the 5’ UTR consists essentially of SEQ ID NO:1 or SEQ ID NO:

2.

53. The method of any one of claims 30-52, wherein the construct further comprises at least a portion of the 3’ UTR of MYB28.

54. The method of claim 53, wherein the 3’ UTR comprises a sequence at least 90% sequence homology to the designated 3’ UTR of SEQ ID NO:9 or SEQ ID NO:

10.

55. The method of claim 53, wherein the 3’ UTR comprises a sequence at least 95% sequence homology to the designated 3’ UTR of SEQ ID NO:9 or SEQ ID NO:10.

56. The method of claim 53, wherein the 3’ UTR comprises a sequence at least 97% sequence homology to the designated 3’ UTR of SEQ ID NO:9 or SEQ ID NO:

10.

57. The method of claim 53, wherein the 3’ UTR comprises a sequence at least 98% sequence homology to SEQ ID NO:1 or SEQ ID NO:

2.

58. The method of claim 53, wherein the 3’ UTR comprises a sequence at least 99% sequence homology to the designated 3’ UTR of SEQ ID NO:9 or SEQ ID NO:

10.

59. The method of claim 53, wherein the 3’ UTR has the designated 3’ UTR of SEQ ID NO:9 or SEQ ID NO:

10.

60. The method of claim 53, wherein the 3’ UTR consists essentially of the designated 3’ UTR of SEQ ID NO:9 or SEQ ID NO:

10.

61. The method of any one of claims 30-60, wherein the construct has constitutive expression.

62. The method of any one of claims 30-60, wherein the construct has tissue-specific expression.

63. A plant cell capable of exhibiting enhanced translation of a gene, wherein said cell comprises an expression construct comprising at least a portion of the 5’ UTR of MYB28 gene upstream of the gene to be expressed.

64. The plant cell of claim 63, wherein the expression construct comprises the expression cassette of any one of claims 1-27.

65. The plant cell of claim 63, wherein the gene has at least 90% sequence homology to MYB28.

66. The plant cell of claim 65, wherein the gene has at least 95% sequence homology to MYB28.

67. The plant cell of claim 66, wherein the gene has at least 97% sequence homology to MYB28.

68. The plant cell of claim 67, wherein the gene has at least 98% sequence homology to MYB28.

69. The plant cell of claim 68, wherein the gene has at least 99% sequence homology toMYB28.

70. The plant cell of claim 69, wherein the gene is MYB28.

71. The plant cell of claim 70, wherein the gene is MYB28 having SEQ ID NO:9 or SEQ ID NO:

10.

72. The plant cell of claim 63, wherein the gene is a reporter gene.

73. The plant cell of any one of claims 63-72, wherein the plant cell is a monocotyledonous plant cell.

74. The plant cell of claim 73, wherein the plant cell is a plant cell selected from an Arabidopsis plant, a tobacco plant, a soybean plant, a canola plant, and a cotton plant.

75. The plant cell of claim 73, wherein the plant cell is a plant cell of the order Brassicale.

76. The plant cell of claim 75, wherein the plant cell is a plant cell of the family Brassicaceae.

77. The plant cell of claim 76, wherein the plant cell is a plant cell of the genus Arabidopsis.

78. The plant cell of claim 77, wherein the plant cell is Arabidopsis thaliana.

79. The plant cell of claim any one of claims 63-78, wherein the 5’ UTR comprises a sequence at least 90% sequence homology to SEQ ID NO:1 or SEQ ID NO:

2.

80. The plant cell of claim 79, wherein the 5’ UTR comprises a sequence at least 95% sequence homology to SEQ ID NO:1 or SEQ ID NO:

2.

81. The plant cell of claim 80, wherein the 5’ UTR comprises a sequence at least 97% sequence homology to SEQ ID NO:1 or SEQ ID NO:

2.

82. The plant cell of claim 81, wherein the 5’ UTR comprises a sequence at least 98% sequence homology to SEQ ID NO:1 or SEQ ID NO:

2.

83. The plant cell of claim 82, wherein the 5’ UTR comprises a sequence at least 99% sequence homology to SEQ ID NO:1 or SEQ ID NO:

2.

84. The plant cell of claim 82, wherein the 5’ UTR has SEQ ID NO:1 or SEQ ID NO: 2.

85. The plant cell of claim 82, wherein the 5’ UTR consists essentially of SEQ ID NO:1 or SEQ ID NO:

2.

86. The plant cell of claim any one of claims 63-85, wherein the construct further comprises at least a portion of the 3’ UTR of MYB28.

87. The plant cell of claim 86, wherein the 3’ UTR comprises a sequence having at least 90% sequence homology to the designated 3’ UTR of SEQ ID NO:9 or SEQ ID NO:

10.

88. The plant cell of claim 86, wherein the 3’ UTR comprises a sequence having at least 95% sequence homology to the designated 3’ UTR of SEQ ID NO:9 or SEQ ID NO:

10.

89. The plant cell of claim 86, wherein the 3’ UTR comprises a sequence having at least 97% sequence homology to the designated 3’ UTR of SEQ ID NO:9 or SEQ ID NO:

10.

90. The plant cell of claim 86, wherein the 3’ UTR comprises a sequence having at least 98% sequence homology to SEQ ID NO:1 or SEQ ID NO:

2.

91. The plant cell of claim 86, wherein the 3’ UTR comprises a sequence having at least 99% sequence homology to the designated 3’ UTR of SEQ ID NO:9 or SEQ ID NO:

10.

92. The plant cell of claim 86, wherein the 3’ UTR comprises a sequence having 99.5% identity to the designated 3’ UTR of SEQ ID NO:9 or SEQ ID NO:

10.

93. The plant cell of claim 86, wherein the 3’ UTR has the designated 3’ UTR of SEQ ID NO:9 or SEQ ID NO:

10.

94. The plant cell of claim 86, wherein the 3’ UTR consists essentially of the designated 3’ UTR of SEQ ID NO:9 or SEQ ID NO:

10.

95. The plant cell of any one of claims 63-94, wherein the construct comprises the gene to be expressed downstream of the 5’ UTR portion.

96. A plant comprising the plant cell of any one of claims 63-95.

Images