Regulation of iron deficiency response in plants

Abstract

The present disclosure provides methods of regulating iron deficiency response in plants, modified plants and plant cells derived therefrom, in particular plants and plant cells with altered iron content, iron transport, iron sensing, or iron uptake.

Description

REGULATION OF IRON DEFICIENCY RESPONSE IN PLANTS

[0001] The present disclosure claims priority from Australian Provisional Patent Application No. 2023902645, the entire content of which is incorporated herein by reference.FIELD OF THE INVENTION

[0002] The present invention relates generally to methods of regulating iron deficiency response in plants, modified plants and plant cells derived therefrom, in particular plants and plant cells with altered iron content, iron transport, iron sensing, or iron uptake.BACKGROUND OF THE INVENTION

[0003] All references, including any patent or patent application cited in this specification are hereby incorporated by reference to enable full understanding of the invention. Nevertheless, such references are not to be read as constituting an admission that any of these documents forms part of the common general knowledge in the art, in Australia or in any other country.

[0004] Iron is an essential element for cellular metabolism for most forms of life, having important roles during electron transfer in metabolic processes, catalysis of enzymatic reactions and as part of co-factors in protein complexes.

[0005] In plants, iron is critical for multiple processes including nucleic acid synthesis, energy transfer, respiration, and photosynthesis. As such, crops that fail to properly uptake, transport, or assimilate iron; or crops growing in iron-deficient conditions, will exhibit poor growth and symptoms of iron deficiency, including leaf chlorosis, which leads to poor plant health. Iron deficiency is a widespread agricultural problem, since it reduces plant growth and crop yield. Furthermore, iron deficiency in plants is often prevalent in important food crop species grown in arid regions such as sorghum and rice, but can still occur when soil iron concentration is high but poorly available for bio-absorption. Plants with improved regulation of iron uptake, transport and content may assist to address poor plant health associated with iron deficiency. Furthermore, crop plants that are more tolerant of iron deficiency are often associated with improved nutritional value, particularly in monocot plants.

[0006] Iron is critical in animal and human metabolism, where it is key component of the blood oxygen carrier, hemoglobin. Dietary iron uptake is an important part of iron homeostasis in animals. Animals and humans that fail to obtain an adequate dietary supplyof iron therefore show symptoms of anaemia. Iron deficiency and / or anaemia is a common nutritional deficiency. For example, anaemia is estimated to affect 33% of the global human population, with half of these cases being attributed to iron deficiency.

[0007] At the same time, there is an increasing global trend towards more plant-based, vegetarian or vegan diets, which may further increase the risk of reduced dietary iron uptake and prevalence of iron deficiency. To address this, some food products are artificially iron- fortified (z.e., bread and breakfest cereal); however, these iron-fortified food products are usually highly processed, which is considered undesirable by many consumers. Because plants are the primary food source for humans, the nutritional benefits of plant-based food is of foundational importance to human health.

[0008] Thus, there remains an urgent need to produce plants that have modified capacity to sense, uptake, transport, assimilate and regulate iron levels, including for the purposes of improving plant health and vigour, and also for providing crops with modified or increased iron levels to enhance the nutritional benefit to the humans or animals that consume them.SUMMARY OF THE INVENTION

[0009] In an aspect disclosed herein, there is provided a method of enhancing the expression of an iron-deficiency response gene in a plant cell, the method comprising introducing a modification to an upstream open reading frame (uORF) of a 5' untranslated region (UTR) of the iron-deficiency response gene, wherein the modification comprises a deletion, addition, or substitution of at least one nucleotide of the uORF.

[0010] In an embodiment, the iron-deficiency response gene encodes a transcription factor.

[0011] In one embodiment, the iron-deficiency response gene is selected from the group consisting of IDEF1, IDEF2 and orthologs of any of the foregoing.

[0012] In a further embodiment, the iron-deficiency response gene is IDEF1 or an ortholog thereof. In a further embodiment, IDEF1 or ortholog thereof comprises a nucleic acid sequence that encodes a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-8, and amino acid sequences having at least 70% sequence identity to any of the foregoing.

[0013] In an embodiment, the 5' UTR of IDEF1 or ortholog thereof comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 17-24, and nucleic acid sequences having at least 70% sequence identity to any of the foregoing.

[0014] In a further embodiment, the iron-deficiency response gene is IDEF2 or an ortholog thereof. In a further embodiment, the IDEF2 or ortholog thereof comprises a nucleic acid sequence that encodes a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 9-16, and amino acid sequences having at least 70% sequence identity to any of the foregoing.

[0015] In a further embodiment, the 5' UTR of IDEF2 or ortholog thereof comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 25-32 and nucleic acid sequences having at least 70% sequence identity to any of the foregoing. In a further embodiment, the 5' UTR of IDEF2 or ortholog thereof comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 54-55 and nucleic acid sequences having at least 70% sequence identity to any of the foregoing.

[0016] In a further embodiment, the 5' UTR of IDEF2 or ortholog thereof comprises an uORF that comprises a nucleic acid sequence that encodes the amino acid sequence of any one of SEQ ID NOs:50-53.

[0017] In a further embodiment, the enhanced expression of the iron-deficiency response gene results in an upregulation of an iron homeostasis gene, wherein the iron homeostasis gene comprise at least one iron deficiency-responsive cis-regulatory element.

[0018] In a further embodiment, the at least one iron deficiency-responsive cis-regulatory element comprises: (i) an IDE1 or an IDE-like motif; or (ii) an IDE2 or an IDE2-like motif.

[0019] In another aspect disclosed herein, there is provided a method for selecting a target modification to be introduced into an upstream open reading frame (uORF) in a 5' untranslated region (UTR) a gene of interest in a plant cell that produces a desirable trait in the plant cell, the method comprising:(i) introducing a modification to the uORF in the 5' UTR to produce a modified uORF, wherein the modification comprises a deletion, addition, or substitution of at least one nucleotide of the uORF;(ii) fusing the modified uORF of step (i) to a polynucleotide that encodes a reporter protein to produce a fusion polynucleotide;(iii) introducing the fusion polynucleotide of step (ii) into a host cell to produce a modified host cell;(iv) exposing the modified host cell of step (iii) to conditions that favour expression of the fusion polynucleotide in the modified host cell;(v) determining the level of expression of the polynucleotide that encodes the reporter protein following step (iv);(vi) comparing the level of expression of the polynucleotide that encodes the reporter protein determined in step (v) with a level of expression of the polynucleotide that encodes the reporter protein in a host cell comprising a corresponding fusion polynucleotide comprising an unmodified uORF and a polynucleotide that encodes the reporter protein; and(iv) selecting tire modification to the uORF that results in an enhanced level of expression of the polynucleotide that encodes the reporter protein for introduction into an endogenous locus of the uORF of plant cell genome.

[0020] In a further embodiment, the modified uORF comprises a nucleic acid sequence having at least 70% sequence identity to any one of SEQ ID NOs: 33-43. In a further embodiment, the modified uORF comprises a nucleic acid sequence encoding the amino acid sequence of any one of SEQ ID NOs:50-53. In a further embodiment, the modified uORF comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 54-59, and nucleic acids having at least 70% sequence identity thereto.

[0021] In a further embodiment, modifying the uORF comprises replacing at least one start codon in the uORF with a stop codon.

[0022] In a further embodiment, modifying the uORF comprises deleting at least one base pair in the uORF.

[0023] In a further embodiment, modifying the uORF comprises deleting at least two base pairs in the uORF.

[0024] In a further embodiment, modifying the uORF comprises deleting at least one start codon in the uORF.

[0025] In a further embodiment, modifying the uORF comprises deleting from about 5 to about 100 base pairs in the uORF.

[0026] In a further embodiment, modifying the uORF comprises deleting the uORF.

[0027] In a further embodiment, modifying tire uORF comprises adding at least one base pair in the uORF.

[0028] In a further embodiment, modifying the uORF comprises substituting at least one base pair in the uORF. In another embodiment, the substitution of the at least one base pah- in the uORF results in a non-synonomous amino acid change or a nonsense mutation.

[0029] In a further embodiment, modifying the uORF comprises substituting from about 5 to about 100 base pairs in the uORF.

[0030] In a further embodiment, modifying the uORF is achieved by means of site-directed nucleases or oligonucleotide-directed mutagenesis.

[0031] In a further embodiment, the mutagenesis is achieved using SDN-1.

[0032] In a further embodiment, said mutagenesis is achieved using Zinc finger nucleases, TALENS and / or CRISPR / Cas gene editing.

[0033] In a further embodiment, said mutagenesis is achieved using CRISPR / Cas9, CRISPR / Casl2 gene editing and / or base-editing.

[0034] In a further embodiment, the plant cell is a leaf cell, a root cell, an embryonic cell, an egg cell, or a pollen cell.

[0035] In a further embodiment, the plant is elected from the group consisting of rice, barley, wheat, soybean, alfalfa, rye, oats, millet, sorghum, triticale, buckwheat, quinoa, spelt, einkom, amaranth, grass, com, canola, wild rice, teff, banana, cassava, brassica, field mustard, cabbage, Aegilops tauschii, brachypodium, Miscanthus, switchgrass, poplar, pine, Eucalyptus, acacia, birch, hazel, willow, bamboo, grape, kiwifruit, apple, pear, avocado, almond, pistachio, walnut, chestnut, a vine, a forage grass, alfalfa, a plant of the genus Prunus, cacao, peas, beans, pulses, legumes, camelina, sweet potato, yam, tomato, potato, eggplant, and plants of the nightshade family.

[0036] In a further embodiment, the plant cell is a rice or a wheat cell.

[0037] The present disclosure also extends to a plant cell produced by the methods disclosed herein.

[0038] In another aspect disclosed herein, there is provided a modified plant cell comprising a polynucleotide capable of controlling iron homeostasis in the plant cell, wherein the polynucleotide comprises an iron-deficiency response gene, wherein the iron- deficiency response gene comprises a nucleic acid sequence comprising a modification to a uORF of a 5' UTR of the iron deficiency response gene, wherein the modification comprises a deletion, addition, or substitution of at least one nucleotide in the uORF when compared to a corresponding nucleic acid sequence of a wild-type uORF of the same plant species, wherein the modification to the uORF results in an increase in the level of expression of the iron deficiency response gene when compared to the level of expression of the irondeficiency response gene in a control plant cell harbouring a corresponding native iron deficiency response gene of the same plant species.

[0039] In a further embodiment, the modified plant cell exhibits a desirable trait selected from the group consisting of increased cellular iron content, increased iron uptake, improved iron homeostasis, increased vigour, darker coloration, increased photosynthesis, increased yield, increased pathogen tolerance, increased abiotic stress tolerance, increased biomass, and increased iron transport.

[0040] In another embodiment, the modified plant cell exhibits increased tolerance to iron deficiency when compared to a plant cell having the corresponding native uORF of the same plant species.

[0041] In yet another aspect disclosed herein, there is provided a plant cell comprising an iron-deficiency response gene having a modified uORF when compared to a corresponding native uORF of the same plant species, wherein the modified uORF comprises a deletion, substitution or insertion of at least one nucleotide when compared to the corresponding wildtype uORF of the same plant species, and wherein said modification enhances expression of the iron-deficiency response gene in the plant cell when compared to a plant cell of the same plant species harbouring the corresponding wild-type uORF of the same plant species.

[0042] In a further embodiment, the iron-deficiency response gene is selected from the group consisting of IDEF1, IDEF2 and orthologs of any of the foregoing.

[0043] In a further embodiment, the iron-deficiency response gene is IDEF1 or an ortholog thereof.In a further embodiment, the iron-deficiency response gene comprises a nucleic acid sequence that encodes a polypeptide selected from the group consisting of SEQ ID NOs: 1- 8 and polypeptides having at least 70% sequence identity to any of the foregoing.

[0044] In a further embodiment, the iron-deficiency response gene is IDEF2 or an ortholog thereof.

[0045] In a further embodiment, the iron-deficiency response gene comprises a nucleic acid that encodes a polypeptide sequence selected from the group consisting of SEQ ID NOs: 9-16 and polypeptides having at least 70% sequence identity to any of the foregoing.

[0046] In a further embodiment, the uORF is modified by replacing at least one start codon of the corresponding wild-type uORF of the same plant species with a stop codon.

[0047] In a further embodiment, the uORF is modified by deleting at least one base pair ofthe corresponding wild-type uORF of the same plant species.

[0048] In a further embodiment, the modified uORF comprises a deletion of at least 2 base pairs of the corresponding wild-type uORF of the same plant species.

[0049] In a further embodiment, the modified uORF comprises a deletion of at least a start codon of the corresponding wild-type uORF of the same plant species.

[0050] In a further embodiment, the entire uORF of the corresponding wild-type uORF of the same plant species has been deleted.

[0051] In a further embodiment, the modified uORF results in enhancing the expression of the gene of interest by at least about 10% when compared to a level of expression of the gene of interest in a plant cell harbouring a corresponding wild-type uORF of the same plant species.

[0052] In yet another aspect disclosed herein, there is provided a polynucleotide comprising a nucleic acid sequence encoding an iron-deficiency response gene having a 5 'UTR comprising a modified uORF, wherein the 5 'UTR comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 33-43, and nucleic acids having at least 70% sequence identity thereto. In a further embodiment, the 5 'UTR comprises a nucleic acid sequence encoding the amino acid sequence of any one of SEQ ID NOs:50-53. In a further embodiment, the 5 'UTR comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 54-59, and nucleic acids having at least 70% sequence identity thereto.

[0053] The present disclosure also extends to polynucleotide constructs comprising the polynucleotides disclosed herein, and to expression vectors comprising the polynucleotides or the polynucleotide constructs disclosed herein.

[0054] The present disclosure also extends to plants comprising the plant cells, the polynucleotides, the polynucleotide constructs, or the expression vectors disclosed herein.

[0055] In a further embodiment, a plant comprising the plant cells, the polynucleotides, the polynucleotide constructs, or the expression vectors disclosed herein exhibits a desirable trait selected from the group consisting of increased cellular iron content, increased iron uptake, improved iron homeostasis, increased vigour, darker coloration, increased photosynthesis, increased yield, increased pathogen tolerance, increased abiotic stress tolerance, increased biomass, and increased iron transport. In another embodiment, the plantexhibits increased tolerance to iron deficiency. In another embodiment the plants exhibits increased tolerance to iron deficiency when compared to a plant of the same plant species harbouring the corresponding wild-type uORF.

[0056] In a further embodiment, the plant is selected from the group consisting of rice, barley, wheat, soybean, alfalfa, rye, oats, millet, sorghum, triticale, buckwheat, quinoa, spelt, einkom, amaranth, grass, com, canola, wild rice, teff, banana, cassava, brassica, field mustard, cabbage, Aegilops tauschii, brachypodium, Miscanthus, switchgrass, poplar, pine, Eucalyptus, acacia, birch, hazel, willow, bamboo, grape, kiwifruit, apple, pear, avocado, almond, pistaccio, walnut, chestnut, a vine, a forage grass, alfalfa, a plant of the genus Prunus, cacao, peas, beans, pulses, legumes, camelina, sweet potato, yam, tomato, potato, eggplant and plants of the nightshade family.

[0057] In a further embodiment, the plant is a rice plant or a wheat plant.

[0058] The present disclosure also extends to seeds produced by tire plants disclosed herein, to plants derived from such seeds, and to plant parts derived from the plants disclosed herein.

[0059] In a further embodiment, the plant part is selected from the group consisting of a seed, grain, fruit leaf, flower, tuber, stalk, rhizome, spore, cutting, nut and root.

[0060] In a further embodiment, the plant part is grain.

[0061] In an embodiment, the grain is selected from the group consisting of wheat grain, rye grain, barley grain or oat grain.

[0062] In an embodiment, the grain is rice.

[0063] The present disclosure also extends to flour produced from the grain disclosed herein.

[0064] The present disclosure also extends to food products comprising the plants, the seeds, the plant parts, the grains or the flour disclosed herein.BRIEF DESCRIPTION OF THE DRAWINGS

[0065] Figure 1 shows the results of a dual luciferase assay of various iron deficiency response gene leader sequences (LS) from rice (Oryza sativa L.) The schematic (left panel) of the WT or mutated (ml / m2) 5’ LS that were fused upstream of the firefly luciferase coding sequence and downstream of a 35S promoter. Putative canonical and non-canonical start codons are indicated (arrowhead) and stop codons (rectangle) within the 5’ LS are indicated. A box plot (right panel) compares luminosity ratios (firefly / renilla) of the WT 5’ LS and their mutated version The p-values compare the mutant and WT LS of the same gene as determined by a two-sample Students t-test assuming unequal variance (n=5). Each biological replicate comprised of three leaf discs (averaged) fiom three infiltration sites on a single leaf. The luminosity ratios (firefly / renilla) were normalised to the 35S vector (positive control containing no 5’ LS). The empty vector contains the firefly luciferase coding sequence with no promoter.

[0066] Figure 2 shows the results of the dual luciferase assay of OsIDEFl 5’ LS fiom rice to identity' the uORF start site or sites. Schematic (left panel) of OsIDEFl 5’ LS containing combinations of mutated ATGs that were fused upstream of the firefly luciferase coding sequence and downstream of a 35S promoter. Putative canonical start codons are indicated (arrowhead) and stop codons (rectangle) within the 5’ LS are indicated. A box plot (right panel) compares luminosity ratios (firefly / renilla) of the various 5’ LS. Letters indicate significant differences (p<0.05) between all LS (excluding the controls) as determined by a one-way ANOVA (post hoc Tukey’s HSD test, n=5). Each biological replicate comprised of three leaf discs from three infiltration sites on a single leaf. The luminosity ratios (firefly / renilla) were normalised to the 35S vector (positive control containing no 5’ LS).

[0067] Figure 3 (a) shows the nucleotide alignment of the uORF-containing region amongst 1DEF2 orthologous sequences in monocots (from top to bottom; 1 : Orzya sativa L (rice); 2: Eragrotis tef (teff); 3: Sorghum bicolor L (sorghum); 4: Zea Mays L (maize); 5: Avena sativa L. (oats); 6: Brachypodium distachyon (brachypodium); 7: Aegilops tauschii (tausch's goatgrass); 8: Triticum aestivumL. (bread wheat) subgenome D; 9: Triticum spelta L. (spelt) subgenome D; 10: Triticum aestivum L (bread wheat) subgenome B; 11: Triticum spelta L. (spelt) subgenome B; 12: Hordeum vulgare L. (barley); 13: Triticum aestivum L. (bread wheat) subgenome A; 14: Triticum spelta L. (spelt) subgenome A). The uORF- containing region located within the 5 ’ end of E2 was identified based on ribosomal profilingand analysis of conserved ORFs (stop-to-stop) within the 5’ LS. A conserved canonical start codon (white arrow) as well as conserved stop codons (black arrow) are indicated as possible start / stop sites of uORFs. The dashed lined rectangle indicates the 5’ LS used for the DLA (13: Triticum aestivum L. (bread wheat) subgenome A), (b) shows a schematic (left panel) of the WT or mutated 5’ LS that were fused upstream of the firefly luciferase coding sequence and downstream of a 35 S promoter. The TalDEF2 5’ LS comprises of two exons (El and E2). The uORF-containing region is indicated (black box). A box plot (right panel) compares luminosity ratios (firefly / renilla) of the various 5’ LS. Letters indicate significant differences (p<0.05) between all LS (excluding the controls) as determined by a one-way ANOVA (post hoc Tukey’s HSD test, n=4). The luminosity ratios (firefly / renilla) were normalised to the 35S vector (positive control containing no 5’ LS). (C) Visual representation of an independently infiltrated N. benthamiana leaf containing the 35S infiltration (top left), empty vector infiltration (top right), TaIDEF2 WT (bottom left), and TaIDEF2 m3 (bottom right).

[0068] Figure 4 provides the gene sequence for rice IDEF1 andiIDEF2, and wheat IDEF2.The 5' leader sequences are underlined, and the uORF regions are in bold.

[0069] Figure 5 shows the generation of TaIDEF2-xiORS mutations in the A and D subgenomes of bread wheat (cv. Fielder, Triticum aestivum L.). (a) The assembled pGGG construct transformed into Agrobacterium tumefeciens and a schematic representation of the T-DNA: NOS, nopaline synthase terminator; Hyg, hygromycin phosphotransferase II; pOsACT, rice Actin promoter; pZmUbi, maize ubiquitin promoter; cas9; TaU6, wheat U6 promoter; gRNA sequences and scaffold; GRF4 and GIF1 chimeric protein, (b) Guide RNA sequences targeting two sites within the uORF. A single nucleotide insertion was isolated in TaIDEF2-D homoeolog (tafe2-1 mutant), and a novel 23-nucleotide sequence replaced the 26-nucleotide wildtype sequence between the two gRNA cut sites in the TalDEF2-A homoeolog (tafe2-2 mutant). Novel nucleotides are indicated (in bold), (c) Predicted amino acid sequences of the wildtype, tafe2-1, and tafe2-2 mutant uORFs. Novel amino acids are indicated (in bold).

[0070] Figure 6 shows the phenotype of hydroponically grown TaIDEF2-uORF mutant and wildtype bread wheat plants, (a) Height and (b) SPAD of 2ndleaf of plants grown for 12 days under Fe sufficient (50 μM) hydroponic conditions, (c) Height, (d) SPAD of 3rdleaf, (e) and SPAD 4thleaf of plants grown for 10 days under Fe deficient (0 μM) hydroponic conditions. Error bars represent the standard error of the mean (n=2, 3 or 4). (f) Thegenotypes grown in the hydroponic experiment are tafe2-1 (black), tafe2-2 (white), and Fielder (donor; star). Mutants are either homozygous (m / m; circle), heterozygous (m / wt; triangle), or wildtype segregants (wt / wt; square).

[0071] Figure 7 shows the phenotype of hydroponically grown TaIDEF2-uORF mutant and wildtype bread wheat plants at day 11 of an iron deficiency treatment. Bar plots of (a) chlorophyll content of the 4thleaf, (b) SPAD of 5thleaf, (c) shoot and (d) root biomass. The genotypes are tafo2-1, tafo2-2, and Fielder (donor). Mutants are either homozygous (m / m), heterozygous (m / wt), or wildty'pe segregants (wt / wt). Letters indicate significant differences between all genotypes apart from Fielder as determined by a one-way ANOVA (post hoc Tukey’s HSD test).

[0072] Figure 8 shows the additional phenotype measurements of hydroponically grown TaIDEF2-uORF mutant and wildtype bread wheat plants at day 11 of an iron deficiency treatment. Bar plots of (a) SPAD of 3rdleaf, (b) SPAD of 4thleaf, (c) shoot and (d) root length, (e) NDG1 of 4thleaf, and (f) tiller number. The genotypes are tafe2-1, tafe2-2, and Fielder (donor). Mutants are either homozygous (m / m; dark colour), heterozygous (m / wt), or wildtype segregants (wt / wt). Letters indicate significant differences between all genotypes apart from Fielder as determined by a one-way ANOVA (post hoc Tukey’s HSD test).

[0073] Figure 9 provides the mutated tafe2-1 leader sequence (SEQ ID NO:54). The 5' leader sequences are in bold and the mutant bases are underlined.

[0074] Figure 10 provides the mutated tafe2-2 leader sequence (SEQ ID NO:55). The 5' leader sequences are in bold and the mutant bases are underlined.DETAILED DESCRIPTION OF THE INVENTION

[0075] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods and materials are described. For the purposes of the present invention, the following terms are defined below.

[0076] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article, unless explicitly stated otherwise. Byway of example, “an element” means one element or more than one element.

[0077] As used herein, “and / or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (or).

[0078] As used herein, the term "about" refers to a quantity, level, value, dimension, size, or amount that varies by as much as 10% (e.g., by 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1%) to a reference quantity, level, value, dimension, size, or amount. The present description uses numerical ranges to quantify certain parameters relating to this disclosure. It should be understood that when numerical ranges are provided, such ranges are to be construed as providing support for claim limitations that recite the lower value of the range as wrell as claim limitations that recite the upper value of the range. For example, a disclosed numerical range of 10 to 100 provides support for a claim reciting “greater than 10” (with no upper bounds) and a claim reciting “less than 100” (with no lower bounds) and provided support for and includes the end points of 10 and 100.

[0079] Throughout this specification, unless the context requires otherwise, the words "comprise", "comprises" and "comprising" will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements.

[0080] All sequence identifiers (e.g., GenBank ID, Addgene reference, EMBL-Bank ID, DNA Data Bank of Japan (DDBJ) ID, etc.) provided herein were current at the filing date.

[0081] The present disclosure is predicated, at least in part, on the inventors' surprising finding that modifying the untranslated open reading frame (uORF) of the 5’ untranslated region (UTR) of an iron-deficiency response gene unexpectedly enhances tire expression of the iron-deficiency response gene. This enhanced expression of the iron-deficiency gene results may result in increased levels of the transcript and / or increased levels of the polypeptide encoded by the iron-deficiency response gene.

[0082] Thus, in one aspect disclosed herein, there is provided a method of enhancing the expression of an iron-deficiency response gene in a plant cell, the method comprising introducing a modification to an upstream open reading frame (uORF) of a 5' untranslated region (UTR) of the iron-deficiency response gene, wherein the modification comprises a deletion, addition, or substitution of at least one nucleotide of the uORF.Iron-deficiency response genes / transcription factors

[0083] Plants respond to abiotic stresses by modulating the expression of target genes through the activity of transcription factors (TFs). The basic Helix-Loop-Helix (bHLH) TF family is one of the largest TF families in plants and plays important roles in many important development and metabolic processes, including anthocyanin biosynthesis, hormone signalling, and nutrient uptake. All bHLH proteins contain a conserved DNA-binding basic region and two alpha helices separated by a variable loop region, and homo / heterodimerise with other TFs prior to binding DNA. Once dimerised, the bHLH protein complex can regulate transcription by' binding to the E-box (5'-CANNTG-3') of gene promoters, and the specificity- of binding is determined by the E-box sequence and its affinity to residues within the basic and loop regions of bHLH domains.

[0084] In rice (Oryza sativa L.), up to 211 bHLH TFs have been annotated within 12 recognised subgroups (see, e.g., http: / / planttfilb.gao-lab.org / accessed on 12 October 2020), including the subgroups lb, IVb, and IVc; bHLH TFs that are considered to be responsible for maintaining cellular iron (Fe) homeostasis and responding to changes in environmental Fe conditions.

[0085] One rice subgroup, IVb bHLH TF, iron-related transcription factor 3 (OsIRO3), is upregulated in response to environmental Fe deficiency, and overexpression of OsIRO3 has been shown to reduce the expression of Fe chelator biosynthetic genes and decreases plant growth and shoot Fe concentrations under Fe deficiency (in this context, these types of transcription factors are also referred to interchangeably as iron-deficiency response genes). For these reasons, OsIRO3 was initially characterised as a putative negative regulator of the Fe deficiency' response in rice. Within the rice Fe homeostasis regulatory' network, OsIRO3 sits downstream of the master regulators iron deficiency-responsive element-binding factors 1 (OsIDEFl) and Hemerythrin motif-containing Really Interesting New Gene (RING) and zinc finger proteins 1 and 2 (OsHRZl and OsHRZ2), which are capable of sensing environmental Fe conditions. The OsHRZ proteins are thought to regulate OsIRO3 expression via 26S proteasome-mediated degradation of Positive Regulator of Iron homeostasis proteins (OsPRIl / OsbHLH60, OsPRI2 / OsbHLH58 and OsPRI3 / OsbHLH59), which are subgroup I Vc bHLH TFs and function as upstream positive regulators of OsIRO3. Putative targets for OsIRO3-mediated transcriptional repression include genes involved in Fe uptake and transport, Yellow Stripe-Like 15 (OsYSLIS), Natural Resistance-Associated Macrophage Protein (OsNRAMP), and Iron Regulated Transporter (OsIRTl), andbiosynthetic genes of the Fe chelators nicotianamine (NA) and 2'-deoxymugineic acid (DMA), Nicotianamine Synthase 1, 2, and 3 (OsNASl, OsNAS2, and OsNAS3) and Nicotianamine aminotransferase 1 (OsNAATl).

[0086] Examples of iron-deficiency response genes (also referred to herein as genes that encode iron-deficiency response transcription factors) will be familiar to persons skilled in the art, illustrative examples of which include IDEF1, IDEF2, NASI, NAS2, NASS, NAAT1, DMAS1, TOMI, T0M2, TSL2. YSL9, YSL13, YSL15, YSL16. YSL18, FRDL1. IRT1, IRT2, PEZ1, PEZ2, IRO2 (e.g., OsIRO2), IRO3, HLH58, HLH59, HLH60, HLH133, HLH156, FRO1, HRZ1, HRZ2, HORZ1, IBP1.1, IBP 1.2, rab6a, RMC, csn6 and IMAI. In an embodiment, the iron-deficiency response gene is selected from a group consisting of IDEF1, IDEF2, NASI, NAS2, NASS, NAAT1, DMAS1, TOMI, T0M2, TSL2, YSL9, YSL13, YSL15, YSL16, YSL18, FRDL1. IRT1, IRT2, PEZ1, PEZ2, IRO2 (e.g., OsIRO2), IRO3, HLH58, HLH59. HIH60, HIH133, HLH156, FRO1, HRZ1, HRZ2, HORZ1, IBP 1.1, IBP1.2, rab6a, RMC, csn6 and IMAI. In one embodiment, the iron-deficiency response gene encodes a transcription factor. In one embodiment, the iron-deficiency response gene is IDEF1 or IDEF2. Examples of IDEF1 and IDEF2 genes in different plant species are provided in Tables 1 and 2.

[0087] In another embodiment, the iron-deficiency response gene is IDEF1. The IDEF1 gene is an IDEF1 gene in any plant disclosed herein, or orthologs thereof. The IDEF1 gene may comprises a nucleic acid sequence that encodes a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-8, and amino acid sequences having at least 40% sequence identity, at least 50% sequence identity, at least 60% sequence identity, at least 70% sequence identity, at least 80% sequence identity, at least 85% sequence identity, or at least 90% sequence identity to any of the foregoing. The IDEF1 gene may comprises a nucleic acid sequence that encodes a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-8, and amino acid sequences having at least 70% sequence identity to any of the foregoing.

[0088] In another embodiment the iron-deficiency response gene is IDEF2. The IDEF2 gene is an IDEF2 gene in any plant disclosed herein, or orthologs thereof. The IDEF2 gene may comprises a nucleic acid sequence that encodes a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 9-16, and amino acid sequences having at least 40% sequence identity, at least 50% sequence identity, at least 60% sequence identity, at least 70% sequence identity, at least 80% sequence identity, atleast 85% sequence identity, or at least 90% sequence identity to any of the foregoing. The IDEF2 gene may comprises a nucleic acid sequence that encodes a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 9-16, and amino acid sequences having at least 70% sequence identity to any of the foregoing.

[0089] Using microarray expression profiling in rice, the basic Helix-Loop-Helix (bHLH) transcription factor (TF), OsIRO2, was identified as a transcription factor that is upregulated upon Fe deficiency. Unlike FER-like iron-deficiency-induced transcription factor (FIT / FER), of which there appears to be no counterpart in rice, IRO2 is typically expressed in both roots and shoots in Fe deficient conditions. When OsIRO2 is overexpressed, plants grow better than wild-type (WT) under Fe-deficient conditions, while RNAi knocked- down OsIRO2 plants do worse than WT. The overexpression lines also have enhanced expression of genes involved in PS synthesis and transport, while the RNAi lines have diminished expression of the same genes. In these plants, OsIRTl levels remain unchanged, suggesting that OsIRO2 is responsible for regulation of PS-mediated Fe uptake, but not Fe(II) uptake. Additionally, not all genes regulated by OsIRO2 have the characteristic consensus IRO2 binding sequence (CACGTGG), so IRO2 may act by regulating other transcription factors. In another recent study, promoter-GUS lines demonstrated that the expression pattern of OsIRO2 is very similar to downstream genes such as OsNASl and OsNAS2 in embryos during seed development and germination and in vascular bundles in roots and leaves during vegetative stages. As previously reported, OsIRO2 overexpression lines exhibited improved tolerance to Fe deficiency. Additionally, seeds accumulated higher levels of Fe compared to WT when plants were grown in alkaline soil. The results of this study suggest that OsIRO2 is synchronously expressed with other genes involved in Fe homeostasis and plays a key role in Fe uptake and Fe transport in both germination and seed development.

[0090] OsIDEFl and OsIDEF2 (IDE binding factors), members of the ABI3 / VP1 and NAC transcription factor families, respectively, have also been identified as positive regulators of the Fe deficiency response based on their ability to bind to sequences that confer Fe regulation on the barley root-specific IDS2 promoter. These sequences are called iron- deficiency-responsive elements 1 and 2, or IDE 1 and IDE2 (Kabayashi et al. 2003, Plant J. 36:780-93). Sequences homologous to IDE1 are also found in other Fe deficiency inducible promoters, such as OsNASl, OsNAS2, OsIRTl, AtIRTl, and AtFRO2. OsIDEFl and OsIDEF2 bind to specific sequences within 1DE1 and IDE2: CATGC andCA[A / C]G[T / C][T / C / A] [T / C / A], respectively. OsIDEFl tianscripts are constitutively expressed and levels do not depend on Fe status (Kobayashi et al. 2010 Ann Bot. 105(7): 1109-1117). OsIDEFl positively regulates the induction of several known Fe uptake and utilization genes in rice, such as OsIRO2, OsYSLIS, OsYSL2, OsIRTl, OsNASl, OsNAS2, and OsNAS3. As Fe deficiency progressed, activation of these genes by OsIDEFl was limited.

[0091] The identification of IDEF1 as an iron-deficiency response gene was reported by Kobayashi et al. in 2009 (Plant J.; 60(6):948-61, the entire contents of which are incorporated herein by reference). The authors noted that IDEF1 regulates the response to iron deficiency in Oryza saliva (rice) by recognizing CATGC sequences within the iron deficiency-responsive cis-acting element IDEL The authors analyzed the response to iron deficiency in transgenic rice plants exhibiting induced or repressed IDEF1 expression and noted that, when exposed to conditions of iron-deficiency, IDEF1 knock-down plants were susceptible to early-stage iron deficiency, in contrast to IDEFl-induced plants. Time-course expression analyses using quantitative reverse-transcriptase PCR revealed that the level of IDEF1 expression was positively correlated with the level of induction of the iron utilization- related genes OsIRO2, OsYSLIS, OsIRTl, OsYSL2, OsNASl, OsNAS2, OsNAS3 and OsDMASl, just after the onset of iron starvation. This overall transactivation mediated by IDEF1 became less evident in subsequent stages. Microarray and in-silico analyses further revealed that genes positively regulated by IDEF1, especially at the early stage, exhibit overrepresentation of CATGC and IDEl-like elements within the proximal promoter regions. These finding indicated the existence of early and subsequent responses to iron deficiency, with the former requiring IDEF1 more specifically. Proximal regions of IDEF1 -regulated gene promoters also showed enrichment of RY elements (CATGCA), which regulate gene expression during seed maturation. The expression of several genes encoding late embryogenesis abundant proteins, including Osem, was induced in iron-deficient roots and / or leaves in an IDEF1 -dependent manner, suggesting a possible function of seed maturation-related genes in iron-deficient vegetative organs.

[0092] As for OsIDEF2, reduction of expression by RNAi results in change in a gene previously implicated in Fe homeostasis: OsYSL2, which encodes a metal-NA transporter.

[0093] The identification of IDEF2 as an iron-deficiency response gene was reported by Ogo et al. in 2008 (J Biol Chern, 283(19): 13407-17, the entire contents of which are incorporated herein by reference). As noted in that report, the authors identified 1DEF2 asa novel transcription factor of rice and barley that specifically binds to tire iron deficiency- responsive cis-acting element 2 (IDE2), as determined by yeast one-hybrid screening. IDEF2 belongs to an uncharacterized branch of the NAC transcription factor family and exhibits novel properties of sequence recognition. An electrophoretic mobility shift assay and cyclic amplification and selection of targets experiment revealed that IDEF2 predominantly recognized CA(A / C)G(T / C)(T / C / A)(T / C / A) within IDE2 as the core-binding site. IDEF2 transcripts are constitutively present in rice roots and leaves (Kobayashi et al. 2010 Ann Bot. 105(7): 1109-1117). Ogo et al. noted that repression of the function of IDEF2 by the RNA interference (RNAi) technique and chimeric repressor gene-silencing technology (CRES-T) caused aberrant iron homeostasis in rice. Several genes up-regulated by iron deficiency, including the Fe(II)-nicotianamine transporter gene OsYSL2, were less induced by iron deficiency in the RNAi rice of IDEF2, suggesting that IDEF2 is involved in the regulation of these genes.Upstream open reading frame (uORF)

[0094] Upstream open reading frames, or “uORFs”, often reside in an mRNA transcript located upstream of protein-coding main ORF (mORF; also referred to herein and in the art as long ORFs or major ORFs). uORFs are a class of small ORFs that typically act as repressors of their downstream mORFs. uORFs may encode evolutionarily conserved functional peptides, such as cis-acting regulatory' peptides, including, for example, through translational repression. uORFs are generally defined by a start codon (any three base pair codon with at least two of the following bases in order: AUG (or ATG in the corresponding DNA sequence)) in the 5'-UTR, with an in frame stop codon: UAA, UAG, UGA (or TAA, TAG, TGA in the corresponding DNA sequence), that is upstream (i.e., in a 5' direction) and not overlapping with the main coding sequence. Alternative non-AUG start codons are known but are very rare in eukaryotic genomes. For example, translation has been shown to be initiated with leucine using a specific leucyl-tRNA corresponding to the codon CUG (see, e.g., Starck et al. 2012 Science 336: 1719-23).

[0095] As described elsewhere herein, the inventors have surprisingly found that the uORFs of iron-deficiency response genes, including the uORF of 1DEF1 and IDEF2, function as translational regulators of these genes.

[0096] The presence of an ORF does not necessarily mean that the region is always translated and / or retained in the mature polypeptide. uORFs, which are located upstream of protein-coding major ORFs (also known as main ORFs; mORFs) in the 5'-untranslatedregions (5’UTR) of mRNAs, can act as cis-acting elements that modify the activity of a downstream sequence that encodes the polypeptide. uORFs may exert their regulatory function by modulate the translation initiation rate of downstream coding sequences (CDSs) by sequestering ribosomes, or by encoding evolutionarily conserved short peptides (sometimes referred to as “uPEPs” that function as cis-acting repressor peptides of the downstream mORF.

[0097] The terms “51untranslated region (UTR)” or “5' UTR” as used herein refer to the leader sequence of a resulting mRNA from a transcribed locus, which comprises the region of the transcript residing 5’, or upstream of, the start codon of the main protein coding ORF within the mRNA. Although the 5' UTR is referred to as an "untranslated region", uORFs in the 5' UTR may be translated into peptides that do not form part of the protein-coding region of the gene. In some embodiments, a uORF within a 5’UTR region may be translated into a short peptide. In other embodiments, a uORF within a 5'UTR is not translated.

[0098] The terms "to enhance the expression", "enhanced expression" and the like, including in the context a polynucleotide / gene or polypeptide of the present disclosure, typically mean an instance where the level of an RNA transcript and / or encoded polypeptide produced is increased when the genomic DNA encoding the uORF of the polynucleotide / gene or polypeptide of the present disclosure is modified, as disclosed herein. The enhanced or increased expression of a polynucleotide / gene or polypeptide of the present disclosure may be an increase of at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, of at least 99% or 100% when compared to the level of expression of the polynucleotide / gene or polypeptide produced from an unmodified (e.g., native) genomic DNA sequence encoding the uORF.

[0099] In an embodiment, the unmodified uORF of rice IDEF1 comprises the nucleic acid sequence of SEQ ID NO: 44. In an embodiment, the unmodified uORF of rice IDEF1 comprises the nucleic acid sequence of SEQ ID NO: 45.

[0100] In an embodiment, the unmodified uORF of rice IDEF2 comprises the nucleic acid sequence of SEQ ID NO: 45.

[0101] In an embodiment, the unmodified uORF of an IDEF2 comprises a nucleic acid sequence of any one of SEQ ID NOs: 47-49. In an embodiment, the unmodified uORF of an IDEF2 comprises a nucleic acid sequence that encodes for a polypeptide having the aminoacid sequence of any one of SEQ ID NOs: 50-53. In an embodiment, the unmodified uORF of wheat IDEF2 comprises the nucleic acid sequence of any one of SEQ ID NOs: 47, 48 and 49. In an embodiment, the unmodified uORF of wheat IDEF2 comprises a nucleic acid sequence that encodes for a polypeptide having the amino acid sequence of any one of SEQ ID NOs: 50-53.Polynucleotides

[0102] In yet another aspect disclosed herein, there is provided a polynucleotide comprising the nucleic acid sequence selected from the group consisting of SEQ ID NOs: 33-43, and variants having at least 70% sequence identify of any of the foregoing. In a further embodiment, the polynucleotide comprises a nucleic acid sequence encoding the amino acid sequence of any one of SEQ ID NOs:50-53. In a further embodiment, the polynucleotide comprises a nucleic acid sequence selected fiom the group consisting of SEQ ID NOs: 54- 59, and nucleic acids having at least 70% sequence identify thereto.

[0103] The present disclosure also extends to polynucleotide constructs comprising the polynucleotides disclosed herein, and to expression vectors comprising the polynucleotides or the polynucleotide constructs disclosed herein.

[0104] The term "polynucleotide", as used herein, means a single or double-stranded deoxyribonucleotide or ribonucleotide polymer of any length, but preferably at least 15 nucleotides in length, and include, as non-limiting examples, coding and non-coding sequences of a gene, sense and antisense sequences complements, exons, introns, genomic DNA, cDNA, pre-mRNA, mRNA, rRNA, siRNA, miRNA, tRNA, ribozymes, recombinant polypeptides, Isolated and purified naturally occurring DNA or RNA sequences, synthetic RNA and DNA sequences, nucleic acid probes, primers and fragments.

[0105] Preferably, the term "polynucleotide" includes both the specified sequence and its complement.

[0106] A "fragment" of a polynucleotide sequence provided herein is a subsequence of contiguous nucleotides, such as, for example, a sequence that is at least 15 nucleotides in length.

[0107] The term "primer" refers to a short polynucleotide, usually having a free 3'OH group, that is hybridized to a template and used for priming polymerization of a polynucleotide complementary to the target.

[0108] In an embodiment, the fragment comprises at least 15 nucleotides, preferably at least 20 nucleotides, more preferably at least 30 nucleotides, more preferably at least 50 nucleotides, more preferably at least 50 nucleotides and most preferably at least 60 nucleotides of contiguous nucleotides of a polynucleotide of the present disclosure.

[0109] In an embodiment, variant polynucleotide sequences preferably exhibit at least 50%, more preferably at least 51%, more preferably at least 52%, more preferably at least 53%, more preferably at least 54%, more preferably at least 55%, more preferably at least 56%, more preferably at least 57%, more preferably at least 58%, more preferably at least 59%, more preferably at least 60%, more preferably at least 61%, more preferably at least 62%, more preferably at least 63%, more preferably at least 64%, more preferably at least 65%, more preferably at least 66%, more preferably at least 67%, more preferably at least 68%, more preferably at least 69%, more preferably at least 70%, more preferably at least 71%, more preferably at least 72%, more preferably at least 73%, more preferably at least 74%, more preferably at least 75%, more preferably at least 76%, more preferably at least 77%, more preferably at least 78%, more preferably at least 79%, more preferably at least 80%, more preferably at least 81%, more preferably at least 82%, more preferably at least 83%, more preferably at least 84%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, and most preferably at least 99% identity to a sequence of the present disclosure. Identity is found over a comparison window of at least 20 nucleotide positions, preferably at least 50 nucleotide positions, more preferably at least 100 nucleotide positions, and most preferably over the entire length of a polynucleotide of the disclosure.

[0110] Polynucleotide sequence identity can be determined by any means known to persons skilled in the art, including by comparing the subject polynucleotide sequence to a candidate polynucleotide sequence using BLASTN (from the BLAST suite of programs, version 2.2.5 [November 2002]) Inbl2seq (Tatiana A. Tatusova, Thomas L. Madden (1999), "Blast 2 sequences— a new tool for comparing protein and nucleotide sequences", FEMS Microbiol. Lett. 174:247-250), which is publicly available from NCBI (ftp colon slash slash file transfer protocol.ncbi.nih.gov / blast / ). The default parameters of bl2seq are utilized except that filtering of low complexity parts should be turned off.

[0111] The identity of polynucleotide sequences may be examined using the following Unix command line parameters: bl2seq -i nucleotideseql -j nucleotideseq2 -F F -p blastn

[0112] The parameter -F F turns off filtering of low complexity sections. The parameter - p selects the appropriate algorithm for the pair of sequences. The bl2seq program reports sequence identity as both the number and percentage of identical nucleotides in a line "Identities-1.

[0113] Polynucleotide sequence identity may also be calculated over the entire length of the overlap between a candidate and subject polynucleotide sequences using global sequence alignment programs (e.g., Needleman, S. B. and Wunsch, C. D. ( 1970) J. Mol. Biol. 48, 443- 453). A full implementation of the Needleman-Wunsch global alignment algorithm is found in the needle program in the EMBOSS package (Rice, P. Longden, I. and Bleasby, A. EMBOSS: The European Molecular Biology Open Software Suite, Trends in Genetics June 2000, vol. 16, No 6. pp. 276-277) which can be obtained from world wide web.hgmp.mrc.ac.uk / Software / EMBOSS / . The European Bioinformatics Institute server also provides the facility to perform EMBOSS-needle global alignments between two sequences on line at world wide web.ebi.ac.uk / emboss / align / .

[0114] Alternatively, the GAP program may be used which computes an optimal global alignment of two sequences without penalizing terminal gaps. GAP is described in the following paper: Huang, X. (1994) On Global Sequence Alignment. Computer Applications in the Biosciences 10, 227-235.

[0115] A preferred method for calculating polynucleotide % sequence identity is based on aligning sequences to be compared using Clustal X (Jeanmougin et al., 1998, Trends Biochem. Sci. 23, 403-5.)

[0116] Polynucleotide variants of the present disclosure also encompass those which exhibit a similarity to one or more of the specifically identified sequences that is likely to preserve the functional equivalence of those sequences and which could not reasonably be expected to have occurred by random chance. Such sequence similarity with respect to polypeptides may be determined using the publicly available bl2seq program from the BLAST suite of programs (version 2.2.5 [November 2002]) from NCBI (ftp colon slash slash file transfer protocol ftn.ncbi .nih, go v / blast / ).

[0117] The similarity' of polynucleotide sequences may be examined using the following Unix command line parameters: bl2seq -i nucleotideseql -j nucleotideseq2 -F F -p tblastx.

[0118] The parameter -F F turns off" filtering of low complexity sections. The parameter - p selects the appropriate algorithm for the pair of sequences. This program finds regions of similarity between the sequences and for each such region reports an "E value" which is the expected number of times one could expect to see such a match by chance in a database of a fixed reference size containing random sequences. The size of this database is set by default in the bl2seq program. For small E values, much less than one, the E value is approximately the probability of such a random match.

[0119] Variant polynucleotide sequences preferably exhibit an E value of less than 1x10-6more preferably less than 1x10-9, more preferably less than 1x10-12, more preferably less than 1x10-15, more preferably less than 1x10-18, more preferably less than 1x10-21, more preferably less than 1x10-30, more preferably less than 1x10-40, more preferably less than 1x10-50, more preferably less than 1x10-60, more preferably less than 1x10-70, more preferably less than 1x10-80, more preferably less than 1x10-80and most preferably less than 1x10-100when compared with any one of the specifically identified sequences.

[0120] Alternatively, variant polynucleotides of the present disclosure hybridize to the specified polynucleotide sequences, or complements thereof under stringent conditions.

[0121] The term "hybridize under stringent conditions", and grammatical equivalents thereof, refers to the ability of a polynucleotide molecule to hybridize to a target polynucleotide molecule (such as a target polynucleotide molecule immobilized on a DNA or RNA blot, such as a Southern blot or Northern blot) under defined conditions of temperature and salt concentration. The ability to hybridize under stringent hybridization conditions can be determined by initially hybridizing under less stringent conditions then increasing the stringency to the desired stringency.

[0122] With respect to polynucleotide molecules greater than about 100 bases in length, typical stringent hybridization conditions are no more than 25 to 30 °C. (for example, 10 °C.) below the melting temperature (Tm) of the native duplex (see generally, Sambrook et al., Eds, 1987, Molecular Cloning, A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press; Ausubel et al., 1987, Current Protocols in Molecular Biology, Greene Publishing,). Tm for polynucleotide molecules greater than about 100 bases can be calculated by the formula Tm=81. 5+0. 41% (G+C-log (Na+). (Sambrook et al., Eds, 1987, Molecular Cloning, A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press; Bolton and McCarthy, 1962, PNAS 84: 1390). Typical stringent conditions for polynucleotide of greaterthan 100 bases in length would be hybridization conditions such as prewashing in a solution of 6x Saline-SodiumCitrate (SSC) , 0.2% SDS; hybridizing at 65 °C., 6xSSC, 0.2% SDS overnight; followed by two washes of 30 minutes each in IxSSC, 0.1% SDS at 65 °C. and two washes of 30 minutes each in 0.2xSSC, 0.1% SDS at 65 °C.

[0123] With respect to polynucleotide molecules having a length less than 100 bases, exemplary stringent hybridization conditions are 5 to 10 °C. below Tm. On average, the Tm of a polynucleotide molecule of length less than 100 bp is reduced by approximately (500 / oligonucleotide length) °C.

[0124] With respect to the DNA mimics known as peptide nucleic acids (PNAs) (Nielsen etal., Science. 1991 Dec. 6; 254(5037): 1497-500) Tm values are higher than those for DNA- DNA or DNA-RNA hybrids, and can be calculated using the formula described in Giesen et al., Nucleic Acids Res. 1998 Nov. 1; 26(21):5004-6. Exemplary stringent hybridization conditions for a DNA-PNA hybrid having a length less than 100 bases are 5 to 10 °C. below the Tm.

[0125] Variant polynucleotides of the present disclosure also encompass polynucleotides that differ from the sequences of the disclosure but that, as a consequence of the degeneracy of the genetic code, encode a polypeptide having similar activity to a polypeptide encoded by a polynucleotide of the present disclosure. A sequence alteration that does not change the amino acid sequence of the polypeptide is a "silent variation". Except for ATG (methionine) and TGG (tryptophan), other codons for the same amino acid may be changed by art recognized techniques, e.g., to optimize codon expression in a particular host organism.

[0126] Polynucleotide sequence alterations resulting in conservative substitutions of one or several amino acids in the encoded polypeptide sequence without significantly altering its biological activity are also included in the present disclosure. A skilled artisan will be aware of methods for making phenotypically silent amino acid substitutions (see, e.g., Bowie et al., 1990, Science 247, 1306).

[0127] Variant polynucleotides due to silent variations and conservative substitutions in the encoded polypeptide sequence may be determined using the publicly available bl2seq program from the BLAST suite of programs (version 2.2.5 [November 2002]) from NCBI (ftp colon slash slash file transfer protocol ftp.ncbi.nih.gov / blast / ) via the tblastx algorithm as previously described.

[0128] The function of a variant polynucleotide of the disclosure may be assessed, for example, by expressing such a sequence in bacteria and testing activity of the encodedprotein as described in the Example section. Function of a variant may also be tested for its ability to alter iron deficiency response gene expression, iron content, a trait, or content of useful compounds, nutrients, or components in plants, also as described in the Examples section herein.Constructs, Vectors and Components Thereof

[0129] The present disclosure also extends to polynucleotide constructs comprising the polynucleotides disclosed herein, and to expression vectors comprising the polynucleotides or the polynucleotide constructs disclosed herein.

[0130] The terms "construct", "genetic construct" and the like refer to a polynucleotide molecule, usually double-stranded DNA, which may have inserted into it another polynucleotide molecule (the insert polynucleotide molecule) such as, but not limited to, a cDNA molecule.

[0131] A construct may contain the necessary elements that permit transcribing the insert polynucleotide molecule, and, optionally, translating the transcript into a polypeptide. The insert polynucleotide molecule may be derived fiom the host cell, or may be derived fiom a different cell or organism and / or may be a recombinant polynucleotide. Once inside the host cell the genetic construct may become integrated in the host chromosomal DNA. The genetic construct may be linked to a vector.

[0132] The term "vector" refers to a polynucleotide molecule, usually double stranded DNA, which is used to transport the genetic construct into a host cell. The vector may be capable of replication in at least one additional host system, such as E. coli.

[0133] The term "expression construct" refers to a genetic construct that includes the necessary elements that permit transcribing the insert polynucleotide molecule, and, optionally, translating the transcript into a polypeptide. An expression construct typically comprises in a 5' to 3' direction: a) a promoter functional in the host cell into which the construct will be transformed, b) the polynucleotide to be expressed, and c) a terminator functional in the host cell into which the construct will be transformed.

[0134] The term "coding region" or "open reading frame" (ORF) refers to the sense strand of a genomic DNA sequence or a cDNA sequence that is capable of producing a transcription product and / or a polypeptide under the control of appropriate regulatory sequences. The coding sequence is identified by the presence of a 5' translation start codon and a 3' translation stop codon. When inserted into a genetic construct, a "coding sequence" iscapable of being expressed when it is operably linked to promoter and terminator sequences.

[0135] "Operably-linked" means that the sequence of interest, such as a sequence to be expressed is placed under the control of, and typically connected to another sequence comprising regulatory elements that may include promoters, tissue-specific regulatory elements, temporal regulatory elements, enhancers, repressors and terminators, 5 -UTR sequences, 5 -UTR sequences comprising uORFs, and uORFs.

[0136] In a preferred embodiment, the regulatory elements include a polynucleotide sequence of the disclosure. Preferably, the sequence of the present disclosure comprises a 5 -UTR sequence. Preferably the 5 -UTR sequence comprises a uORF.

[0137] The term "noncoding region" refers to untranslated sequences that are upstream of the translational start site and downstream of the translational stop site of amain ORF. These sequences are also referred to respectively as the 5 -UTR and the 3'-UTR. These regions include elements required for transcription initiation and termination and for regulation of translation efficiency.

[0138] A 5'-UTR sequence is the sequence between the transcription initiation site, and the translation start site.

[0139] The 5 -UTR sequence is an mRNA sequence encoded by the genomic DNA. However as used herein the term 5 -UTR sequence includes the genomic sequence encoding the 5 -UTR sequence, and the complement of that genomic sequence, and the 5 -UTR mRNA sequence.

[0140] Terminators are sequences, which terminate transcription, and are found in the 3' untranslated ends of genes downstream of the translated sequence. Terminators are important determinants of mRNA stability and in some cases have been found to have spatial regulator)' functions.

[0141] The terms "to alter expression of and "altered expression" of a polynucleotide or polypeptide of the present disclosure, are intended to encompass the situation where genomic DNA corresponding to a polynucleotide of the present disclosure is modified thus leading to altered expression of a polynucleotide or polypeptide of the present disclosure. Modification of the genomic DNA may be through genetic transformation or other methods known in the art for inducing mutations. The "altered expression" can be related to an increase or decrease in the amount of messenger RNA transcribed and / or polypeptide translated and may also result in altered activity of a polypeptide due to alterations in thesequence of a polynucleotide and polypeptide produced.

[0142] The term “introduced targeted genetic modification” or “targeted genetic modification” refers to a change in the DNA sequence of a plant at a specific chromosomal position (also known as a locus) in the genome which is chosen by a skilled practitioner (such as plant breeder or molecular biologist) and which change is introduced by a process of gene editing and / or selection using a specific complementary nucleic acid molecule sequence as a guide or probe to enable the process.

[0143] The term “native genomic locus” refers to a gene or DNA sequence that is present in the genome of a wild-type plant at particular chromosomal position of a given species. The “native genomic locus” typically comprises a region spanning a start to stop codon, along with any intervening introns, that is transcribed to generate a main ORF that encodes a long polypeptide that is typically around 100 amino acids or more in length, as well as the associated upstream regulatory elements including the promoter region and any elements that control the activity of the mORF such as uORFs. A uORF is present in the same mRNA transcript as the mORF that the uORF regulates; both the uORF and the mORF can therefore be considered part of the same overall native genomic locus. A native genomic locus is often specified by reference to an accession number, deposited in GenBank, which, for example, indicates the DNA sequence and encoded polypeptide that is present at that position. It should also be noted that a locus may encode multiple protein variants that result from alternative splicing of mRNA and these variants are represent by different “gene models” that are denoted by the accession number followed by a dot and a number (see, e.g. Liang et al. U.S. Pat. No. 9,648,813).

[0144] The term -‘non-native allele of a gene” refers to a sequence variant of a gene (where the term gene includes both the protein coding region as well as upstream control elements such as uORFs) from a given plant species that results from a human intervention such as gene editing or selection and / or a sequence of nucleotides which is not found in nature in either the genome of a wild-type plant of that species or in the genome of a plant of that species taken from a naturally-occurring wild population.

[0145] The terms “complementary” and “complementarity” refer to polynucleotides (i.e., a sequence of nucleotides) related by the base-pairing rules. For example, the sequence “A- G-T,” is complementary to the sequence “T-C-A.” Complementarity may be “partial,” in which only some of the nucleic acids’ bases are matched according to the base pairing rales. Or, there may be “complete” or “total” complementarity between the nucleic acids. Thedegree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands.

[0146] The term “corresponding” as used herein in reference to a particular gene or nucleic acid sequence is intended to mean an analogous or equivalent or comparable gene or nucleic acid sequence. For example, where reference is made to a corresponding nucleic acid sequence, it is intended to mean the analogous, equivalent or comparable naturally-occinring nucleic acid sequence. Where reference is made to a corresponding exogenous nucleic acid sequence, it is intended to mean an analogous, equivalent or comparable exogenous nucleic acid sequence. In some embodiments, the corresponding nucleic acid sequence has analogous or equivalent function or having sequence similarity. In one embodiment, the corresponding nucleic acid sequence may be identical in function and / or sequence. In another embodiment, the corresponding nucleic acid sequence may have about the same function or activity. In another embodiment, the corresponding nucleic acid sequence may have reduced function or activity. In another embodiment, the corresponding nucleic acid sequence may have lost function or activity. In some embodiments, the phrase “corresponds to” or “corresponding to” is meant a nucleic acid sequence that displays substantial sequence identity to a reference nucleic acid sequence. In general the nucleic acid sequence will display at least about 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or even up to 100% sequence identity to the reference nucleic acid sequence.

[0147] The term “expression”, as used herein, typically refers to any step involved in the production of the product of gene including an RNA molecule or a polypeptide, such as by transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0148] The term “gene” is used herein to refer to a unit of inheritance that comprises a coding sequence and optionally transcriptional and / or translational regulatory sequences and / or non-translated sequences (z.e., introns, 5’ and 3’ untranslated sequences) whether or not such regulatory sequences are adjacent to coding and / or transcribed sequences. Accordingly, a gene may include or encode upstream open reading frames (uORF), promoter sequences, signal peptides, 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. In some embodiments the gene may comprise coding sequences and non-coding sequences. In otherembodiments, the gene may comprise only coding sequences. In some embodiments the gene may comprise translated and untranslated sequences. In other embodiments, the gene may comprise only untranslated sequences.

[0149] As used herein, the term “nucleic acid”, “nucleic sequence”, “polynucleotide”, “oligonucleotide” and “nucleotide sequence” as used herein refers to mRNA, RNA, cRNA, rRNA, cDNA, or DNA, or a combination thereof. The term typically refers to polymeric form of nucleotides, either ribonucleotides or deoxynucleotides or a modified form of either type of nucleotide. The term includes single-, double- or triple- stranded forms of DNA and RNA. It can be of recombinant, artificial and / or synthetic origin and it can comprise modified nucleotides, comprising for example a modified bond, a modified purine or pyrimidine base, or a modified sugar. The nucleic acids of the present disclosure can be in isolated or purified form, and made, isolated and / or manipulated by techniques known per se in the art, e.g., cloning and expression of cDNA libraries, amplification, enzymatic synthesis or recombinant technology. The nucleic acids can also be synthesized in vitro by well-known chemical synthesis techniques, as described in, e.g., Belousov (1997) Nucleic Acids Res. 25:3440-3444.

[0150] The term “gene product” or “expression product” as used herein refers to an RNA or protein that results from expression of a gene. For example, the gene product may be an RNA, such as rnRNA, rRNA, tRNA, miRNA or siRNA, or the gene product may be a polypeptide product.

[0151] The terms “modification”, “alteration”, and the like, as used herein in relation to a nucleotide or an amino acid residue, typically mean that the nucleotide or amino acid or in the particular position has been modified compared to the nucleotide or amino acid of the wild-type or parent polynucleotide or polypeptide. Modification of a nucleotide or an amino acid may be deletion of the nucleotide or an amino acid. Modification of a nucleotide or an amino acid may be insertion of one or more nucleotide / s or an amino acid / s. Modification of a nucleotide or an amino acid may be substitution of the nucleotide or an amino acid with a nucleotide or an amino acid that is not the same as the original nucleotide or an amino acid. The terms “mutant” and “variant” and “modified” may be used interchangeably herein, to refer to a non-wild-type organism, strain, expression pattern or expression level, gene / polynucleotide sequence or amino acid sequence.

[0152] The present disclosure also extends to homologs and orthologs of the polynucleotide and polypeptide sequences disclosed herein. The term “homolog” or“homologue” as further described and used herein means a polynucleotide or polypeptide from the same species or a different species which has a substantial level of identity within either its conserved domain and / or across its entire sequence, wherein the level of identity' is at least 30% or at least 35%, or at least 40%, or at least 45%, or at least 50%, or at least 55%, or at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%, or about 100% identity as compared to the polynucleotide or polypeptide sequences disclosed herein. Suitable homologs may be identified at the DNA level by identifying polynucleotides that hybridize, such as at a high stringency, to an Arabidopsis iron-deficiency response gene product. “Orthologs”, as used herein, means evolutionarily-related genes that have similar sequence and similar functions. Orthologs are typically structurally related genes in different species that are derived by a speciation event.Polypeptides

[0153] The term "polypeptide", as used herein, encompasses an amino acid sequence comprising a plurality of consecutive polymerized amino acid residues e.g., at least about 15 consecutive polymerized amino acid residues, optionally at least about 30 consecutive polymerized amino acid residues, at least about 50 consecutive polymerized amino acid residues. In many instances, a polypeptide comprises a polymerized amino acid residue sequence that is a transcription factor or a domain or portion or fragment thereof. Additionally, the polypeptide may comprise 1) a localization domain, 2) an activation domain, 3) a repression domain, 4) an oligomerization domain, or 5) a DNA-binding domain, or the like. The polypeptide optionally comprises modified amino acid residues, naturally occurring amino acid residues not encoded by a codon, non-naturally occurring amino acid residues.

[0154] A "fragment" of a polypeptide is a subsequence of the polypeptide. Preferably, the fragment is a functional fragment, insofar as it performs a function that is required for the biological activity and / or provides three-dimensional structure of the polypeptide.

[0155] The term "isolated", as applied to the polynucleotide or polypeptide sequences disclosed herein, is used to refer to sequences that are removed from their natural cellular environment. In one embodiment the sequence is separated from its flanking sequences as found in nature. An isolated molecule may be obtained by any method or combination of methods including biochemical, recombinant, and synthetic techniques.

[0156] The term "recombinant" refers to a polynucleotide sequence that is removed from sequences that surround it in its natural context and / or is recombined with sequences that are not present in its natural context.

[0157] A "recombinant" polypeptide sequence is produced by translation from a "recombinant" polynucleotide sequence.

[0158] The term "derived from" with respect to polynucleotides or polypeptides of the present disclosure being derived from a particular genera or species, means that the polynucleotide or polypeptide has the same sequence as a polynucleotide or polypeptide found naturally in that genera or species. The polynucleotide or polypeptide, derived from a particular genera or species, may therefore be produced synthetically or recombinantly.

[0159] The term "variant" with reference to polypeptides encompasses naturally occurring, recombinantly and synthetically produced polypeptides. Variant polypeptide sequences preferably exhibit at least 50%, more preferably at least 51%, more preferably at least 52%, more preferably at least 53%, more preferably at least 54%, more preferably at least 55%, more preferably at least 56%, more preferably at least 57%, more preferably at least 58%, more preferably at least 59%, more preferably at least 60%, more preferably at least 61%, more preferably at least 62%, more preferably at least 63%, more preferably at least 64%, more preferably at least 65%, more preferably at least 66%, more preferably at least 67%, more preferably at least 68%, more preferably at least 69%, more preferably at least 70%, more preferably at least 71%, more preferably at least 72%, more preferably at least 73%, more preferably at least 74%, more preferably at least 75%, more preferably at least 76%, more preferably at least 77%, more preferably at least 78%, more preferably at least 79%, more preferably at least 80%, more preferably at least 81%, more preferably at least 82%, more preferably at least 83%, more preferably at least 84%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, and most preferably at least 99% identity to a sequences of the present disclosure. Identity is found over a comparison window of at least 20 amino acid positions, preferably at least 50 amino acid positions, more preferably at least 100 amino acid positions, and most preferably over tire entire length of a polypeptide of the disclosure.

[0160] "Identity" or "similarity" refers to sequence similarity between two polynucleotidesequences or between two polypeptide sequences, with identity being a stricter comparison. The phrases "percent identity" and "% identity" refer to the percentage of sequence identity found in a comparison of two or more polynucleotide sequences or two or more polypeptide sequences. "Sequence similarity" refers to the percent similarity in base pair sequence (as determined by any suitable method) between two or more polynucleotide sequences. Two or more sequences can be anywhere from 0-100% similar, or any integer value therebetween. Identity or similarity' can be determined by comparing a position in each sequence that may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same nucleotide base or amino acid, then the molecules are identical at that position. A degree of similarity or identity between polynucleotide sequences is a function of the number of identical or matching nucleotides at positions shared by the polynucleotide sequences. A degree of identity of polypeptide sequences is a function of the number of identical amino acids at positions shared by the polypeptide sequences. A degree of homology or similarity of polypeptide sequences is a function of the number of amino acids at positions shared by the polypeptide sequences.

[0161] The term “sequence identity” as used herein refers to the extent that sequences are identical on a nucleotide-by-nucleotide basis or an amino acid-by-amino acid basis over a window of comparison (e.g. over 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200 or more nucleotides or amino acids residues). Thus, a “percentage of sequence identity” is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G) or the identical amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Vai, Leu, De, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gin, Cys and Met) 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 window of comparison (z.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. For the purposes of the present disclosure, “sequence identity” will be understood to mean the “match percentage” calculated by an appropriate method. For example, sequence identity analysis may be carried out using the DNASIS computer program (Version 2.5 for windows; available from Hitachi Software engineering Co., Ltd., South San Francisco, California, USA) using standard defaults as used in the reference manual accompanying the software. Sequences may be aligned using a global alignment algorithms (e.g., Needleman and Wunsch algorithm; Needleman and Wunsch, 1970), which aligns the sequences optimallyover the entire length, while sequences of substantially different lengths are preferably aligned using a local alignment algorithm (e.g., Smith and Waterman algorithm (Smith and Waterman, 1981) or Altschul algorithm (Altschul et al., 1997; Altschul et al., 2005)). Alignment for the purposes of determining percent amino acid sequence identity can be achieved by any means available to persons skilled in the art, illustrative examples of which include publicly available computer software, such as is available at http: / / blast.ncbi nlm.nih .gov / or http: / / www.ebi .ac.uk / Tools / emboss / ). Persons skilled in the art can readily determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. As used herein, % sequence identity' typically refers to values generated using pair wise sequence alignment that creates an optimal global alignment of two sequences (e.g., using the Needleman-Wunsch algorithm).

[0162] Polypeptide sequence identity can be determined in the following manner. The subject polypeptide sequence is compared to a candidate polypeptide sequence using BLASTP (fiom the BLAST suite of programs, version 2.2.5 [November 2002]) in bl2seq, which is publicly available fiom NCBI (ftp colon slash slash file transfer protocol ftp.ncbi.nih.gov / blast / ). The default parameters of bl2seq are utilized except that filtering of low complexity regions should be turned off.

[0163] Polypeptide sequence identity may also be calculated over the entire length of the overlap between a candidate and subject polynucleotide sequences using global sequence alignment programs. EMBOSS-needle (available at world wide web.ebi.ac.uk / emboss / align / ) and GAP (Huang, X. (1994) On Global Sequence Alignment. Computer Applications in the Biosciences 10, 227-235.) as discussed above are also suitable global sequence alignment programs for calculating polypeptide sequence identity.

[0164] A preferred method for calculating polypeptide % sequence identity is based on aligning sequences to be compared using Clustal X (Jeanmougin et al., 1998, Trends Biochem. Sci. 23, 403-5.)

[0165] Polypeptide variants of the present disclosure also encompass those which exhibit a similarity to one or more of the specifically identified sequences that is likely to preserve the functional equivalence of those sequences and which could not reasonably be expected to have occurred by random chance. Such sequence similarity with respect to polypeptides may be determined using the publicly available bl2seq program from the BLAST suite of programs (version 2.2.5 [November 2002]) from NCBI (ftp colon slash slash file transferprotocol flp.ncbi.nih.gov / blast / ). The similarity of polypeptide sequences may be examined using the following Unix command line parameters: bl2seq -i peptide seql -j peptideseq2 -F F -p blastp

[0166] Variant polypeptide sequences preferably exhibit an E value of less than 1x10-6more preferably less than 1x10-9, more preferably less than 1x10-12, more preferably less than 1x10-15, more preferably less than 1x10-18, more preferably less than 1x10-21, more preferably less than 1x10-30, more preferably less than 1x10-40, more preferably less than 1x10-50, more preferably less than 1x10-60, more preferably less than 1x10-70, more preferably less than 1x10-80more preferably less than 1x10-90and most preferably 1x10-100when compared with any one of the specifically identified sequences.

[0167] The parameter -F F turns off filtering of low complexity sections. The parameter - p selects the appropriate algorithm for the pair of sequences. This program finds regions of similarity between the sequences and for each such region reports an "E value" which is the expected number of times one could expect to see such a match by chance in a database of a fixed reference size containing random sequences. For small E values, much less than one, this is approximately the probability of such a random match.

[0168] A variant polypeptide includes a polypeptide wherein the amino acid sequence differs from a polypeptide herein by one or more conservative amino acid substitutions, deletions, additions, or insertions which do not affect the biological activity of the peptide. Conservative substitutions typically include the substitution of one amino acid for another with similar characteristics, e.g., substitutions within the following groups: valine, glycine, alanine; valine, Isoleucine, leucine; aspartic acid, glutamic acid; asparagines, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.

[0169] Non-conservative substitutions will entail exchanging a member of one of these classes for a member of another class.

[0170] Analysis of evolved biological sequences has shown that not all sequence changes are equally likely, reflecting at least in part the differences in conservative versus nonconservative substitutions at a biological level. For example, certain amino acid substitutions may occur frequently, whereas others are very rare. Evolutionary' changes or substitutions in amino acid residues can be modelled by a scoring matrix also referred to as a substitution matrix. Such matrices are used in bioinformatics analysis to identify relationships between sequences, one example being the BLOSUM62 matrix [Henikoff and Henikoff, 1992], TheBLOSUM62 matrix is used to generate a score for each aligned amino acid pair found at the intersection of the corresponding column and row. For example, the substitution score from a glutamic acid residue (E) to an aspartic acid residue (D) is 2. The diagonal show scores for amino acids which have not changed. Most substitutions changes have a negative score. The matrix contains only whole numbers.

[0171] Determination of an appropriate scoring matrix to produce the best alignment for a given set of sequences is believed to be within the skill of in the art. The BLOSUM62 matrix is also used as the default matrix in BLAST searches, although not limited thereto.

[0172] Other variants include peptides with modifications which influence peptide stability. Such analogs may contain, for example, one or more non-peptide bonds (which replace the peptide bonds) in the peptide sequence. Also included are analogs that include residues other than naturally occurring L-amino acids, e.g., D-amino acids or non-naturally occurring synthetic amino acids, e.g., beta or gamma amino acids and cyclic analogs.

[0173] The function of a polypeptide variant, as a variant of a polypeptide encoded by an iron deficiency response gene, may be assessed by the methods described in the Example section herein.Methods for Isolating or Producing Polynucleotides

[0174] The polynucleotide molecules of the present disclosure can be isolated by using a variety of techniques known to those of ordinary skill in the art, an illustrative example of includes the use of the polymerase chain reaction (PCR; as described, e.g., in Mullis et al., Eds. 1994 The Polymerase Chain Reaction, Birkhauser, incorporated herein by reference). The polypeptides of the present disclosure can be amplified using primers, as defined herein, derived from the polynucleotide sequences of the present disclosure.

[0175] Further methods for isolating polynucleotides of the present disclosure include use of all, or portions of, the polypeptides having the sequence set forth herein as hybridization probes. The technique of hybridizing labelled polynucleotide probes to polynucleotides immobilized on solid supports such as nitrocellulose filters or nylon membranes, can be used to screen the genomic or cDNA libraries. Exemplary hybridization and wash conditions are: hybridization for 20 hours at 65 °C. in 5. OxSSC, 0. 5% sodium dodecyl sulfete, IxDenhardt's solution; washing (three washes of twenty minutes each at 55 °C) in 1. OxSSC, 1% (w / v) sodium dodecyl sulfete, and optionally one wash (for twenty minutes) in 0. 5xSSC, 1% (w / v) sodium dodecyl sulfate, at 60° C. An optional further wash (for twenty minutes) can beconducted under conditions of 0. IxSSC, 1% (w / v) sodium dodecyl sulfete, at 60 °C.

[0176] The polynucleotide fragments of the present disclosure may be produced by techniques well-known in the art, illustrative examples of which include restriction endonuclease digestion, oligonucleotide synthesis and PCR amplification.

[0177] A partial polynucleotide sequence may be used in methods known to persons skilled in the art to identify the corresponding full length polynucleotide sequence. Illustrative examples of suitable methods include PCR-based methods, 5’RACE (see, e.g., Frohman M A, 1993, Methods Enzymol. 218: 340-56) and hybridization-based method, computer / database-based methods. Further, by way of illustrative example, inverse PCR permits acquisition of unknown sequences, flanking the polynucleotide sequences disclosed herein, starting with primers based on a known region (see, e.g., Triglia et al., 1998, Nucleic Acids Res 16, 8186, incorporated herein by reference). The method uses several restriction enzymes to generate a suitable fragment in the known region of a gene. The fragment is then circularized by intramolecular ligation and used as a PCR template. Divergent primers are designed from the known region. In order to physically assemble full-length clones, standard molecular biology approaches can be utilized (see, e.g., Sambrook et al. , Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press, 1987).

[0178] In some embodiments, it may be beneficial, when producing a transformed plant from a particular species, to transform such a plant with a sequence or sequences derived from that species. The benefit may be to alleviate public concerns regarding cross-species transformation in generating transformed organisms. Additionally, when down-regulation of a gene is the desired result, it may be necessary to utilize a sequence identical (or at least highly similar) to that in the plant, for which reduced expression is desired. For these reasons among others, it is desirable to be able to identify and isolate orthologs of a particular gene in several different plant species.Methods for Identifying Variants

[0179] Variants (including homologs and orthologs) may be identified by the methods described. For example, variant polypeptides may be identified using PCR-based methods (see, e.g., Mullis et al., Eds. 1994 The Polymerase Chain Reaction, Birkhauser). Typically, the polynucleotide sequence of a primer, useful to amplify variants of polynucleotide molecules of the present disclosure by PCR, may be based on a sequence encoding a conserved region of the corresponding amino acid sequence.

[0180] Alternatively, library screening methods, known to persons skilled in the art, may be employed (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press, 1987). When identifying variants of the probe sequence, hybridization and / or wash stringency will typically be reduced relatively to when exact sequence matches are sought.

[0181] Polypeptide variants may also be identified by physical methods, for example by screening expression libraries using antibodies raised against polypeptides of the present disclosure (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press, 1987) or by identifying polypeptides from natural sources with the aid of such antibodies.

[0182] The variant sequences of the present disclosure, including both polynucleotide and polypeptide variants, may also be identified by computer-based methods known to persons skilled in the art, such as by using public domain sequence alignment algorithms and sequence similarity search tools to search sequence databases (public domain databases include GenBank, EMBL, Swiss-Prot, PIR and others). See, e.g., Nucleic Acids Res. 29: 1- 10 and 11-16, 2001 for examples of online resources. Similarity searches retrieve and align target sequences for comparison with a sequence to be analyzed (i.e., a query sequence). Sequence comparison algorithms use scoring matrices to assign an overall score to each of the alignments.

[0183] Illustrative examples of suitable programs useful for identifying variants in sequence databases include the BLAST suite of programs (version 2.2.5 [November 2002]) including BLASTN, BLASTP, BLASTX, tBLASTN and tBLASTX, which are publicly available from (ftp colon slash slash file transfer protocol ftp.ncbi.nih.gov / blast / ) or from the National Center for Biotechnology Information (NCBI), National Library of Medicine, Building 38A, Room 8N805, Bethesda, Md. 20894 USA. The NCBI server also provides the facility to use the programs to screen a number of publicly available sequence databases. BLASTN compares a nucleotide query sequence against a nucleotide sequence database. BLASTP compares an amino acid query sequence against a protein sequence database. BLASTX compares a nucleotide query sequence translated in all reading frames against a protein sequence database. tBLASTN compares a protein query sequence against a nucleotide sequence database dynamically translated in all reading frames. tBLASTX compares the six-frame translations of a nucleotide query sequence against the six-frame translations of a nucleotide sequence database. The BLAST programs may be used withdefault parameters or tire parameters may be altered as required to refine the screen.

[0184] The use of the BLAST family of algorithms, Including BLASTN, BLASTP, and BLASTX, is described by Altschul et al. (Nucleic Acids Res. 25: 3389-3402, 1997).

[0185] The "hits" to one or more database sequences by a queried sequence produced by BLASTN, BLASTP, BLASTX, tBLASTN, tBLASTX, or a similar algorithm, align and identify similar portions of sequences. The hits are arranged in order of the degree of similarity and the length of sequence overlap. Hits to a database sequence generally represent an overlap over only a fraction of the sequence length of the queried sequence.

[0186] The BLASTN, BLASTP, BLASTX, tBLASTN and tBLASTX algorithms also produce "Expect" values for alignments. The Expect value (E) Indicates the number of hits one can "expect" to see by chance when searching a database of the same size containing random contiguous sequences. The Expect value is used as a significance threshold for determining whether the hit to a database indicates true similarity. For example, an E value of 0.1 assigned to a polynucleotide hit is interpreted as meaning that in a database of the size of the database screened, one might expect to see 0.1 matches over the aligned portion of the sequence with a similar score simply by chance. For sequences having an E value of 0.01 or less over aligned and matched portions, the probability of finding a match by chance in that database is 1% or less using the BLASTN, BLASTP, BLASTX, tBLASTN or tBLASTX algorithm.

[0187] Multiple sequence alignments of a group of related sequences can be carried out with CLUSTALW (see, e.g., Thompson, J. D., Higgins, D. G. and Gibson, T. J. (1994) CLUSTALW: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and wreight matrix choice. Nucleic Acids Research, 22:4673-4680, world wide web -igbmc.u- strasbg.fr / BioInfo / ClustalW / Top.html) or T-COFFEE (see, e.g., Cedric Notredame, Desmond G. Higgins, Jaap Heringa, T-Coffee: A novel method for fast and accurate multiple sequence alignment, J. Mol. Biol. (2000) 302: 205-217)) or PILEUP, which uses progressive, pairwise alignments (see, e.g., Feng and Doolittle, 1987, J. Mol. Evol. 25, 351).

[0188] Pattern recognition software applications are available for finding motifs or signature sequences. For example, MEME (Multiple Em for Motif Elicitation) finds motifs and signature sequences in a set of sequences, and MAST (Motif Alignment and Search Tool) uses these motifs to identify similar or the same motifs in query sequences. The MASTresults are provided as a series of alignments with appropriate statistical data and a visual overview of the motifs found. MEME and MAST were developed at the University of California, San Diego.

[0189] PROSITE (see, e.g., Bairoch and Bucher, 1994, Nucleic Acids Res. 22, 3583; and Hofmann et al., 1999, Nucleic Acids Res. 27, 215) is a method of identifying the functions of uncharacterized proteins translated from genomic or cDNA sequences. The PROSITE database (world wide web.expasy.org / prosite) contains biologically significant patterns and profiles and is designed so that it can be used with appropriate computational tools to assign a new sequence to a known family of proteins or to determine which known domain(s) are present in the sequence (Falquet et al ., 2002, Nucleic Acids Res. 30, 235). Prosearch is a tool that can search SWISS-PROT and EMBL databases with a given sequence pattern or signature.Methods for Isolating Polypeptides

[0190] The polypeptides of the present disclosure, including variant polypeptides, may be prepared using peptide synthesis methods known to persons skilled in the art, such as direct peptide synthesis using solid phase techniques (see, e.g , Stewart et al., 1969, in Solid-Phase Peptide Synthesis, WH Freeman Co, San Francisco Calif, or automated synthesis, for example using an Applied Biosystems 431 A Peptide Synthesizer (Foster City, Calif.). Mutated forms of the polypeptides may also be produced during such syntheses.

[0191] The polypeptides and variant polypeptides of the present disclosure may also be purified from natural sources using a variety of techniques that are well known in the art (see, e.g., Deutscher, 1990, Ed, Methods in Enzymology, Vol. 182, Guide to Protein Purification).

[0192] Alternatively, the polypeptides and variant polypeptides of the present disclosure may be expressed recombinantly in suitable host cells and separated from the cells as discussed below.Methods for Modifying Sequences

[0193] Suitable methods for modifying the sequence of proteins, or the polynucleotide sequences encoding them, will be known to persons skilled in the art. The sequence of a protein may be conveniently modified by altering / modifying the sequence encoding the protein and expressing the modified protein. Approaches such as site-directed mutagenesis may be applied to modify existing polynucleotide sequences. Alternatively, restrictionendonucleases may be used to excise parts of existing sequences. Altered polynucleotide sequences may also be conveniently synthesized in a modified form.

[0194] In an embodiment, modifying the uORF comprises site-directed nucleases or oligonucleotide-directed mutagenesis. In an embodiment, the mutagenesis is achieved using SDN-1. In an embodiment, said mutagenesis is achieved using Zinc finger nucleases, TALENS and / or CRISPR / Cas gene editing. In an embodiment, said mutagenesis is achieved using CRISPR / Cas9 or CRISPR / Cas 12 gene editing or so called “base editing”.Methods for Producing Constructs and Vectors

[0195] The genetic constructs of the present disclosure comprise one or more polynucleotide sequences of the present disclosure and / or polynucleotides encoding polypeptides of the present disclosure, and may be useful for transforming, for example, bacterial, fungal, insect, mammalian or plant organisms. The genetic constructs of the present disclosure are intended to include expression constructs as herein defined.

[0196] Methods for producing and using genetic constructs and vectors are well known in the art and are described generally in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press, 1987; and in Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing, 1987).Methods for Producing Host Cells Comprising Polynucleotides, Constructs or Vectors

[0197] The present disclosure also provides a host cell which comprises a genetic construct or vector of the present disclosure. Host cells may be derived from, for example, bacterial, fungal, insect, mammalian or plant organisms. In an embodiment, the host cell is a plant cell. Suitable plant cells will be familiar to persons skilled in the art, an illustrative example of which includes cells of a plant selected from the group consisting of plant is elected from the group consisting of rice, barley, wheat, soybean, alfalfa, rye, oats, millet, sorghum, triticale, buckwheat, quinoa, spelt, einkom, amaranth, grass, com, canola, wild rice, teff, banana, cassava, brassica, field mustard, cabbage, Aegilops tauschii, brachypodium, Miscanthus, switchgrass, poplar, pine, Eucalyptus, acacia, birch, hazel, willow, bamboo, grape, kiwifruit, apple, pear, avocado, almond, pistachio, walnut, chestnut, a vine, a forage grass, alfalfa, a plant of the genus Primus, sweet potato, yam and a nightshade family plant. In an embodiment, the plant cell is a rice plant cell. In an embodiment, the plant cell is a wheat plant cell.

[0198] Host cells comprising genetic constructs, such as expression constructs, of theinvention are useful in methods well known in the art (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press, 1987; Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing, 1987) for recombinant production of polypeptides of the invention. Such methods may involve the culture of host cells in an appropriate medium in conditions suitable for or conducive to expression of a polypeptide of the invention. The expressed recombinant polypeptide, which may optionally be secreted into the culture, may then be separated from the medium, host cells or culture medium by methods known to persons skilled in the art (see, e.g., Deutscher, Ed, 1990, Methods in Enzymology, Vol 182, Guide to Protein Purification).Methods for Producing Plant Cells and Plants Comprising Constructs and Vectors

[0199] The present disclosure also extends to plant cells comprising a genetic construct of the invention, and plant cells modified to alter expression of a polynucleotide or polypeptide of the invention. Plants comprising such cells also form an aspect of the invention.

[0200] Methods for transforming plant cells, plants and portions thereof with polypeptides will be known to persons skilled in the art, an illustrative example of which is described in Draper et al., 1988, Plant Genetic Transformation and Gene Expression. A Laboratory Manual. Blackwell Sci. Pub. Oxford, p. 365; Potrykus and Spangenburg, 1995, Gene Transfer to Plants. Springer-Veriag, Berlin.; and Gelvin et al., 1993, Plant Molecular Biol. Manual. Kluwer Acad. Pub. Dordrecht. A review of transgenic plants, including transformation techniques, is provided in Galun and Breiman, 1997, Transgenic Plants. Imperial College Press, London.Methods for Genetic Manipulation of Plants and Plant Cells

[0201] A number of suitable plant transformation strategies are available (see, e.g., Birch, 1997, Ann Rev Plant Phys Plant Mol Biol, 48, 297; Hellens R P, et al (2000) Plant Mol Biol 42: 819-32; Hellens R et al (2005) Plant Meth 1: 13). For example, strategies may be designed to increase expression of a polynucleotide / polypeptide in a plant cell, organ and / or at a particular developmental stage where / when it is normally expressed or to ectopically express a polynucleotide / polypeptide in a cell, tissue, organ and / or at a particular developmental stage which / when it is not normally expressed. The expressed polynucleotide / polypeptide may be derived from the plant species to be transformed or may be derived from a different plant species.

[0202] Transformation strategies may be designed to reduce expression of a polynucleotide / polypeptide in a plant cell, tissue, organ or at a particular developmental stage which / when it is normally expressed. Such strategies are known as gene silencing strategies.

[0203] Genetic constructs for expression of genes in transformed plants typically include promoters for driving the expression of one or more cloned polynucleotide, terminators and selectable marker sequences to detest presence of the genetic construct in the transformed plant.

[0204] The promoters suitable for use in the constructs of this present disclosure are functional in a cell, tissue or organ of a monocot or dicot plant and include cell-, tissue- and organ-specific promoters, cell cycle specific promoters, temporal promoters, Inducible promoters, constitutive promoters that are active in most plant tissues, and recombinant promoters. Choice of promoter will depend upon the temporal and spatial expression of the cloned polynucleotide, so desired. The promoters may be those normally associated with a transgene of interest, or promoters which are derived from genes of other plants, viruses, and plant pathogenic bacteria and fungi. Persons skilled in the art will, without undue experimentation, be able to select promoters that are suitable for use in modifying and modulating plant traits using genetic constructs comprising the polynucleotide sequences of the present disclosure. Examples of constitutive plant promoters include the CaMV 35S promoter, the nopaline synthase promoter and the octopine synthase promoter, and the Ubi 1 promoter from maize. Plant promoters which are active in specific tissues, respond to internal developmental signals or external abiotic or biotic stresses are described in the scientific literature. Exemplary' promoters are described, e.g., in WO 02 / 00894, which is herein incorporated by reference.

[0205] Exemplary terminators that are commonly used in plant transformation genetic construct include, e.g., the cauliflower mosaic vims (CaMV) 35S terminator, the Agrobacterium tumefaciens nopaline synthase or octopine synthase terminators, the Zea mays zein gene terminator, the Oryza saliva ADP-glucose pyrophosphorylase terminator and the Solanum tuberosum PI-II terminator.

[0206] Selectable markers commonly used in plant transformation include the neomycin phophotransferase II gene (NPT II) which confers kanamycin resistance, the aadA gene, which confers spectinomycin and streptomycin resistance, the phosphinothricin acetyl transferase (bar gene) for Ignite (AgrEvo) and Basta (Hoechst) resistance, and thehygromycin phosphotransferase gene (hpt) for hygromycin resistance.

[0207] Use of genetic constructs comprising reporter genes (coding sequences which express an activity that is foreign to the host, usually an enzymatic activity and / or a visible signal, e.g., luciferase (LUC), ^-glucuronidase (GUS_, or green fluorescent protein (GFP) which may be used for promoter expression analysis in plants and plant tissues are also contemplated. The reporter gene literature is reviewed in Herrera-Estrella et al., 1993, Nature 303, 209, and Schrott, 1995, In: Gene Transfer to Plants (Potrykus, T., Spangenberg. Eds) Springer Veriag. Berline, pp. 325-336.

[0208] Gene silencing strategies may be focused on the gene itself or regulatory elements which effect expression of the encoded polypeptide. "Regulatory elements" is used here in the widest possible sense and includes other genes which interact with the gene of interest.

[0209] Genetic constructs designed to decrease or silence the expression of a polynucleotide / polypeptide of the present disclosure may include an antisense copy of a polynucleotide of the present disclosure. In such constructs the polynucleotide is placed in an antisense orientation with respect to the promoter and terminator.

[0210] An "antisense" polynucleotide is obtained by inverting a polynucleotide or a segment of the polynucleotide so that the transcript produced will be complementary to the mRNA transcript of the gene, e.g.,• 5'GATCTA 3' (coding strand); 3'CTAGAT 5' (antisense strand)• 3'CUAGAU 5' (mRNA); 5'GAUCUCG 3' antisense RNA[0211 [ Genetic constructs designed for gene silencing may also include an inverted repeat. An 'Inverted repeat' is a sequence that is repeated where the second half of the repeat is in the complementary strand, e.g.,S'-GATCTA...TAGATC-3'• 3'-CTAGAT...ATCTAG-5'

[0212] The transcript formed may undergo complementary base pairing to form a hairpin structure. Usually, a spacer of at least 3-5 bp between the repeated region is required to allow hairpin formation.

[0213] Another silencing approach involves the use of a small antisense RNA targeted to the transcript equivalent to an miRNA (Llave et al., 2002, Science 297, 2053). Use of suchsmall antisense RNA corresponding to polynucleotide of tire invention is expressly contemplated.

[0214] The term genetic construct as used herein also includes small antisense RNAs and other such polypeptides effecting gene silencing.

[0215] Transformation with an expression construct, as herein defined, may also result in gene silencing through a process known as sense suppression (see, e.g., Napoli et al., 1990, Plant Cell 2, 279; and de Carvalho Niebel et al., 1995, Plant Cell, 7, 347). In some cases, sense suppression may involve over-expression of the whole or a partial coding sequence but may also involve expression of non-coding region of the gene, such as an intron or a 5' or 3' untranslated region (UTR). Chimeric partial sense constructs can be used to coordinately silence multiple genes (see, e.g., Abbott et al., 2002, Plant Physiol. 128(3): 844- 53; and Jones et al., 1998, Planta 204: 499-505). The use of such sense suppression strategies to silence the expression of a polynucleotide of the present disclosure is also contemplated herein.

[0216] The polynucleotide inserts in genetic constructs designed for gene silencing may correspond to coding sequence and / or non-coding sequence, such as promoter and / or intron and / or 5' or 3 -UTR sequence, or the corresponding gene.

[0217] Other gene silencing strategies include dominant negative approaches and the use of ribozyme constructs (see, e.g., McIntyre, 1996, Transgenic Res, 5, 257).

[0218] Pre-transcriptional silencing may be brought about through mutation of the gene itself or its regulatory elements. Such mutations may include point mutations, frameshifts, insertions, deletions and substitutions.

[0219] The following are representative publications disclosing genetic transformation protocols that can be used to genetically transform the following plant species: Rice (Alam et al., 1999, Plant Cell Rep. 18, 572); apple (Yao et al., 1995, Plant Cell Reports 14, 407- 412); maize (U.S. Pat. Nos. 5,177,010 and 5,981,840); wheat (Ortiz et al., 1996, Plant Cell Rep. 15, 1996, 877); tomato (U.S. Pat. No. 5,159,135); potato (Kumar et al., 1996 Plant J. 9, 821); cassava (Li et al., 1996 Nat. Biotechnology 14, 736); lettuce (Michelmore et al., 1987, Plant Cell Rep. 6, 439); tobacco (Horsch et al., 1985, Science 227, 1229); cotton (U.S. Pat. Nos. 5,846,797 and 5,004,863); grasses (U.S. Pat. Nos. 5,187,073 and 6,020,539); peppermint (Niu et al., 1998, Plant Cell Rep. 17, 165); citrus plants (Pena et al., 1995, Plant Sci. 104, 183); caraway (Krens et al., 1997, Plant Cell Rep, 17, 39); banana (U.S. Pat. No.5,792,935); soybean (U.S. Pat. Nos. 5,416,011; 5,569,834; 5,824,877; 5,563,04455 and 5,968,830); pineapple (U.S. Pat. No. 5,952,543); poplar (U.S. Pat. No. 4,795,855); monocots in general (U.S. Pat. Nos. 5,591,616 and 6,037,522); brassica (U.S. Pat. Nos. 5,188,958; 5,463,174 and 5,750,871); cereals (U.S. Pat. No. 6,074,877); pear (Matsuda et al., 2005, Plant Cell Rep. 24(l):45-51); Primus (Ramesh et al., 2006 Plant Cell Rep. 25(8): 821 -8; Song and Sink 2005 Plant Cell Rep. 2006; 25(2): 117-23; Gonzalez Padilla et al., 2003 Plant Cell Rep. 22(l):38-45); strawberry (Oosumi et al., 2006 Planta. 223(6): 1219-30; Folta et al., 2006 Planta April 14; PMID: 16614818), rose (Li et al., 2003), Rubus (Graham et al., 1995 Methods Mol Biol. 1995; 44: 129-33), tomato (Dan et al., 2006, Plant Cell Reports V25:432- 441), apple (Yao et al., 1995, Plant Cell Rep. 14, 407-412) and Actinidia eriantha (Wang et al., 2006, Plant Cell Rep. 25, 5: 425-31). Transformation of other species is also contemplated by the disclosure. Suitable methods and protocols are available in the scientific literature.

[0220] Several further methods known in the art may be employed to alter expression of a nucleotide and / or polypeptide of the disclosure. Such methods include but are not limited to Tilling (Till et al., 2003, Methods Mol Biol, 2%, 205), so called "Deletagene" technology (Li et al., 2001, Plant Journal 27(3), 235) and the use of artificial transcription factors such as synthetic zinc finger transcription factors, (e g., Jouvenot et al., 2003, Gene Therapy 10, 513). Additionally, antibodies or fragments thereof, targeted to a particular polypeptide may also be expressed in plants to modulate the activity of that polypeptide (Jobling et al., 2003, Nat. Biotechnol. 21(1), 35). Transposon tagging approaches may also be applied. Additionally, peptides interacting with a polypeptide of the disclosure may be identified through technologies such as phase-display (Dyax Corporation). Such interacting peptides may be expressed in or applied to a plant to affect activity of a polypeptide of the disclosure. Use of each of the above approaches in alteration of expression of a nucleotide and / or polypeptide of the disclosure is specifically contemplated.Methods for Modifying Endogenous DNA Sequences in Plant

[0221] Methods for modifying endogenous genomic DNA sequences in plants are known to those skilled in the art, illustrative examples of which may involve the use of sequencespecific nucleases that generate targeted double-stranded DNA breaks in genes of interest. Illustrative examples of such methods for use in plants include: zinc finger nucleases (Curtin et al., 2011. Plant Physiol. 156:466-473.; Sander, et al., 2011. Nat. Methods 8:67-69.), transcription activator-like effector nucleases or "TALENs" (Cerrnak et al., 2011, NucleicAcids Res. 39:e82; Mahfouz era / ., 2011 Proc. Natl. Acad. Sci. USA 108:2623-2628; Li et al., 2012 Nat. Biotechnol. 30:390-392), and LAGLIDADG homing endonucleases, also termed "meganucleases" (Tzfira era / ., 2012. Plant Biotechnol. J. 10:373-389).

[0222] In certain embodiments, TALENs or a Zinc finger nucleases can be used to modify one or more base pairs in the uORF in order to, at least partially, disable or render it inoperative, so it is no longer translatable. In an embodiment, the first base pair of the ACG start codon is changed to TCG to accomplish this. Without being bound by theory or by a particular mode of application, it is understood that this would at least partially inactivate the negative regulation of iron deficiency response gene translation and allow' for increased iron uptake, transport, and / or accumulation in the plant.

[0223] Alternatively, a codon for a highly conserved amino acid in the uORF can be changed to stop the uORF from functioning in downregulating translation of the iron deficiency response gene. For example, a His residue in the conserved region of the uORF can be changed to a Leu. In one embodiment, a start codon can be changed into a stop codon. In another embodiment, one or more nucleotides can be inserted, deleted or substituted to disrupt the uORF.

[0224] In a further embodiment, an early base pair in the uORF is altered to introduce a stop codon, and cause early termination of the uORF which stops inhibition of the translation of iron deficiency response gene.

[0225] In an embodiment, modifying the uORF comprises site-directed nucleases or oligonucleotide-directed mutagenesis. In an embodiment, the mutagenesis is achieved using SDN-1. In an embodiment, said mutagenesis is achieved using Zinc finger nucleases, TALENS and / or CRISPR / Cas gene editing. In an embodiment, said mutagenesis is achieved using CRISPR / Cas9 or CRISPR / Cas 12 gene editing or so called “base editing”.

[0226] Persons skilled in the art will thus appreciate that there are numerous ways in which the uORF can be disrupted to remove or at least partially inhibit negative regulation and to increase iron deficiency response gene expression. Any such method is included within the scope of the present disclosure.Plants, plant parts and plant cells

[0227] The present disclosure also extends to a plant cell produced by the methods disclosed herein.

[0228] In another aspect disclosed herein, there is provided a plant cell, including a modified plant cell, comprising a polynucleotide capable of controlling iron homeostasis in the plant cell, wherein the polynucleotide comprises an iron-deficiency response gene, wherein the iron-deficiency response gene comprises a nucleic acid sequence comprising a modification to a uORF of a 5' UTR of the iron deficiency response gene, wherein the modification comprises a deletion, addition, or substitution of at least one nucleotide in the uORF when compared to a corresponding nucleic acid sequence of a wild-type uORF of the same plant species, wherein the modification to the uORF results in an increase in the level of expression of the iron deficiency response gene when compared to the level of expression of the iron deficiency response gene in a control plant cell harbouring a corresponding native iron deficiency response gene of the same plant species.

[0229] In an embodiment, the modified plant cell exhibits a desirable trait selected from the group consisting of increased cellular iron content, increased iron uptake, dark coloration, increased photosynthesis, improved iron homeostasis, increased SPAD, increased chlorophyll, increased vigour, increased growth, increased iron transport, increased tolerance to an abiotic stress and increased tolerance to a pathogen, for example, when compared to an unmodified plant of the same species or when compared to a plant of the same plant species harbouring the corresponding wild-type uORF. In an embodiment, the plant comprising a modified plant cell as disclosed herein, exhibits a desirable trait selected from the group consisting of increased cellular iron content, increased iron uptake, dark coloration, increased photosynthesis, improved iron homeostasis, increased SPAD (an indirect measure of chlorophyll content), increased vigour, increased iron transport, increased tolerance to an abiotic stress and increased tolerance to a pathogen. In an embodiment, the plant comprising a modified plant cell as disclosed herein, exhibits increased tolerance to iron deficiency / iron-deficient growing conditions when compared to a plant of the same plant species harbouring the corresponding wild-type uORF.

[0230] In yet another aspect disclosed herein, there is provided a plant cell comprising an iron-deficiency response gene having a modified uORF wrhen compared to a corresponding native uORF of the same plant species, wherein the modified uORF comprises a deletion, substitution or insertion of at least one nucleotide when compared to the corresponding wildtype uORF of the same plant species, and wherein said modification enhances expression of the iron-deficiency response gene in the plant cell when compared to a plant cell of the same plant species harbouring the corresponding wild-type uORF of the same plant species.

[0231] In an embodiment, the iron-deficiency response gene is selected from the group consisting of IDEF1, IDEF2 and orthologs of any of the foregoing. In an embodiment, the iron-deficiency response gene is IDEF1 or an ortholog thereof. In an embodiment, the iron- deficiency response gene comprises a nucleic acid sequence encoding a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-8 and amino acid sequences having at least 70% sequence identity to any of the foregoing.

[0232] In an embodiment, the iron-deficiency response gene is IDEF2 or an ortholog thereof. In an embodiment, the iron-deficiency response gene comprises a nucleic acid sequence encoding a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 9-16 and amino acid sequences having at least 70% sequence identity to any of the foregoing.

[0233] In an embodiment, the uORF has been modified by replacing at least one start codon of the corresponding wild-type uORF of the same plant species with a stop codon. In an embodiment, the uORF has been modified by deleting at least one base pair of the corresponding wild-type uORF of the same plant species. In an embodiment, the modified uORF comprises a deletion of at least 2 base pairs of the corresponding wild-type uORF of the same plant species. In an embodiment, the modified uORF comprises a deletion of at least a start codon of the corresponding wild-type uORF of the same plant species. In an embodiment, the entire uORF of the corresponding wild-type uORF of the same plant species has been deleted. In an embodiment, the uORF has been modified by inserting at least one base pair into the corresponding wild-type uORF of the same plant species. In an embodiment, the uORF has been modified by substituting at least one base pair into the corresponding wild-type uORF of the same plant species. In another embodiment, the substitution of the at least one base pair into the corresponding wild-type uORF of the same plant species results in a non-synonomous amino acid change or a nonsense mutation. In an embodiment, the modified uORF results in enhancing the expression of the gene of interest by at least about 10% when compared to a level of expression of the gene of interest in a plant cell harbouring a corresponding wild-type uORF of the same plant species.

[0234] The present disclosure also extends to plants comprising the plant cells, the polynucleotides, the polynucleotide constructs, or the expression vectors disclosed herein.

[0235] The present disclosure also extends to plants, plant cells, transgenic plants or transgenic plant cells comprising an iron-deficiency response gene having a modified uORF in its 5'UTR, as disclosed herein. The uORF modification may comprises a deletion,addition, or substitution of at least one nucleotide in the uORF when compared to a corresponding nucleic acid sequence of a wild-type uORF of the same plant species, wherein the modification to the uORF results in an increase in the level of expression of the iron deficiency response gene when compared to the level of expression of tire iron deficiency response gene in a control plant cell harbouring a corresponding native iron deficiency response gene of the same plant species. The transgenic plant or plant cells may comprise the polynucleotides disclosed herein.

[0236] In one embodiment, the plant, plant cell, transgenic plant or transgenic plant cell is a monocot plant.

[0237] In an embodiment, the plant is selected from the group consisting of rice, barley, wheat, soybean, alfalfa, rye, oats, millet, sorghum, triticale, buckwheat, quinoa, spelt, einkom, amaranth, grass, com, canola, wild rice, teff, banana, cassava, brassica, field mustard, cabbage, Aegilops tauschii, brachypodium, Miscanthus, switchgrass, poplar, pine, Eucalyptus, acacia, birch, hazel, willow, bamboo, grape, kiwifruit, apple, pear, avocado, almond, pistaccio, walnut, chestnut, a vine, a forage grass, alfalfa, a plant of the genus Prunus, a leguminous plant, sweet potato, yam and a plant of the nightshade family.

[0238] In an embodiment, the plant is a rice or wheat plant.

[0239] The present disclosure also extends to seeds produced by the plants disclosed herein, to plants derived flora such seeds, and to plant parts derived from the plants disclosed herein.

[0240] In an embodiment, the plant part is selected from the group consisting of a seed, grain, fruit, leaf, flower, tuber, stalk, rhizome, spore, cutting, nut, and root. In an embodiment, the plant part is grain. In an embodiment, the grain is selected from the group consisting of wheat grain, rye grain, barley grain or oat grain. In an embodiment, the grain is rice.

[0241] The present disclosure also extends to flour produced from the grain disclosed herein. The present disclosure also extends to food products comprising the plants, the seeds, the plant parts, the grains or the flour disclosed herein.

[0242] The term "plant" is intended to include a whole plant, any part of a plant, propagules and progeny of a plant. The term “propagule” means any part of a plant that may be used in reproduction or propagation, either sexual or asexual, including seeds and cuttings.

[0243] The plants of the disclosure may be grown and either self-ed or crossed with a different plant strain and the resulting off-spring from two or more generations also form an aspect of the present disclosure, provided they maintain the transgene or modification of the invention.

[0244] The present disclosure may also be said broadly to consist in the parts, elements and features referred to or indicated in the specification of the application, Individually or collectively, and any or all combinations of any two or more said parts, elements or features, and where specific integers are mentioned herein which have known equivalents in the art to which this disclosure relates, such known equivalents are deemed to be incorporated herein as if individually set forth.

[0245] In an embodiment, the plant is elected from the group consisting of rice, barley, wheat, soybean, alfalfa, rye, oats, millet, sorghum, triticale, buckwheat, quinoa, spelt, einkom, amaranth, grass, com, canola, wild rice, teff, banana, cassava, brassica, field mustard, cabbage, Aegilops tauschii, brachypodium, Miscanthus, switchgrass, poplar, pine, Eucalyptus, acacia, birch, hazel, willow, bamboo, grape, kiwifruit, apple, pear, avocado, almond, pistachio, walnut, chestnut, a vine, a forage grass, alfalfa, a plant of the genus Primus, sweet potato, yam, a leguminous plant and a plant of the nightshade family. In an embodiment, the plant is a rice plant or a wheat plant.

[0246] The term "plant", as used herein, is intended to include a whole plant, any part of a plant, shoot vegetative organs / structures (e.g., leaves, stems and tubers), roots, flowers and floral organs / structures (e.g., bracts, sepals, petals, stamens, carpels, anthers and ovules), seed (including embryo, endosperm, and seed coat) and fruit (the mature ovary), plant tissue (e.g., vascular tissue, ground tissue, and the like) and cells (e g., guard cells, egg cells, and the like), and propagules and progeny of a plant. The term “propagule” means any part of a plant that may be used in reproduction or propagation, either sexual or asexual, including seeds and cuttings. In one embodiment, the plant is an embryo, a seed, a seedling, a juvenile plant or a mature plant. In another embodiment, the plant is a plant derived from a seed of a plant generated by the methods disclosed herein, or a plant propagated from the plant tissue of a plant generated by the methods disclosed herein. The class of plants that can be used in the method of the present disclosure is generally as broad as the class of higher and lower plants amenable to transformation techniques, including angiosperms (monocotyledonous and dicotyledonous plants), gymnosperms, fems, horsetails, psilophytes, lycophytes, bryophytes, and multicellular algae. See for example, Daly et al. (2001) Plant Physiol. 127:1328-1333; Ku et al. (2000) Proc. Natl. Acad. Sci. 97: 9121-9126; and see also Tudge, in The Variety' of Life, Oxford University' Press, New York, NY (2000) pp. 547-606.

[0247] The disclosure as disclosed here in extends to plant parts derived from the plants generated by the methods disclosed herein. In an embodiment, the plant part is a seed, grain, fruit, leaf, flower, tuber, stalk, rhizome, spore, cutting, nut, or root produced by the plant generated by the methods disclosed herein.

[0248] The class of plants that can be used in the method of the present disclosure is generally as broad as the class of higher and lower plants amenable to transformation techniques, including angiosperms (monocotyledonous and dicotyledonous plants), gymnosperms, fems, horsetails, psilophytes, lycophytes, bryophytes, and multicellular algae. See for example, Daly et al. (2001) Plant Physiol. 127: 1328-1333; Ku et al. (2000) Proc. Natl. Acad. Sci. 97: 9121-9126; and see also Tudge, in The Variety of Life, Oxford University Press, New York, NY (2000) pp. 547-606.

[0249] In one embodiment, the plant is rice, barley (Hordeum vulgare), wheat (Triticum aestivum), soybean, alfalfa, rye (Secale cereal), oats (Avena saliva), millet, sorghum, triticale, buckwheat, quinoa, einkom, amaranth, spelt (Triticum spelta), grass, com, canola or wild rice, teff (Eragrostis tef), banana, cassava, brassica, field mustard, cabbage, Aegilops tauschii, brachypodium, Miscanthus, switchgrass, poplar, pine, Eucalyptus, acacia, birch, hazel, willow, bamboo, grape, kiwifruit, apple, pear, avocado, almond, pistaccio, walnut, chestnut, a vine, a forage grass, alfalfa, a plant of the genus Primus, a legume, a brassica plant, or a nightshade family plant. Examples of brassica plants include bok choy, broccoli, cauliflower, cabbage, choy sum, kohlrabi, napa cabbage, rutabaga and turnips. Some examples of nightshade plants include tomatoes, potatoes, eggplant, bell and chili peppers.

[0250] In a preferred embodiment, the plant is a rice plant. In another preferred embodiment, the plant is a Pooideae or cereal grass plant. In another preferred embodiments, the plant is a wheat, barley, oat, rye or spelt plant.

[0251] The plants of the present disclosure may be grown and either selfed or crossed with a different plant strain. The resulting offspring from two or more generations also form an aspect of the present disclosure, preferably where such generations retain the modification to the uORF, as described elsewhere herein.

[0252] A “trait” refers to a physiological, morphological, biochemical, or physical characteristic of a cell or organism, including of a plant or of a particular plant material orof a plant cell . In some instances, this characteristic is visible to the human eye, such as seed or plant size, or pigmentation, or can be measured by biochemical techniques, such as detecting the protein, starch, or oil content of seed or leaves, or by observation of a metabolic or physiological process, e.g., by measuring uptake of carbon dioxide, or by the observation of the expression level of a gene or genes, e.g., by employing Northern analysis, RT-PCR, microarray gene expression assays, RNA Seq or reporter gene expression systems, or by agricultural observations such as stress tolerance or pathogen tolerance. Any technique can be used to measure the amount of, comparative level of, or difference in any selected chemical compound or macromolecule in the transformed plants, however.

[0253] ‘Trait modification” refers to a detectable difference in a characteristic in a plant ectopically expressing a polynucleotide or polypeptide of the present disclosure relative to a plant not doing so, such as a wild-type plant. In some cases, the trait modification can be evaluated quantitatively. For example, the trait modification can entail at least about a 2% increase or decrease in an observed trait (difference), at least a 5% difference, at least about a 10% difference, at least about a 20% difference, at least about a 30%, at least about a 50%, at least about a 70%, at least about an 85%, or about a 100%, or an even greater difference compared with a wild-type plant. It is known that there can be a natural variation in the modified trait. Therefore, the trait modification observed entails a change of the normal distribution of the trait in the plants compared with the distribution observed in wild-type plant.A “crop” plant includes cultivated plants or agricultural produce, and may be a grain, vegetables, or fruit plant, generally considered as a group. A crop plant may be grown in commercially usefill numbers or amounts. In a crop plant a desirable “trait” may include, for example, one or more of the following, as compared to a control plant: increased vigor, increased “yield”, darker coloration, increased organ size, improved root growth, increased photosynthesis, increased SPAD, increased iron transport, increased cellular and / or or tissue iron content, increased iron uptake, improved water use efficiency, improved pathogen tolerance, and improved abiotic stress tolerance (where abiotic stress may include, for example, drought, heat, cold, freezing, salinity, the presence of heavy metal ions, or low nutrient conditions). In an embodiment, the desirable trait is increased tolerance for iron- deficiency or iron-deficient growing conditions. The term “yield” typically refers to the harvestable portion of the plant such as grain, fruit, biomass or an edible vegetative part such as a stem, leaf or tuber.

[0254] In one embodiment, the desirable trait is increased cellular and / or or tissue ironcontent. In one embodiment, the plant or plant cell comprising an iron-deficiency response gene having a modified uORF, wherein the modified uORF comprises a deletion, substitution or insertion of at least one nucleotide when compared to the corresponding wildtype uORF of the same plant species, the modification enhances the iron content in the plant or plant cell, by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 100%, when compared to a plant or plant cell comprising the corresponding native uORF of the same plant species, when grown or cultured under the same conditions.

[0255] In one embodiment, the selected plant has an iron content of about 20μg-100μg of iron per gram of leaf tissue (dry weight) and / or 50μg-200μg of iron per gram of root tissue (dry' weight). In another embodiment, the selected plant has at least 20μg, at least 30μg, at least 40μg or at least 50μg of iron per gram of leaf tissue (dry weight). In another embodiment, the selected plant has at least 50μg, at least 60μg, at least 70μg, at least 80μg, at least 90μg, at least lOOμg, at least 120μg, at least 130μg or at least 150μg of iron per gram of leaf tissue (dry weight).

[0256] In one embodiment, the plant tissue is leaf tissue, stem tissue, root tissue, seed tissue or fruit tissue.

[0257] In one embodiment, the iron-deficiency response gene having a modified uORF is IDEF1. In one embodiment, the iron-deficiency response gene having a modified uORF is IDEF2.

[0258] The term “recombinant”, as used herein, refer to a biomolecule, e.g., a gene or protein, or to a cell or microorganism. The term “recombinant” may be used in reference to cloned DNA isolates, chemically synthesized polynucleotides, or polynucleotides that are biologically synthesized by heterologous systems, as well as proteins or polypeptides encoded by such nucleic acids, e.g. enzymes. A “recombinant” nucleic acid is a nucleic acid linked to a nucleotide or polynucleotide to which it is not linked in nature. For example, the recombinant polynucleotide may be in the form of an expression vector. As use herein, a “recombinant protein” refers to a protein that is expressed from an exogenously introduced nucleic acid. The amino acid sequence of the recombinant protein may be identical to its corresponding native protein, or it may contain amino acid sequence modifications / substitutions / mutations. As use herein, a “recombinant cell” refers to a cell that has introduced into it exogenous nucleic acid, typically exogenous DNA, such as a vector orother polynucleotides. The term includes the progeny of the original cell into which the exogenous DNA has been introduced. Thus, a “recombinant cell" as used herein generally refers to a cell that has been transformed, transfected or transduced with exogenous DNA. The host cell may be transformed, transfected or transduced in a transient or stable manner. The exogenous nucleic acid is typically introduced into a host cell so that it is maintained as a chromosomal integrant or as a self-replicating extra-chromosomal vector. The term “recombinant cell” encompasses any progeny of a parent host cell that is not identical to the parent host cell due to the alterations introduced.

[0259] The term “wild-type” is used herein to denote an organism, gene, or gene product, or the expression pattern or expression level of the gene or gene product in a non-modified organism; that is, as it appears in nature, or that which is most frequently observed in a population and is thus arbitrarily designed the “normal” or “wild-type” form. In regard to the term “variants” and “derivatives”, these terms are taken to refer to a biological equivalent of the sequence from which it was derived.

[0260] The present invention is further described by reference to the following nonlimiting examples.EXAMPLESExample 1. OsFEl and OsFE2 in rice (Oryza sativa L.) contain translationally repressive uORFs

[0261] The development of ribosomal profiling, which allows the detection of cryptic translation events, has been particularly useful for uORF identification. But predicting the exact start site of uORFs from ribo-seq datasets is challenging particularly as uORFs can contain non-canonical start codons which could be present in any frame. The three- nucleotide periodicity of elongating ribosomes can theoretically be leveraged to determine the exact P-site of the SOS ribosome and thus the translation frame and translated peptide, however single nucleotide resolution ribo-seq datasets are difficult to produce. Here, ribosomal profiling was used to estimate the approximate location of uORFs followed by sequence and conservation analysis of all possible ORFs with non-canonical start sites (i.e., stop codon to stop codon)

[0262] The ribosomal profiles of iron homeostasis genes and their splice variants were analysed, and evidence of translation events upstream of the mAUG was observed in 9genes: OsHRZl, OsHRZ2, OsIDEFl, OsIDEF2, OsIMAl, OsIRO2, OsPRI2, OsYSL16, and OsGGP. Ofthe nine genes that exhibited ribosomal peaks within the 5’ LS, six (OslMA, OsIRO2, OsNAS3, OsPRI2, OsIDEFl, and OsIDEF2) contained uORF start sites or conserved ORFs in the 5’ LS that were testable using a dual luciferase assay (DLA).

[0263] To investigate if these identified genes were subject to uORF control, wild-type (WT) and mutated leader sequences (LS) were fused with the firefly luciferase coding sequence. Those fusion constructs were then transiently expressed in Nicotiana benthamiana leaves, according to the method of Hellens et al., 2005 (Plant Methods 1, 13.), as described below.1. Vector construction

[0264] The pGreen II 0800-LUC modified to have a CaMV 2x35S promoter driving the firefly luciferase coding sequence and CaMV 35S promoter driving renilla luciferase (as an internal control) was provided by Dr. Ronan Broad at the John Innes Centre (Hellens et al., 2005). LSs were PCR amplified from synthesised sequences and cloned downstream of the CaMV 2x35S promoter and upstream of the firefly luciferase coding sequence using Ncol and Sall restriction enzymes. Ligated plasmids were grown on selection media containing kanamycin (50 μg / mL) and validated using a diagnostic restriction digest (Kpnl and ApaLI) and sanger sequencing (primer sequence: GCCTTATGCAGTTGCTCTCCA) (SEQ ID NO:65). Mutated LSs were either synthesised (Twist Bioscience) or mutated from WT sequences using the Q5® Site-Directed Mutagenesis Kit (NEB).2. Agroinfiltration

[0265] The reporter plasmids were co-transformed (electroporation) into agrobacterium (GV3101) with the helper plasmid pSOUP (Hellens et al., 2005). Transformants were grown on selection media containing kanamycin (50 μg / mL), rifampicin (50 μg / mL) and tetracycline (10 μg / mL) at 28°C. Single colonies were grown in liquid media overnight, washed in agroinfiltration solution (10 mM MgCh and 10 mM MES) and resuspended in agroinfiltration solution containing acetosyringone (200 μM). The cultures were left to incubate (gentle shaking) at room temperature in the dark for at least two hours and then normalised to 0.2 OD600in a 10 mL volume. Each construct was infiltrated into the underside of six or eleven leaves of 4-week-old Nicotiana benthamiana plants (three leaves per plant) with four infiltration sites per leaf. Plants were grown in grown cabinets at 22°C with a 16- hour light period.3. Leaf disc analysis

[0266] After five days of growth, nine of the eleven leaves were chosen for leaf disc analysis (n=9). Leaf discs (5 mm in diameter) were taken from areas adjacent to three of the four infiltration sites (representing three technical replicates). Leaf discs were placed in 100 μL of PBS buffer within a 96-well flat-bottom plate and ground using the Geno / Grinder® (SPEX SamplePrep). Background luminosity was measured using the GloMax Multi Detection System (Promega). Luminosity was measured using the DualGlo® Luciferase Assay System (Promega). For firefly luminosity, 75 μL of DualGlo® Luciferase Reagent was added to the wells and incubated (gentle shaking) at room temperature for 10 minutes, then luminosity was measured using the plate reader. For renilla luminosity, 75 μL of DualGlo® Stop & Gio® Reagent was added to the wells and incubated (gentle shaking) at room temperature for 10 minutes, then luminosity was measured using the plate reader. Background measurements were subtracted from firefly and renilla readings. The three technical replicates were averaged to form one biological replicate and firefly luminosity values were divided by renilla to get the final luminosity ratios.4. Results

[0267] For the mutated (m) OsIMAl ml, OsIRO2 ml, OsNAS3 ml, and OsPRI2 ml sequences, the putative uORF start site was determined by the psORF database and then mutated to a stop codon (TAG, TGA, or TAA). These mutated 5’ LS did not show statistically significant differences in mORF translation efficiency relative to WT sequences, indicating that no translationally repressive uORF is present at the selected non-canonical start site (Figure 1).

[0268] However, when a single non-canonical start codon predicted by psORF was mutated in OsIDEFl uORF (OsIDEFl ml) sequence was a mutated, no statistically significant differences in mORF translation efficiency relative to WT sequence was observed.

[0269] However, when all four canonical start codons (ATGs) within the 5’ LS were mutated into stop codons (OsIDEFl m2), translation of the mORF increases by 2.18-fold (p=0.0031) (Figure la).

[0270] A region of high conservation was detected within the 5 ’ LS of OsIDEFl orthologs. Within the conserved region there is an ATG and a non-canonical start codon (CTG) predicted by psORF (an online database that predicts the start sites of uORFs;http: / / psorf. whu.edu.en / # / ; see also Chen et al. 2020 Plant Biotechnol 1, 18:2158-60) which were both mutated into stop codons (OsIDEF2 ml), leading to a 1.99-fold increase in mORF translation (p=0.0007) (see Figure la).Example 2: OsIDEFl contains a non-conserved uORF

[0271] To identify which ATGs are the start site of a translationally repressive uORF within the OsIDEFl 5 ’ LS we created a series of OsIDEFl 5 ’ LS with different combinations of mutations. As shown in Figure 2, modification of OsFel LSs resulted in statistically different luminosity when compared to WT LS comprising constructs. The mutant (ml-m8) constructs containing stop codons instead of their putative uORF start sites. The OsFEl m2 LS (wherein 4 predicted start codons were mutated into stop codons) had about a two-fold higher luminosity (firefly / renilla) compared to the OsFEl WT LS (p <0.001). The OsFEl m3 also had provided a significantly increased luminosity (about a 1 .85 fold increase; p- value=0.001). Mutating the first two ATGs into stop codons (OsIDEFl m3) resulted in a 1.85-fold derepression in mORF expression relative to the WT sequence (padjusted=<0.001). Interestingly, mutating the second ATG alone (OsIDEFl m6) resulted in a 1.49-fold derepression in mORF expression relative to the WT sequence (padjusted=0.059).

[0272] OsFEl ml, m4, m7 and m8 constructs provided marginal increase in luminosity.

[0273] Statistical significance was calculated using by a Student’s Two-tailed T-test assuming unequal variance (between two 5’ LS) or a one-way ANOVA with Tukey’s HSD test of multiple comparisons (amongst more than two 5’ LS). All luminosity values were normalised against the 35 S construct.Table 1: Non-limiting examples of IDEF1 orthologsOrthologous species Gene ID of IDEF1 orthologOryza sativa (rice) L0c_0s08g01090 / 0s08g0101000Aegilops tauschii (tausch’sAET7Gv20819000 goalgrass)Triticum spelta (spelt)TraesTSP7B01G246500 subgenome B*Triticum spelta (spelt)TraesTSP7D0IG359I00 subgenome D*Eragrostis tef(tefi)* Et_s5804-1.35-l.pathlSorghum bicolor (sorghum) SORBI 3007G000400Zea mays (maize) Zm00001ebl98710 T002Brachypodium distachyonBRADI_3gl2910v3 (brachypodium)Avena sativa (oats) AVESA.0000Ib.r3.7Ag0002777Hordeum vulgare (bailey) HORVU.MOREX.r3.7HG0708130Secale cereale (rye)* SECCE7RvlG0494460Triticum aestivum (bread wheat)TraesCS7A02G329600 subgenome ATriticum spelta (spelt)TraesTSP7A01G356200 subgenome A*Triticum aestivum (bread wheat)TraesCS7B02G230100 subgenome BTriticum aestivum (bread wheat)TraesCS7D02G326300 subgenome DExample 3. Elucidation of the Control of TaIDEF2 in bread wheat (Triticum aestivum L.)

[0274] The uORF-containing region in the IDEF2 is highly conserved amongst monocots (Figure 3a) but not eudicots. The uORF-containing region in monocots comprise the amino acid sequence of SEQ ID NO: 53; MSX1EX2X3X4X5X6PYX7X8X9CYFWFIYCIKFRX10FT, wherein Xi is A, F, or L, X2 is V or L, X3 is A, S, T, or R, X4 is G or E, X5 is G or E, Xe is H, S, or Y, X7is F or L, Xg is L orH, X9 is S or T and Xio is D or E.

[0275] To assess if the IDEF2 uORF repressive function was conserved in other monocots, the leader sequence in IDEF2 in breadwheat was investigated. Wheat IDEF2 LS of A, B andD subgenomes appear to comprise a highly conserved uORF (see SEQ ID NO:26-28 of Table 3, see also Figure 3a), which when translated, would comprise the amino acid sequence of SEQ ID NO: 50; MSFEVSEGSPYFX1SCYFWFIYCIKFRX2F, wherein Xi is H or L and X2 is E or D.

[0276] To investigate if the bread wheat TaIDEF2 gene is subject to uORF control, wildtype (WT) and mutated leader sequences (LS) of TaIDEF2-Al with the firefly luciferase coding sequence and expressed the construct transiently in Nicotiana benthamiana leaves according to the method of Hellens et al, 2005 (Plant Methods 1, 13.), as described below.Vector construction

[0277] The pGreen II 0800-LUC modified to have a CaMV 2x35S promoter driving the firefly luciferase coding sequence and CaMV 35S promoter driving renilla luciferase (as an internal control) was provided by Dr. Ronan Broad at the John Innes Centre (Hellens et al, 2005). LSs o£TaIDEF2 from subgenome A were PCR amplified from synthesised sequences and cloned downstream of the CaMV 2x35S promoter and upstream of the firefly luciferase coding sequence using Ncol and Sall restriction enzymes. Ligated plasmids were grown on selection media containing kanamycin (50 μg / mL) and validated using a diagnostic restriction digest (Kpnl and ApaLI) and sanger sequencing (primer sequence: GCCTTATGCAGTTGCTCTCCA) (SEQ ID NO: 65). Mutated LSs were either synthesised (Twist Bioscience) or mutated from WT sequences using the Q5® Site-Directed Mutagenesis Kit (NEB).2. Agroinfiltration

[0278] The reporter plasmids were co-transformed (electroporation) into agrobacterium (GV3101) with the helper plasmid pSOUP (Hellens et al., 2005). Transformants were grown on selection media containing kanamycin (50 μg / mL), rifampicin (50 μg / mL) and tetracycline (10 μg / mL) at 28°C. Single colonies were grown in liquid media overnight, washed in agroinfiltration solution (10 mM MgC12 and 10 mM MES) and resuspended in agroinfiltration solution containing acetosyringone (200 μM). The cultures were left to incubate (gentle shaking) at room temperature in the dark for at least two hours and then normalised to 0.2 OD600 in a 10 mL volume. Each construct was infiltrated into the underside of six or eleven leaves of 4-week-old Nicotiana benthamiana plants (three leaves per plant) with four infiltration sites per leaf. Plants were grown in growth cabinets at 22°Cwith a 16-hour light period.3. Leaf disc analysis

[0279] After five days of growth, four of the six leaves were chosen for leaf disc analysis (n=5). Leaf discs (5 mm in diameter) were taken from areas adjacent to three of the four infiltration sites (representing three technical replicates). Leaf discs were placed in 100 μL of PBS buffer within a 96-well flat-bottom plate and ground using the Geno / Grinder® (SPEX Sample Prep). Background luminosity was measured using a Cytation 5 Cell Imaging Multimode Reader (BioTek). Luminosity was measured using the DualGio® Luciferase Assay System (Promega). For firefly luminosity, 75 μL of DualGio® Luciferase Reagent was added to the wells and incubated (gentle shaking) at room temperature for 10 minutes, then luminosity was measured using the plate reader. For renilla luminosity, 75 μL of DualGlo® Stop & Gio® Reagent was added to the wells and incubated (gentle shaking) at room temperature for 10 minutes, then luminosity was measured using the plate reader. Background measurements were subtracted from firefly and renilla readings. The three technical replicates were averaged to form one biological replicate and firefly luminosity values were divided by renilla to get the final luminosity ratios.4. Results'.

[0280] The TaIDEF2-Al 5’ LS contains two exons (El and E2), and the uORF-containing region was located nearthe 5’ end of E2.

[0281] As shown in Figure 3b, modified TaIDEF2 LS that did not contain the predicted uORF containing region resulted in a large derepression of the mORF relative to WT sequence: 4.16-fold for TaIDEF2 El LS (deletion of the uORF-containing region and E2) (padjusted=<0.001), 4.78-fold for TaIDEF2 m3 (deletion of the uORF-containing region) (padjusted=<0.001), and 4.41-fold for TaIDEF2 m4 (deletion of the uORF-containing region and El) (padjustcd=<0.001), indicating one or several translationally repressive uORFs were severely disrupted.

[0282] Smaller mutations in TalDEF2 ml (1 bp deletion in the uORF region) and TaIDEF2 m2 (ATG deletion in the uORF region) resulted in a 2.06-fold (padjusted=0.514) and 2.79-fold (padjusted=0.058) derepression of the mORF relative to the WT sequence, respectively.

[0283] The predicted peptide from the uORF in the TaIDEF2 m 1 sequence was 7 aa shorterand had different peptide composition relative to the WT sequence, suggesting that peptide length and sequence is important for translational repression.

[0284] The derepression of the TaIDEF2 m3 sequence was 1.71-fold higher than the TaIDEF2 m2 sequence (padjusted=0.028), suggesting that other ribosomal binding sites or multiple uORFs may be present in the 5’ LS.Table 2: Non-limiting examples of IDEF2 orthologsOrthologous species Gene ID of IDEF2 orthologBrassica juncea (Chinese mustard) BjuA06gl5940SSolanum lycopersicum (tomato) Solyc02g036430Manihot esculenta (cassava) MANES 13G083400Example 4: TaIDEF2-uORF mutations generated in bread wheat CRISPR-Cas9 gene editing

[0285] A CRISPR-Cas9 construct containing two gRNA sequences targeting the 5’ end of the highly conserved uORF region in the three bread wheat (Triticum aestivum L.) homologs: TaIDEF2-Al / TaFE2-Al, TaIDEF2-Bl / TaFE2-Bl, and TaIDEF2-Dl / TaFE2-Dl (Figure 5) was constructed. The construct contained the GRF4-GIF1 chimeric protein to enhance bread wheat regeneration efficiency from tissue culture (Figure 5a). The CRISPR- Cas9 construct was transformed into Agrobacterium tumefaciens and cocultured with immature wheat embryos (cv. Fielder). Following callus induction, T-DNA-containing To plantlets was regenerated using hygromycin to select for presence of the hygromycin phosphotransferase II (HPTII) gene. Low copy number (1-6) To plants were selected forgrowth to maturity to allow easy segregation of the T-DNA in subsequent generations. 490 Ti grains were germinated; and amplified and sequenced 691 TaFE2-Al, TaFE2-Bl or TaFE2-Dl-uOKF regions within the 490 plant samples to identify mutations induced by the gRNA / Cas9 complex.

[0286] We identified two plants with stable heterozygous mutations (tafe2-1 and tafe2-2) and four plants with chimeric mutations that were not maintained until maturity or in the next generation (Figure 5b). The tafe2-1 mutant contained a single nucleotide insertion in the TalDEF2-D1-uORF, which is predicted to extend the uORF length with novel amino acids (Figure 5c). The tafe2-2 mutant contained a novel 23-nucleotide sequence that replaced a 26-nucleotide wild-type sequence in the TaIDEF2-X 1 -uORF. The tafe2-2 mutant sequence is predicted to cause a premature stop codon in the TalDEF2-uORF. The sequences of tafe2- 1 and tafe2-2 mutant are provided in Figure 9.

[0287] Individual isolated Ti heterozygous mutants and wild-type plants were grown to maturity in a John Innes Centre glasshouse. Peris'' staining was conducted on the flag leaves and T2grain. The heterozygous tafe2-1 and tafe2-2 plants grew similarly to wild-type plants grown under the same conditions. Peris’ Prussian Blue staining revealed no difference in Fe content in the mature flag leaves of heterozygous tafe 2-1 or tafe2-2 plants compared to wildtype leaves when grown under the same conditions. No significant difference in grain iron content was detected using Peris’ Prussian Blue staining.Example 5: Mutations in the TaIDEF2-uORF conferred tolerance to iron deficiency

[0288] A hydroponic growth experiment was performed using at The University of Melbourne using T2 grain shipped from the John Innes Centre (UK). Following germination, the zygosity (homozygous mutant, heterozygous, or homozygous wild-type) of each plant was determined, and each genotype was grown in separate boxes. The plants were grown under Fe sufficient hydroponic conditions for 12 days, and then Fe deficient hydroponic conditions for 11 days.

[0289] The heterozygous and homozygous tafe2-1 and tafe2-2 mutants grew similarly to their respective wild-type segregants under the iron-sufficient treatment (Figure 6a-b).

[0290] Under iron-deficiency, plant height remained similar for all genotypes apart from the homozygous tafe2-1 mutant, which continued to grow throughout the entire Fe deficiency period (Figure 6c). The SPAD (indirect measurement of chlorophyl content) of leaves 3 and 4 began to diverge following 6 days of Fe deficiency, with the homozygoustafe2-1 and tafe2-2 mutants having the highest SPAD values (Figure 6d-e). At day 7 of Fe deficiency, the wild-type plants had visible chlorosis in the leaves, whereas the homozygous tafe2-1 and tafe2-2 mutants showed no visible chlorosis.

[0291] At 11 days of Fe deficiency, destructive measurements of all hydroponically grown plants were taken. The homozygous tafe2-1 mutant had a 4.59-fold higher chlorophyl content in the 4thleaf compared to the wild-type segregant (padjusted<0.001) (Figure 7a). The homozygous tafe2-2 mutant had a 4.89-fold higher chlorophyl content in the 4thleaf compared to the wild-type segregant (padjusted=0.012). The homozygous tafe2-1 mutant had a 2.49-fold higher SPAD value in the 5thleaf compared to the wild-type segregant (padjusted=0.004) (Fig. 7b). The homozygous tafe2-2 mutant had a 7.83-fold higher SPAD value in the 5thleaf compared to the wild-type segregant (padjusted<0.001). The homozygous tafe2-1 mutant had a 1.53-fold higher shoot biomass compared to the wild-type segregant (padjusted=0.003), but no statistically significant difference for root biomass.

[0292] The tafe2-2 mutant had no statistically significant difference in shoot or root biomass compared to the wild-type segregant. Other measurements at 11 days of Fe deficiency showed no major growth differences between the tafe2 mutants and their respective wild-type segregants. Peris’ Prussian Blue staining on the 1stleaf following 1 day of Fe deficiency revealed no dramatic differences in Fe content of homozygous and heterozygous tafe2-1 or tafe2-2 mutants compared to their respective wild-type segregants.

[0293] The data suggests that any disruption to the TaFE2-uORF peptide (such as the insertion of novel amino acids and a delayed stop codon ( TaFE2-1), or a premature stop codon ( tafe2-2), results in enhanced plant tolerance to iron-deficient growth conditions. This is demonstrated by the increased chlorophyl content and plant growth compared to wild-type plants when grown under the same iron-deficient conditions.

[0294] This increased tolerance to iron-deficient conditions also appears to have a dosagedependent / cumulative effect, such that chlorophyl and SPAD values were highest in homozygous mutants, and lowest in wild-type segregants, with heterozygous mutants having chlorophyl and SPAD values between the homozygous mutants and wild-type segregants.

[0295] This will have significant implications for generating mutations across multiple homologs. For example, we could cross tafe2-1 and tafe 2-2, which contain mutations in TaFE2-D1 and TaFE2-A1, respectively, to generate a double mutant, which may enhance stress tolerance when compared to a modification in a single homolog.

[0296] Interestingly, the mechanism of iron deficiency tolerance does not seem to be associated with significantly increased iron content in the vegetative shoot tissue, based on Peris’ Prussian Blue staining. This is likely beneficial for plant health as unregulated increases in tissue Fe concentrations can produce reactive oxygen species, which leads to necrosis. Enhanced Fe deficiency tolerance is often associated with improved grain nutrition in monocots (Kobayashi et al. 2013 Nat Commun. 4; Masuda 2013 Front. Plant Set. 4: 132; Briat et al. 2015 Trends in Plant Science. 20: 33-40).Table 3: Sequences of IDEF1 and IDEF2 related sequencesSEQ Description Sequence IDNO (uORF sequences are underlined, where applicable)1 Rice 1DEF1 MGQMDGGDGGGGGHPYHYQALLAAVHQQTVPFPNPFPAPS polypeptide SGAEPPHPHNHNHNHNHNHNIHNSHNHNHNHNAAPHPCHTPTPTPTPRGFADWSASTSAFTSLAAHSSTAPSNAVHYSFSPCYLOC_ps08g01 AFWTHYMLNKNAYPTSFPAPHDDHLRLANNNHPRDAPGPA 090.1 SSYGVESFTSPSMAPNICTHMPPIEGPISAKEDKKPEILPRWK(MSU Rice SSDELETRNSNVEFHSETVGTLPESKQGHDSRATKLLNSGEY Genome QVILRKELTKSDVGNVGRTVLPKKDAEASLPPLLQRDPLILHAnnotation MDDMVLPVTWKFKYRYWPNNKSRMYILDSAGEFLKTHGLProject) QAGDVIIIYKNLAPGKFIIRGEKAIHQQTTNP2 Wheat IDEF1 MGGDGRPSDGGGGGGAGAGAGHPHQFQYQALLAAVHTQNPNHS polypeptide HNLPFPLPPLNGPGPDASTHNAVRQPPTPRGFADWSASTSAFTSLA (A VQSTPSTATANAYHYSLSPCYAFWTHYMLNKNAYSYYPAPNQEH THPFSLDNNQAKDPGSIPSFGIESFNTTSLAPNMSAHMPPMEGPLPT subgenome) KEPEAPEDMPARVATIKDEMDARNGVELKCETVDALPELKQGHE SCASKFNSGEYQVILRKELTKSDVANVGRIVLPKKDAEASLPPLCETraesCS7A02RDPVILQMDDMVLPITWKFKYRFWPNNKSRMYILDSTSEFVKTHGG329600.1 LQAGDALIIYKNPVPGKYIVRGEKAIQQTN(IWGSC)3 Wheat IDEF1 MGGDGRPSDGGAGGGGGAGHPHQFQYQALLAAVHTQNPN polypeptide HSHNLPFPLPPLNGPGPDASTHNAARQPPTPRGFADWSASTS (B subgenome) AFTSLAVQSTPSAATANAYHYSLSPCYAFWTHYMLNKNAYSYYPAPNQEHTHHFSLDNNQAKDPGSIPSFGIESFNTTSLAPNTraesCS7B02 MSAHMPPMEGPLPTKEPEAPEDMPARVATIKDEMDARNGVG230100.2 ELKCETVDALPELKQGHESCASKFNSGEYQVILRKELTKSDV(IWGSC) ANVGR1VLPKKDAEASLPPLCERDPV1LQMDDMVLPITWKF KYRFWPNNKSRMYILDSTSEFVKTHGLQAGDALIIYKNPVPG KYIVRGEKAIQQTN4 WheatlDEFl MGGDGRPSDGGGGGGGAGHPHQFQYQALLAAVHTQNPNH polypeptide SHNLPFPLPPLNGPGPDASTHNAARQPPTPRGFADWSASTSA (D FTSLAVQSTPSTATANAYHYSLSPCYAFWTHYMLNKNAYSY subgenome) YPAPNQEHIHPFSLDNNQAKDPGSIPSFGIESFNTTSLAPNMS AHMPPMEGPLPTKEPEAPEDMPARVATIKDEMDARNGVELTraesCS7D02 KCETVDALPELKQGHESCASKFNSGEYQVILRKELTKSDVAG326300.2 NVGRIVLPKKDAEASLPPLCERDPVILQMDDMVLPITWKFK(IWGSC) YRFWPNNKSRMYILDSTSEFVKTHGLQAGDALIIYKNPVPGK YIVRGEKAIQQTN5 Barley IDEF1 MGGDGRPGDGGGGGAGHPHQFQYQALLAAVHTQNPNHNH polypeptide NHNHNHNLPFPLPPLNGPGPDASTHNAARQPPTPRGFADWSASTSAFTSLAVQSTPSTATANAYHYSLSPCYAFWTHYMLNKHORVU.MOR NAYSYYPAPNQEHITIPFSLDNNQAKDPGSIPNFGIESFNTTSLEX.r3.7HG070 APNMSAHMPPMEGPLSTKEPEASEDMPARVVAIKDERDARN8130.1 GIELKCETVDALPELKQGHESCATKFNSGEYQVILRKELTKS(IPK) DVANVGRTVLPKKDAEASLPPLCERDPVILQMDDMVLPVTWKFKYRFWPNNKSRMYILDSTSEFVKTHGLQAGDAUIYKNPVPGKYIVRGEKAIQQTN6 Oat IDEFl MGGDGRPSDGGGGGHPHQFQYQALLAAVHTQNPNNTLPFP polypeptide LPPLNGPGADSPPTNSVARQPPTPRGFTDWSASTSAFTSLAVQSPPSSNATNAYHYSLSPCYAFWTHYMLNKNAYSYYPAPNAVESA.00001 QEHTHPFYHDNNQAKDPGSLPSFGFESFNTASLAPNMSAHM b.r3.7Ag00027 PPMEPPMEGPLSVKEPEISEDVPARVVRIKDEMETRNGGELK77.1 FETVDTLPELKKNHESCATKFNSGEYQVILRKELTKSDVANV(EnsemblPlant GRIVLPKKDAEASLPPLCERDPVILQMDDMVLPVTWKFKYR s) FWPNNKSRMYILDSTTEFVKTHGLQAGDAIVIYKNPVPGKYIVRGEKATNH7 Rye IDEF1 MGGDGRPSDGGGGGGGAGHPHQFQYQALLAAVHTQNPNH polypeptide SHNLPFPLPPLNGPGPDASTHNAARQPPTPRGFADWSASTSA FTSLAVQSTPSTAAANAYHYSLSPCYAFWTHYMLNKNAYSSECCE7RvlG YYPAPNQEHTHPFSLDNNQAKDPGSIPSFGIESFNTTSLAPNM0494460.1 SAHMPPMEGPLSTKESEAPEDMPARWPIKDEMDARNGVEL(PCBG) KSETVDTLPELKQGHESCAGKFNSGEYQVILRKELTKSDVA NVGRIVLPKKDAEACLPPLCERDPVILQMDDMVLPVTWKFK YRFWPNNKSRMYILDSTSEFVKTHGLQAGDALIIYKNPVPGK YTVRGEKAIQQTN8 Com IDEF1 MGQMGGPDGDGDGGAGPHHQYHYQALLAAVQNPSQGLHP polypeptide FPLPFHVPLHAGAGAPAAGPGPGADADASTHNANAAHHSQP PRGFTDWSASNSAFAAVASQPAPATTNTPFHYNLSQSYALWZm00001ebl9 THYMLNKNVSYSTYPTPHEEHPHPLRHTHIQENPHPLRHTHI8710 T002 PDKDSGCASSLGFDSFTTMSLGPNICSHMTPMEGSISAKEPE(EnsemblPlant NSEDLPATVRSSDEMDTRNSGKVHRDTVGTLPESKQSHESCA s) SVNNKFNSGEYQVILRKELTKSDVANSGRIVLPKKDAEAGLPPLVQGDPLILQMDDMVLPIIWKFKYRFWPNNKSRMYILEAAGEFVKTHGLQAGDALIIYKNSVPGKFnRGEKSIQQTNP9 Rice IDEF2 MAQTCLPPGFRFHPTDVELVSYYLKRKIMGKKPLIQAISDVE polypeptide LYKFAPWDLPAQSCLQSRDLEWFFFCPRDKKYPNGSRTNRSTPNGYWKTSGKDRTIELNSRIVGSKKTLIFHEGKAPKGNRTDLOC_Os05g35 WVMYEYKMEDNQLVSAGFSKDDFVLCKIFKKSGLGPRIGEQ170.1 YGAPFNEEEWEHADAEMFPLLPNVETSVFPLLPSSEVVNSTD(MSU Rice DTRVQPSVAARAIEELPVQHLPHVCAGNGSTYQNITVTGESA Genome LMELPSQHSVESIGDEWSVDNCSNWNNADSPVIEGLVLEEAnnotation LSRFLTDSPHHGNPVGEHSGLPPMSEAEAHAFEVSTNDLYNEProject) LAGLAELGVPNGDGFSPSNAGVTEQQPTYFGVPNSENYVNMDDIFAPDTRLSYAYPLPNNQFWHYPMDQFTYSTTLSAAFPSGDSRPTMRTVDDLPAAANNGGFASKPSMQFPLS10 Wheat IDEF2 MAQTCLPPGFRFHPTDVELVSYYLKRKIMGKKLFVEAISEVE polypeptide (A LYKFAPWDLPDKSCLQSKDLEWFFFCPRDKKYPKGSRTNRA subgenome) TPNGYWKTSGKDRTIELNSRIVGLKKTLIFHEGKAPKGNRTDWVMYEYKMEDETLVSAGFSKDAYVLCKIFKKSGLGPRIGEQTraesCSlA02 YGAPFDENEWENLDVGSSIFSFAPSSGVEDPQVESSALATAIVG266300.2 IHEPSAPQQSVQFSEHVNICSDQVNDAPPEIDGILLDELTKFL(IWGSC) NDSPNHDNSGLPPMSEREAQAFEINTAELYDQLAVLAQSGD MPAANFPASNGDVIENNLQQTNSGLAMDDDYIGLDDLFAPGETFSYDFSGETFSYDLPGGTFSYLSVPNNQFSQYPLDQSSNGL HYGDGATQSTFLASGPLPPLEPMPSTFDDMPSVSNKPASSYC LNPAMKDPFS11 Wheat IDEF2 MAQACLPPGFRFHPTDVELVSYYLKRKIMGKKLFVEAISEVE polypeptide (B LYKFAPWDLPDKSCLQSKDLEWFFFCPRDKKYPKGSRTNRA subgenome) TPNGYWKTSGKDRTIELNSRIVGLKKTLIFHEGKAPKGNRTDWVMYEYKMEDETLVSAGFSKDAYVLCKIFKKSGLGPRIGEQTraesCSlB02 YGAPFDENEWENLDVGSSIFSFAPSSGVDDPQVNDVPPEIDGIG277300.2 LLDELTKFLDDSPNHDWFPENSGLPPMSELEAQAFEINSAEL(IWGSC) YDQLAGLTQSGDMSAVNFPASNGDVIENNLQQTNSALAMDDDYIGLDDLLAPGETFSYDFSGETFSYDLPGGSFSYDLSVPNNQFLQYPLDQSTNGSHYGVGATQSTFEASGSLPPMQPMPSTFDDMPSVSRLLNDAVIFWIGAE12 Wheat IDEF2 MAQACLPPGFRFHPTDVELVSYYLKRKIMGKKLFVEAISEVE polypeptide (D LYKFAPWDLPDKSCLQSKDLEWFFFCPRDKKYPKGSRTNRA subgenome) TPNGYWKTSGKDRTIELNSRIVGLKKTLIFHEGKAPKGNRTDWVMYEYKMEDETLVSAGFSKDAYVLCKIFKKSGLGPRIGEQTraesCSlD02 YGAPFDENEWENLDVGSSIFSFAPSSGVEDPQVNDAPPEIDGIG266500.1 LLDELAKFLDDSPNDDWLPENSGLPPMSELEAQAFEINTAE(IWGSC) LYDQLAGLSQSGDMSTVNFPASNGDVIENNLQQANSGLAMDDDYIGLDDLLAPGETFSYDFSGETFSYDLPGGSFSYDLSVPNNQFLLYPLDQSTSGSHYGVGATQSTFEASGTLPPMQPMPSTFNDMPSVSNCLNPAMKDPFS13 Barley IDEF2 MAQTCLPPGFRFHPTDVELVSYYLKRKIMGKKLFVEAISEVE polypeptide LYKFAPWDLPDKSCLQSKDLEWFFFCPRDKKYPKGSRTNRATPNGYWKTSGKDRTIELNSRTVGLKKTLIFUEGKAPKGNRTDHORVU.MOR WVMYEYKMEDETLDAAGFSKDAYVLCKIFKKSGLGPRIGEEX.r3.1HG006 QYGAPFDENEWENLDVGSSIFSFAPSSGVEDPQVESSALATA7040.1 VIQEPFVPQQSVQFSEHVNICSNEDNNAPPEIDGIWLEELALF(IPK) LNDSPNHDIALPENSGLPPMSELEAQAFEMNTAELYDQLAGLAQSGDMCNVNFPAGDVGVTENDFQQSNSGFAMDDDYIELDDLFAPGETFSYDFSGETFSYDLTGGTFSYDLSVPNNQFLQYPLDQSTNGSHYGDGATQSTFEASGSLPPMPSTFDDMPSVSNK PANSNCLNPTMKDPFS14 Oat IDEF2 MAQACLPPGFRFHPTDVELVSYYLKRKIMGKKLFVEAISEVE polypeptide LYKFPPWDLPDKSCLRSKDLEWFFFCPRDKKYPKGSRTNRATPNGYWKTSGKDRTIELNSRTVGLKKTUFHEGKAPKGNRTAVESA.00001 DWVMYEYKMEDDNLVSAGFSKDAYVLCKIFKKSGLGPRIG b.r3.1Cg00017 EQYGAPFDENEWENMDGGTSLFAFAPSSWDDPQLERSVLA89.1 TATDIQEPPVPSQQSVPFSDDANISSIEVNEAPPEIDGILLDELT(EnsemblPlant MFLNEPAHNDIPFAENSGLPPISEFEAQALEMNTSDLYNELA s) GLAQAGSTSNVDYRASNVDVTENNLENTYSFDDYLELDDLL APGETFSYGLPDDQFLQYPMDPATYGSHIDFGSSLSAFDACT SLPPLPSFFDDMPSVPNNLANSN15 Rye IDEF2 MAQTCLPPGFRFHPTDVELVSYYLKRKIMGKKLFVEAISEVE polypeptide LYKFAPWDLPDKSCLQSKDLEWFFFCPRDKKYPKGSRTNRATPNGYWKTSGKDRTIELNSCIVGLKKTLIFHEGKAPKGNRTDSECCElRvlG WVMYEYKMEDETLVSAGFSKDAYVLCKIFKKSGLGPRIGEQ0038480.1 YGAPFDENEWENLDVGSSIFSFAPSSGVEDPQVESSGLATAIV(PSGB) IQEPAAPQQSVQFSEKVDICSNEDSNVPPEIDGILLEELTMFLN DSPNHDTVLPENSGLPPMSELEAQAFEINTAELYDQLAGLAH SGDMSNVNFPVSDGGVIENNFQHANSGLAMDNDYIELDDLF APGETFSYDVSGETFSYDLPGGTFSYDLSVPNNQFLQYPLDQ STNGCHYSDSATQSTFQASGSLPPMQPMPSTFDDMPSVSNKP ASSNCLNPFMKDPFS16 Com IDEF2 MAQTSLPPGFRFHPTDVELVSYYLKRKIMGKKHIVDAIAEVD polypeptide LYKFPPWDLCDKSSLRSKDLEWFFFCPRDKKYPNGSRTNRATPNGYWKTSGKDRHMLNSRTVGMKKTLIFHEGKAPKGDRTZm00001eb34 DWVMYEYRMEDEDLVSAGFSKDAYVLCKIFKKSGLGPRIGE9010 T001 QYGAPFNEDEWDNAEVETSLFPLTCSEVNPTEGQRAQPTVL AGALLEPEPPLHNSSSTCAGEESSFGHATAEDLTFSHSVASSA(EnsemblPlant IQDTPARMSGDGWSVNNISNVANDMRSPRDCDEFLSELSRF s) MNDSPLRNTPLGECSGLPPMSEAEAHAFEVNSFGLYNELSELVGLGCADNNLNTSNVETTDYLMLPPDRELSANDYMELNDLLAPDPSFPSEFSAQDNQYMQHPLAQYNGHYDLSGPSEPTMPSIFDVFPPNNGVLATDDPANYLDPAMQFPFS17 Rice IDEF1 AGATGAGATGAGATAGAGAAGAGAGATAGAGAGAGAGGLS CAGGCTGGTAGAACGGGTCACCTCACCACCTTCGCCTCCGCCTCCTCCTTCGCCTCCGTCTCCACCTCTCCTCTCCTCTTCCLOC_Os08g01TCTTCTTCCTCCTCCTGGGGCCTAGCCGCCTAGGGGGGTT090.1GTGGCCTGCGACTGCGACTTCTTCCTCTGCCTTCTGCTCTC(MSU RiceTGCCTCTGAGTATATGGAATATGGTGTGTGCGTGAAGAGAGenomeAATTAAGTGAGGGAGGGGAAAGCGAGCGAACAGGGCAAAnnotationCAProject)18 Wheat IDEF1 GAGGCCAGAGGAGAGAGAGAAGCCAGGGCTTGGCTGGTALS GATGCGCGAGTAGAGGTGTCTGCCAAAGGCCAAGGGGA(A subgenome)TraesCS7A02G329600.1(IWGSC)19 Wheat IDEF1 AAGGGAAGGAAAGGATAGATAGAAGAGAAGAGGTCAGTLS GACCACCACCCTTCCCTCCCCTCCCCTCACTCCCGTGCCCC(B subgenome) CGCCTCTGCCTGCCTGGGTTACCCTACCACCCCTCCTCCCACCCATAGCCGAGGCCAGAGGAGAGAGAGAGAGAAGCCATraesCS7B02 GGGCTTGGCTGGTAGATGCGCGAGTAGCGGTGTCTGCCAAG230100.2 AGGCCAAGGGGA(IWGSC)20 Wheat IDEF1 AAGGGAAGGAAAGGATAGATAGAAGAGAAGAGGTCAGTLS GACCACCACCCTTCCCTCCCCTCCCCTCAGTCCCGTGCCCC(D CCGCCTCTGCCTGCCTGGGTTACCCTACCACCCCTCCTCCC subgenome) ACCCATAGCCGAGGCCAGAGGAGAGAGAGAGAGAAGCCAGGGCTTGGCTGGTAGATGCGCGAGTAGCGGTGTCTGCCATraesCS7D02 AAGGCCAAGGGGAG326300.2(IWGSC)21 Barley IDEF1 GTGACCACCACCCTTCCCTCCCCTCCCCTCACTCCCCCCGC LS CTCTGCCTGCCCGGGTTACCCTACCACCCCTCCTCCCACCCATAGCCGAGGCAGGCCAGAGGAGAGAGAGAGAGAGAAAHORVU.MOR GAGAAGCCAGGGCTTGGCTGGTAGATGCGCGAGTAGCGGEX.r3.7HG070 TGCCTGCCAAGGCCAAGGGGA8130.1(IPK)22 OatIDEFl LS AGGAAAGGATAGATAGAAGAGACGAGGTCAGTGAGACCACCACCCTTCCCTCCCCTCCCCTCACTCCCCCCCTACCTCTAVESA.00001 GCCCGGGTTACCCTACCTCCCCTCCTCCCACCCATAGCTG b.r3.7Ag00027 GAGAGAGAAAAAGAGGGCAGGGTTTGGCTGGTAGATGCG77.1 CAAGTAGCGGTGTCTGCCAACGCAAGGGGA(EnsemblPlant s)23 Rye IDEF1 LS AAGGATAGATAGAAGAGAAGAGGTCAGTGACCACCACCC (200bp taken TrCCCTCCCCTCCCTTCACTCCCCCCCGCCTCTGCCTGCCT upstream of GGGTTACCCTACCACCCCTCCTCCAACCCATAGCCGAGGC translational CAGAGGAGAGAGAGAGAAAGAGAAGCCAGGGCTTGGCT start site) GGTAGATGCGCGAGTAGCGGTGTCTGCCAAAGGCCAAGGGGASECCE7RvlG0494460.1(PCBG)24 Com IDEF1 CTTGGCCCTTGGCCTCCTTTCGTCGAGAGAGAGAGAGCTGLS TGCTGGCTGCTGCTGCCCCGGGTCCGTCGGGACGGGTAGATGAGGAGGAAGAGAACAGCTCGTGACTGACTGACTCCCTZm00001ebl9 CCCTCAGCGTGAGTGGGTTGCGGACGACCACATCCATCCG8710 T002 TTCCCACCATTCCCTTCTCCCACCTCGCCTCCTCCCCTTGCCCTTGCCCTTCCACAGCCGGAATCAGGGGGTA(EnsemblPlant s)25 Rice IDEF2 GCGGTTGCTTCCTCCGAAAATCCTCATCCTCTCCATTGACCLS (intron AGACGGCGGCGCAGGCGCAGGCGCTCACCTCCCTCCTCCT removed) CCGCGGCATCCCCAATCGACCAGCTGGCTCCCCTGCCTCCTCCGCGATTTTGATCCTAATCACCCATGTCTGCTGAAGTCLOC_Os05g35 AGCGAGGAATATCCTTATTTCCTCTCCTGCTACTTCTGGTT170.1 CATATATTGCATAAAATTCCGAGATTTCACTTGATACAAG(MSU Rice TGGATCACCTGGAGTTTTGTGCTAATACTTCCTATCATGTGGenome AATCAGTTGTAGTTAGCATAATTGGCAAGACTCAAGTTTTAnnotation CTCTCATCTGGGTTCAGAAACATTTGTCGGTTGAGTGCTCProject) GATCTAAACTCTGCTTGAGTCTTGCCGAAGATTGTTTCTCAACAATCACGTCTTCTGTTCTCTCTGAGGTTGGACCAATCTCCCTTTTGGTCTGATCAACCTCCTGATCTCCCTTAAGTGAACCGTCCTGCTGTGTTCACGCAATACAATCTTCTGAAGTCCTCTCTTGTTCCTTCTGTGCTCAAACATCAAAAGCAGCTCTCTCCTAAGTTCTCTTGATTGTTTGTGCTCCTAAATTCTGTTGTTTGAGCACTCAAACATTTCCCTTACTGGTCAATCTTAGATCACTTGTTGATTATTGGTATTGGCATTATTTGGTGAAGCATTTCTGCTTGTACCTTAAACAAAGGTCTCTGTTGTCTTATTGGTGCTGAAGGCAAGATATACACTGCAGTG26 Wheat IDEF2 GCTTCCCCCAAAACCTCACCTCATCTCATCTCCTCCACAATLS (intron TGACCAGACGGCGGCGCTCACCTCCTCCCCCTCGGCATCC removed) (A CCCATCGTCCTGCTGGCTTCCCCGGTTCTCCCTCGTTnTG subgenome) ATCTCAGTCACTCATGTCTTTTGAAGTCAGCGAGGGATCCCCTTATTTCCACTCGTGCTACTTCTGGTTCATATATTGCATTraesCSlA02 AAAATTCCGAGAATTCACTTGATACAAGTGGATCATCTGGG266300.2 AGTTTTGTGCGAGTACTTCCTATCATGTGAATCAGTGGTA(IWGSC) GTTGGTTTAATTGGCAAGACTCAAACTTCTCTCATCTGAGTTCAGAAACAnTGTCGCTTGAGTCCTTGGTGTACTGCnTCCTTGAGTCTTGCCAAGCCTGGTTTTCCATAAGTTGTTTCTCCrCCAGCTTTTGGTTGGACCATTCTCTCCTCTGGTCTAATCTCATCCATCCTTTGATCCTCCCTTCAGAATAGTTGTCATAACGTGAGCTGTCCTCTGCTCACACGGCAATCTTCTGAAGCTCTCTTCTCTGGTTCTTGTTTGTTCTCTTGAGAGCTCCTCTCCTTAAGTTTGGTTGATCTGCGGCCTTGAATTCTGTCTTTCGAGGACTGAAGCTTTCTTGCCAAACTTGATATTTCCCTGATTTACTGGTATTGTTAATTGCATAATCTGGTGAAGCAATTCTGGTTGTGTTTCACCTCTTGTGTTCTTCTGCTGATACTGAGCGCAAGACACCCACTGCAGTG27 Wheat IDEF2 ATTGCTTCCCCCCAAACCTCACCTCATCTCCTCCACCATTGLS (intron ACCAGACGGCGGCGCTCACCTCCTCCCCCTCGGCCTCCCC removed) (B CATCGTCCTGCTGGCTTCCCCGGTTCTTTCTCGTTTTTGAT subgenome) CTTAGTCACTCATGTCTTTTGAAGTCAGCGAGGGATCCCCTTATTTCCTCTCGTGCTACTTCTGGTTCATATATTGCATAATraesCSlB02 AATTCCGGGACTTCACTTGATACAAGTGGATCATCTGGAGG277300.2 TTTTGTGTGAATACTTCCTATCATGTGAATCAGTGGTAGTT(IWGSC) GGTTTAATTGGCAAGACTCAAACTTCTCATCTGAGTTCAGAAACATTTGTTGCTTGAGTCTTTGGTGGACTGCTTTCCTTGAGTCTTGCCAAGCCTGGTTTCCCATCAGTTGTTTCTCCTCCAGCTTTCGGTTGGACCATTCTCTCTTCTGGTCTAATCTCATCAATCTTTTCATCCTCCCTTCAGAATAGTrGTTTAACGTGAGCTTGTCCTACTCTGCTCACACTGCAATCTTCTGAAGCTCTCTTCTCTGGTTCTTGTCTGTTCTCCAAGCATTTGAGAGCTCCTCTCTCCTTAAGTTTGGTTGATCTGTGGCCCTGAATTCTGTCCTTTGAGGGCTAAAGCATATCCCTTTCTGGCCAAACTTGATATTTCCCTGATTACTGGTATTGTTAGTTGCATAATCTGCTGAAGCATTTCTTGTTGTGTCTCTTGTGTTCTTCTCTTGATACTGAGCACAAGAGACCCACTGCAGTG28 Wheat IDEF2 GTTTTTGATCTCAGTCACTCATGTCTTTTGAAGTCAGCGAGLS (D GGATCCCCTTATTTCCTCTCGTGCTACTTCTGGTTCATATA subgenome) TTGCATAAAATTCCGGGACTTCACTTGATACAAGTGGATCATCTGGAGTTTTGTGCGAATACTTCCTATCATGTGAATCATraesCSlD02 GTGGTAGTTGGTTTAATTGGCAAGACTCAAACTTCTCTCAG266500.1 TCTGAGTTCAGAAACATTTGTTGCTTGAGTCTTTGGTGTAC(IWGSC) TGCTTTCCTTGAGTCTTGCCAAGCCTGGTTTCCCATCAGTTGTTTCTCCTCCAGCTTTCGGTTGGACCATTCTCTCTTCTGGTCTAATCTCATCAATCTTTTGATCCTCCCTTCAGAATAGTTGTTTAACGTGAGCTGTCCTACTCTGCTCACACTGCAATCTTCTGAAGCTCTCTTCTCTGGTTCTTGTCTGTTCTCCAAGCATTTGAGAGCTCCTCTCTCCTTAAGTTTGGTTGATCTGTGGCCCTGAATTCTGTCCTITCAGGGCTAAAGCATATCCCTTTCTGGCCAGACTTGATATTTCCCTGATTACTGGTATTGTTAATTGCCTAATCTGGTGAACCATTTCTGGTTGTGTTTCACCTCTTGTGTTCTTCTCTTGATACTGAGCACAAGAGACCCACTGCAGTG29 Barley IDEF2 CCTCACCTCCTCTCCTCCCCCCATTGACCAGACGGCGGCG LS CCCACCCCCTCCTCCTCGGCCTCCCCCATCGTCCTGCTGGCTTCCCCGGTTCTTCCCCGTTTTTGATCTGAGTCACTCATGTHORVU.MOR CTnTGAAGTCAGCGAGGGATCCCCTTATTTCCTCTCGTGCEX.r3.1HG006 TACTTCTGGTTCATATATTGCATAAAATTCCGAGATTTCAC7040.1 TTGATACAAGTGGAACATCTGGAGTTTTGTGCGAATACTT(IPK) CCTATCATGTGAATCAGTGGTAGTTGGTTTAATTGGGAAGACTCAAACTTCTCTCATCTCAGTTCAGAAACATTTGTCGCTTGAGTCCTTGGTTTACTGCTTTCCTTGAGTCTTGCCAAGCCTGGTTTCCCATCAGTTGCGTCTCCTCAAGCTTTCGGTTGGACCATTCTCTCCTCTGGTCTAATCTCATCAATCCTTTGATTGATCCTCCCTTCAGAATAGTTGTCATAACGTGAGCTCTCCTACTCTGCTCACACTGCAATATTCTGAAGCTCTCTTCTCTGGTTCTTGTTTGCTCTCTTGAGAGCTCCTCTCTCCTTAAGTTTGGTTGACCTGTGGCCTTGAATTCTGTCTTTTGAGGACCGAAGCATTCAACTTTCTGGCCAAACTTGATATTTTCCTGATTTACTGGTATTGTTAATTGCATAATCTGGTGAAGCAATTCTGGTTGTGTTTCACCTCTTGTGTTCTTCTGTTGATACTGAGCACAAGACACCCACTGCAGTG30 Oat IDEF2 LS GAAATTGCTTCCCCCAAAACCTTCACATCCTCTCATCCTCCCCATTGACCAGACGGCGGCGCTCACCTCCTCCTCGTCGATCTCCCCAATCGTCCTGCTGGCTTCCCCGCGTCCTCCGCGATTTTGAACTTGATCACCCATGTCTnTGAAGTCCGCGAGGGAVESA.00001 ATCTCCTTATTTCCTCACGTGCTACTTCTGGTTCATATATT b.r3.1Cg00017 GCATAAAATTCCGAGATTTCACTTGATACAAGTGGACCAC89.1 CTGGAGTTTTGTGCAAGTATCATGTGAATCAGTGGTAGTT(EnsemblPlant GGTTTAATTGGCAAGACTCAACCTCTCTCATCTGAGTTCA s) GAAACGTTTGCTCCTCGAGTCCTTTGTGTACTGCTTTCCCTGAGTCTTGCCAAGAGTGGTTTCCCATCAGTTCCTTTCTCTGCTCTrGCTGTCATTGGACCAATATCTCTGTrGGTCCTAGTCTCATCAAGATTTCCACCCTCTCTTCAGAACTGTTGTTAAAACGTGAGCTGCCCTACTCTGCTCACACTGCAATCTTCTGAAGCTCTCTCCTCTGATTCTTCTGCTCTCAAAACATTTGAGAGCTAGTCTCTCGTTCTGAACTTTGGTTGATTTGTTGTCCTTGATACACTTTCTTGAGGACTGAAACAGCATCCCCTTCTCAAGCCAAACCTGATATTTCTCTGATTGGCATTGATAATTGCATAATCTGGTGAAGCATTTCTACTTGTGTTTCACGCCCAGATCTCTCTGTCCCTTGTTCTTACTGAGCGCAAGACACCCGCCGTGGTG31 Rye IDEF2 LS ATTTTGAACTTGATCACCCATGTCTTTTGAAGTCCGCGAG (200bp taken GGATCTCCTTATTTCCTCACGTGCTACTTCTGGTTCATATA upstream of TTGCATAAAATTCCGAGATTTCACTTGATACAAGTGGACC translational ACCTGGAGTTTTGTGCAAGTATCATGTGAATCAGTGGTAG start site) TTGGnTAATTGGCAAGACTCAACCTCTCTCATCTGAGTTCAGAAACGTTTGCTCCTCGAGTCCTTTGTGTACTGCTTTCCCSECCElRvlG TGAGTCTTGCCAAGAGTGGTTTCCCATCAGTTCCTTTCTCT0038480.1 GCTCTTGCTGTCATTGGACCAATATCTCTGTTGGTCCTAGT(PSGB) CTCATCAAGATTTCCACCCTCTCTTCAGAACTGTTGTTAAAACGTGAGCTGCCCTACTCTGCTCACACTGCAATCTTCTGAAGCTCTCTCCTCTGATTCTTCTGCTCTCAAAACATTTGAGAGCTAGTCTCTCGTTCTGAACTTTGGTTGATTTGTTGTCCTTGATACACTTTCTTGAGGACTGAAACAGCATCCCCTTCTCAAGCCAAACCTGATATTTCTCTGATTGGCATTGATAATTGCATAATCTGGTGAAGCATTTCTACTTGTGTTTCACGCCCAGATCTCTCTGTCCCTTGTrCTrACTGAGCGCAAGACACCCGCCGTGG32 Com IDEF2 CCACACCCACACAACCGCACCTCGCACCCATTGACCAGACLS GGCGGCGCTCATCTCCTCCCCCACGGTTCTCCCCATCGGCCTGCTGGCTTCTCCCCGGCTTCCCCGATACTGATCCTCATCZm00001eb34 ACTCATGTCTGCTGAAGTTGCTGGAGGACACCCTTATTTC9010 T001 CTCTCGTGCTACTTCTGGTTCATATACTGCATAAAATTCCG(EnsemblPlant AGATTTTACTTGATACAAGTGGAACACTCAGAGTTTTGTG s) CGAATACTTCCTACCATGTGAATCAGTTGTAGCTGGTGAATTTGGCAAGACTCAATTTCTCTCATCTGAGTTCAGAAACGTTTGCTTGGTCTCCTGCTTCCCTTGAGTCTTGCCAACCATAGTTTTCTCTCATCTCTTTCTGTGACTTGACCAACATCTCTTTTGGTCCTGTCAGTCTCGCAATACTCCCTTCCAAAGACTTGTCTAGAGGTTGGATTGCCCTGCTCTCTGCTGGCACTGCAGCCTGCTGAAGTCTTTACTTGCTGCATTCTGTGCTCACTTTTGAAAGCTGCTCTCTTCTGCTCTGCGTTGTTGATTTCTGCACTCAAGCTTCTCTTGCCTGTGCACTCAAGCTTCTCTCTTCCTGGGCAACTAGTGATCTCCTTGAAGCATTTCAGCTTTTACCTAGAGATCTTTCTGTTGTTGAAGGCAAGAGAGGCTTTAGGGTG33 OsIDEFl / OsF AGATGAGATGAGATAGAGAAGAGAGATAGAGAGAGAGGEl ml uORF CAGGCTGGTAGAACGGGTCACCTCACCACCTTCGCCTCCG comprising LS CCTCCTCCTTCGCCTCCGTCTCCACCTCTCCTCTCCTCTTCCTCTTCTTCCTCCTCTAGGGGCCTAGCCGCCTAGGGGGGTT(The start>stop GTGGCCTGCGACTGCGACTTCTTCCTCTGCCTTCTGCTCTC mutated codon TGCCTCTGAGTATATGGAATATGGTGTGTGCGTGAAGAGA is shown in AATTAAGTGAGGGAGGGGAAAGCGAGCGAACAGGGCAA bold) CA34 OsIDEFl / OsF AGTAGAGTAGAGATAGAGAAGAGAGATAGAGAGAGAGGEl m2 uORF CAGGCTGGTAGAACGGGTCACCTCACCACCTTCGCCTCCG comprising LS CCTCCTCCTTCGCCTCCGTCTCCACCTCTCCTCTCCTCTTCCTCTTCTTCCTCCTCCTGGGGCCTAGCCGCCTAGGGGGGTr(The stait>stop GTGGCCTGCGACTGCGACTTCTTCCTCTGCCTTCTGCTCTC mutated codon TGCCTCTGAGTATTAGGAATTAGGTGTGTGCGTGAAGAGis shown in AAATTAAGTGAGGGAGGGGAAAGCGAGCGAACAGGGCA bold) ACA35 OsIDEFl / OsF AGTAGAGTAAGATAGAGAAGAGAGATAGAGAGAGAGGCEl m3 uORF AGGCTGGTAGAACGGGTCACCTCACCACCTTCGCCTCCGC comprising LS CTCCTCCTTCGCCTCCGTCTCCACCTCTCCTCTCCTCTTCCTCTTCTTCCTCCTCCTGGGGCCTAGCCGCCTAGGGGGGTTG(The stait>stop TGGCCTGCGACTGCGACTTCTTCCTCTGCCTTCTGCTCTCT mutated codon GCCTCTGAGTATATGGAATATGGTGTGTGCGTGAAGAGAA is shown in ATTAAGTGAGGGAGGGGAAAGCGAGCGAACAGGGCAAC bold) A36 OsIDEFl / OsF AGATGAGATGAGATAGAGAAGAGAGATAGAGAGAGAGGEl m4 uORF CAGGCTGGTAGAACGGGTCACCTCACCACCTTCGCCTCCG comprising LS CCTCCTCCTTCGCCTCCGTCTCCACCTCTCCTCTCCTCTTCCTCTTCTTCCTCCTCCTGGGGCCTAGCCGCCTAGGGGGGTT(The start>stop GTGGCCTGCGACTGCGACTTCTTCCTCTGCCTTCTGCTCTC mutated codon TGCCTCTGAGTATTAGGAATTAGGTGTGTGCGTGAAGAG is shown in AAATTAAGTGAGGGAGGGGAAAGCGAGCGAACAGGGCA bold) ACA37 OsIDEF2 / OsF GATTTTGATCCTAATCACCCTAGTTAGCTGAAGTCAGCGAE2 ml uORF GGAATATCCTTATTTCCTCTCCTGCTACTTCTGGTTCATAT comprising LS ATTGCATAAAATTCCGAGATTTCArATACAAGTGGATCACCTGGAGTTTTGTGCTAATACTTCCTATCATGTGAATCA(The start>stop GTTGTAGTTAGCATAATTGGCAAGACTCAAGTTTTCTCTC mutated codon ATCTGGGTTCAGAAACATTTGTCGGTTGAGTGCTCGATCT is shown in AAACTCTGCTTGAGTCTTGCCGAAGATTGTTTCTCAACAA bold) TCACGTCTTCTGTTCTCTCTGAGGTTGGACCAATCTCCCTTTTGGTCTGATCAACCTCCTGATCTCCCTTAAGTGAACCGTCCTGCTGTGTTCACGCAATACAATCTTCTGAAGTCCTCTCTTGTTCCTTCTGTGCTCAAACATCAAAAGCAGCTCTCTCCTAAGTTCTCTTGATTGTTTGTGCTCCTAAATTCTGTTGTTTGAGCACTCAAACAnTCCCTTACTGGTCAATCTTAGATCACTTGTTGATTATTGGTATTGGCATTATTTGGTGAAGCATTTCTGCTTGTACCTTAAACAAAGGTCTCTGTTGTCTTATTGGTGCTGAAGGCAAGATATACACTGCAGTG38 TaIDEF2 / TaFe GCTTCCCCCAAAACCTCACCTCATCTCATCTCCTCCACAAT2 El uORF TGACCAGACGGCGGCGCTCACCTCCTCCCCCTCGGCATCC comprising LS CCCATCGTCCTGCTGGCTTCCCCGGTTCTCCCTC39 TaIDEF2 / TaFe GTTTTTGATCTCAGTCACTCATGTCTTTTGAAGTCAGCGAG2 E2 uORF GGATCCCCTTATTTCCACTCGTGCTACTTCTGGTTCATATA comprising LS TTGCATAAAATTCCGAGAATTCACTTGATACAAGTGGATCATCTGGAGTTTTGTGCGAGTACTTCCTATCATGTGAATCAGTGGTAGTTGGTTTAATTGGCAAGACTCAAACTTCTCTCATCTGAGTTCAGAAACATTTGTCGCTTGAGTCCTTGGTGTACTGCTTTCCTTGAGTCTTGCCAAGCCTGGTTTTCCATAAGTTGTTTCTCCTCCAGCTTTTGGTTGGACCATTCTCTCCTCTGGTCTAATCTCATCCATCCTTTGATCCTCCCTTCAGAATAGTTGTCATAACGTGAGCTGTCCTCTGCTCACACGGCAATCTTCTGAAGCTCTCTTCTCTGGTTCTTGTTTGTTCTCTTGAGAGCTCCTCTCCTTAAGTTTGGTTGATCTGCGGCCTTGAATTCTGTCnTCGAGGACTGAAGCnTCTTGCCAAACTTGATATITCCCTGATTTACTGGTATTGTTAATTGCATAATCTGGTGAAGCAATTCTGGTTGTGTTTCACCTCTTGTGTTCTTCTGCTGATACTGAGCGCAAGACACCCACTGCAGTG40 TaIDEF2 / TaFe GCTTCCCCCAAAACCTCACCTCATCTCATCTCCTCCACAAT2 ml uORF TGACCAGACGGCGGCGCTCACCTCCTCCCCCTCGGCATCC comprising LS CCCATCGTCCTGCTGGCTTCCCCGGTTCTCCCTCGTmTGATCTCAGTCACTCATGTCTTTTGAAGTCAGCGAGGGATCCCCTTATTTCCACTGTGCTACTTCTGGTTCATATATTGCATAAAATTCCGAGAATTCACTTGATACAAGTGGATCATCTGGAGTTTTGTGCGAGTACTTCCTATCATGTGAATCAGTGGTAGTTGGTTTAATTGGCAAGACTCAAACTTCTCTCATCTGAGTTCAGAAACATTTGTCGCTTGAGTCCTTGGTGTACTGCTTTCCTTGAGTCTTGCCAAGCCTGGTTTTCCATAAGTTGTTTCTCCTCCAGCmTGGTTGGACCATTCTCTCCTCTGGTCTAATCTCATCCATCCTTTGATCCTCCCTTCAGAATAGTTGTCATAACGTGAGCTGTCCTCTGCTCACACGGCAATCTTCTGAAGCTCTCTTCTCTGGTTCTTGTTTGTTCTCTTGAGAGCTCCTCTCCTTAAGTTTGGTTGATCTGCGGCCTTGAATTCTGTCTTTCGAGGACTGAAGCTITCTTGCCAAACTTGATATTTCCCTGATTTACTGGTATTGTTAATTGCATAATCTGGTGAAGCAATTCTGGTTGTGTTTCACCTCTrGTGTTCTTCTGCTGATACTGAGCGCAAGACACCCACTGCAGTG41 TaIDEF2rTaFe GCTTCCCCCAAAACCTCACCTCATCTCATCTCCTCCACAAT2 m2 uORF TGACCAGACGGCGGCGCTCACCTCCTCCCCCTCGGCATCC comprising LS CCCATCGTCCTGCTGGCTTCCCCGGTrCTCCCTCGTnTTGATCTCAGTCACTCTCTTTTGAAGTCAGCGAGGGATCCCCTTATTTCCACTCGTGCTACTTCTGGTTCATATATrGCATAAAATTCCGAGAATTCACTTGATACAAGTGGATCATCTGGAGTTTTGTGCGAGTACTTCCTATCATGTGAATCAGTGGTAGTTGGTTTAATTGGCAAGACTCAAACTTCTCTCATCTGAGTTCAGAAACATTTGTCGCTTGAGTCCTTGGTGTACTGCTTTCCTTGAGTCTTGCCAAGCCTGGTTTTCCATAAGTTGTTTCTCCTCCAGCTTTTGGTTGGACCATTCTCTCCTCTGGTCTAATCTCATCCATCCTTTGATCCTCCCTTCAGAATAGTTGTCATAACGTGAGCTGTCCTCTGCTCACACGGCAATCTTCTGAAGCTCTCTTCTCTGGTTCTTGTTTGTTCTCTTGAGAGCTCCTCTCCTTAAGTTTGGTrGATCTGCGGCCTIGAATTCTGTCTTTCGAGGACTGAAGCTTTCTTGCCAAACTTGATATTTCCCTGATTTACTGGTATTGTTAATrGCATAATCTGGTGAAGCAATTCTGGTTGTGTTTCACCTCTTGTGTTCTTCTGCTGATACTGAGCGCAAGACACCCACTGCAGTG42 TaIDEF2 / TaFe GCTTCCCCCAAAACCTCACCTCATCTCATCTCCTCCACAAT2 2 m3 uORF TGACCAGACGGCGGCGCTCACCTCCTCCCCCTCGGCATCC comprising LS CCCATCGTCCTGCTGGCTTCCCCGGTTCTCCCTCGTTTTTGATTGGnTAATTGGCAAGACTCAAACTTCTCTCATCTGAGTTCAGAAACATTTGTCGCTTGAGTCCTTGGTGTACTGCTTTCCTTGAGTCTTGCCAAGCCTGGTTTTCCATAAGTTGTTTCTCCrCCAGCTTTTGGTTGGACCATTCTCTCCTCTGGTCTAATCTCATCCATCCTTTGATCCTCCCTTCAGAATAGTTGTCATAACGTGAGCTGTCCTCTGCTCACACGGCAATCTTCTGAAGCTCTCTTCTCTGGTTCTTGTTTGTTCTCTTGAGAGCTCCTCTCCTTAAGTTTGGTTGATCTGCGGCCTTGAATTCTGTCTTTCGAGGACTGAAGCTTTCTTGCCAAACTTGATATTTCCCTGATTTACTGGTATTGTTAATTGCATAATCTGGTGAAGCAATTCTGGTTGTGTTTCACCTCTTGTGTTCTTCTGCTGATACTGAGCGCAAGACACCCACTGCAGTG43 TaIDEF2 / TaFe TTGGTTTAATTGGCAAGACrCAAACTTCTCTCATCTGAGTT2 m4 uORF CAGAAACATTTGTCGCTTGAGTCCTTGGTGTACTGCTTTCC comprising LS TTGAGTCTTGCCAAGCCTGGTTTTCCATAAGTTGTTTCTCCTCCAGCTTTTGGTTGGACCATTCTCTCCTCTGGTCTAATCTCATCCATCCTTTGATCCTCCCTTCAGAATAGTTGTCATAACGTGAGCTGTCCTCTGCTCACACGGCAATCTTCTGAAGCTCTCTTCTCTGGTTCTTGTTTGTTCTCTTGAGAGCTCCTCTCCTTAAGTTTGGTTGATCTGCGGCCTTGAATTCTGTCTTTCGAGGACTGAAGCTTTCTTGCCAAACTTGATATTTCCCTGATTTACTGGTATTGTTAATTGCATAATCTGGTGAAGCAATTCTGGTTGTGTTTCACCTCTTGTGTTCTTCTGCTGATACTGAGCGCAAGACACCCACTGCAGTG44 Rice IDEF1 ATGAGATGAGATAGAGAAGAGAGATAG uORF 145 Rice IDEF1 ATGGAATATGGTGTGTGCGTGAAGAGAAATTAAGTGA uORF 246 Rice IDEF2 ATGTCTGCTGAAGTCAGCGAGGAATATCCTTATTTCCTCT uORF CCTGCTACTTCTGGTTCATATATTGCATAAAATTCCGAGATTTCACTTGATACAAGTGGATCACCTGGAGTnTGTGCTAATACTTCCTATCATGTGAATCAGTTGTAG47 Wheat IDEF2- ATGTCTTTTGAAGTCAGCGAGGGATCCCCTTATTTCCACTCA uORF GTGCTACTTCTGGTTCATATATTGCATAAAATTCCGAGAATTCACTTGATACAAGTGGATCATCTGGAGTTTTGTGCGAGTACTTCCTATCATGTGAATCAGTGGTAG48 Wheat IDEF2- ATGTCTTTTGAAGTCAGCGAGGGATCCCCTTATTTCCTCTCB uORF GTGCTACTTCTGGTTCATATATTGCATAAAATTCCGGGACTTCACTTGATACAAGTGGATCATCTGGAGTTTTGTGTGAATA 2CTATCATGTGAATCAGTGGTAG49 Wheat IDEF2- ATGTCTTTTGAAGTCAGCGAGGGATCCCCTrATTTCCTCTCD uORF GTGCTACTTCTGGTTCATATATTGCATAAAATTCCGGGACTTCACTTGATACAAGTGGATCATCTGGAGmTGTGCGAATACTTCCTATCATGTGAATCAGTGGTAG50 Wheat IDEF2 MSFEVSEGSPYFX1SCYFWFIYCIKFRX2F wherein Xi is H or L, uORF peptide X2is E or D(partial)51 Wheat IDEF2- MSFEVSEGSPYFHSCYFWFIYCIKFREFTA uORF peptide(partial)52 Wheat IDEF2- MSFEVSEGSPYFLSCYFWFIYCIKFRDFTB uORF peptide(partial)53 Monocot MSX1EX2X3X4X5X6PYX7X8X9CYFWFIYCIKFRX10FT whereinIDEF2 uORF Xi is A, F, or L, X2is V or L, X3is A, S, T, or R, X4 is G or E, X5is peptide G or E, X6 is H, S, or Y, X7is F or L, Xg is L or H, X9is S or T and(partial) X10 is D or E.54 tafe2-1 leader GTTTTTGATCTCAGTCACTCATGTCTTTTGAAGTCAGA sequence CGAGGGATCCCCTTATTTCCTCTCGTGCTACTTCTGGTTCATATATTGCATAAAATTCCGGGACTTCACTTGATAC(Leader AAGTGGATCATCTGGAGTTTTGTGCGAATACTTCCTAT sequence is CATGTGAATCAGTGGTAGTTGGTTTAATTGGCAAGACT bold; CAAACTTCTCTCATCTGAGTTCAGAAACATTTGTTGCTMutant bases TGAGTCTTTGGTGTACTGCTTTCCTTGAGTCTTGCCAAG are underlined) CCTGGTTTCCCATCAGTTGTTTCTCCTCCAGCTTTCGGTTGGACCATTCTCTCTTCTGGTCTAATCTCATCAATCTnTGATCCTCCCTTCAGAATAGTTGTTTAACGTGAGCTGTCCTACTCTGCTCACACTGCAATCTTCTGAAGCTCTCTTCTCTGGTTCTTGTCTGTTCTCCAAGCATTTGAGAGCTCCTCTCTCCTTAAGTTTGGTTGATCTGTGGCCCTGAATTCTGTCCTTTCAGGGCTAAAGCATATCCCTTTCTGGCCAGACTTGATATTTCCCTGATTACTGGTATTGTTAATTGCCTAATCTGGTGAACCATTTCTGGTTGTGTTTCACCTCTTGTGTTCTTCTCTTGATACTGAGCACAAGAGACCCACTGCAGTGATGGCTCAAGCTTGCCTGCCACCTGGCnTCGTTTCCATCCAACTGATGTTGAGCTTGTTTCTTACTACTTGAAAAGGAAGATTATGGGGAAGAAACTTTTTGTTGAAGCTATTTCAGAGGTTGAGCTGTACAAATTTGCTCCTTGGGACCTTCCTGGTAATTTTCCTCTATTGTTITGATTTTTCTTGATGTAAGAATTGCACGCAAACGAGTTGGAAACTAATCTAGAATTTTTCAAATGCTTCCACTTTAGTTGAGTTTAGCTCTTACTTTTGTTGCAACCTTGTATATTTCCGTGCAGATAAATCCTGTCTCCAGAGCAAAGATCTTGAGTGGTTCTTCTTTTGTCCCCGTGACAAAAAATATCCTAAAGGGTCTAGGACGAACCGTGCCACACCAAATGGTTATTGGAAAACAAGTGGAAAAGACAGAACAATTGAGCTGAACTCTCGCATTGTTGGGTTGAAGAAAACATTGATTTTTCATGAAGGCAAGGCACCCAAAGGCAATAGGACTGACTGGGTGATGTATGAATACAAAATGGAAGATGAAACTTTGGTGTCTGCTGGTTTCTCTAAGGTACCAATAACTCTTATCTAATGTTTTTGTTTGTTTCACTTTATGTTAAATATGTTCAGTACAGTATATGAAAGATTCTACCATACTGGAATATGTTTTGAGGCATGCTCTTTTGCTTGTGCCTGAAATTGTGCGTGGTAAGTTAGCCTTTATATTTAGTTAGAAAGTTGTGATCCTAGTATTTCTTTTAAATTCTCTGCAAGTTTATTTAAATGTATTTGTTGTTTGTTTGTTTCAGGATGCTTATGTACTCTGCAAGATATTTAAAAAGAGTGGGCTTGGCCCAAGGATCGGCGAGCAATATGGGGCTCCTTTTGATGAAAATGAATGGGAGAATTTAGATGTTGGATCTTCTATCTrCTCTnTGCACCGTCGTCAGGTGTAGAGGATCCACAGGTTGAAAGTTCTGCCTTGGCTACTGCCATAGTTATGCAGGAACCATCCGCTCCTCAGCAGTCTGTTCAATnTCTGAACATGTTAATATCTGTTCTGATGAGGTCAATGACGCGCCTCCTGAAATTGATGGAATATTGTTGGACGAACTGGCAAAGTTnTGGATGATTCTCCGAACGATGATGTAGTTTTGCCAGAGGTATTGGCTTGCTGACTTCI7GCTrAACATGGTCTGTTTGCTTGTACCCAAGGAATTACAAATTATATACTAACCTTTATACATCTTACAGAATTCTGGTCrCCCrCCAATGTCTGAGCTTGAGGCTCAAGCTnTGAGATAAATACTGCTGAGCTCTATGACCAATTGGCAGGACTTTCCCAGTCAGGAGATATGTCTACTGTAAATTTCCCTGCTAGCAACGGGGATGTTATTGAAAACAATTTGCAGCAAGCAAACTCTGGACTTGCTATGGATGATGATTATATAGGGCTGGATGATCTGCTTGCTCCTGGGGAAACCTTCTCCTATGATTTTTCTGGTGAAACCTTTTCCTATGATCTGCCTGGTGGAAGCTTCTCCTATGATTTGTCTGTGCCAAACAATCAGTTTTTGCTGTACCCACTTGATCAATCAACTAGTGGCAGTCACTACGGTGTTGGTGCTACTCAATCAACTTTCGAAGCAAGTGGAACTCTTCCACCAATGCAACCAATGCCAAGCACTTTTAATGACATGCCTTCTGTTTCCAATTGCTTGAACCCAGCCATGAAAGACCCATTCTCTTAGACAGCAACTTTTGAGGTATAAGTCTGTTATCTTTGTGGTCATTGGTGCTGGAAGATGCTGCCGTTTCTGGTTTGGTTGCCCATCATTGTCTGATGAGGCTCTrGGTGGATCTTGGATGTTGGTCCCTGCTGTAGATGGTCCAACAATGGTGTTTGTATCATGGCATGGCTGCTGCTCAATTGAAGAAGCCGGAACTTGGTTGTGTCTTTGATCACGAGCACAATACTGGACAAGTGACCGAGCTrAGCTTACTGCTTrACCGCGGAGGAACTGTTTCTGCTGGTTTGGAAGATGTCTTATCTGGGTGGGGATGCTGCAGTCCACCGTAGTrCnTCTGTAATAACTGCCATGTTAGATGCCGAGTTGTCCATAAACCTAATGACAACTTCGTAGCTTAAGTCGCATCTTCTATCTATTTTCTGCCAATTTACTGTATTTGAACCATCGTCATTATTATTATTATTGTAATCGTGCCC55 tafe2-2 leader GCTTCCCCCAAAACCTCACCTCATCTCATCTCCTCCAC sequence AATTGACCAGACGGCGGCGCTCACCTCCTCCCCCTCGGCATCCCCCATCGTCCTGCTGGCTTCCCCGGTTCTCCC(Leader TCGTAAGCGCCCCTCCCGATCTCCCCCAATCCCTTCCTTCG sequence is ATCGATCTGGGTAGATCGGGGCCGATTGTTGCGAGTCATG bold; GTTCGTGTCGATCCGATGGCCAATTCGGGCGATCTGCGTCMutant bases CGATCCGTCGATTCTGAGGTGGTGGTTAAGCAATCGCCGC are underlined) ACGGATTGGTCTGGTCTTCCGTCCGTCGAAGTTTACGGCCGATTAGGGTTTGTGGTCGATCGATCGGTCGATCTACGCGTTGATTTGGGGTTATGCGATCCAGATGTTGTAGTAGCGTCGCGAAAGTTTGAGATTTGGTCGGGAATTGGATTGTTCAGCGTCTATTTAGGCTCGATTCCTTTGGGTGCCTTGTGATGGATCGGTGGATTCTATTAGATGGTTGAACTAATGGTCCGGAACTGATCCAAGGCAGTTTATTCTAGGAAACATCCGTTAGTTTTTTTTTTCCGTITGTGGGAAAAATTACTTTTGTTTTGTTAATAATATCATCTGTGCCTATGAAGTATCATAGCGTGTAAGTTTTGGTTAGATCGTAGGTTGGATGCTAATGTGCTTATTATATATTTTTATTTCTGCTGCACTGATGTGGTTTGTGATTAAGCTCAATATnTGGGGTACCTGAACTTGGTGAAAATATATTAGGTGCTCACAATGGTTTGATCTTTGGTTATCTAAGTGCAGCGCTGAAAGTCCTGTATGATCCTTTTCACAGTITrCTrTTTGTCAACTAGTTTTTTCATGAGTTACATCCGAGCCAATATGAAATATACTAGTAAAATCTGGTGAAGCGATGGTGTAGGTTAGATTCATGTTGTTAATGCGCTATTTCACnTGAATTGCGTACAATGTAAAATTCACTTGCTATATGCAGTCTTTTCTTTGCTTCCTGGGACTTTAGTTATTTGGATCGAAGGATAAAACATAGAACACATGTATTCTGTATTTTTCTGCTGTTTATGTTTTTCCTTTCTGACGATGTTTTCTAATCTCATGCAGGTTTTTGATCTCAGTCACTCATGTCTTTTGAAGTCACTTGATACAAGTGGATCATCTATTGCTACTTCTGGTTCATATATTGCATAAAATTCCGAGAATTCACTTGATACAAGTGGATCATCTGGAGTTTTGTGCGAGTACTTCCTATCATGTGAATCAGTGGTAGTTGGTTTAATTGGCAAGACTCAAACTTCTCTCATCTGAGTTCAGAAACATTTGTCGCTTGAGTCCTTGGTGTACTGCTTTCCTTGAGTCTTGCCAAGCCTGGTnTCCATAAGTTGTTTCTCCrCCAGCTTTTGGTTGGACCATTCTCTCCTCTGGTCTAATCTCATCCATCCTTTGATCCTCCCTTCAGAATAGTTGTCATAACGTGAGCTGTCCTCTGCTCACACGGCAATCTTCTGAAGCTCTCTTCTCTGGTTCTTGTTTGTTCTCTTGAGAGCTCCTCTCCTTAAGTTTGGTTGATCTGCGGCCTTGAATTCTGTCTTTCGAGGACTGAAGCTTTCTTGCCAAACTTGATATTTCCCTGATTTACTGGTATTGTTAATTGCATAATCTGGTGAAGCAATTCTGGTTGTGTTTCACCTCTTGTGTTCTTCTGCTGATACTGAGCGCAAGACACCCACTGCAGTGATGGCTCAAACTTGCCTGCCACCTGGCnTCGTTrCCATCCAACTGATGTrGAGCTTGTTTCTTACTACTTGAAAAGGAAGATTATGGGGAAGAAACTmTGTTGAAGCTATCTCAGAGGTTGAGCTGTACAAATTTGCTCCTTGGGACCTTCCTGGTAATTTTCATCTATTGTTTTATTTTTTATATGTAATAATTTCACCCAAATTGGTTGGAAATTAATCTATATTTCTCATAATGCTCTCACTTTGGTTGAATTTAGTTCTCAACTTTGTTGTGACCTTCTGTnTrCTGTGCAGATAAATCATGTCTTCAGAGCAAAGATCTTGAGTGGTTCTTCTmGCCCCCGTGACAAGAAGTATCCTAAAGGGTCTAGGACGAACCGTGCCACACCAAATGGTTATTGGAAAACAAGTGGAAAAGACAGAACAATTGAGCTGAACTCTCGCATTGTTGGGTTGAAGAAAACATTGATTTTTCATGAAGGCAAGGCACCGAAAGGCAACAGGACTGATTGGGTGATGTATGAATACAAAATGGAAGATGAAACTTTGGTGTCTGCTGGTTTCTCGAAGGTACTTTAAACTCTTATCTAATGTTTTTGTTTGTTTCACTTTTTTGAAGTATGTTCACTGTATATGAAAGATTCTACCATGCTGGAATATGTTTTGAGGCATGCTCTTTTGCTTATGGCTGAAACTGAGCGTGATTAGTTGCCTTTATATTTAGTTAGAAAGCTGTGATCCTAGTATTTCTTTCAAATTCTCTGCAAGCTTAATTAAATATATTTGCTGTCTGTTTGTTTCAGGATGCTTATGTACTCTGCAAGATATTTAAAAAGAGTGGGCTTGGCCCAAGGATTGGTGAGCAATATGGGGCTCCTTTTGATGAAAACGAATGGGAGAATTTAGATGTTGGATCTTCTATCTTCTCTnTGCACCGTCCTCAGGTGTAGAGGATCCACAGGTCGAAAGTTCTGCCTTGGCTACTGCAATAGTTATTCACGAACCATCCGCTCCTCAGCAATCTGTrCAATnTCCGAACATGTTAATATCTGTTCTGATCAGGTCAATGATGCGCCTCCTGAAATTGATGGGATATTGTTGGACGAACTGACAAAGTTTTTGAATGATTCTCCCAACCATGACGTACGTTCGCCGGAGGTATTAGCTTGCTGACTTCTTGCTTATCATATTCTGTATGnTGTACCGAAGGAATTATATTTTTITAATAACCTTTITACATCTnTGTTGTAGAATTCTGGTCTGCCTCCAATGTCTGAGCGTGAGGCTCAAGCTTTTGAGATAAATACTGCTGAGCTCTATGACCAATTGGCAGTACTTGCCCAGTCAGGAGATATGCCTGCTGCAAATTTCCCTGCTAGCAACGGGGATGTTATTGAAAACAATTTGCAGCAAACAAACTCTGGGCTTGCTATGGATGATGATTATATAGGGTTGGATGATCTGTTTGCTCCTGGGGAAACTTTCTCCTATGATnTTCTGGTGAAACATrCTCCTATGATTTGCCTGGTGGAACCTTCTCCTATTTGTCTGTGCCAAACAATCAGTTTTCGCAGTACCCACTGGATCAATCATCTAATGGCCTTCACTACGGTGATGGTGCCACTCAATCAACTTTCTTAGCAAGTGGACCGCTTCCACCATTGGAACCAATGCCTAGCACTTTTGATGACATGCCTTCTGTTTCCAATAAGCCTGCAAGCTCCTACTGCTTGAACCCAGCCATGAAAGACCCATTCTCATAGATAGCAACTTTTGAGGTACCAAGTCTGTTACCTTTGTGGTCATTGATGTTGGAAGTTGCTGCTGATTCTGGTTTTGGTTGTCCATCATTGTCTGATGTGACTCTTGATGGATCTTGGATGTTGGTCCCTGCTCGAGATGACCCAACAATGGTGTTTGTATCATGGCATGGCTGCTGCTCTATTGAAGAAGCCGGAACTTGGTTGTGTCTCAATCACGAGCACAGACCGAGCTTAGCTTACTGCTATACCGCGGAGAAACTGTTTTCTGCTGGTTTGGAAGATGTCTTGTCTGGGTGGAGATGCTGCAGTCCATCTTGTTCTTTCTGTAATAACTGCCATGTTTGATGCGTTGTCCATAAACCTAGTGACAACTTAATAGCTrAAGTCGCATCTTCTATCTATnTCTGTCAAAGTAGTACTACTGTATTTGAACCATCGTCATTAATATTATTATTATTATTATTGTAAACCATCATTATGTTTTTGTCGCCACAATGCTTGATGTCTTC56 OsIDEFl / OsF AGTAGAGATGAGATAGAGAAGAGAGATAGAGAGAGAGGEl m5 CAGGCTGGTAGAACGGGTCACCTCACCACCTTCGCCTCCG comprising CCTCCTCCTTCGCCTCCGTCTCCACCTCTCCTCTCCTCTTCCLS: TCTTCTTCCTCCTCCTGGGGCCTAGCCGCCTAGGGGGGTTGTGGCCTGCGACTGCGACTTCTTCCTCTGCCTTCTGCTCTC(The stait>stop TGCCTCTGAGTATATGGAATATGGTGTGTGCGTGAAGAGA mutated codon AATTAAGTGAGGGAGGGGAAAGCGAGCGAACAGGGCAA is shown in CA bold)57 OsIDEFl / OsF AGATGAGTAGAGATAGAGAAGAGAGATAGAGAGAGAGGEl m6 CAGGCTGGTAGAACGGGTCACCTCACCACCTTCGCCTCCG comprising CCTCCTCCTTCGCCTCCGTCTCCACCTCTCCTCTCCTCTTCCLS: TCTTCTTCCTCCTCCTGGGGCCTAGCCGCCTAGGGGGGTTGTGGCCTGCGACTGCGACTTCTTCCTCTGCCTTCTGCTCTC(The start>stop TGCCTCTGAGTATATGGAATATGGTGTGTGCGTGAAGAGA mutated codon AATTAAGTGAGGGAGGGGAAAGCGAGCGAACAGGGCAA is shown in CA bold; uORF sequences are underlined)58 OsIDEFl / OsF AGATGAGATGAGATAGAGAAGAGAGATAQAGAGAGAGGEl m7 CAGGCTGGTAGAACGGGTCACCTCACCACCTTCGCCTCCG comprising CCTCCTCCTTCGCCTCCGTCTCCACCTCTCCTCTCCTCTTCCLS: TCTTCTTCCTCCTCCTGGGGCCTAGCCGCCTAGGGGGGTTGTGGCCTGCGACTGCGACTTCTTCCTCTGCCTTCTGCTCTC(The start>stop TGCCTCTGAGTATTAGGAATATGGTGTGTGCGTGAAGAGA mutated codon AATTAAGTGAGGGAGGGGAAAGCGAGCGAACAGGGCAA is shown in CA bold; uORF sequences are underlined)59 OsIDEFl / OsF AGATGAGATGAGATAGAGAAGAGAGATAGAGAGAGAGGEl m8 CAGGCTGGTAGAACGGGTCACCTCACCACCTTCGCCTCCGcomprising CCTCCTCCTTCGCCTCCGTCTCCACCTCTCCTCTCCTCTTCCLS: TCTTCTTCCTCCTCCTGGGGCCTAGCCGCCTAGGGGGGTTGTGGCCTGCGACTGCGACTTCTTCCTCTGCCTTCTGCTCTC(The start>stop TGCCTCTGAGTATATGGAATTAGGTGTGTGCGTGAAGAGA mutated codon AATTAAGTGAGGGAGGGGAAAGCGAGCGAACAGGGCAA is shown in CA bold; uORF sequences are underlined)

Claims

CLAIMS:

1. A method of enhancing the expression of an iron-deficiency response gene in a plant cell, the method comprising introducing a modification to an upstream open reading frame (uORF) of a 5' untranslated region (UTR) of the iron-deficiency response gene, wherein the modification comprises a deletion, addition, or substitution of at least one nucleotide of the uORF.

2. The method of claim 1, wherein the iron-deficiency response gene is selected from the group consisting of IDEF1, IDEF2 and orthologs of any of the foregoing.

3. The method of claim 2, wherein the iron-deficiency response gene is IDEF1 or an ortholog thereof.

4. The method of claim 3, wherein the IDEF1 or ortholog thereof comprises a nucleic acid sequence that encodes a polypeptide having an amino acid sequence selected fiom the group consisting of SEQ ID NOs: 1-8, and amino acid sequences having at least 70% sequence identity to any of the foregoing.

5. The method of any one of claims 2-4, wherein the 5' UTR of the 1DEF1 or ortholog thereof comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 17-24 and nucleic acid sequences having at least 70% sequence identity to any of the foregoing.

6. The method of claim 2, wherein the iron-deficiency response gene is IDEF2 or an ortholog thereof.

7. The method of claim 6, wherein the IDEF2 or ortholog thereof comprises a nucleic acid sequence that encodes a polypeptide having an amino acid sequence selected fiom the group consisting of SEQ ID NOs: 9-16 and amino acid sequences having at least 70% sequence identity to any of the foregoing.

8. The method of claim 6 or claim 7, wherein the 5' UTR of the IDEF2 or ortholog thereof comprises a nucleic acid sequence selected fiom the group consisting of SEQ ID NOs: 25- 32 and nucleic acid sequences having at least 70% sequence identity to any of the foregoing.

9. The method of any one of claims 1 to 8, wherein the enhanced expression of the iron- deficiency response gene results in an upregulation of an iron homeostasis gene, wherein theiron homeostasis gene comprise at least one iron deficiency-responsive cis-regulatory element.

10. The method of claim 9, wherein the at least one iron deficiency-responsive cis- regulatory element comprises:(a) an IDE1 or an IDE-like motif; or(b) an IDE2 or an IDE2-like motif.

11. A method for selecting a target modification to be introduced into an upstream open reading frame (uORF) of a 5' untranslated region (UTR) of a gene of interest in a plant cell, wherein said modification produces a desirable trait in the plant cell, the method comprising:(i) introducing a modification to the uORF of the 5' UTR to produce a modified uORF, wherein the modification comprises a deletion, addition, or substitution of at least one nucleotide of the uORF;(ii) fusing the modified uORF of step (i) to a polynucleotide that encodes a reporter protein to produce a fusion polynucleotide;(iii) introducing the fusion polynucleotide of step (ii) into a host cell to produce a modified host cell;(iv) exposing the modified host cell of step (iii) to conditions that favour expression of the fusion polynucleotide in the modified host cell;(v) determining the level of expression of the polynucleotide that encodes the reporter protein following step (iv);(vi) comparing the level of expression of the polynucleotide that encodes the reporter protein determined in step (v) with a level of expression of the polynucleotide that encodes the reporter protein in a host cell comprising a corresponding fusion polynucleotide comprising an unmodified uORF and a polynucleotide that encodes the reporter protein; and(iv) selecting the modification to the uORF that results in an enhanced level of expression of the polynucleotide that encodes the reporter protein for introduction into an endogenous locus of the uORF of plant cell genome.

12. The method of claim 11, wherein the 5' UTR comprising the modified uORF comprises a nucleic acid sequence having at least 70% sequence identity to any one of SEQ ID NOs: 33-43.

13. The method of any one of claims 1-12, wherein modifying the uORF comprises replacing at least one start codon in the uORF with a stop codon.

14. The method of any one of claims 1-13, wherein modifying the uORF comprises deleting at least one base pair in the uORF.

15. The method of claim 14, wherein modifying the uORF comprises deleting at least two base pairs in the uORF.

16. The method of claim 15, wherein modifying the uORF comprises deleting at least one start codon in the uORF.

17. The method of any one of claims 14-16, wherein modifying the uORF comprises deleting from about 5 to about 100 base pairs in the uORF.

18. The method of any one of claims 14-17, wherein modifying the uORF comprises deleting the uORF.

19. The method of any one of claims 1-18, wherein modifying the uORF comprises site- directed nucleases or oligonucleotide-directed mutagenesis.

20. The method of claim 19, wherein said mutagenesis is achieved using SDN-1.

21. The method of claim 19, wherein said mutagenesis is achieved using Zinc finger nucleases, TALENS and / or CRISPR / Cas gene editing and / or base editing.

22. The method of claim 21, wherein said mutagenesis is achieved using CRISPR / Cas9 or CRISPR / Casl2 gene editing.

23. The method of any claims 1-22, wherein the plant cell is a leaf cell, a root cell, an embryonic cell, an egg cell or a pollen cell.

24. The method of any one of claims 1-23, wherein the plant is elected from the group consisting of rice, barley, wheat, soybean, alfalfa, rye, oats, millet, sorghum, triticale, buckwheat, quinoa, spelt, einkom, amaranth, grass, com, canola, wild rice, teff, banana, cassava, brassica, field mustard, cabbage, Aegilops tauschii, brachypodium, Miscanthus, switchgrass, poplar, pine, Eucalyptus, acacia, birch, hazel, willow, bamboo, grape, kiwifruit, apple, pear, avocado, almond, pistaccio, walnut, chestnut, a vine, a forage grass, alfalfa, a plant of the genus Prunus, sweet potato, yam, a leguminous plant, and a plant of the nightshade family.

25. The method of any one of claims 1-24, wherein the plant cell is a wheat cell or a rice cell.

26. A plant cell produced by the method of any one of claims 1-25.

27. A modified plant cell comprising a polynucleotide capable of controlling iron homeostasis in the plant cell, wherein the polynucleotide comprises an iron-deficiency response gene, wherein the iron-deficiency response gene comprises a nucleic acid sequence comprising a modification to a uORF in the 5' UTR of the iron deficiency response gene, wherein the modification comprises a deletion, addition, or substitution of at least one nucleotide in the uORF when compared to a corresponding nucleic acid sequence of a wildtype uORF of the same plant species, wherein the modification to the uORF results in an increase in the level of expression of the iron deficiency response gene when compared to the level of expression of the iron deficiency response gene in a control plant cell harbouring a corresponding native iron deficiency response gene of the same plant species.

28. The modified plant cell of claim 27, wherein the modified plant cell exhibits a desirable trait selected from the group consisting of increased cellular iron content, increased iron uptake, darker coloration, increased photosynthesis, increased size, increased vigour, increased yield, increased tolerance to abiotic stress, increased tolerance to biotic stress, and increased iron transport.

29. A plant cell comprising an iron-deficiency response gene having a modified uORF when compared to a corresponding native uORF of the same plant species, wherein the modified uORF comprises a deletion, substitution or insertion of at least one nucleotide when compared to the corresponding wild-type uORF of the same plant species, and wherein said modification enhances expression of the iron-deficiency response gene in the plant cellwhen compared to a plant cell of the same plant species harbouring the corresponding wildtype uORF of the same plant species.

30. The plant cell of any one of claims 26-29, wherein the iron-deficiency response gene is selected from the group consisting of 1DEF1, IDEF2 and orthologs of any of the foregoing.

31. The plant cell of claim 30, wherein the iron-deficiency response gene is IDEF1 or an ortholog thereof.

32. The plant cell of claim 31, w'herein the iron-deficiency response gene comprises a nucleic acid sequence that encodes a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-8 and amino acid sequences having at least 70% sequence identity to any of the foregoing.

33. The plant cell of claim 30, wherein the iron-deficiency response gene is IDEF2 or an ortholog thereof.

34. The plant cell of claim 33, wherein the iron-deficiency response gene comprises a nucleic acid sequence that encodes a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 9-16 and amino acid sequences having at least 70% sequence identity to any of the foregoing.

35. The plant cell of any one of claims 26-34, wherein the uORF has been modified by replacing at least one start codon of the corresponding wild-type uORF of the same plant species with a stop codon.

36. The plant cell of any one of claims 26-35, wherein the uORF has been modified by deleting at least one base pair of the corresponding wild-type uORF of the same plant species.

37. The plant cell of claim 36, wherein the modified uORF comprises a deletion of at least 2 base pairs of the corresponding wild-type uORF ofthe same plant species.

38. The plant cell of claim 36 or claim 37, wherein the modified uORF comprises a deletion of at least a start codon of the corresponding wild-type uORF of the same plant species.

39. The plant cell of any one of claims 36-38, wherein the entire uORF ofthe corresponding wild-type uORF of the same plant species has been deleted.

40. The plant cell of any one of claims 26-39, wherein the modified uORF results in enhancing the expression of the gene of interest by at least about 10% when compared to a level of expression of the gene of interest in a plant cell harbouring a corresponding wildtype uORF of the same plant species.

41. The plant cell of any one of claims 26-40, wherein the plant cell exhibits an enhanced iron content by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9% or at least 10% higher when compared to the iron content of a plant cell harbouring a corresponding wild-type uORF of the same plant species.

42. A polynucleotide comprising the nucleic acid sequence selected from the group consisting of SEQ ID NOs: 33-43 and nucleic acid sequences having at least 70% sequence identity thereto.

43. A polynucleotide construct comprising the polynucleotide of claim 42.

44. An expression vector comprising the polynucleotide of claim 42 or the polynucleotide construct of claim 43.

45. A plant comprising the plant cell of any one of claims 26-41, the polynucleotide of claim 42, the polynucleotide construct of claim 43, or the expression vector of claim 44.

46. The plant of claim 45, wherein the plant is selected from the group consisting of rice, barley, wheat, soybean, alfalfa, rye, oats, millet, sorghum, triticale, buckwheat, quinoa, spelt, einkom, amaranth, grass, com, canola, wild rice, teff, banana, cassava, brassica, field mustard, cabbage, Aegilops tauschii, brachypodium, Miscanthus, switchgrass, poplar, pine, Eucalyptus, acacia, birch, hazel, willow, bamboo, grape, kiwifruit, apple, pear, avocado, almond, pistachio, walnut, chestnut, a vine, a forage grass, alfalfa, a plant of the genus Primus, sweet potato, yam, a leguminous plant and a plant of the nightshade family.

47. The plant of claim 46, wherein the plant is a wheat plant or a rice plant.

48. A seed produced by the plant of claim 46 or claim 476.

49. A plant derived from the seed of claim 48.

50. A plant part derived from the plant of any one of claims 45-47 and 49.

51. The plant part of claim 50, wherein the plant part is selected from the group consisting of a seed, grain, fruit, leaf, flower, tuber, stalk, rhizome, spore, cutting, nut, and root.

52. The plant part of claim 51 , wherein the plant part is grain.

53. The plant part of claim 52, wherein the grain is selected from the group consisting of wheat grain, rye grain, barley grain or oat grain.

54. The plant part of claim 52, wherein the grain is rice.

55. A flour produced from the grain of any one of claims 51-53.

56. A food product comprising the plant of any one of claims 44-46 and 48, the seed of claim 47, the plant part of claim 50, the grain of any one of claims 51-53, or the flour of claim 54.

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