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WO2025236045A1 - Regulation of gene expression - Google Patents

Regulation of gene expression

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Publication number
WO2025236045A1
WO2025236045A1 PCT/AU2025/050512 AU2025050512W WO2025236045A1 WO 2025236045 A1 WO2025236045 A1 WO 2025236045A1 AU 2025050512 W AU2025050512 W AU 2025050512W WO 2025236045 A1 WO2025236045 A1 WO 2025236045A1
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WIPO (PCT)
Prior art keywords
uorf
plant
ggp
modified
amino acid
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PCT/AU2025/050512
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French (fr)
Inventor
Alexander Arthur Theodore JOHNSON
Ronan Charles BROAD
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University of Melbourne
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University of Melbourne
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Priority claimed from AU2024901433A external-priority patent/AU2024901433A0/en
Application filed by University of Melbourne filed Critical University of Melbourne
Publication of WO2025236045A1 publication Critical patent/WO2025236045A1/en
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8262—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield involving plant development
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/07—Nucleotidyltransferases (2.7.7)
    • C12Y207/07069—GDP-L-galactose phosphorylase (2.7.7.69)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
    • A01H6/46—Gramineae or Poaceae, e.g. ryegrass, rice, wheat or maize
    • A01H6/4636—Oryza sp. [rice]

Definitions

  • the present invention relates generally to control and manipulation of expression of GDP-L-Galactose phosphorylase (GGP) and ascorbate production in improving plant growth and agronomical traits.
  • GGP GDP-L-Galactose phosphorylase
  • the invention also relates to sequence elements controlling GGP expression and production, and methods of their use.
  • BACKGROUND OF THE INVENTION [0002] 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.
  • L-ascorbic acid also known as ascorbate or vitamin C, and hereafter referred to ascorbate
  • Ascorbate is a reducing agent capable of donating electrons and primarily functions as a cellular antioxidant and enzymatic co-factor.
  • Ascorbate is the most abundant water-soluble antioxidant and is well- known for its important role in photosynthetic functions and stress tolerance.
  • Crop plants such as citrus plants, tomatoes, and plants with starchy tuberous roots such as yams, potatoes and cassava have relatively high ascorbate content.
  • cereal grain plants (for example, rice; wheat; barley; and sorghum) provide more food energy worldwide than any other type of crop but have low or negligible ascorbate levels.
  • rice and wheat are staple crops in many parts of the world; and is cultivated on more land than any other crops.
  • a modified genome of a cereal grass plant comprising a polynucleotide comprising a modification to an upstream open reading frame (uORF) in a 5' untranslated region (UTR) of a GDP-L-galactose phosphorylase (GGP) gene or an ortholog thereof, wherein the modification comprises a deletion, addition, or substitution of at least one base pair in the 5' UTR of the GGP gene.
  • the expression of the GGP gene is not silenced.
  • the modified plant has increased grain crop yield.
  • the increase in grain crop yield is increased grain weight per plant.
  • the modified plant has an increase in the number of panicles per plant. In another embodiment, the modified plant has increased foliar ascorbate levels [0012]
  • the GGP gene 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: 59-81, and amino acid sequences having at least 70% sequence identity to any of the foregoing.
  • the 5' UTR of GGP gene or ortholog thereof comprises a nucleic acid sequence selected from the group of SEQ ID NOs:11, 15-17, 21-23, 25, 28, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 50-51, and 54-55, and nucleic acid sequences having at least 70% sequence identity to any of the foregoing.
  • the modified uORF of GGP comprises at least one deletion or non-conservative substitution of at least one base pair in the uORF.
  • the modified uORF of GGP comprises deletion or non- conservative substitution of 1 to 100 base pairs in the uORF.
  • the modified uORF of GGP comprises deletion or non-conservative substitution of 2 to 80 base pairs in the uORF. In another embodiment, wherein the modified uORF of GGP comprises deletion or non-conservative substitution of 5 to 70 base pairs in the uORF. [0016] In some embodiments, the modified uORF of GGP comprises non-conservative substitution of 1 base pair in the uORF. In another embodiment, the modified uORF of GGP comprises deletion of 5 base pairs in the uORF. In another embodiment, the modified uORF of GGP comprises deletion of 63 base pairs in the uORF.
  • the modified uORF of GGP results in disruption of the reading frame of the GGP uORF, wherein the GGP uORF comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 12-14, 18-20, 24, 26-27, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48-49, and 52-53, and amino acid sequences having at least 70% sequence identity to any of the foregoing.
  • the disruption of the uORF reading frame results in amino acid substitution, deletion or insertion of one or more amino acid residues from position 30 to position 61 the GGP uORF peptide.
  • the disruption of the uORF reading frame comprises substitution of an amino acid sequence comprising the amino acid residues at position 57-61 of SEQ ID NO:10. In some embodiments, the disruption of the uORF reading frame comprises deletion of an amino acid sequence comprising the amino acid residues at position 55-56 of SEQ ID NO: 10. In some embodiments, the disruption of the uORF reading frame comprises deletion of an amino acid sequence comprising the amino acid residues at position 37-57 of SEQ ID NO:10. [0019] In some embodiments, the modified GGP uORF peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 56-58.
  • modifying the uORF comprises site-directed nucleases or oligonucleotide-directed mutagenesis.
  • the mutagenesis is achieved using SDN-1.
  • the mutagenesis is achieved using Zinc finger nucleases, TALENS and / or CRISPR/Cas gene editing and/or base editing.
  • the mutagenesis is achieved using CRISPR/Cas9 or CRISPR/Cas12 gene editing.
  • the cereal grass plant is selected from the group consisting of rice, barley, wheat, rye, oats, millet, sorghum, triticale, buckwheat, quinoa, spelt, einkorn, amaranth, grass, corn, wild rice, teff; Aegilops tauschii, brachypodium, Miscanthus, switchgrass or a forage grass.
  • the cereal grass plant is a wheat plant or a rice plant. In another embodiment, the cereal grass plant is a rice plant.
  • a polynucleotide construct comprising a modification to an upstream open reading frame (uORF) in a 5' untranslated region (UTR) of a GDP-L-galactose phosphorylase (GGP) gene or an ortholog thereof, wherein the modification comprises a deletion, addition, or substitution of at least one base pair of in the 5' UTR of the GGP gene.
  • the GGP gene 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: 59-81, and amino acid sequences having at least 70% sequence identity to any of the foregoing.
  • the 5' UTR of the GGP gene or ortholog thereof comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 11, 15-17, 21- 23, 25, 28, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 50-51, and 54-55 and nucleic acid sequences having at least 70% sequence identity to any of the foregoing.
  • the present disclosure also extends to a modified cereal grass plant comprising a polynucleotide comprising a modification to an upstream open reading frame (uORF) in a 5' untranslated region (UTR) of a GDP-L-galactose phosphorylase (GGP) gene or an ortholog thereof, wherein the modification comprises a deletion, addition, or substitution of at least one base pair of in the 5' UTR of the GGP gene.
  • uORF upstream open reading frame
  • UTR 5' untranslated region
  • GGP GDP-L-galactose phosphorylase
  • a method of modifying the genome of a cereal grass plant comprising introducing a modification to an upstream open reading frame (uORF) in a 5' region (UTR) of a GDP-L-galactose phosphorylase (GGP) gene or an ortholog thereof, wherein the modification comprises a deletion, addition, or substitution of at least one base pair of in the 5' UTR of the GGP gene.
  • uORF upstream open reading frame
  • UTR 5' region
  • GGP GDP-L-galactose phosphorylase
  • a method of increasing the grain crop yield of a cereal grass plant comprising introducing a modification to an upstream open reading frame (uORF) in a 5' untranslated region (UTR) of a GDP-L-galactose phosphorylase (GGP) gene or an ortholog thereof, wherein the modification comprises a deletion, addition, or substitution of at least one base pair of in the 5' UTR of the GGP gene.
  • the increase in grain crop yield is increased grain weight per plant.
  • the increase in grain crop yield is the result of an increase in the number of panicles per plant.
  • a method of increasing the number of panicles of a cereal grass plant comprising introducing a modification to an upstream open reading frame (uORF) in a 5' untranslated region (UTR) of a GDP-L- galactose phosphorylase (GGP) gene or an ortholog thereof.
  • uORF upstream open reading frame
  • UTR 5' untranslated region
  • GGP GDP-L- galactose phosphorylase
  • the modified plant has increased grain crop yield.
  • the increase in grain crop yield is increased grain weight per plant.
  • the modified plant has an increase in the number of panicles per plant.
  • the modified plant has increased foliar ascorbate levels
  • the GGP gene 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: 59-81, and amino acid sequences having at least 70% sequence identity to any of the foregoing.
  • the 5' UTR of GGP gene or ortholog thereof comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 11, 15-17, 21-23, 25, 28, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 50-51, and 54-55 and nucleic acid sequences having at least 70% sequence identity to any of the foregoing.
  • the modified uORF of GGP comprises at least one deletion or non-conservative substitution of at least one base pair in the uORF.
  • the modified uORF of GGP comprises deletion or non- conservative substitution of 1 to 100 base pairs in the uORF.
  • the modified uORF of GGP comprises or non-conservative substitution of 2 to 80 base pairs in the uORF.
  • the modified uORF of GGP comprises deletion or non-conservative substitution of 5 to 70 base pairs in the uORF.
  • the modified uORF of GGP comprises non-conservative substitution of 1 base pair in the uORF.
  • the modified uORF of GGP comprises deletion of 5 base pairs in the uORF. In another embodiment, the modified uORF of GGP comprises deletion of 63 base pairs in the uORF. [0037] In some embodiments, the modified uORF of GGP results in disruption of the reading frame of the GGP uORF, wherein the GGP uORF comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 12-14, 18-20, 24, 26-27, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48-49, and 52-53, and amino acid sequences having at least 70% sequence identity to any of the foregoing.
  • the disruption of the uORF reading frame results in amino acid substitution, deletion or insertion of one or more amino acid residues from position 30 to position 61 the GGP uORF peptide.
  • the disruption of the uORF reading frame comprises substitution of an amino acid sequence comprising the amino acid residues at position 57-61 of SEQ ID NO:10.
  • the disruption of the uORF reading frame comprises deletion of an amino acid sequence comprising the amino acid residues at position 55-56 of SEQ ID NO :10.
  • the disruption of the uORF reading frame comprises deletion of an amino acid sequence comprising the amino acid residues at position 37-57 of SEQ ID NO: 10.
  • the modified GGP uORF peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 56-58. [0040] In some embodiments, wherein the modified GGP uORF results in reduced ribosomal stalling in the uORF. [0041] In some embodiments, modifying the uORF comprises site-directed nucleases or oligonucleotide-directed mutagenesis. In some embodiments, the mutagenesis is achieved using SDN-1. In some embodiments, the mutagenesis is achieved using Zinc finger nucleases, TALENS and / or CRISPR/Cas gene editing and/or base editing.
  • the mutagenesis is achieved using CRISPR/Cas9 or CRISPR/Cas12 gene editing.
  • the plant is selected from the group consisting of rice, barley, wheat, rye, oats, millet, sorghum, triticale, buckwheat, quinoa, spelt, einkorn, amaranth, grass, corn, wild rice, teff; Aegilops tauschii, brachypodium, Miscanthus, switchgrass or a forage grass.
  • the cereal grass plant is a wheat plant or a rice plant. In another embodiment, the cereal grass plant is a rice plant.
  • the present disclosure also extends to a plant cell produced by the methods disclosed herein. [0044] The present disclosure also extends to a plant comprising any one of the modified genomes or the polynucleotides disclosed herein. [0045] The present disclosure also extends to seeds produced by the plants disclosed herein, to plants derived from such seeds, and to plant parts derived from the plants disclosed herein. [0046] 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, a panicle and root. [0047] In a further embodiment, the plant part is grain.
  • the plant part is a germinated grain.
  • the grain is selected from the group consisting of rice grain, wheat grain, rye grain, barley grain or oat grain.
  • the grain is rice.
  • 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. BRIEF DESCRIPTION OF THE DRAWINGS [0052]
  • Figure 1 shows the ascorbate-glutathione cycle—a major antioxidant system of plants cells. In this cycle electrons flow from NADPH to H2O2.
  • FIG. 1 shows production and characterization of independent rice transformation events constitutively overexpressing CDS.
  • RB right border; 2 x 35S, dual CaMV 35S promoter; OsGGP, OsGGP CDS; nos T, nopaline synthase terminator; 2 x 35S enhanced, dual CaMV 35S promoter enhanced; hptII, hygromycin phosphotransferase II; pA, CaMV poly(A) signal; LB, left border.
  • Ascorbate concentrations in BR and GBR of T3 homozygous NS and 35S-OsGGP shown in grey). Bars represent mean ⁇ SEM of three independent replicates of approximately fifty grains.
  • Figure 3 shows salt tolerance assessment of NS and 35S-OsGGP plants during the vegetative growth phase. Foliar ascorbate concentrations of (a) control and (b) salt-stressed T4 homozygous NS-1 and 35S-OsGGP-1 plants and T3 homozygous NS-2 and 35S- OsGGP-2 plants at DAP 40. Bars represent mean ⁇ SEM of six biological replicates.
  • Asterisks indicate statistically significant differences between NS and 35S-OsGGP plants (two-sample t-test; ** p-value ⁇ 0.01; *** p-value ⁇ 0.001).
  • the sPSA RGR of (c) control and (d) salt-stressed T4 homozygous NS-1 and 35S-OsGGP-1 plants and of (e) control and (f) salt-stressed T3 homozygous NS-2 and 35S-OsGGP-2 plants. Salt was applied at DAP 20 and 23. Values represent mean ⁇ half least significant (5%) pairwise difference of six biological replicates. Non-overlapping error bars indicate significant differences at ⁇ 0.05.
  • Figure 4 shows CRISPR/Cas9-targeted mutagenesis of the OsGGP uORF. Schematic representation of the (a) p35S-Cas9-uORF and (b) pUbi-Cas9-uORF T-DNA.
  • LB left border
  • pA CaMV poly(A) signal
  • hptII hygromycin phosphotransferase II
  • 35S CaMV 35S promoter
  • tRNA transfer RNA
  • sgRNA single guide RNA
  • 2 x 35S dual CaMV 35S promoter
  • Ubi maize ubiquitin promoter
  • hSpCas9 human codon optimized Streptococcus pyogenes Cas9; nos, nopaline synthase terminator
  • RB right border.
  • Figure 5 shows nucleotide alignment of the parental T0 CRISPR/Cas9- induced uorfOsGGP mutants used in this chapter.
  • the sgRNA target site is underlined.
  • the protospacer-adjacent motif, sequence changes, and deleted nucleotides are shown in bold, red, and as hyphens, respectively.
  • Figure 6 shows nucleotide and peptide sequence alignment of the CRISPR/Cas9- induced homozygous, transgene-free uorfOsGGP mutants used in this chapter. (a) Nucleotide sequence alignment.
  • FIG. 7 shows transcript analysis of WT and uorfOsGGP plants. Transcript levels of the (a) OsGGP, (b) OsGME1, (c) OsGME2, and (d) OsGPP genes in T2 homozygous, transgene-free WT and uorfOsGGP plants. Bars represent mean ⁇ SEM of six biological replicates.
  • Figure 8 shows ascorbate concentrations of WT and uorfOsGGP plants.
  • the sPSA AGR of (c) control and (d) salt- stressed T2 homozygous, transgene-free WT and uorfOsGGP-1 plants. Salt was applied at DAP 20 and 23. Values represent mean ⁇ half least significant (5%) pairwise difference of six biological replicates. Non-overlapping error bars indicate significant differences at ⁇ 0.05.
  • 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 that recite the lower value of the range as well as claim limitations that recite the upper value of the range.
  • 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.
  • 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 a GGP gene or an ortholog thereof, wherein the modification comprises a deletion, addition, or substitution of at least one base pair of in the 5' UTR of the GGP gene, unexpectedly results in increased grain crop yield, increased grain weight per plant; increased number of panicles and/or increased foliar ascorbate levels.
  • UORF untranslated open reading frame
  • UTR untranslated region
  • a modified genome of a cereal grass plant comprising a polynucleotide comprising a modification to an upstream open reading frame (uORF) in a 5' untranslated region (UTR) of a GDP-L-galactose phosphorylase (GGP) gene or an ortholog thereof, wherein the modification comprises a deletion, addition, or substitution of at least one base pair of in the 5' UTR of the GGP gene.
  • uORF upstream open reading frame
  • UTR 5' untranslated region
  • GGP GDP-L-galactose phosphorylase
  • a modified cereal grass plant comprising a polynucleotide comprising a modification to an upstream open reading frame (uORF) in a 5' untranslated region (UTR) of a GDP-L-galactose phosphorylase (GGP) gene or an ortholog thereof, wherein the modification comprises a deletion, addition, or substitution of at least one base pair of in the 5' UTR of the GGP gene.
  • uORF upstream open reading frame
  • UTR 5' untranslated region
  • GGP GDP-L-galactose phosphorylase
  • UTR upstream open reading frame
  • GGP GDP-L-galactose phosphorylase
  • Ascorbate is a reducing agent capable of donating electrons and primarily functions as a cellular antioxidant and enzymatic co-factor.
  • Ascorbate is the most abundant water- soluble antioxidant and is well-known for its important role in photosynthetic functions and stress tolerance.
  • Ascorbate is an essential micronutrient and has key roles in human physiology, such as promoting the dietary uptake of iron (Fe) in the gut. It is thought that increasing ascorbate concentrations in crops may have the potential to improve both the abiotic stress tolerance of crops.
  • the L-galactose pathway which converts D-fructose-6-P to ascorbate via eight enzymatic steps (by phosphomannose isomerase (PMI), phosphomannose mutase (PMM), GDP-D-mannose pyrophosphorylase (GMP), GDP-D- mannose-3′,5′-epimerase (GME), GDP-L-galactose phosphorylase (GGP), L-galactose-1- phosphate phosphatase (GPP), L-galactose dehydrogenase (L-GalDH), and L-galactono- 1,4-lactone dehydrogenase (L-GalLDH)), is the dominant pathway leading to ascorbate biosynthesis in many plants including Arabidopsis thaliana, tomato and rice.
  • PMI phosphomannose isomerase
  • PMM phosphomannose mutase
  • GMP GDP-D-mannose pyrophosphorylase
  • GGP GDP-L-galactose phosphorylase
  • GGP gene has increased ascorbate concentrations 2.9- to 4.1-fold in Arabidopsis thaliana; 3.1-fold in potato; 2.1-fold in strawberry and 2.0- to 6.2-fold in tomato (Zhou et al. 2012 Biol Plantarum; Bulley et al.2012 Plant Biotechnol J). Increased ascorbate levels in some plants have been associated with in abnormalities in fruit development, seed deformities and/or delayed transition into reproductive stage (i.e. fruiting stage) (see Deslous et al. 2021 Journal of Experimental Botany 72: 3091–3107; Bulley et al 2012 Plant Biotechnology Journal 10: 390-397).
  • a graminaceous plant is a plant from the Gramineae family (also known as Poaceae family) of monocotyledonous flowering plants commonly known as grasses (including cereal grass plants, bamboos and grass species associated with grassland, pasture).
  • a cereal grass plant as described herein refers to an edible grain grass plant.
  • a cereal grass plant may be a grass plant that is cultivated for its edible grain, wherein the grain is edible by humans.
  • a cereal grass plant may be a grass plant that is cultivated for its edible grain, wherein the grain is edible by animals, including ruminant animals.
  • Examples of graminaceous cereal grass plants include rice, wheat, rye, oats, barley, millet and maize.
  • Other types of edible grain plants include buckwheat, quinoa and chia.
  • Other examples of cereal grass plants include sorghum, triticale, spelt, einkorn, amaranth, wild rice, teff, Aegilops tauschii, brachypodium, and forage grass.
  • the cereal grass plant is a rice, barley, wheat, rye, oats, millet, sorghum, triticale, buckwheat, quinoa, spelt, einkorn, amaranth, grass, corn, wild rice, teff; Aegilops tauschii, brachypodium, Miscanthus, switchgrass or a forage grass plant.
  • the cereal grass plant is a graminaceous cereal grass plant.
  • the cereal grass plant is a rice, wheat, rye, oats, barley, millet or maize plant.
  • the cereal grass plant is a rice or wheat plant.
  • Table 1 provides a list of GGP genes and their corresponding sequence records.
  • Table 1 Non-limiting examples of GGP genes and orthologs Species Gene ID(s) SEQ ID SEQ ID ide) TaGGP2-B (Wheat 64 87 subgenome B) GG upstream open readng rame (uO )
  • uORF upstream open reading frame
  • the GGP uORF is proposed to initiate from a non-canonical AUC or ACG start- codon, and encode a 60- to 65-residue long peptide. Disruption of the GGP uORF increased ascorbate concentrations when a GGP promoter-uORF-GGP construct was transiently transformed in Nicotiana benthamiana (Laing et al.2015 Plant Cell).
  • uORFs upstream open reading frames, or “uORFs”, as used herein refers to elements located upstream of the 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.
  • 5' untranslated region refers 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. In some within a 5’UTR region may be translated into a short peptide. In other embodiments, a uORF within a 5'UTR is not translated.
  • the regulation of translation by uORFs may be a form of dynamic regulatory mechanism for the control of protein translation.
  • uORFs repress translation of the mORF by allowing fewer ribosomes to reach the mORF, for example by causing ribosome stalling in response to metabolite levels.
  • 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′-untranslated regions (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).
  • CDSs downstream coding sequences
  • uPEPs evolutionarily conserved short peptides
  • the inventors have found that the uORFs of GGP genes in grass plants, such as cereal grass plants, function as translational regulators of these genes; and that modification of GGP uORFs, wherein the modification comprises a deletion, addition, or substitution of at least one base pair of in the 5' UTR of the GGP gene, surprisingly results in increased yield, in particular grain crop yield or panicle numbers.
  • the unmodified uORF peptide of the GGP gene comprises amino acid sequence of any one of SEQ ID NO: 10, 12-14, 18-20, 24, 26-27, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48-49, or 52-53, or an amino acid sequence having at least 70% sequence identity to any of the foregoing.
  • the unmodified uORF of the GGP gene comprises the nucleic acid sequence encoding the amino acid sequence of any one of SEQ ID NO: 10, 12-14, 18-20, 24, 26-27, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48-49, or 52-53; or any one of SEQ ID NOs: 11, 15-17, 21-23, 25, 28, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 50- 51, or 54-55; or nucleotide sequence having at least 70% sequence identity to any of the foregoing.
  • Unmodified uORF of GGP GGP gene uORF amino acid uORF nucleotide sequence sequence SE ID NO AeGGP2 (Aegilops Wilmingtonii) 54 AeGGP1 (Ae ilo s tauschii) 53 55 g p gion, potentially encoding a 60-65 amino acid peptide. Within the uORF region, there were regions of high amino acid sequence conservation (see Laing et al.2015 Plant Cell; and Broad et al.2019 BMC Plant Biol.).
  • the unmodified GGP uORF peptide comprises the amino acid sequence "GGGRL" (SEQ ID NO: 4) at the C-terminal end of the peptide.
  • the unmodified GGP uORF peptide comprises the amino acid sequence "LAGGG” (SEQ ID NO: 5) at the C-terminal end of the peptide. In one embodiment, the unmodified GGP uORF peptide comprises the amino acid sequence "GGGRTL” (SEQ ID NO: 6) at the C-terminal end of the peptide. In one embodiment, the unmodified GGP uORF peptide comprises the amino acid sequence "GGGRLIL" (SEQ ID NO: 7) at the C-terminal end of the peptide.
  • the unmodified GGP uORF peptide comprises the amino acid sequence "GGGRLLL” (SEQ ID NO: 8) at the C-terminal end of the peptide. In one embodiment, the unmodified GGP uORF peptide comprises the amino acid sequence "GGGRILL” (SEQ ID NO: 9) at the C-terminal end of the peptide. In one embodiment, the unmodified GGP uORF peptide comprises the amino acid sequence "GGRGALPSXGGSPSDLLFLAG" [SEQ ID NO: 1], wherein X can be any amino acid.
  • the unmodified GGP uORF peptide comprises the amino acid sequence "GGRGALPSXGGSPSDLLFLAGGG” [SEQ ID NO: 105], wherein X can be any amino acid.
  • the X in SEQ ID NO: 1 and SEQ ID NO:105 is A.
  • the unmodified GGP uORF peptide comprises the amino acid sequence "GGRGALPSAGGSPSDLLFLAG” [SEQ ID NO: 2].
  • the unmodified GGP uORF peptide comprises the amino acid sequence "GGRGALPSAGGSPSDLLFLAGGG” [SEQ ID NO: 106].
  • the X in SEQ ID NO: 1 and SEQ ID NO:105 is E.
  • the unmodified GGP uORF peptide comprises the amino acid sequence "GGRGALPSEGGSPSDLLFLAG” [SEQ ID NO: 3]. In another embodiment, the unmodified GGP uORF peptide comprises the amino acid sequence "GGRGALPSEGGSPSDLLFLAGGG” [SEQ ID NO: 107]. [0089] In an embodiment, the unmodified uORF peptide of rice GGP comprises the amino acid sequence of SEQ ID NO: 10. In an embodiment, the unmodified uORF of rice GGP comprises the nucleic acid sequence SEQ ID NO:10 or the nucleic acid sequence of SEQ ID NO:11.
  • the unmodified uORF peptide of wheat GGP may be TaGGP1.
  • the unmodified uORF peptide of TaGGP1 comprises the amino acid sequence of any one of SEQ ID NOs: 12-14.
  • the unmodified uORF of TaGGP1 comprises the nucleic acid sequence encoding any one of SEQ ID NOs: 12-14 or the nucleic acid sequence of any one of SEQ ID NOs: 15-17.
  • the unmodified uORF peptide of wheat GGP may be TaGGP2.
  • the unmodified uORF peptide of TaGGP2 comprises the amino acid sequence of any one of any one of SEQ ID NOs: 18-20. In an embodiment, the unmodified uORF of TaGGP2 comprises the nucleic acid sequence encoding any one of SEQ ID NOs: 18-20 or the nucleic acid sequence of any one of SEQ ID NOs: 21-23.
  • Modified uORFs [0092] Generating mutations or modifications that disrupt the reading frame or delete residues within the GGP uORF peptide offers insight into the function of the uORF peptide that cannot otherwise be gained when the start codon is disrupted (as was the case for the GGP uORFs that were edited in Arabidopsis, lettuce, and tomato (Zhang et al.2018 Nat Biotechnol.; Li et al., 2018 Nat Biotechnol.); for example, deletions or modifications within the conserved regions of the GGP uORF peptides, such as "GGGRL” (SEQ ID NO: 4); "LAGGG” (SEQ ID NO: 5); “GGGRTL” (SEQ ID NO: 6); GGGRLIL (SEQ ID NO: 7); GGGRLLL (SEQ ID NO: 8); GGGRILL (SEQ ID NO: 9); "GGRGALPSXGGSPSDLLFLAG", wherein X can be any amino acid (S
  • the deletion or modification results in a modified GGP uORF that affects the transcription of the uORF region. In an embodiment, the deletion or modification results in a modified GGP uORF that affects the translation of the GGP uORF peptide. In an embodiment, the deletion or modification results in a modified uORF that affects the transcription of the GGP mORF. In an embodiment, the deletion or modification results in a modified uORF that affects the transcription level of the GGP mORF. In an embodiment, the deletion or modification results in a modified uORF that increases the transcription level of the GGP mORF.
  • the deletion or modification results in a modified uORF that reduces level of the GGP mORF. In an embodiment, the deletion or modification results in a modified uORF that affects the translation of the GGP mORF. In an embodiment, the deletion or modification results in a modified uORF that increases the translation of the GGP mORF. In an embodiment, the deletion or modification results in a modified uORF that reduces the translation level of the GGP mORF. In an embodiment, the deletion or modification results in expression of a functional GGP transcript. In an embodiment, the deletion or modification results in expression of a functional GGP polypeptide. In an embodiment, the deletion or modification results in functional expression level of the GGP gene.
  • the deletion or modification does not result in silencing of the GGP gene.
  • the term “silencing of a gene”, as used herein, is to be understood to mean that the expression of the gene is disrupted or suppressed to a level or degree such that the expression of that gene is undetectable.
  • the expression of the GGP gene is not silenced in a plant having a modified genome comprising a polynucleotide comprising a modification to an upstream open reading frame (uORF) in a 5' untranslated region (UTR) of a GDP-L-galactose phosphorylase (GGP) gene or an ortholog thereof, wherein the modification comprises a deletion, addition, or substitution of at least one base pair of in the 5' UTR of the GGP gene.
  • the deletion or modification in the GGP uORF results in increased ribosome engagement.
  • the deletion or modification in the GGP uORF results in reduced ribosomal stalling in the GGP uORF.
  • the modification in the GGP uORF may be a conservative amino acid substitution.
  • the modification in the GGP uORF may be a non-conservative amino acid substitution.
  • the modification in the GGP uORF may be a conservative nucleotide substitution.
  • the modification in the GGP uORF may be a non-conservative nucleotide substitution.
  • the modification in the GGP uORF may be an insertion of one or more amino acids.
  • the modification in the GGP uORF may be an insertion of one or more nucleotides.
  • the modification in the GGP uORF may be a deletion of one or more nucleotides.
  • the modified uORF peptide may comprise a deletion or modification of 1 to 65 amino acids in the uORF peptide.
  • the modified uORF peptide may comprise a deletion or modification of 1 to 60 amino acids, 1 to 59 amino acids, 1 to 58 amino acids, 1 to 57 amino acids, 1 to 56 amino acids, 1 to 55 amino acids, 1 to 54 amino acids, 1 to 53 amino acids, 1 to 52 amino acids, 1 to 51 amino acids, 1 to 50 amino acids, 1 to 49 amino acids, 1 to 48 amino acids, 1 to 47 amino acids, 1 to 46 amino acids, 1 to 45 amino acids, 1 to 44 amino acids, 1 to 43 amino acids, 1 to 42 amino acids, 1 to 41 amino acids, 1 to 40 amino acids, 1 to 39 amino acids, 1 to 38 amino acids, 1 to 37 amino acids, 1 to 36 amino acids, 1 to 35 amino acids, 1 to 34 amino acids, 1 to 33 amino acids, 1 to 32 amino acids, 1 to 31 amino acids, 1 to 30 amino acids, 1 to 29 amino acids, 1 to 28 amino acids, 1 to 27 amino acids, 1 to 26 amino acids, 1 to 25 amino acids, 1 to 24 amino acids, 1 to 23 amino acids, 1 to 22
  • the modified uORF peptide comprises a deletion or modification of at least one amino acid in the uORF peptide. In another embodiment, the modified uORF peptide comprises a deletion or modification of at least two amino acids in the uORF peptide. In one embodiment, the modified uORF peptide comprises a deletion or modification of at least three amino acids in the uORF peptide. In one embodiment, the modified uORF peptide comprises a deletion or modification of at least four amino acids in the uORF peptide. In one embodiment, the modified uORF peptide comprises a deletion or modification of at least five amino acids in the uORF peptide.
  • the modified uORF peptide comprises a deletion or modification of one amino acid in the uORF peptide. In another embodiment, the modified uORF peptide comprises a deletion or substitution of two amino acids in the uORF peptide. In one embodiment, the modified uORF peptide comprises a deletion or modification of three amino acids in the uORF peptide. In one embodiment, the modified uORF peptide comprises a deletion or modification of 4 amino acids in the uORF peptide. In one embodiment, the modified uORF peptide comprises a deletion or modification of 5 amino acids in the uORF peptide.
  • the modified uORF peptide comprises a deletion or of 6 amino acids in the uORF peptide. In one embodiment, the modified uORF peptide comprises a deletion or modification of 7 amino acids in the uORF peptide. In one embodiment, the modified uORF peptide comprises a deletion or modification of 8 amino acids in the uORF peptide. In one embodiment, the modified uORF peptide comprises a deletion or modification of 9 amino acids in the uORF peptide. In one embodiment, the modified uORF peptide comprises a deletion or modification of 10 amino acids in the uORF peptide.
  • the modified uORF peptide comprises a deletion or modification of up to 15 amino acids in the uORF peptide. In one embodiment, the modified uORF peptide comprises a deletion or modification of up to 18 amino acids in the uORF peptide. In one embodiment, the modified uORF peptide comprises a deletion or modification of up to 20 amino acids in the uORF peptide. [0097] The modified uORF region may comprise a deletion or non-conservative modification of 1 to 200 nucleotides.
  • the modified uORF region may comprise a deletion or non-conservative modification of 1 to 200 nucleotides, 1 to 190 nucleotides, 1 to 180 nucleotides, 1 to 170 nucleotides, 1 to 160 nucleotides, 1 to 150 nucleotides, 1 to 140 nucleotides, 1 to 130 nucleotides, 1 to 120 nucleotides, 1 to 110 nucleotides, 1 to 100 nucleotides, 1 to 99 nucleotides, 1 to 98 nucleotides, 1 to 97 nucleotides, 1 to 96 nucleotides, 1 to 95 nucleotides, 1 to 94 nucleotides, 1 to 93 nucleotides, 1 to 92 nucleotides, 1 to 91 nucleotides, 1 to 90 nucleotides, 1 to 89 nucleotides, 1 to 88 nucleotides, 1 to 87 nucleotides, 1 to
  • the modified uORF region comprises a deletion or non- conservative substitution of at least one amino acid in the uORF region. In another embodiment, the modified uORF region comprises a deletion or non-conservative substitution of at least two nucleotides in the uORF region. In one embodiment, the modified uORF region comprises a deletion or non-conservative substitution of at least three nucleotides in the uORF region. In one embodiment, the modified uORF region comprises a deletion or non-conservative substitution of at least four nucleotides in the uORF region. In one embodiment, the modified uORF region comprises a deletion or non-conservative substitution of at least five nucleotides in the uORF region.
  • the modified uORF region comprises a deletion or non-conservative substitution of one nucleotide in the uORF region. In another embodiment, the modified uORF region comprises a deletion or non-conservative substitution of two nucleotides in the uORF region. In one embodiment, the modified uORF region comprises a deletion or non-conservative substitution of three nucleotides in the uORF region. In one embodiment, the modified uORF region comprises a deletion or non-conservative substitution of 4 nucleotides in the uORF region. In one embodiment, the modified uORF region comprises a deletion or non-conservative substitution of 5 nucleotides in the uORF region.
  • the modified uORF region comprises a deletion or non-conservative substitution of 6 nucleotides in the uORF region. In one embodiment, the modified uORF region comprises a deletion or non- conservative substitution of 7 nucleotides in the uORF region. In one embodiment, the modified uORF region comprises a deletion or non-conservative substitution of 8 nucleotides in the uORF region. In one embodiment, the modified uORF region comprises a deletion or non-conservative substitution of 9 nucleotides in the uORF region. In one embodiment, the modified uORF region comprises a deletion or non-conservative substitution of 10 nucleotides in the uORF region.
  • the modified uORF region comprises a deletion or non-conservative substitution of up to 15 nucleotides in the uORF region. In one embodiment, the modified uORF region comprises a deletion or non- conservative substitution of up to 20 nucleotides in the uORF region. In one embodiment, the modified uORF region comprises a or non-conservative substitution of up to 25 nucleotides in the uORF region. In one embodiment, the modified uORF region comprises a deletion or non-conservative substitution of up to 30 nucleotides in the uORF region. In one embodiment, the modified uORF region comprises a deletion or non-conservative substitution of up to 35 nucleotides in the uORF region.
  • the modified uORF region comprises a deletion or non-conservative substitution of up to 40 nucleotides in the uORF region. In one embodiment, the modified uORF region comprises a deletion or non-conservative substitution of up to 45 nucleotides in the uORF region. In one embodiment, the modified uORF region comprises a deletion or non-conservative substitution of up to 50 nucleotides in the uORF region. In one embodiment, the modified uORF region comprises a deletion or non-conservative substitution of up to 55 nucleotides in the uORF region.
  • the modified uORF region comprises a deletion or non-conservative substitution of up to 60 nucleotides in the uORF region. In one embodiment, the modified uORF region comprises a deletion or non-conservative substitution of up to 65 nucleotides in the uORF region. In one embodiment, the modified uORF region comprises a deletion or non-conservative substitution of up to 63 nucleotides in the uORF region. In one embodiment, the modified uORF region comprises a deletion or non-conservative substitution of up to 70 nucleotides in the uORF region.
  • the modified uORF of GGP comprises at least one deletion or non-conservative substitution of at least one base pair in the uORF. In another embodiment, the modified uORF of GGP comprises a deletion or non-conservative substitution of 1 to 100 base pairs. In another embodiment, the modified uORF of GGP comprises a deletion or non-conservative substitution of 2 to 80 base pairs. In another embodiment, the modified uORF of GGP comprises a deletion or non-conservative substitution of 5 to 70 base pairs in the uORF. [0099] In another embodiment, the modified uORF of GGP comprises a non-conservative substitution of 1 base pair in the uORF.
  • the modified uORF of GGP comprises a deletion of 5 base pairs in the uORF. In another embodiment, the modified uORF of GGP comprises deletion of 63 base pairs in the uORF. [0100] In another embodiment, the modified uORF of GGP comprises an amino acid substitution, deletion or insertion of one or more amino acid residues at amino acid positions corresponding to amino acid positions 30 to 61 of the GGP uORF peptide of SEQ ID NO: 10. In another embodiment, the uORF of GGP comprises an amino acid substitution, deletion or insertion of one or more amino acid residues at amino acid positions corresponding to amino acid positions 57 to 61 of the GGP uORF peptide of SEQ ID NO: 10.
  • the modified uORF of GGP an amino acid substitution, deletion or insertion of one or more amino acid residues at amino acid positions corresponding to amino acid positions 55 to 56 of the GGP uORF peptide of SEQ ID NO: 10.
  • the modified uORF of GGP comprises an amino acid substitution, deletion or insertion of one or more amino acid residues at amino acid positions corresponding to amino acid positions 37 to 57 of the GGP uORF peptide of SEQ ID NO: 10.
  • the modified GGP uORF may comprise a deletion or modification of regions of high amino acid sequence conservation in GGP gene uORF (Laing et al.2015 Plant Cell; and Broad et al.2019 BMC Plant Biol.).
  • the modified GGP uORF comprises a deletion or modification within the amino acid sequence "GGGRL” (SEQ ID NO: 4); “LAGGG” (SEQ ID NO: 5); “GGGRTL” (SEQ ID NO: 6); GGGRLIL (SEQ ID NO: 7); GGGRLLL (SEQ ID NO: 8); GGGRILL (SEQ ID NO: 9); "GGRGALPSXGGSPSDLLFLAG", wherein X can be any amino acid (SEQ ID NO: 1); or "GGRGALPSAGGSPSDLLFLAGGG” wherein X can be any amino acid (SEQ ID NO: 106), at the C-terminal end of the GGP uORF peptide.
  • the modified uORF of GGP comprises an amino acid substitution, deletion or insertion of one or more amino acid residues at amino acid positions corresponding to amino acid positions 30 to 61 of the GGP uORF peptide of SEQ ID NO: 10, wherein the GGP uORF comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 12-14, 18-20, 24, 26-27, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48-49, and 52-53, and amino acid sequences having at least 70% sequence identity to any of the foregoing.
  • the modified uORF of GGP comprises an amino acid substitution, deletion or insertion of one or more amino acid residues at amino acid positions corresponding to amino acid positions 57 to 61 of the GGP uORF peptide of SEQ ID NO: 10, wherein the GGP uORF comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 12-14, 18-20, 24, 26-27, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48-49, and 52-53, and amino acid sequences having at least 70% sequence identity to any of the foregoing.
  • the modified uORF of GGP an amino acid substitution, deletion or insertion of one or more amino acid residues at amino acid positions corresponding to amino acid positions 56 of the GGP uORF peptide of SEQ ID NO: 10, wherein the GGP uORF comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 12-14, 18-20, 24, 26-27, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48-49, and 52-53, and amino acid sequences having at least 70% sequence identity to any of the foregoing.
  • the modified uORF of GGP comprises an amino acid substitution, deletion or insertion of one or more amino acid residues at amino acid positions corresponding to amino acid positions 37 to 57 of the GGP uORF peptide of SEQ ID NO: 10, wherein the GGP uORF comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 12-14, 18-20, 24, 26-27, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48-49, and 52-53, and amino acid sequences having at least 70% sequence identity to any of the foregoing.
  • the modified uORF of GGP comprises an amino acid substitution, deletion or insertion of one or more amino acid residues from position 30 to 61 of the GGP uORF peptide of SEQ ID NO: 10. In another embodiment, the modified uORF of GGP comprises an amino acid substitution, deletion or insertion of one or more amino acid residues from position 57 to 61 of the GGP uORF peptide of SEQ ID NO: 10. In another embodiment, the modified uORF of GGP comprises an amino acid substitution, deletion or insertion of one or more amino acid residues from position 55 to 56 of the GGP uORF peptide of SEQ ID NO: 10.
  • the modified uORF of GGP comprises an amino acid substitution, deletion or insertion of one or more amino acid residues from position 37 to 57 of the GGP uORF peptide of SEQ ID NO: 10.
  • the modified uORF of GGP comprises a substitution of one or more amino acids in the amino acid sequence from position 30 to 61 the GGP uORF peptide of SEQ ID NO: 10.
  • the modified uORF of GGP comprises a substitution of one or more amino acids in the amino acid sequence from position 57 to 61 of SEQ ID NO: 10.
  • the modified uORF of GGP comprises a substitution of one or more amino acids in the amino acid sequence from position 55 to 56 of SEQ ID NO: 10.
  • the modified uORF of GGP comprises a substitution of one or more amino acids in the amino acid sequence from position 37 to 57 of SEQ ID NO: 10.
  • the modified uORF of GGP comprises a substitution of the amino acid sequence from position 30 to 61 the GGP uORF peptide of SEQ ID NO: 10.
  • the modified comprises a substitution of the amino acid sequence from position 57 to 61 of SEQ ID NO: 10.
  • the modified uORF of GGP comprises a substitution of the amino acid sequence from position 55 to 56 of SEQ ID NO: 10.
  • the modified uORF of GGP comprises a substitution of the amino acid sequence from position 37 to 57 of SEQ ID NO: 10.
  • the modified uORF of GGP comprises a deletion of one or more amino acids at the amino acid sequence from position 30 to 61 the GGP uORF peptide of SEQ ID NO: 10. In another embodiment, the modified uORF of GGP comprises a deletion of one or more amino acids at the amino acid sequence from position 57 to 61 of SEQ ID NO: 10. In another embodiment, the modified uORF of GGP comprises a deletion of one or more amino acids at the amino acid sequence from position 55 to 56 of SEQ ID NO: 10. In another embodiment, the modified uORF of GGP comprises a deletion of one or more amino acids at the amino acid sequence from position 37 to 57 of SEQ ID NO: 10.
  • the modified uORF of GGP comprises a deletion of the amino acid sequence from position 30 to 61 the GGP uORF peptide of SEQ ID NO: 10. In another embodiment, the modified uORF of GGP comprises a deletion of the amino acid sequence from position 57 to 61 of SEQ ID NO: 10. In another embodiment, the modified uORF of GGP comprises a deletion of the amino acid sequence from position 55 to 56 of SEQ ID NO: 10. In another embodiment, the modified uORF of GGP comprises a deletion of the amino acid sequence from position 37 to 57 of SEQ ID NO: 10.
  • the modified uORF of GGP comprises an insertion of one or more amino acids at the amino acid sequence from position 30 to 61 the GGP uORF peptide of SEQ ID NO: 10. In another embodiment, the modified uORF of GGP comprises an insertion of one or more amino acids at the amino acid sequence from position 57 to 61 of SEQ ID NO: 10. In another embodiment, the modified uORF of GGP comprises an insertion of one or more amino acids at the amino acid sequence from position 55 to 56 of SEQ ID NO: 10. In another embodiment, the modified uORF of GGP an insertion of one or more amino acids at the amino acid sequence from position 37 to 57 of SEQ ID NO: 10.
  • the deletion or modification of the uORF of GGP results in disruption of the reading frame of the GGP uORF, wherein the GGP uORF comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 12-14, 18-20, 24, 26-27, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48-49, and 52-53, and amino acid sequences having at least 70% sequence identity of the foregoing.
  • the deletion or modification of the uORF of GGP results in disruption of the reading frame of the GGP uORF
  • the GGP uORF comprises the nucleic acid sequence encoding any one of SEQ ID NOs: 10, 12-14, 18-20, 24, 26-27, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48-49, and 52-53 or the nucleic acid sequence of SEQ ID NO: 11, 15-17, 21-23, 25, 28, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 50-51, and 54-55.
  • the modified GGP uORF peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 56-58.
  • the modified GGP uORF comprises a nucleic acid sequence encoding any one of SEQ ID NOs: 56-58.
  • 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 or of a plant cell.
  • 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.
  • 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.
  • the trait modification can be evaluated quantitatively.
  • 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 or unmodified 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 useful numbers or amounts.
  • the crop plant is a cereal grass plant.
  • a desirable “trait” may include, for example, one or more of the following, as compared to a control plant: increased vigour, increased “yield”, darker coloration, increased organ size, improved root growth, increased photosynthesis, increased SPAD, increased nutrient content, increased nutrient 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).
  • the plant having a modified genome comprising a polynucleotide comprising a modification to an upstream open reading frame (uORF) in a 5' untranslated region (UTR) of a GDP-L-galactose phosphorylase (GGP) gene or an ortholog thereof, wherein the modification comprises a deletion, addition, or substitution of at least one base pair of in the 5' UTR of the GGP gene, has increased or enhanced foliar ascorbate levels.
  • uORF upstream open reading frame
  • UTR 5' untranslated region
  • GGP GDP-L-galactose phosphorylase
  • the plant having a modified genome comprising a polynucleotide comprising a modification to an upstream open reading frame (uORF) in a 5' untranslated region (UTR) of a GDP-L-galactose phosphorylase (GGP) gene or an ortholog thereof, wherein the modification comprises a deletion, addition, or substitution of at least one base pair of in the 5' UTR of the GGP gene, has increased or enhanced root ascorbate levels.
  • uORF upstream open reading frame
  • UTR 5' untranslated region
  • GGP GDP-L-galactose phosphorylase
  • the plant having a modified genome comprising a polynucleotide comprising a modification to an upstream open reading frame (uORF) in a 5' untranslated region (UTR) of a GDP-L-galactose phosphorylase (GGP) gene or an ortholog thereof, wherein the modification comprises a deletion, addition, or substitution of at least one base pair of in the 5' UTR of the GGP gene, has increased or enhanced foliar ascorbate levels.
  • uORF upstream open reading frame
  • UTR 5' untranslated region
  • GGP GDP-L-galactose phosphorylase
  • the plant having a modified genome comprising a polynucleotide comprising a modification to an upstream open reading frame (uORF) in a 5' untranslated region (UTR) of a GDP-L-galactose phosphorylase (GGP) gene or an ortholog thereof, wherein the modification comprises a deletion, addition, or substitution of at least one base pair of in the 5' UTR of the GGP gene, results in increased ascorbate levels in grain.
  • uORF upstream open reading frame
  • UTR 5' untranslated region
  • GGP GDP-L-galactose phosphorylase
  • having a modified genome comprising a polynucleotide comprising a modification to an upstream open reading frame (uORF) in a 5' untranslated region (UTR) of a GDP-L-galactose phosphorylase (GGP) gene or an ortholog thereof, wherein the modification comprises a deletion, addition, or substitution of at least one base pair of in the 5' UTR of the GGP gene, results in increased ascorbate levels in germinated grain.
  • uORF upstream open reading frame
  • UTR 5' untranslated region
  • GGP GDP-L-galactose phosphorylase
  • the modification enhances the ascorbate foliar content in the plant or plant tissue, including foliar, root or germinated grain tissue 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.
  • the plant having a modified genome comprising a polynucleotide comprising a modification to an upstream open reading frame (uORF) in a 5' untranslated region (UTR) of a GDP-L-galactose phosphorylase (GGP) gene or an ortholog thereof, wherein the modification comprises a deletion, addition, or substitution of at least one base pair of in the 5' UTR of the GGP gene, has the same or improved salt tolerance, 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, wherein the improvement of salt tolerance is 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
  • a plant having a modified genome comprising a polynucleotide comprising a modification to an upstream open reading frame (uORF) in a 5' untranslated region (UTR) of a GDP-L-galactose phosphorylase (GGP) gene or an ortholog thereof, wherein the modification comprises a deletion, addition, or substitution of at least one base pair of in the 5' UTR of the GGP gene, is capable of increased yield.
  • uORF upstream open reading frame
  • UTR 5' untranslated region
  • GGP GDP-L-galactose phosphorylase
  • Also disclosed herein are methods of modifying the genome of a cereal grass plant comprising introducing a modification to an reading frame (uORF) in a 5' untranslated region (UTR) of a GDP-L-galactose phosphorylase (GGP) gene or an ortholog thereof, wherein the modification comprises a deletion, addition, or substitution of at least one base pair of in the 5' UTR of the GGP gene, is capable of increased yield.
  • Yield typically refers to the portion of the plant that is harvestable such as grain, fruit, biomass; or part of the plant that generates the harvestable grain fruit or biomass such as flowers or panicles; or an edible vegetative part such as a stem, leaf or tuber.
  • the increased yield is increased grain crop yield.
  • the increase in grain crop yield is the result of increased grain weight per plant.
  • the increased yield is an increase in the number of panicles per plant.
  • the increase in grain crop yield is the result of increased grain weight per plant. In an embodiment, the increase in grain crop yield is the result of an increase in the number of panicles per plant. [0122] In an embodiment, the modified uORF results in an increase in the grain crop yield of a plant or a cereal grass plant by at least about 1%, by at least 2%, by at least 3%, by at least 4%, by at least 5%, by at least 6%, by at least 7%, by at least 8%, by at least 9%, by at least 10%, by at least 15%, by at least 20%, by at least 21%, by at least 22%, by at least 23%, by at least 24%, by at least 25%, by at least 26%, by at least 27%, by at least 28%, by at least 29%, by at least 30%, by at least 31%, by at least 32%, by at least 33%, by at least 34%, by at least 35%, by at least 36%, by at least 37%, by at least 38%, by
  • the modified uORF results in at least a 1.1 fold increase in the grain crop yield of a plant or a cereal grass plant, when compared to the grain crop yield of a plant or a cereal grass plant harbouring a corresponding wild-type uORF of the same plant species. In an embodiment, the modified uORF results in at least a 1.2 fold increase in the grain crop yield of a plant or a cereal grass plant, when compared to the grain crop yield of a plant or a cereal grass plant harbouring a corresponding wild-type uORF of the same plant species.
  • the modified uORF results in a 1.3 fold increase in the grain crop yield of a plant or a cereal grass plant, when compared to the grain crop yield of a plant or a cereal grass plant harbouring a corresponding wild-type uORF of the same plant species. In an embodiment, the modified uORF results in at least a 1.3 fold increase in the grain crop yield of a plant or a cereal grass plant, when compared to the grain crop yield of a plant or a cereal grass plant harbouring a corresponding wild-type uORF of the same plant species.
  • the modified uORF results in at least a 1.4 fold increase in the grain crop yield of a plant or a cereal grass plant, when compared to the grain crop yield of a plant or a cereal grass plant harbouring a corresponding wild-type uORF of the same plant species. In an embodiment, the modified uORF results in at least a 1.5 fold increase in the grain crop yield of a plant or a cereal grass plant, when compared to the grain crop yield of a plant or a cereal grass plant harbouring a corresponding wild-type uORF of the same plant species.
  • the modified uORF results in at least a 2 fold increase in the grain crop yield of a plant or a cereal grass plant, when compared to the grain crop yield of a plant or a cereal grass plant harbouring a corresponding wild-type uORF of the same plant species.
  • the modified uORF results in at least a 3 fold increase in the grain crop yield of a plant or a cereal grass plant, when compared to the grain crop yield of a plant or a cereal grass plant harbouring a corresponding wild-type uORF of the same plant species.
  • the modified uORF results in at least a 4 fold increase in the grain crop yield of a plant or a cereal grass plant, when compared to the grain crop yield of a plant or a cereal grass plant harbouring a corresponding wild-type uORF of the same plant species.
  • the modified uORF results in at least a 5 fold increase in the grain crop yield of a plant or a cereal grass plant, when compared to the grain crop yield of a plant or a cereal grass plant harbouring a corresponding wild-type uORF of the same plant species.
  • the modified uORF results in an increase in the grain weight per plant of a plant or a cereal grass plant by at least about 1%, by at least 2%, by at least 3%, by at least 4%, by at least 5%, by at by at least 7%, by at least 8%, by at least 9%, by at least 10%, by at least 15%, by at least 20%, by at least 21%, by at least 22%, by at least 23%, by at least 24%, by at least 25%, by at least 26%, by at least 27%, by at least 28%, by at least 29%, by at least 30%, by at least 31%, by at least 32%, by at least 33%, by at least 34%, by at least 35%, by at least 36%, by at least 37%, by at least 38%, by at least 39%, by at least 40%, by at least 41%, by at least 42%, by at least 43%, by at least 44%, by at least 45%, by at least 46%, by at least
  • the modified uORF results in at least a 1.1 fold increase in the grain weight per plant of a plant or a cereal grass plant, when compared to the grain weight per plant of a plant or a cereal grass plant harbouring a corresponding wild-type uORF of the same plant species. In an embodiment, the modified uORF results in at least a 1.2 fold increase in the grain weight per plant of a plant or a cereal grass plant, when compared to the grain weight per plant of a plant or a cereal grass plant harbouring a corresponding wild- type uORF of the same plant species.
  • the modified uORF results in a 1.3 fold increase in the grain weight per plant of a plant or a cereal grass plant, when compared to the grain weight per plant of a plant or a cereal grass plant harbouring a corresponding wild-type uORF of the same plant species. In an embodiment, the modified uORF results in at least a 1.3 fold increase in the grain weight per plant of a plant or a cereal grass plant, when compared to the grain weight per plant of a plant or a cereal grass plant harbouring a corresponding wild-type uORF of the same plant species.
  • the modified uORF results in at least a 1.4 fold increase in the grain weight per plant of a plant or a cereal grass plant, when compared to the grain weight per plant of a plant or a cereal grass plant harbouring a corresponding wild-type uORF of the same plant species. In an embodiment, the modified uORF results in at least a 1.5 fold increase in the grain weight per plant of a plant or a cereal grass plant, when compared to the grain weight per plant of a plant or a cereal grass plant harbouring a corresponding wild-type uORF of the same plant species.
  • the modified uORF results in at least a 2 fold increase in the grain weight per plant of a plant or a plant, when compared to the grain weight per plant of a plant or a cereal grass plant harbouring a corresponding wild-type uORF of the same plant species. In an embodiment, the modified uORF results in at least a 3 fold increase in the grain weight per plant of a plant or a cereal grass plant, when compared to the grain weight per plant of a plant or a cereal grass plant harbouring a corresponding wild- type uORF of the same plant species.
  • the modified uORF results in at least a 4 fold increase in the grain weight per plant of a plant or a cereal grass plant, when compared to the grain weight per plant of a plant or a cereal grass plant harbouring a corresponding wild-type uORF of the same plant species. In an embodiment, the modified uORF results in at least a 5 fold increase in the grain weight per plant of a plant or a cereal grass plant, when compared to the grain weight per plant of a plant or a cereal grass plant harbouring a corresponding wild-type uORF of the same plant species.
  • a method of increasing the number of panicles of a cereal grass plant comprising introducing a modification to an upstream open reading frame (uORF) in a 5' untranslated region (UTR) of a GDP-L-galactose phosphorylase (GGP) gene or an ortholog thereof.
  • uORF upstream open reading frame
  • UTR 5' untranslated region
  • GGP GDP-L-galactose phosphorylase
  • the modified uORF results in an increase in the number of panicles per plant of a plant or a cereal grass plant by at least about 1%, by at least 2%, by at least 3%, by at least 4%, by at least 5%, by at least 6%, by at least 7%, by at least 8%, by at least 9%, by at least 10%, by at least 15%, by at least 20%, by at least 21%, by at least 22%, by at least 23%, by at least 24%, by at least 25%, by at least 26%, by at least 27%, by at least 28%, by at least 29%, by at least 30%, by at least 31%, by at least 32%, by at least 33%, by at least 34%, by at least 35%, by at least 36%, by at least 37%, by at least 38%, by at least 39%, by at least 40%, by at least 41%, by at least 42%, by at least 43%, by at least 44%, by at least 45%, by at least 46%,
  • the modified uORF results in at least a 1.1 fold increase in the number of panicles per plant of a plant or a cereal grass plant, when compared to the number of panicles per plant of a plant or a cereal grass plant harbouring a corresponding wild-type uORF of the plant species. In an embodiment, the modified uORF results in at least a 1.2 fold increase in the number of panicles per plant of a plant or a cereal grass plant, when compared to the number of panicles per plant of a plant or a cereal grass plant harbouring a corresponding wild-type uORF of the same plant species.
  • the modified uORF results in a 1.3 fold increase in the number of panicles per plant of a plant or a cereal grass plant, when compared to the number of panicles per plant of a plant or a cereal grass plant harbouring a corresponding wild-type uORF of the same plant species. In an embodiment, the modified uORF results in at least a 1.3 fold increase in the number of panicles per plant of a plant or a cereal grass plant, when compared to the number of panicles per plant of a plant or a cereal grass plant harbouring a corresponding wild-type uORF of the same plant species.
  • the modified uORF results in at least a 1.4 fold increase in the number of panicles per plant of a plant or a cereal grass plant, when compared to the number of panicles per plant of a plant or a cereal grass plant harbouring a corresponding wild-type uORF of the same plant species. In an embodiment, the modified uORF results in at least a 1.5 fold increase in the number of panicles per plant of a plant or a cereal grass plant, when compared to the number of panicles per plant of a plant or a cereal grass plant harbouring a corresponding wild-type uORF of the same plant species.
  • the modified uORF results in at least a 2 fold increase in the number of panicles per plant of a plant or a cereal grass plant, when compared to the number of panicles per plant of a plant or a cereal grass plant harbouring a corresponding wild-type uORF of the same plant species. In an embodiment, the modified uORF results in at least a 3 fold increase in the number of panicles per plant of a plant or a cereal grass plant, when compared to the number of panicles per plant of a plant or a cereal grass plant harbouring a corresponding wild-type uORF of the same plant species.
  • the modified uORF results in at least a 4 fold increase in the number of panicles per plant of a plant or a cereal grass plant, when compared to the number of panicles per plant of a plant or a cereal grass plant harbouring a corresponding wild-type uORF of the same plant species. In an embodiment, the modified uORF results in at least a 5 fold increase in the number of panicles per plant of a plant or a cereal grass plant, when compared to the number of panicles per plant of a plant or a cereal grass plant harbouring a corresponding wild-type uORF of the same plant species.
  • the modified uORF results in an increase in the grain weight per plant of a plant or a cereal grass plant by at least about 1%, by at least 2%, by at least 3%, by at least 4%, by at least 5%, by at by at least 7%, by at least 8%, by at least 9%, by at least 10%, by at least 15%, by at least 20%, by at least 21%, by at least 22%, by at least 23%, by at least 24%, by at least 25%, by at least 26%, by at least 27%, by at least 28%, by at least 29%, by at least 30%, by at least 31%, by at least 32%, by at least 33%, by at least 34%, by at least 35%, by at least 36%, by at least 37%, by at least 38%, by at least 39%, by at least 40%, by at least 41%, by at least 42%, by at least 43%, by at least 44%, by at least 45%, by at least 46%, by at least
  • polynucleotides [0126] The present disclosure extends to polynucleotides encoding the polypeptides and uORFs disclosed herein. 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. The present disclosure also extends to polynucleotides encoding the polypeptides and uORFs disclosed herein.
  • polynucleotide 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.
  • polynucleotide includes both the specified sequence and its complement.
  • 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.
  • primer refers to 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.
  • 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.
  • 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%,
  • Polynucleotide sequence 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.
  • 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]) In bl2seq (Tatiana A. Tatusova, Thomas L.
  • the parameter - p selects the appropriate algorithm for the pair of sequences.
  • 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).
  • 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 online at world-wide web.ebi.ac.uk/emboss/align/.
  • 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.
  • 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.)
  • 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.
  • sequence similarity with respect to polypeptides may be determined 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/).
  • the similarity of polynucleotide sequences may be examined using the following Unix command line parameters: bl2seq -i nucleotideseq1 -j nucleotideseq2 -F F -p tblastx.
  • 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.
  • Variant polynucleotide sequences preferably exhibit an E value of less than 1x10 -6 more 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 -90 and most preferably less than 1x10 -100 when compared with any one of the specifically identified sequences.
  • variant polynucleotides of the present disclosure hybridize to the specified polynucleotide sequences, or complements thereof under stringent conditions.
  • hybridize under stringent conditions 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.
  • Tm melting temperature
  • Typical stringent conditions for polynucleotide of greater than 100 bases in length would be hybridization conditions such as prewashing in a solution of 6x Saline-Sodium Citrate (SSC) , 0.2% SDS; hybridizing at 65 °C., 6xSSC, 0.2% SDS overnight; followed by two washes of 30 minutes each in 1xSSC, 0.1% SDS at 65 °C. and two washes of 30 minutes each in 0.2xSSC, 0.1% SDS at 65 °C. [0146] With respect to polynucleotide molecules having a length less than 100 bases, exemplary stringent hybridization conditions are 5 to 10 °C. below Tm.
  • Tm of a polynucleotide molecule of length less than 100 bp is reduced by approximately (500/oligonucleotide length) °C.
  • PNAs peptide nucleic acids
  • 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.
  • 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.
  • Variant polynucleotides due 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.
  • 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 encoded protein as described in the Example section.
  • Modified Genomes, Constructs, Vectors and Components Thereof The present disclosure also extends to modified genomes and polynucleotide constructs comprising the polynucleotides disclosed herein, and to expression vectors comprising the polynucleotides or the polynucleotide constructs disclosed herein.
  • modified genome and the like refer to genomes that have been modified when compared to wild-type genomes of the corresponding plant species.
  • construct refers 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.
  • 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 from the host cell, or may be derived from a different cell or organism and/or may be a recombinant polynucleotide.
  • the genetic construct may become integrated in the host chromosomal DNA.
  • the genetic construct may be linked to a vector.
  • 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.
  • expression 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.
  • 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.
  • a "coding sequence” When inserted into a genetic construct, a “coding sequence” is capable of being expressed when it is operably linked to promoter and terminator sequences.
  • "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.
  • the regulatory elements include a polynucleotide sequence of the disclosure.
  • the sequence of the present disclosure comprises a 5'-UTR sequence.
  • the 5'-UTR sequence comprises a uORF.
  • noncoding region refers to untranslated sequences that are upstream of the translational start site and downstream of the translational stop site of a main 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.
  • 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 regulatory functions.
  • 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 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 the sequence of a polynucleotide and polypeptide produced.
  • 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).
  • 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.
  • the terms “complementary” and “complementarity” refer to polynucleotides (i.e., a sequence of nucleotides) related by the base-pairing rules.
  • 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 rules. Or, there may be “complete” or between the nucleic acids. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands.
  • 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-occurring nucleic acid sequence.
  • a corresponding exogenous nucleic acid sequence it is intended to mean an analogous, equivalent or comparable exogenous nucleic acid sequence.
  • the corresponding nucleic acid sequence has analogous or equivalent function or having sequence similarity.
  • the corresponding nucleic acid sequence may be identical in function and/or sequence.
  • the corresponding nucleic acid sequence may have about the same function or activity.
  • the corresponding nucleic acid sequence may have reduced function or activity.
  • the corresponding nucleic acid sequence may have lost function or activity.
  • the phrase “corresponds to” or “corresponding to” is meant a nucleic acid sequence that displays substantial sequence identity to a reference nucleic acid sequence.
  • 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.
  • expression 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.
  • a 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 (i.e., introns, 5’ and 3’ untranslated sequences) whether or not such regulatory sequences are adjacent to coding and/or transcribed sequences.
  • 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 sites, and locus control regions.
  • uORF upstream open reading frames
  • the gene may comprise coding sequences and non-coding sequences. In other embodiments, 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. [0170] 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.
  • the term “gene product” or “expression product” as used herein refers to an RNA or protein that results from expression of a gene.
  • the gene product may be an RNA, such as mRNA, rRNA, tRNA, miRNA or siRNA, or the gene product may be a polypeptide product.
  • RNA such as mRNA, rRNA, tRNA, miRNA or siRNA
  • the gene product may be a polypeptide product.
  • 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 sequence. [0173] The present disclosure also extends to homologs and orthologs of the polynucleotide and polypeptide sequences disclosed herein.
  • 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 [0174] 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.
  • 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.
  • a "fragment" of a polypeptide is a subsequence of the polypeptide.
  • 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.
  • isolated as applied to the polynucleotide or polypeptide sequences disclosed herein, is used to refer to 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.
  • 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.
  • 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.
  • the recombinant polynucleotide may be in the form of an expression vector.
  • 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.
  • a “recombinant cell” refers to a cell that has introduced into its exogenous nucleic acid, typically exogenous DNA, such as a vector or other polynucleotides. The term includes the progeny of the original cell into which the exogenous DNA has been introduced.
  • 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.
  • mutant cell encompasses any progeny of a parent host cell that is not identical to the parent host cell due to the alterations introduced.
  • a "recombinant" polypeptide sequence is produced by translation from a "recombinant” polynucleotide sequence.
  • 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.
  • variants and “derivatives”, are taken to refer to a biological equivalent of the sequence from which it was derived.
  • 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
  • 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 the entire length of a polypeptide of the disclosure.
  • Identity or “similarity” refers to sequence similarity between two polynucleotide sequences 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 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 there between. 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.
  • sequence identity 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).
  • 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, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, 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 (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.
  • the identical nucleic acid base e.g., A, T, C, G
  • the identical amino acid residue e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His
  • sequence identity will be understood to mean the “match percentage” calculated by an appropriate method.
  • 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 optimally over 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 et al., 1997; Altschul et al., 2005)).
  • a global alignment algorithms e.g., Needleman and Wunsch algorithm; Needleman and Wunsch, 1970
  • a local alignment algorithm e.g., Smith and Waterman algorithm (Smith and Waterman, 1981) or 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). [0184] Polypeptide sequence identity can be determined in the following manner.
  • the subject polypeptide sequence is compared to a candidate polypeptide sequence using BLASTP (from the BLAST suite of programs, version 2.2.5 [November 2002]) in bl2seq, which is publicly available from NCBI (ftp colon slash slash file transfer protocol ftp.ncbi.nih.gov/blast/).
  • NCBI ftp colon slash slash file transfer protocol ftp.ncbi.nih.gov/blast/.
  • 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/
  • GAP Human, 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.
  • 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.
  • 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.
  • 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 ftp.ncbi.nih.gov/blast/).
  • Variant polypeptide sequences preferably exhibit an E value of less than 1x10 -6 more 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 -90 and most preferably 1x10 -100 when compared with any one of the specifically identified sequences.
  • 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.
  • Non-conservative substitutions will entail exchanging a member of one of these classes for a member of another class.
  • 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 non- conservative substitutions at a biological level.
  • substitution matrix also referred to as a substitution matrix.
  • BLOSUM62 matrix is used to generate a score for each aligned amino acid pair found at the intersection of the corresponding 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.
  • polypeptide variant as a variant of a polypeptide encoded by a GGP gene, may be assessed by the methods described in the Example section herein.
  • Methods for Isolating or Producing Polynucleotides [0196]
  • 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).
  • PCR polymerase chain reaction
  • polypeptides of the present disclosure can be amplified using primers, as defined herein, derived from the polynucleotide sequences of the present disclosure.
  • 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.
  • the polynucleotide 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.
  • 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.
  • 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.
  • Variants 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.
  • 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.
  • variant sequences of the present disclosure 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.
  • BLAST suite of programs 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.
  • NCBI National Center for Biotechnology Information
  • 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
  • the BLAST programs may be used with default parameters or the parameters may be altered as required to refine the screen.
  • 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.
  • 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.
  • the probability of finding a match by chance in that database is 1% or less using the BLASTN, BLASTP, BLASTX, tBLASTN or tBLASTX algorithm.
  • CLUSTALW 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 weight matrix choice.
  • MEME Multiple Em for Motif Elicitation
  • MAST Motif Alignment and Search Tool
  • the MAST results 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.
  • 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.
  • 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.
  • polypeptides of the present disclosure 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 431A Peptide Synthesizer (Foster City, Calif.). Mutated forms of the polypeptides may also be produced during such syntheses.
  • 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., Guide to Protein Purification).
  • 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 [0215] 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 the sequence encoding the protein and expressing the modified protein.
  • modifying the uORF comprises site-directed nucleases or oligonucleotide-directed mutagenesis.
  • the mutagenesis is achieved using SDN-1.
  • said mutagenesis is achieved using Zinc finger nucleases, TALENS and / or CRISPR/Cas gene editing.
  • 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.
  • 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, rye, oats, millet, sorghum, triticale, buckwheat, quinoa, spelt, einkorn, amaranth, grass, corn, 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, sweet potato, yam and family plant.
  • Host cells comprising genetic constructs, such as expression constructs, of the invention 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., Guider, Ed, 1990, Methods in Enzymology, Vol 182, Guide to Protein Purification).
  • Methods for Producing Plant Cells and Plants Comprising Constructs and Vectors [0221]
  • 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.
  • 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 the plant species to be transformed or may be derived from a different plant species.
  • 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.
  • 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.
  • 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.
  • Exemplary terminators that are commonly used in plant transformation genetic construct include, e.g., the cauliflower mosaic virus (CaMV) 35S terminator, the Agrobacterium tumefaciens nopaline synthase or octopine synthase terminators, the Zea mays zein gene terminator, the Oryza sativa ADP-glucose pyrophosphorylase terminator and the Solanum tuberosum PI-II terminator.
  • CaMV cauliflower mosaic virus
  • Agrobacterium tumefaciens nopaline synthase or octopine synthase terminators the Zea mays zein gene terminator
  • the Oryza sativa ADP-glucose pyrophosphorylase terminator and the Solanum tuberosum PI-II terminator.
  • NPT II neomycin phophotransferase II gene
  • aadA gene confers spectinomycin and streptomycin resistance
  • phosphinothricin acetyl transferase bar gene
  • Ignite AgrEvo
  • Basta Basta
  • hpt hygromycin phosphotransferase gene
  • 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.
  • LOC luciferase
  • GUS_ ⁇ -glucuronidase
  • GFP green fluorescent protein
  • 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.
  • 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.
  • 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 [0233] 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., ⁇ 5'-GATCTA...TAGATC-3' ⁇ 3'-CTAGAT...ATCTAG-5'
  • 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.
  • Another silencing approach involves the use of a small antisense RNA targeted to the transcript equivalent to a miRNA (Llave et al., 2002, Science 297, 2053). Use of such small antisense RNA corresponding to polynucleotide of the invention is expressly contemplated.
  • genetic construct as used herein also includes small antisense RNAs and other such polypeptides effecting gene silencing.
  • 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).
  • 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 co- ordinately 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.
  • 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.
  • Transformation of other species is also contemplated by the disclosure. Suitable methods and protocols are available in the scientific literature.
  • 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).
  • 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.
  • 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 Plants are known to those skilled in the art, illustrative of which may involve the use of sequence- specific 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.
  • TALENs or a Zinc finger nucleases can be used to modify one or more base pairs in the uORF in order to change the encoded amino acid, or at least partially, disable or render it inoperative, so it is no longer translatable.
  • a codon for a highly conserved amino acid in the uORF can be changed to stop the uORF from functioning in downregulating transcription or translation of the downstream coding sequence.
  • a His residue in the conserved region of the uORF can be changed to a Leu.
  • an early base pair in the uORF is altered to introduce a stop codon and cause early termination of the uORF.
  • the uORF may be altered to remove a stop codon.
  • modifying the uORF comprises site-directed nucleases or oligonucleotide-directed mutagenesis.
  • the mutagenesis is achieved using SDN-1.
  • said mutagenesis is achieved using Zinc finger nucleases, TALENS and / or CRISPR/Cas gene editing.
  • said mutagenesis is achieved using CRISPR/Cas9 or CRISPR/Cas12 gene editing or so called “base editing”.
  • a plant, plant cell, or plant part including a modified plant, plant cell or plant part, comprising a comprising a modification to an upstream open reading frame (uORF) in a 5' untranslated region (UTR) of a GDP-L- galactose phosphorylase (GGP) gene or an ortholog thereof, wherein the modification comprises a deletion, addition, or substitution of at least one base pair of in the 5' UTR of the GGP gene.
  • the expression of GGP gene is not silenced.
  • the modification is capable of increasing foliar ascorbate levels.
  • the modification is capable of increased grain crop yield.
  • the increase in grain crop yield is increased grain weight per plant.
  • the modification is capable of generating increased number of panicles per plant.
  • the modified plant or plant cell exhibits a desirable trait selected from the group consisting of increased cellular ascorbate levels, increased foliar ascorbate content, increased root ascorbate content, increased fruit ascorbate content, increased grain ascorbate content, increased germinated grain ascorbate content, increased cellular iron content, increased photosynthesis, improved increased SPAD, increased vigour, increased tolerance to an abiotic stress, such as increased salt tolerance and increased tolerance to a pathogen.
  • the GGP gene 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: 59-81, and amino acid sequences having at least 70% sequence identity to any of the foregoing.
  • the 5' UTR of the GGP gene or ortholog thereof comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 11, 15-17, 21-23, 25, 28, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 50-51, and 54-55and nucleic acid sequences having at least 70% sequence identity to any of the foregoing.
  • the modified uORF of the GGP gene comprises at least one deletion or non- conservative substitution of at least one base pair in the uORF
  • the modified plant comprises a modified genome, comprises a modified GGP gene comprising a modified GGP uORF as disclosed herein.
  • the modified uORF of GGP comprises at least one deletion or non-conservative substitution of at least one base pair in the uORF.
  • the modified uORF of GGP comprises deletion or non-conservative substitution of 1 to 100 base pairs in the uORF.
  • the modified uORF of GGP comprises deletion or non-conservative substitution of 2 to pairs in the uORF.
  • the modified uORF comprises deletion or non-conservative substitution of 5 to 70 base pairs in the uORF. In one embodiment, the modified uORF of GGP comprises non-conservative substitution of 1 base pair in the uORF. In another embodiment the modified uORF of GGP comprises deletion of 5 base pairs in the uORF. In another embodiment the modified uORF of GGP comprises deletion of 63 base pairs in the uORF.
  • the modified uORF of GGP results in disruption of the reading frame of the GGP uORF, wherein the GGP uORF comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 12-14, 18-20, 24, 26-27, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48-49, and 52- 53, and amino acid sequences having at least 70% sequence identity to any of the foregoing.
  • the modified uORF results in the disruption of the uORF reading frame results in amino acid substitution, deletion or insertion of one or more amino acid residues from position 30 to position 61 of SEQ ID NO: 10.
  • the disruption of the uORF reading frame comprises substitution of an amino acid sequence comprising the amino acid residues at position 57-61 of SEQ ID NO: 10. In an embodiment, the disruption of the uORF reading frame comprises deletion of an amino acid sequence comprising the amino acid residues at position 55-56 of SEQ ID NO: 10. In another embodiment the disruption of the uORF reading frame comprises deletion of an amino acid sequence comprising the amino acid residues at position 37-57 of SEQ ID NO: 10. In some embodiments, the modified GGP uORF peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 56-58. In some embodiments, the entire GGP uORF has been modified or deleted.
  • the modified GGP uORF results in reduced ribosomal stalling in the uORF.
  • the modified uORF results in enhancing the expression of GGP by at least about 1%, by at least 2%, by at least 3%, by at least 4%, by at least 5%, by at least 6%, by at least 7%, by at least 8%, by at least 9%, by at least 10%, by at least 15%, by at least 20%, by at least 25%, by at least 30%, by at least 35%, by at least 40%, by at least 45%, by at least 50%, by at least 55%, by at least 60%, by at least 65%, by at least 70%, by at least 75%, by at least 80%, by at least 85%, by at least 90%, by at least 95%, or by at least 100%,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.
  • the present disclosure also to plants comprising the plant cells, the polynucleotides, the polynucleotide constructs, or the expression vectors disclosed herein.
  • the present disclosure also extends to plants, plant cells, transgenic plants or transgenic plant cells comprising a comprising a modification to an upstream open reading frame (uORF) in a 5' untranslated region (UTR) of a GDP-L-galactose phosphorylase (GGP) gene or an ortholog thereof, wherein the modification comprises a deletion, addition, or substitution of at least one base pair of in the 5' UTR of the GGP gene.
  • uORF upstream open reading frame
  • UTR 5' untranslated region
  • GGP GDP-L-galactose phosphorylase
  • 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 GGP gene or ortholog thereof when compared to the level of expression of the GGP gene or ortholog thereof in a control plant cell harbouring a corresponding native the GGP gene or ortholog thereof of the same plant species.
  • the transgenic plant or plant cells may comprise the polynucleotides disclosed herein. [0260] In one embodiment, the plant, plant cell, transgenic plant or transgenic plant cell is a cereal grass plant.
  • the plant is selected from the group consisting of rice, barley, wheat, rye, oats, millet, sorghum, triticale, buckwheat, quinoa, spelt, einkorn, amaranth, grass, corn, 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, a leguminous plant, sweet potato, yam and a plant of the nightshade family.
  • the plant is a rice or wheat plant.
  • the present disclosure also extends to seeds produced by the plants disclosed herein, to plants derived from such seeds, and to plant parts derived from the plants disclosed herein.
  • the plant part is selected from the group consisting of a seed, grain, fruit, leaf, flower, tuber, stalk, rhizome, spore, cutting, nut, and root.
  • the plant part is grain.
  • the grain is selected from the group consisting of rice grain, wheat grain, rye grain, barley grain or oat grain.
  • the grain is rice grain.
  • wheat grain is wheat grain.
  • 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.
  • 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.
  • 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.
  • 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.
  • the plant is elected from the group consisting of rice, barley, wheat, rye, oats, millet, sorghum, triticale, buckwheat, quinoa, spelt, einkorn, amaranth, grass, corn, 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, sweet potato, yam, a leguminous plant and a plant of the nightshade family.
  • the plant is a rice plant or a wheat plant.
  • 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.
  • 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
  • the term “propagule” means any part of a plant that may be used in either sexual or asexual, including seeds and cuttings.
  • the plant is an embryo, a seed, a seedling, a juvenile plant or a mature plant.
  • 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, ferns, horsetails, psilophytes, lycophytes, bryophytes, and multicellular algae.
  • angiosperms monocotyledonous and dicotyledonous plants
  • gymnosperms ferns
  • horsetails psilophytes, lycophytes, bryophytes, and multicellular algae.
  • the disclosure as disclosed here in extends to plant parts derived from the plants generated by the methods disclosed herein.
  • 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.
  • the plant part is a seed or a grain.
  • 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, ferns, horsetails, psilophytes, lycophytes, bryophytes, and multicellular algae.
  • angiosperms monocotyledonous and dicotyledonous plants
  • gymnosperms ferns
  • horsetails psilophytes, lycophytes, bryophytes, and multicellular algae.
  • the plant is rice, barley (Hordeum vulgare), wheat (Triticum aestivum), rye (Secale cereal), oats (Avena sativa), millet, sorghum, triticale, buckwheat, quinoa, einkorn, amaranth, spelt (Triticum spelta), grass, corn, 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, pistachio, walnut, chestnut, a vine, a forage grass, alfalfa, a plant of the genus Prunus, a
  • Examples of brassica plants include bok choy, broccoli, cauliflower, cabbage, choy sum, kohlrabi, napa cabbage, rutabaga and turnips. Some examples of nightshade plants include potatoes, eggplant, bell and chili peppers.
  • the plant is a rice plant.
  • the plant is a Pooideae or cereal grass plant.
  • the plant is a wheat, barley, oat, rye or spelt plant.
  • 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.
  • the plant tissue is leaf tissue, stem tissue, root tissue, seed tissue or fruit tissue.
  • the present invention is further described by reference to the following non- limiting examples. EXAMPLES Methods Plant growth conditions [0278] Oryza sativa cv. Nipponbare was used for all experiments.
  • Rice grain were surface sterilized with 70% (v/v) ethanol (Chem-Supply, SA, Australia) for 1 min and 30% (v/v) bleach (White King, NSW, Australia) with a few drops of Tween-20 (Sigma-Aldrich, MO, USA) for 30 min and washed three times with sterile dH2O.
  • Surface sterilized rice grain were germinated in a petri dish with moist sterile filter paper (Whatman, UK) for 7 to 9 days prior to transplanting to 1 L pots filled with potting mix in a glasshouse maintained at 26°C and 70% relative humidity at The University of Melbourne (Melbourne, VIC, Australia).
  • the potting mix was prepared by mixing one part washed fine sand (Col Smith, VIC, Australia), one part propagating sand (Brunnings, VIC, Australia), two parts premium vermiculate (Exfoliators, VIC, Australia), and one part General Mix potting media (Australian Growing Solutions, VIC, Australia) fertilized with Osmocote Exact Standard 8- 9 M (ICL, NSW, Australia) at a rate of 6 g/L.
  • Vector construction for constitutive OsGGP expression in rice [0279] The OsGGP (LOC_Os12g08810) CDS was PCR-amplified from rice cv.
  • Agrobacterium-mediated transformation of rice callus was carried out using established protocols (Sallaud et al.2003 Theor Appl Genet) Vector construction for mutated OsGGP uORF in rice
  • the sgRNAs were inserted downstream of the OsU3 promoter using the tRNA-gRNA system and BsaI cloning strategy as previously described (Xie et al 2015 PNAS).
  • the p35S-Cas9 sgRNA was assembled in two fragments with primers S1 (5’-GGTCTCATGGCAACAAAGCACCAGTGGTCTAG- 3’) (SEQ ID NO: 108) and AS1 (5’- TAGGTCTCAGGCTGGGCTGGATGCACCAGCCGGGAATC-3’) (SEQ ID NO: 109) and S2 (5’- TAGGTCTCCAGCCCCGCACGGGTTTTAGAGCTAGAAATAG-3’) (SEQ ID NO: 110) and AS2 (5’- TAGGCATGACTATGGTCTCTGTAGTAAAAAAAAGCACCGACTCGGTGCCAC 3’) (SEQ ID NO: 111), respectively.
  • the pUbi-Cas9 sgRNA was assembled in two fragments with primers S3 (5’-GGTCTCATGGCAACAAAGCACCAGTGGTCTAG- 3’) (SEQ ID NO: 112) and AS3 (5’- TAGGTCTCAGAAGAGGAGGTCTGCACCAGCCGGGAATC-3’) (SEQ ID NO: 113) and S4 (5’- TAGGTCTCCCTTCCTCGCCGGGTTTTAGAGCTAGAAATAG-3’) (SEQ ID NO: 114) and AS4 (5’TAGGCATGACTATGGTCTCTGTAGTAAAAAAAAAAGCACCGACTCGGTGCC AC-3’) (SEQ ID NO: 115), respectively.
  • the resulting vectors were named p35S-Cas9- uORF and pUbi-Cas9-uORF, respectively, and were mobilised into Agrobacterium tumefaciens (strain AGL1) by electroporation prior to rice transformation.
  • Rice transformation [0281] Transformation of rice callus was performed using established protocols [198]. Briefly, seed callus was induced on for 4 weeks (28°C, dark) followed by multiplication of embryonic units on fresh NB medium for a further 10-14 d (28°C, dark). Calli were then co-cultured with A. tumefaciens and placed on R2-CS medium for 3 d (25°C, dark).
  • Transformed calli were then selected on R2-S medium for 14 d (28°C, dark), proliferated on NBS medium for 2-3 weeks (28°C, dark), and matured on PR-AG medium for 7-10 d (28°C, dark) prior to regeneration on RN medium for 2 d (28°C, dark) and then a further 4-6 weeks (28°C, 12 h light/12 h dark). Plantlets were then rooted on P medium for 3-4 weeks (28°C, 12 h light/12 h dark) prior to greenhouse acclimation in Jiffy peat pellets for 15 d, followed by transfer to soil pots.
  • BR samples were immediately snap-frozen in liquid nitrogen, lyophilized, and ground using a Tube Mill with a 40 mL grinding chamber (IKA), whereas GBR samples were imbibed in sterile dH2O for 12 h in the dark at 28°C, washed three times with sterile dH2O, and spread in a petri dish with moist sterile filter paper (Whatman) for 72 h in the dark at 28°C. The GBR was then washed three times with sterile dH2O, snap-frozen in liquid nitrogen, lyophilized, and ground using a Tube Mill with a 40 mL grinding chamber (IKA).
  • IKA moist sterile filter paper
  • the plants were automatically watered daily so that approximately 500 mL of water was maintained in the soil.
  • the salt plants were treated with saline solution on DAP 20 and 23 (100 mL of 225mM NaCl) to reach a final concentration of 90 mM NaCl in the soil solution, while the control plants received equivalent water applications.
  • Imaging was carried out daily from DAP 14 to 40 inclusive. From these images the PSA of the plant, as viewed using an RGB camera, was obtained as previously described. For analysis purposes, PSA was defined as the sum of the areas as measured (in six side/oblique camera views plus twice the shoot area (top view).
  • the data was prepared for analysis using growthPheno [205], a package for the R statistical computing environment.
  • RNA from foliar tissue was isolated using the Direct-zolTM RNA MiniPrep Kit (Zymo Research, CA, USA) according to manufacturer’s instructions.
  • To isolate total RNA from BR and GBR sub-samples of snap-frozen BR and GBR were ground using a Tube Mill with a chilled 40 mL grinding chamber (IKA, Germany) and suspended in an extraction buffer containing 0.1M glycine-NaOH (Chem-Supply), 100 mM NaCl (Chem- Supply), 10 mM EDTA (Chem-Supply), 2% SDS (Sigma-Aldrich), and 1% sodium lauryl- sarcosine (Sigma-Aldrich) at pH 9 for 7 min at 1,400 RPM [200].
  • RNA was then purified from the upper aqueous phase using the Direct-zolTM RNA MiniPrep Kit (Zymo Research) according to manufacturer’s instructions. Quantification, DNase treatment, and reverse 57 transcription of the RNA was performed using the QuantiFluor® RNA System (Promega, WI, USA), RQ1 RNase-Free DNase (Promega), and the Tetro cDNA Synthesis Kit (Bioline), respectively, according to manufacturer’s instructions.
  • the quantitative reverse transcription-PCR (qRT-PCR) primers were designed to amplify the OsGGP, 35S-OsGGP, OsGME1, OsGME2, and OsGPP genes using Primer 3 software [201, 202]. Primer efficiency was ⁇ 93% for all primer pairs.
  • the qRT-PCR analysis was carried out on a CFX ConnectTM Real-Time System (Bio-Rad, CA, USA) with Hard-Shell® 96-Well PCR Plates (Bio-Rad).
  • the qRT-PCRs were performed in a final volume of 10 ⁇ L for KAPA SYBR® FAST (Kapa Biosystems, MA, USA) according to manufacturer’s instructions, each with four technical replicates.
  • the absolute quantification of transcript copy number was determined using a 10-fold serial dilution of 108 –102 copies for each PCR product and a standard curve generated with the Bio-Rad CFX Manager 3.1 software (Bio-Rad).
  • PCR products were purified and quantified with a DNA Clean & ConcentratorTM-5 Kit (Zymo Research) and the QuantiFluor® ONE dsDNA System (Promega), respectively, prior to serial dilution.
  • the geometric mean expression of three housekeeping genes selected from OsGAPDH, OsELF1, OsACT1 and OsUBQ5 using the NormFinder Excel add-in was used to normalize the expression of the 35S-OsGGP, OsGGP, OsGME1, OsGME2, and OsGPP genes. Quantification of total ascorbate [0285] Total ascorbate was extracted and measured as previously described with modifications [199].
  • total ascorbate was extracted from ground, lyophilized tissue homogenised in extraction fluid containing 8% metaphosphoric acid, 2 mM EDTA, and 2 mM TCEP (Sigma-Aldrich) at 40°C for 2 h.
  • the extract was centrifuged and 5 ⁇ L of supernatant was injected onto a C183 ⁇ m 33 x 7 mm Alltima Rocket column (Hichrom Limited, UK) maintained at 40°C with a flow rate of 1 mL/min.
  • the concentration was determined by reverse phase chromatography on a Shimadzu Nexera UHPLC system (Shimadzu, Japan) with simultaneous UV and MS detection.
  • Mobile phase A consisted of MS grade water with 0.1% formic acid (ThermoFisher Scientific, MA, USA).
  • the elution procedure utilized a 1 min gradient from 0% to 90% of mobile phase B: MS grade acetonitrile with 0.1% formic acid (ThermoFisher Scientific).
  • the UV absorption signal was acquired using a Shimadzu SPD-20A detector at 245 nm wavelength.
  • the MS data was acquired on a Shimadzu LCMS-8050 triple quadrupole mass spectrometer equipped with a DUIS source.
  • the MS instrument was operated in Multiple Reaction Monitoring mode monitoring ions in both positive (177>95, 177>85) and negative (175 >115, 175>71, 175>59) ionisation modes.
  • Example 1 Constitutive overexpression of OsGGP in rice [0286] Rice cv. Nipponbare transformants constitutively overexpressing the OsGGP CDS were generated through Agrobacterium tumefaciens-mediated transformation of a T-DNA containing the OsGGP CDS under transcriptional control of the dual CaMV 35S promoter.
  • a total of 27 independent, hemizygous 35S-OsGGP transformation events were regenerated from tissue culture and ascorbate concentrations were measured in the T1 BR. Ascorbate concentrations were negligible in both wild-type (WT) and 35S-OsGGP brown rice (BR) but was significantly increased up to 5.0-fold in 35S-OsGGP germinated brown rice (GBR) relative to WT.
  • Table 3 Ascorbate concentrations of T135S-OsGGP GBR and the number of T1 35S-OsGGP seedlings with the 35SGGP T-DNA present or absent. Genotype Total ascorbate No. of seedlings No.
  • Salt stress was applied in two steps to the plants at days after planting (DAP) 20 and 23 and the plants were imaged daily from DAP 14 to 40 inclusive.
  • DAP days after planting
  • foliar ascorbate concentrations were significantly fold in 35S-OsGGP-1 plants relative to NS- 1 at DAP 40, however foliar ascorbate concentrations did not differ significantly between NS-2 and 35S-OsGGP-2 plants ( Figure 3a).
  • foliar ascorbate concentrations were significantly increased 1.3-fold in both 35S-OsGGP-1 and 35S- OsGGP-2 plants relative to NS-1 and NS-2, respectively, at DAP 40 ( Figure 3b).
  • sPSA smoothed projected shoot area
  • AGR sPSA absolute growth rate
  • RGR sPSA relative growth rate
  • Example 3 Production of rice with CRISPR/Cas9-induced mutations in the OsGGP uORF [0294] Two CRISPR/Cas9 vectors, p35S-Cas9-uORF and pUbi-Cas9-uORF, were transformed into rice cv. Nipponbare via A.
  • the uorfOsGGP-1 mutation disrupts the reading frame replacing the final 5 residues and introduces a delayed stop codon extending the peptide from 61 to 72 residues in length ( Figure 6b).
  • the uorfOsGGP-2 mutation deletes two residues and disrupts the reading frame replacing the final 5 residues and introduces a delayed stop codon extending the peptide from 61 to 70 residues in length ( Figure 6b).
  • the uorfOsGGP-3 mutation deletes 21 residues whilst maintaining the reading frame reducing the peptide from 61 to 40 residues in length ( Figure 6b).
  • transcript levels of the GDP-D-mannose-3′, 5′-epimerase 1 and 2 (OsGME1 and OsGME2) genes which encode the enzyme responsible for the fourth enzymatic step of the L-galactose pathway
  • the L-galactose-1- phosphate phosphatase (OsGPP) gene which encodes the sixth enzymatic step of the L-galactose pathway
  • Example 5 Ascorbate levels in uorf OsGGP mutants [0298] Foliar ascorbate concentrations were significantly increased 1.2- and 1.3-fold in the T2 homozygous, transgene-free uorfOsGGP-2 and uorfOsGGP-3 mutants, respectively, relative to WT ( Figure 8a). Similarly, foliar ascorbate concentrations were increased 1.2- fold in the T2 homozygous, transgene-free uorfOsGGP-1 mutant relative to WT but did not differ significantly (Figure 8a).
  • Foliar ascorbate concentrations were also increased 1.2- to 1.7-fold in a range of T1 heterozygous, homozygous, and bi-allelic, transgene-free uorfOsGGP-2 and uorfOsGGP-3 mutants relative to WT (Figure 8b). Ascorbate concentrations were negligible in both T3 homozygous, transgene-free WT and uorfOsGGP- 1 brown rice and did not differ significantly (Figure 8c). Ascorbate concentrations were significantly decreased 1.4-fold in uorfOsGGP-1 brown rice germinated for 24 h relative to WT, but otherwise did not differ significantly in brown rice germinated for 48 and 72 h.
  • Example 6 Shoot growth uorf OsGGP mutants
  • sPSA smoothed PSA
  • the sPSA absolute growth rate (AGR) and sPSA relative growth rate (RGR) were calculated at the following DAP intervals: 16-30, 30-44, 44-58, 58-79, 79- 101, and 101-128.
  • the WT and uorfOsGGP-1 plants did not differ significantly with respect to sPSA AGR values at any of the calculated DAP intervals ( Figure 9b).
  • the uorfOsGGP-1 mutant had a significantly higher sPSA RGR value for the DAP interval 16-30 relative to WT, but otherwise did not differ significantly at any of the other calculated DAP intervals (Figure 9c).
  • Example 7 Salt stress tolerance assessment of the uorf OsGGP mutants
  • Shoot growth measurements of control and salt-stressed T2 homozygous, transgene-free WT and uorfOsGGP-1 plants were carried out using automated imaging. Salt stress was applied in two steps to the plants at DAP 20 and 23 and the plants were imaged daily from DAP 14 to 40 inclusive. Foliar ascorbate concentrations were significantly increased 1.3-fold in both the control and salt-stressed uorfOsGGP-1 mutants relative to WT ( Figure 10a, b).
  • the sPSA AGR and sPSA RGR were calculated at the following DAP intervals: 16-20, 20-24, 24-30, 30-35, and 35-40 ( Figure 10 and Figure 11).
  • the sPSA RGR declined more rapidly in salt-stressed plants than control plants ( Figure 10c, d).
  • a similar trend in the sPSA RGR of both the control and salt-stressed uorfOsGGP-1 mutant relative to WT was observed, with the uorfOsGGP-1 mutant having a significantly higher sPSA RGR for the DAP intervals 16-20, 20-24, and 24- 30 relative to WT, but otherwise did not differ significantly for the DAP intervals 30-35 and 35-40.
  • Example 8 Agro-morphological performance of the uorfOsGGP mutants [0301]
  • the T2 homozygous, transgene-free uorfOsGGP-1 mutant had a significant 1.2- fold decrease in panicle length, 1.1-fold decrease in the number of primary branches per panicle, and 1.2-fold decrease in the number of spikelets per panicle relative to WT but had a significant 1.3-fold increase in both of panicles per plant and grain weight per plant (Table 4).
  • the WT and uorfOsGGP-1 plants did not differ significantly with respect to straw biomass, the number of fertile spikelets per panicle, spikelet fertility, and thousand grain weight (Table 4).
  • the uorfOsGGP-1 mutant had a minor significant increase in the grain length to width ratio relative to WT, but otherwise did not differ significantly with respect to the percentage of chalky grain, grain length, grain width, and grain area (Table 5).
  • Table 4 Agro-morphological performance of T2 homozygous, transgene-free WT and uorfOsGGP-1 plants WT uorf OsGGP -1 Straw biomass (g DW) 36.72 ⁇ 1.05 35.33 ⁇ 0.71 No. of panicles per plant 20.33 ⁇ 1.02 27.33 ⁇ 0.76*** Panicle length (cm) 16.45 ⁇ 0.57 13.49 ⁇ 0.28** No.
  • Asterisks indicate significant differences between the WT and uorf OsGGP plants (two-sample t-test, * p-value ⁇ 0.05, ** p-value ⁇ 0.01, *** p-value ⁇ 0.001).
  • Table 5 Quality parameters of T3 homozygous, transgene-free WT and uorfOsGGP-1grain. Genotype Chalky grains (%) WT 4.60 ⁇ 1.32 uorf OsGGP -1 3.72 ⁇ 1.23 Values represent mean ⁇ SEM of six biological replicates. Asterisks indicate significant differences between the WT and uorfOsGGP plants (two-sample t-test, * p-value ⁇ 0.05).

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Abstract

The present disclosure relates generally to modifications of an uORF in the 5' untranslated region (UTR) of a GDP-L-galactose phophorylase (GGP) gene or orthologs thereof in cereal grass plants, for improving plant growth and agronomical traits.

Description

REGULATION GENE EXPRESSION FIELD OF THE INVENTION [0001] The present invention relates generally to control and manipulation of expression of GDP-L-Galactose phosphorylase (GGP) and ascorbate production in improving plant growth and agronomical traits. The invention also relates to sequence elements controlling GGP expression and production, and methods of their use. BACKGROUND OF THE INVENTION [0002] 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. [0003] Providing adequate, safe, and nutritious food for the growing human population under changing climatic conditions represents one of the greatest challenges of the 21st century. Adverse environmental conditions such as extreme temperatures, drought, and soil salinity are the primary cause of crop losses worldwide and are predicted to be exacerbated by climate change. The need for more abiotic stress tolerant crops is critical for ensuring global food security. [0004] Essential vitamin and mineral deficiency is a major threat to the health and development of populations worldwide; and its effects are often acute and long-lasting - leading to impaired mental and physical development, poor health. Improving the yield and/or nutritional content of crops through agronomic practices, plant breeding, or biotechnology - a process known as biofortification - offers a low-cost and sustainable strategy to combat nutritional deficiencies. [0005] L-ascorbic acid (also known as ascorbate or vitamin C, and hereafter referred to as ascorbate) is an important multifunctional molecule for both plants and mammals. Ascorbate is a reducing agent capable of donating electrons and primarily functions as a cellular antioxidant and enzymatic co-factor. [0006] In plants, ascorbate is the most abundant water-soluble antioxidant and is well- known for its important role in photosynthetic functions and stress tolerance. This is largely due to the ability of ascorbate to counteract oxidative stress produced by normal or stressed cellular metabolism, either directly as oxygen species (ROS), or through the ascorbate-glutathione cycle (major antioxidant system of plants cells). [0007] Crop plants such as citrus plants, tomatoes, and plants with starchy tuberous roots such as yams, potatoes and cassava have relatively high ascorbate content. However, cereal grain plants, (for example, rice; wheat; barley; and sorghum) provide more food energy worldwide than any other type of crop but have low or negligible ascorbate levels. In particular, rice and wheat are staple crops in many parts of the world; and is cultivated on more land than any other crops. It is thought that by increasing the amount of vitamins such as ascorbate in such staple food crops would go somewhat to addressing nutrition deficiencies in human populations and improving global food security by improving plant tolerance to abiotic stresses brought on by changing climate. [0008] Thus, there is a need to produce plants that have elevated ascorbate levels to improve plant tolerance of environmental stressors, and also for providing crops with modified or increased ascorbate 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 modified genome of a cereal grass plant comprising a polynucleotide comprising a modification to an upstream open reading frame (uORF) in a 5' untranslated region (UTR) of a GDP-L-galactose phosphorylase (GGP) gene or an ortholog thereof, wherein the modification comprises a deletion, addition, or substitution of at least one base pair in the 5' UTR of the GGP gene. [0010] In one embodiment, the expression of the GGP gene is not silenced. [0011] In one embodiment, the modified plant has increased grain crop yield. In a further embodiment, the increase in grain crop yield is increased grain weight per plant. In a further embodiment, the modified plant has an increase in the number of panicles per plant. In another embodiment, the modified plant has increased foliar ascorbate levels [0012] In an embodiment, the GGP gene 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: 59-81, and amino acid sequences having at least 70% sequence identity to any of the foregoing. [0013] In an embodiment, the 5' UTR of GGP gene or ortholog thereof comprises a nucleic acid sequence selected from the group of SEQ ID NOs:11, 15-17, 21-23, 25, 28, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 50-51, and 54-55, and nucleic acid sequences having at least 70% sequence identity to any of the foregoing. [0014] In one embodiment, the modified uORF of GGP comprises at least one deletion or non-conservative substitution of at least one base pair in the uORF. [0015] In one embodiment, the modified uORF of GGP comprises deletion or non- conservative substitution of 1 to 100 base pairs in the uORF. In another embodiment, the modified uORF of GGP comprises deletion or non-conservative substitution of 2 to 80 base pairs in the uORF. In another embodiment, wherein the modified uORF of GGP comprises deletion or non-conservative substitution of 5 to 70 base pairs in the uORF. [0016] In some embodiments, the modified uORF of GGP comprises non-conservative substitution of 1 base pair in the uORF. In another embodiment, the modified uORF of GGP comprises deletion of 5 base pairs in the uORF. In another embodiment, the modified uORF of GGP comprises deletion of 63 base pairs in the uORF. [0017] In some embodiments, the modified uORF of GGP results in disruption of the reading frame of the GGP uORF, wherein the GGP uORF comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 12-14, 18-20, 24, 26-27, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48-49, and 52-53, and amino acid sequences having at least 70% sequence identity to any of the foregoing. [0018] In a further embodiment, the disruption of the uORF reading frame results in amino acid substitution, deletion or insertion of one or more amino acid residues from position 30 to position 61 the GGP uORF peptide. In some embodiments, the disruption of the uORF reading frame comprises substitution of an amino acid sequence comprising the amino acid residues at position 57-61 of SEQ ID NO:10. In some embodiments, the disruption of the uORF reading frame comprises deletion of an amino acid sequence comprising the amino acid residues at position 55-56 of SEQ ID NO: 10. In some embodiments, the disruption of the uORF reading frame comprises deletion of an amino acid sequence comprising the amino acid residues at position 37-57 of SEQ ID NO:10. [0019] In some embodiments, the modified GGP uORF peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 56-58. [0020] In some embodiments, wherein the modified GGP uORF results in reduced ribosomal stalling in the uORF. [0021] In some embodiments, modifying the uORF comprises site-directed nucleases or oligonucleotide-directed mutagenesis. In some embodiments, the mutagenesis is achieved using SDN-1. In some embodiments, the mutagenesis is achieved using Zinc finger nucleases, TALENS and / or CRISPR/Cas gene editing and/or base editing. In some embodiments, the mutagenesis is achieved using CRISPR/Cas9 or CRISPR/Cas12 gene editing. [0022] In some embodiments, the cereal grass plant is selected from the group consisting of rice, barley, wheat, rye, oats, millet, sorghum, triticale, buckwheat, quinoa, spelt, einkorn, amaranth, grass, corn, wild rice, teff; Aegilops tauschii, brachypodium, Miscanthus, switchgrass or a forage grass. In some embodiments, the cereal grass plant is a wheat plant or a rice plant. In another embodiment, the cereal grass plant is a rice plant. [0023] In another aspect disclosed herein, there is provided a polynucleotide construct comprising a modification to an upstream open reading frame (uORF) in a 5' untranslated region (UTR) of a GDP-L-galactose phosphorylase (GGP) gene or an ortholog thereof, wherein the modification comprises a deletion, addition, or substitution of at least one base pair of in the 5' UTR of the GGP gene. [0024] In a further embodiment, the GGP gene 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: 59-81, and amino acid sequences having at least 70% sequence identity to any of the foregoing. [0025] In a further embodiment, the 5' UTR of the GGP gene or ortholog thereof comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 11, 15-17, 21- 23, 25, 28, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 50-51, and 54-55 and nucleic acid sequences having at least 70% sequence identity to any of the foregoing. [0026] The present disclosure also extends to a modified cereal grass plant comprising a polynucleotide comprising a modification to an upstream open reading frame (uORF) in a 5' untranslated region (UTR) of a GDP-L-galactose phosphorylase (GGP) gene or an ortholog thereof, wherein the modification comprises a deletion, addition, or substitution of at least one base pair of in the 5' UTR of the GGP gene. [0027] In another aspect disclosed herein, there is provided a method of modifying the genome of a cereal grass plant, comprising introducing a modification to an upstream open reading frame (uORF) in a 5' region (UTR) of a GDP-L-galactose phosphorylase (GGP) gene or an ortholog thereof, wherein the modification comprises a deletion, addition, or substitution of at least one base pair of in the 5' UTR of the GGP gene. [0028] In another aspect disclosed herein, there is provided a method of increasing the grain crop yield of a cereal grass plant, comprising introducing a modification to an upstream open reading frame (uORF) in a 5' untranslated region (UTR) of a GDP-L-galactose phosphorylase (GGP) gene or an ortholog thereof, wherein the modification comprises a deletion, addition, or substitution of at least one base pair of in the 5' UTR of the GGP gene. [0029] In one embodiment, the increase in grain crop yield is increased grain weight per plant. In one embodiment, the increase in grain crop yield is the result of an increase in the number of panicles per plant. [0030] In another aspect disclosed herein, there is provided a method of increasing the number of panicles of a cereal grass plant, the method comprising introducing a modification to an upstream open reading frame (uORF) in a 5' untranslated region (UTR) of a GDP-L- galactose phosphorylase (GGP) gene or an ortholog thereof. [0031] In one embodiment, the modified plant has increased grain crop yield. In a further embodiment, the increase in grain crop yield is increased grain weight per plant. In a further embodiment, the modified plant has an increase in the number of panicles per plant. In another embodiment, the modified plant has increased foliar ascorbate levels [0032] In an embodiment, the GGP gene 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: 59-81, and amino acid sequences having at least 70% sequence identity to any of the foregoing. [0033] In an embodiment, the 5' UTR of GGP gene or ortholog thereof comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 11, 15-17, 21-23, 25, 28, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 50-51, and 54-55 and nucleic acid sequences having at least 70% sequence identity to any of the foregoing. [0034] In one embodiment, the modified uORF of GGP comprises at least one deletion or non-conservative substitution of at least one base pair in the uORF. [0035] In one embodiment, the modified uORF of GGP comprises deletion or non- conservative substitution of 1 to 100 base pairs in the uORF. In another embodiment, the modified uORF of GGP comprises or non-conservative substitution of 2 to 80 base pairs in the uORF. In another embodiment, wherein the modified uORF of GGP comprises deletion or non-conservative substitution of 5 to 70 base pairs in the uORF. [0036] In some embodiments, the modified uORF of GGP comprises non-conservative substitution of 1 base pair in the uORF. In another embodiment, the modified uORF of GGP comprises deletion of 5 base pairs in the uORF. In another embodiment, the modified uORF of GGP comprises deletion of 63 base pairs in the uORF. [0037] In some embodiments, the modified uORF of GGP results in disruption of the reading frame of the GGP uORF, wherein the GGP uORF comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 12-14, 18-20, 24, 26-27, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48-49, and 52-53, and amino acid sequences having at least 70% sequence identity to any of the foregoing. [0038] In a further embodiment, the disruption of the uORF reading frame results in amino acid substitution, deletion or insertion of one or more amino acid residues from position 30 to position 61 the GGP uORF peptide. In some embodiments, the disruption of the uORF reading frame comprises substitution of an amino acid sequence comprising the amino acid residues at position 57-61 of SEQ ID NO:10. In some embodiments, the disruption of the uORF reading frame comprises deletion of an amino acid sequence comprising the amino acid residues at position 55-56 of SEQ ID NO :10. In some embodiments, the disruption of the uORF reading frame comprises deletion of an amino acid sequence comprising the amino acid residues at position 37-57 of SEQ ID NO: 10. [0039] In some embodiments, the modified GGP uORF peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 56-58. [0040] In some embodiments, wherein the modified GGP uORF results in reduced ribosomal stalling in the uORF. [0041] In some embodiments, modifying the uORF comprises site-directed nucleases or oligonucleotide-directed mutagenesis. In some embodiments, the mutagenesis is achieved using SDN-1. In some embodiments, the mutagenesis is achieved using Zinc finger nucleases, TALENS and / or CRISPR/Cas gene editing and/or base editing. In some embodiments, the mutagenesis is achieved using CRISPR/Cas9 or CRISPR/Cas12 gene editing. [0042] In some embodiments, the plant is selected from the group consisting of rice, barley, wheat, rye, oats, millet, sorghum, triticale, buckwheat, quinoa, spelt, einkorn, amaranth, grass, corn, wild rice, teff; Aegilops tauschii, brachypodium, Miscanthus, switchgrass or a forage grass. In some embodiments, the cereal grass plant is a wheat plant or a rice plant. In another embodiment, the cereal grass plant is a rice plant. [0043] The present disclosure also extends to a plant cell produced by the methods disclosed herein. [0044] The present disclosure also extends to a plant comprising any one of the modified genomes or the polynucleotides disclosed herein. [0045] The present disclosure also extends to seeds produced by the plants disclosed herein, to plants derived from such seeds, and to plant parts derived from the plants disclosed herein. [0046] 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, a panicle and root. [0047] In a further embodiment, the plant part is grain. In a further embodiment, the plant part is a germinated grain. [0048] In an embodiment, the grain is selected from the group consisting of rice grain, wheat grain, rye grain, barley grain or oat grain. [0049] In an embodiment, the grain is rice. [0050] The present disclosure also extends to flour produced from the grain disclosed herein. [0051] 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 [0052] Figure 1 shows the ascorbate-glutathione cycle—a major antioxidant system of plants cells. In this cycle electrons flow from NADPH to H2O2. Dashed arrows indicate non-enzymatic disproportionation. APX, ascorbate peroxidase; MDAR, monodehydroascorbate reductase; DHAR, dehydroascorbate reductase; GR, glutathione reductase. [0053] Figure 2 shows production and characterization of independent rice transformation events constitutively overexpressing CDS. (a) Schematic representation of the T-DNA used for constitutive overexpression of the OsGGP CDS. RB, right border; 2 x 35S, dual CaMV 35S promoter; OsGGP, OsGGP CDS; nos T, nopaline synthase terminator; 2 x 35S enhanced, dual CaMV 35S promoter enhanced; hptII, hygromycin phosphotransferase II; pA, CaMV poly(A) signal; LB, left border. (b) Ascorbate concentrations in BR and GBR of T3 homozygous NS and 35S-OsGGP (shown in grey). Bars represent mean ± SEM of three independent replicates of approximately fifty grains. (c) Foliar and (d) root ascorbate concentrations of T3 homozygous NS and 35S-OsGGP (shown in grey) plants at the vegetative growth phase. (e) Foliar ascorbate concentrations and transcript levels of the (f) endogenous OsGGP gene, (g) 35S-OsGGP transgene, (h) OsGME1, (i) OsGME2, and (j) OsGPP genes of T2 homozygous NS and 35S-OsGGP (shown in grey) plants at the reproductive growth phase. Bars represent mean ± SEM of three biological replicates. Asterisks indicate statistically significant differences between NS and 35S-OsGGP plants (two-sample t-test; * p-value ≤0.05; ** p-value ≤0.01; *** p-value ≤0.001). [0054] Figure 3 shows salt tolerance assessment of NS and 35S-OsGGP plants during the vegetative growth phase. Foliar ascorbate concentrations of (a) control and (b) salt-stressed T4 homozygous NS-1 and 35S-OsGGP-1 plants and T3 homozygous NS-2 and 35S- OsGGP-2 plants at DAP 40. Bars represent mean ± SEM of six biological replicates. Asterisks indicate statistically significant differences between NS and 35S-OsGGP plants (two-sample t-test; ** p-value ≤0.01; *** p-value ≤0.001). The sPSA RGR of (c) control and (d) salt-stressed T4 homozygous NS-1 and 35S-OsGGP-1 plants and of (e) control and (f) salt-stressed T3 homozygous NS-2 and 35S-OsGGP-2 plants. Salt was applied at DAP 20 and 23. Values represent mean ± half least significant (5%) pairwise difference of six biological replicates. Non-overlapping error bars indicate significant differences at α = 0.05. [0055] Figure 4 shows CRISPR/Cas9-targeted mutagenesis of the OsGGP uORF. Schematic representation of the (a) p35S-Cas9-uORF and (b) pUbi-Cas9-uORF T-DNA. LB, left border; pA, CaMV poly(A) signal; hptII, hygromycin phosphotransferase II; 35S, CaMV 35S promoter; OsU3, rice U3 promoter; tRNA, transfer RNA; sgRNA, single guide RNA; 2 x 35S, dual CaMV 35S promoter; Ubi, maize ubiquitin promoter; hSpCas9, human codon optimized Streptococcus pyogenes Cas9; nos, nopaline synthase terminator; RB, right border. (c) Nucleotide sequence of the OsGGP uORF. The sgRNA target sites are underlined and the protospacer-adjacent motifs are shown in bold. [0056] Figure 5 shows nucleotide alignment of the parental T0 CRISPR/Cas9- induced uorfOsGGP mutants used in this chapter. The sgRNA target site is underlined. The protospacer-adjacent motif, sequence changes, and deleted nucleotides are shown in bold, red, and as hyphens, respectively. [0057] Figure 6 shows nucleotide and peptide sequence alignment of the CRISPR/Cas9- induced homozygous, transgene-free uorfOsGGP mutants used in this chapter. (a) Nucleotide sequence alignment. The sgRNA target site is underlined. The protospacer- adjacent motif, sequence changes, and deleted nucleotides are shown in bold, red, and as hyphens, respectively. (b) Peptide sequence alignment. Sequence changes and deleted residues are shown in red and as hyphens, respectively. [0058] Figure 7 shows transcript analysis of WT and uorfOsGGP plants. Transcript levels of the (a) OsGGP, (b) OsGME1, (c) OsGME2, and (d) OsGPP genes in T2 homozygous, transgene-free WT and uorfOsGGP plants. Bars represent mean ± SEM of six biological replicates. No statistically significant differences were detected between WT and uorfOsGGP plants (two-sample t-test). [0059] Figure 8 shows ascorbate concentrations of WT and uorfOsGGP plants. (a) Foliar ascorbate concentrations of T2 homozygous, transgene-free WT and uorfOsGGP plants. Bars represent mean ± SEM of six biological replicates. (b) Foliar ascorbate concentrations of T1 heterozygous, homozygous, and bi-allelic, transgene-free WT and uorfOsGGP plants. Bars represent mean ± SEM of one to five biological replicates. (c) Ascorbate concentrations of T3 homozygous, transgene-free WT and uorfOsGGP-1 brown rice and germinated brown rice. Values represent mean ± SEM of three independent replicates of approximately 50 grain. Asterisks indicate statistically significant differences between WT and uorfOsGGP plants (two-sample t-test; * p-value ≤0.05; ** p-value ≤0.01). [0060] Figure 9 shows shoot growth measurements of WT and uorfOsGGP-1 plants using automated imaging over the course of a full lifecycle. The (a) sPSA, (b) sPSA AGR, and (c) sPSA RGR of T2 homozygous, transgene-free WT and uorfOsGGP-1 plants. Values represent mean ± half least significant (5%) pairwise difference of six biological replicates. Non-overlapping error bars indicate significant differences at α = 0.05. [0061] Figure 10 salt stress tolerance assessment of WT and uorfOsGGP-1 plants. Foliar ascorbate concentrations of (a) control and (b) salt-stressed T2 homozygous, transgene-free WT and uorfOsGGP-1 plants at DAP 40. Bars represent mean ± SEM of six biological replicates. Asterisks indicate significant differences between WT and uorfOsGGP plants (two-sample t-test; ** p-value ≤0.01; *** p-value ≤0.001). The sPSA RGR of (c) control and (d) salt-stressed T2 homozygous, transgene-free WT and uorfOsGGP-1 plants. Salt was applied at DAP 20 and 23. Values represent mean ± half least significant (5%) pairwise difference of six biological replicates. Non-overlapping error bars indicate significant differences at α = 0.05. [0062] Figure 11 shows the sPSA and sPSA AGR of control and salt-stressed WT and uorfOsGGP-1 plants. The sPSA of (a) control and (b) salt-stressed T2 homozygous, transgene-free WT and uorfOsGGP-1 plants. The sPSA AGR of (c) control and (d) salt- stressed T2 homozygous, transgene-free WT and uorfOsGGP-1 plants. Salt was applied at DAP 20 and 23. Values represent mean ± half least significant (5%) pairwise difference of six biological replicates. Non-overlapping error bars indicate significant differences at α = 0.05. DETAILED DESCRIPTION OF THE INVENTION [0063] 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. [0064] 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. By way of example, “an element” means one element or more than one element. [0065] 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). [0066] 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 that recite the lower value of the range as well 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. [0067] 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. [0068] 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 a GGP gene or an ortholog thereof, wherein the modification comprises a deletion, addition, or substitution of at least one base pair of in the 5' UTR of the GGP gene, unexpectedly results in increased grain crop yield, increased grain weight per plant; increased number of panicles and/or increased foliar ascorbate levels. [0069] Thus, in one aspect disclosed herein, there is provided a modified genome of a cereal grass plant comprising a polynucleotide comprising a modification to an upstream open reading frame (uORF) in a 5' untranslated region (UTR) of a GDP-L-galactose phosphorylase (GGP) gene or an ortholog thereof, wherein the modification comprises a deletion, addition, or substitution of at least one base pair of in the 5' UTR of the GGP gene. In another aspect disclosed herein, there is provided, a modified cereal grass plant comprising a polynucleotide comprising a modification to an upstream open reading frame (uORF) in a 5' untranslated region (UTR) of a GDP-L-galactose phosphorylase (GGP) gene or an ortholog thereof, wherein the modification comprises a deletion, addition, or substitution of at least one base pair of in the 5' UTR of the GGP gene. [0070] In another aspect disclosed herein, there is disclosed are methods of modifying the genome of a cereal grass plant; methods of increasing the grain crop yield of a cereal grass plant; and methods of increasing the number of panicles of a cereal grass plant, wherein the methods comprise introducing a modification to an upstream open reading frame (uORF) in a 5' untranslated region (UTR) of a GDP-L-galactose phosphorylase (GGP) gene or an ortholog thereof, wherein the modification comprises a deletion, addition, or substitution of at least one base pair of in the 5' UTR of the GGP gene. Ascorbate in plants [0071] Ascorbate is an important multifunctional molecule for both plants and mammals. Ascorbate is a reducing agent capable of donating electrons and primarily functions as a cellular antioxidant and enzymatic co-factor. In plants, ascorbate is the most abundant water- soluble antioxidant and is well-known for its important role in photosynthetic functions and stress tolerance. In humans, ascorbate is an essential micronutrient and has key roles in human physiology, such as promoting the dietary uptake of iron (Fe) in the gut. It is thought that increasing ascorbate concentrations in crops may have the potential to improve both the abiotic stress tolerance of crops. [0072] Four pathways for ascorbate biosynthesis have been proposed in plants: the L- galactose, L-gulose, myo-inositol, and D-galacturonate pathways. All four pathways share an aldonolactone as the direct precursor to ascorbate (L-galactono-1,4-lactone for the L- galactose and D-galacturonate pathways and L-gulono-1,4-lactone for the L-gulose and myo-inositol pathways). The L-galactose pathway, which converts D-fructose-6-P to ascorbate via eight enzymatic steps (by phosphomannose isomerase (PMI), phosphomannose mutase (PMM), GDP-D-mannose pyrophosphorylase (GMP), GDP-D- mannose-3′,5′-epimerase (GME), GDP-L-galactose phosphorylase (GGP), L-galactose-1- phosphate phosphatase (GPP), L-galactose dehydrogenase (L-GalDH), and L-galactono- 1,4-lactone dehydrogenase (L-GalLDH)), is the dominant pathway leading to ascorbate biosynthesis in many plants including Arabidopsis thaliana, tomato and rice. In addition to de novo biosynthesis of ascorbate, there is an ascorbate recycling pathway where oxidized ascorbate can be regenerated via the recycling enzymes MDAR and DHAR to maintain cellular ascorbate levels. Little is known how ascorbate biosynthesis and recycling occur in plants, and it is becoming clear that ascorbate biosynthesis and recycling is tightly regulated at the transcriptional, translational, and post-translational level (Bulley et al.2016 Curr Opin Plant Biol). GDP-L-galactose phosphorylase (GGP) gene [0073] Substantial genetic evidence supports the L-galactose pathway - which converts D- fructose-6-P to ascorbate via eight enzymatic steps - as the predominant pathway toward ascorbate biosynthesis in plants. [0074] The GPD-L-galactose phosphorylase (GGP, also known as VTC2/VTC5) gene encodes the fifth enzymatic step of pathway catalysing the conversion of GDP-L-galactose to L-galactose1-P and represents the first committed step toward ascorbate biosynthesis. [0075] Splice junction and missense mutations in the Arabidopsis GGP gene reduced ascorbate to 10% of wild-type levels, supporting its critical role in L-galactose ascorbate biosynthesis, exhibit a seedling-lethal phenotype without the supplementation of L-galactose or ascorbate (e.g. Dowdle et al.2007 Plant J.). Relative to other genes from the L-galactose pathway, increased expression of the GGP gene consistently results in the largest increases in ascorbate concentrations in a wide range of species, suggesting GGP as the rate-limiting enzymatic step of the L-galactose pathway.; e.g. increased expression of the GGP gene has increased ascorbate concentrations 2.9- to 4.1-fold in Arabidopsis thaliana; 3.1-fold in potato; 2.1-fold in strawberry and 2.0- to 6.2-fold in tomato (Zhou et al. 2012 Biol Plantarum; Bulley et al.2012 Plant Biotechnol J). Increased ascorbate levels in some plants have been associated with in abnormalities in fruit development, seed deformities and/or delayed transition into reproductive stage (i.e. fruiting stage) (see Deslous et al. 2021 Journal of Experimental Botany 72: 3091–3107; Bulley et al 2012 Plant Biotechnology Journal 10: 390-397). Studies that have increased the expression of ascorbate genes have been shown to increase foliar ascorbate concentrations in rice (e.g. Zhang et al.2016 PLoS One) but it is unclear whether these strategies alter ascorbate concentrations in the rice grain and/or other tissues. [0076] Moreover, as the first committed step toward ascorbate biosynthesis in the L- galactose pathway, the catalysis of GDP-L-galactose to Lgalactose1-P by GGP is the most strongly regulated step in the L-galactose pathway, with the production of GGP thought to be post-transcriptionally regulated through a highly conserved cis-acting upstream open reading frame (uORF) in the GGP mRNA, in addition to transcriptional regulation. [0077] Recently, GGP genes were reported and characterised in an important crop plant, bread wheat. Further analyses indicated that plant GGP genes could be broadly categorised in two clades of GGP proteins, belonging to either graminaceous or non-graminaceous species (Broad et al.2019 BMC Plant Biol). In particular, it was noted that GGP genes in graminaceous species were highly conserved. A graminaceous plant is a plant from the Gramineae family (also known as Poaceae family) of monocotyledonous flowering plants commonly known as grasses (including cereal grass plants, bamboos and grass species associated with grassland, pasture). [0078] A cereal grass plant as described herein refers to an edible grain grass plant. A cereal grass plant may be a grass plant that is cultivated for its edible grain, wherein the grain is edible by humans. A cereal grass plant may be a grass plant that is cultivated for its edible grain, wherein the grain is edible by animals, including ruminant animals. Examples of graminaceous cereal grass plants include rice, wheat, rye, oats, barley, millet and maize. Other types of edible grain plants (sometimes called pseudocereals) include buckwheat, quinoa and chia. Other examples of cereal grass plants include sorghum, triticale, spelt, einkorn, amaranth, wild rice, teff, Aegilops tauschii, brachypodium, and forage grass. In an embodiment, the cereal grass plant is a rice, barley, wheat, rye, oats, millet, sorghum, triticale, buckwheat, quinoa, spelt, einkorn, amaranth, grass, corn, wild rice, teff; Aegilops tauschii, brachypodium, Miscanthus, switchgrass or a forage grass plant. In another embodiment, the cereal grass plant is a graminaceous cereal grass plant. In another embodiment, the cereal grass plant is a rice, wheat, rye, oats, barley, millet or maize plant. In another embodiment, the cereal grass plant is a rice or wheat plant. [0079] Table 1 provides a list of GGP genes and their corresponding sequence records. Table 1: Non-limiting examples of GGP genes and orthologs Species Gene ID(s) SEQ ID SEQ ID ide) TaGGP2-B (Wheat 64 87 subgenome B) GG upstream open readng rame (uO ) [0080] The translation of GGP has been discovered to be post-transcriptionally regulated through a highly conserved, cis-acting upstream open reading frame (uORF) in the long 5’ leader sequence upstream of the major (mORF) (see Laing et al.2015 Plant Cell; and Broad et al.2019 BMC Plant Biol). [0081] The GGP uORF is proposed to initiate from a non-canonical AUC or ACG start- codon, and encode a 60- to 65-residue long peptide. Disruption of the GGP uORF increased ascorbate concentrations when a GGP promoter-uORF-GGP construct was transiently transformed in Nicotiana benthamiana (Laing et al.2015 Plant Cell). It is thought, that under high ascorbate concentrations, the GGP uORF is proposed to be translated and stall the ribosome, thereby preventing translation of the GGP mORF (major ORF), while under low ascorbate concentrations the GGP uORF is either skipped or does not cause the ribosome to stall, thereby allowing the GGP mORF to be translated. The precise mechanism of how ascorbate influences the translation of the GGP uORF or mORF, however, has yet to be determined. Recently, disruption of the GGP uORF was shown to increase ascorbate concentrations in Arabidopsis, lettuce and tomato. Moreover, disruption of the lettuce GGP uORFs was associated with enhanced tolerance to methyl viologen-induced stress. (see Zhang et al.2018 Nat Biotechnol.; Li et al., 2018 Nat Biotechnol.) However, the role of GGP and specifically GGP uORF in other plants, such as grass plants, are less well characterised. [0082] The term upstream open reading frames, or “uORFs”, as used herein refers to elements located upstream of the 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). [0083] The terms “5' untranslated 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. In some within a 5’UTR region may be translated into a short peptide. In other embodiments, a uORF within a 5'UTR is not translated. [0084] The regulation of translation by uORFs may be a form of dynamic regulatory mechanism for the control of protein translation. Generally, uORFs repress translation of the mORF by allowing fewer ribosomes to reach the mORF, for example by causing ribosome stalling in response to metabolite levels. [0085] 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′-untranslated regions (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). [0086] As described elsewhere herein, the inventors have found that the uORFs of GGP genes in grass plants, such as cereal grass plants, function as translational regulators of these genes; and that modification of GGP uORFs, wherein the modification comprises a deletion, addition, or substitution of at least one base pair of in the 5' UTR of the GGP gene, surprisingly results in increased yield, in particular grain crop yield or panicle numbers. [0087] In an embodiment, the unmodified uORF peptide of the GGP gene comprises amino acid sequence of any one of SEQ ID NO: 10, 12-14, 18-20, 24, 26-27, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48-49, or 52-53, or an amino acid sequence having at least 70% sequence identity to any of the foregoing. In an embodiment, the unmodified uORF of the GGP gene comprises the nucleic acid sequence encoding the amino acid sequence of any one of SEQ ID NO: 10, 12-14, 18-20, 24, 26-27, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48-49, or 52-53; or any one of SEQ ID NOs: 11, 15-17, 21-23, 25, 28, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 50- 51, or 54-55; or nucleotide sequence having at least 70% sequence identity to any of the foregoing. Table 2: Unmodified uORF of GGP GGP gene uORF amino acid uORF nucleotide sequence sequence SE ID NO AeGGP2 (Aegilops tauschii) 54 AeGGP1 (Ae ilo s tauschii) 53 55 g p gion, potentially encoding a 60-65 amino acid peptide. Within the uORF region, there were regions of high amino acid sequence conservation (see Laing et al.2015 Plant Cell; and Broad et al.2019 BMC Plant Biol.). In one embodiment, the unmodified GGP uORF peptide comprises the amino acid sequence "GGGRL" (SEQ ID NO: 4) at the C-terminal end of the peptide. In one embodiment, the unmodified GGP uORF peptide comprises the amino acid sequence "LAGGG" (SEQ ID NO: 5) at the C-terminal end of the peptide. In one embodiment, the unmodified GGP uORF peptide comprises the amino acid sequence "GGGRTL" (SEQ ID NO: 6) at the C-terminal end of the peptide. In one embodiment, the unmodified GGP uORF peptide comprises the amino acid sequence "GGGRLIL" (SEQ ID NO: 7) at the C-terminal end of the peptide. In one embodiment, the unmodified GGP uORF peptide comprises the amino acid sequence "GGGRLLL" (SEQ ID NO: 8) at the C-terminal end of the peptide. In one embodiment, the unmodified GGP uORF peptide comprises the amino acid sequence "GGGRILL" (SEQ ID NO: 9) at the C-terminal end of the peptide. In one embodiment, the unmodified GGP uORF peptide comprises the amino acid sequence "GGRGALPSXGGSPSDLLFLAG" [SEQ ID NO: 1], wherein X can be any amino acid. In one embodiment, the unmodified GGP uORF peptide comprises the amino acid sequence "GGRGALPSXGGSPSDLLFLAGGG" [SEQ ID NO: 105], wherein X can be any amino acid. In one embodiment, the X in SEQ ID NO: 1 and SEQ ID NO:105 is A. In one embodiment, the unmodified GGP uORF peptide comprises the amino acid sequence "GGRGALPSAGGSPSDLLFLAG" [SEQ ID NO: 2]. In one embodiment, the unmodified GGP uORF peptide comprises the amino acid sequence "GGRGALPSAGGSPSDLLFLAGGG" [SEQ ID NO: 106]. In one embodiment, the X in SEQ ID NO: 1 and SEQ ID NO:105 is E. In another embodiment, the unmodified GGP uORF peptide comprises the amino acid sequence "GGRGALPSEGGSPSDLLFLAG" [SEQ ID NO: 3]. In another embodiment, the unmodified GGP uORF peptide comprises the amino acid sequence "GGRGALPSEGGSPSDLLFLAGGG" [SEQ ID NO: 107]. [0089] In an embodiment, the unmodified uORF peptide of rice GGP comprises the amino acid sequence of SEQ ID NO: 10. In an embodiment, the unmodified uORF of rice GGP comprises the nucleic acid sequence SEQ ID NO:10 or the nucleic acid sequence of SEQ ID NO:11. [0090] In an embodiment, the unmodified uORF peptide of wheat GGP may be TaGGP1. In an embodiment, the unmodified uORF peptide of TaGGP1 comprises the amino acid sequence of any one of SEQ ID NOs: 12-14. In an embodiment, the unmodified uORF of TaGGP1 comprises the nucleic acid sequence encoding any one of SEQ ID NOs: 12-14 or the nucleic acid sequence of any one of SEQ ID NOs: 15-17. [0091] In an embodiment, the unmodified uORF peptide of wheat GGP may be TaGGP2. In an embodiment, the unmodified uORF peptide of TaGGP2 comprises the amino acid sequence of any one of any one of SEQ ID NOs: 18-20. In an embodiment, the unmodified uORF of TaGGP2 comprises the nucleic acid sequence encoding any one of SEQ ID NOs: 18-20 or the nucleic acid sequence of any one of SEQ ID NOs: 21-23. Modified uORFs [0092] Generating mutations or modifications that disrupt the reading frame or delete residues within the GGP uORF peptide offers insight into the function of the uORF peptide that cannot otherwise be gained when the start codon is disrupted (as was the case for the GGP uORFs that were edited in Arabidopsis, lettuce, and tomato (Zhang et al.2018 Nat Biotechnol.; Li et al., 2018 Nat Biotechnol.); for example, deletions or modifications within the conserved regions of the GGP uORF peptides, such as "GGGRL" (SEQ ID NO: 4); "LAGGG" (SEQ ID NO: 5); "GGGRTL" (SEQ ID NO: 6); GGGRLIL (SEQ ID NO: 7); GGGRLLL (SEQ ID NO: 8); GGGRILL (SEQ ID NO: 9); "GGRGALPSXGGSPSDLLFLAG", wherein X can be any amino acid (SEQ ID NO: 1); or "GGRGALPSAGGSPSDLLFLAGGG" wherein X can be any amino acid (SEQ ID NO: 106). [0093] In an embodiment, the deletion or modification results in a modified GGP uORF that affects the transcription of the uORF region. In an embodiment, the deletion or modification results in a modified GGP uORF that affects the translation of the GGP uORF peptide. In an embodiment, the deletion or modification results in a modified uORF that affects the transcription of the GGP mORF. In an embodiment, the deletion or modification results in a modified uORF that affects the transcription level of the GGP mORF. In an embodiment, the deletion or modification results in a modified uORF that increases the transcription level of the GGP mORF. In an embodiment, the deletion or modification results in a modified uORF that reduces level of the GGP mORF. In an embodiment, the deletion or modification results in a modified uORF that affects the translation of the GGP mORF. In an embodiment, the deletion or modification results in a modified uORF that increases the translation of the GGP mORF. In an embodiment, the deletion or modification results in a modified uORF that reduces the translation level of the GGP mORF. In an embodiment, the deletion or modification results in expression of a functional GGP transcript. In an embodiment, the deletion or modification results in expression of a functional GGP polypeptide. In an embodiment, the deletion or modification results in functional expression level of the GGP gene. In an embodiment, the deletion or modification does not result in silencing of the GGP gene. In the absence of any statement to the contrary, either explicit or implicit, the term “silencing of a gene”, as used herein, is to be understood to mean that the expression of the gene is disrupted or suppressed to a level or degree such that the expression of that gene is undetectable. In an embodiment disclosed herein, the expression of the GGP gene is not silenced in a plant having a modified genome comprising a polynucleotide comprising a modification to an upstream open reading frame (uORF) in a 5' untranslated region (UTR) of a GDP-L-galactose phosphorylase (GGP) gene or an ortholog thereof, wherein the modification comprises a deletion, addition, or substitution of at least one base pair of in the 5' UTR of the GGP gene. [0094] In one embodiment, the deletion or modification in the GGP uORF results in increased ribosome engagement. In one embodiment, the deletion or modification in the GGP uORF results in reduced ribosomal stalling in the GGP uORF. Methods for assaying ribosome engagement or ribosome stalling, and methods for identifying genomic motifs associated with ribosome engagement or ribosome stalling are known to those skilled in the art, illustrative examples of which include. ribosome profiling methods and those described in Laing et al.2015 Plant Cell; and Juntawong et al 2014 PNAS, the entire contents of which are incorporated herein by reference. [0095] The modification in the GGP uORF may be a conservative amino acid substitution. The modification in the GGP uORF may be a non-conservative amino acid substitution. The modification in the GGP uORF may be a conservative nucleotide substitution. The modification in the GGP uORF may be a non-conservative nucleotide substitution. The modification in the GGP uORF may be an insertion of one or more amino acids. The modification in the GGP uORF may be an insertion of one or more nucleotides. The modification in the GGP uORF deletion of one or more amino acids. The modification in the GGP uORF may be a deletion of one or more nucleotides. [0096] The modified uORF peptide may comprise a deletion or modification of 1 to 65 amino acids in the uORF peptide. In one embodiment, the modified uORF peptide may comprise a deletion or modification of 1 to 60 amino acids, 1 to 59 amino acids, 1 to 58 amino acids, 1 to 57 amino acids, 1 to 56 amino acids, 1 to 55 amino acids, 1 to 54 amino acids, 1 to 53 amino acids, 1 to 52 amino acids, 1 to 51 amino acids, 1 to 50 amino acids, 1 to 49 amino acids, 1 to 48 amino acids, 1 to 47 amino acids, 1 to 46 amino acids, 1 to 45 amino acids, 1 to 44 amino acids, 1 to 43 amino acids, 1 to 42 amino acids, 1 to 41 amino acids, 1 to 40 amino acids, 1 to 39 amino acids, 1 to 38 amino acids, 1 to 37 amino acids, 1 to 36 amino acids, 1 to 35 amino acids, 1 to 34 amino acids, 1 to 33 amino acids, 1 to 32 amino acids, 1 to 31 amino acids, 1 to 30 amino acids, 1 to 29 amino acids, 1 to 28 amino acids, 1 to 27 amino acids, 1 to 26 amino acids, 1 to 25 amino acids, 1 to 24 amino acids, 1 to 23 amino acids, 1 to 22 amino acids, 1 to 21 amino acids, 1 to 20 amino acids, 1 to 19 amino acids, 1 to 18 amino acids, 1 to 17 amino acids, 1 to 16 amino acids, 1 to 15 amino acids, 1 to 14 amino acids, 1 to 13 amino acids, 1 to 12 amino acids, 1 to 11 amino acids, 1 to 10 amino, 1 to 9 amino acids, 1 to 8 amino acids, 1 to 7 amino acids, 1 to 6 amino acids, 1 to 5 amino acids, 1 to 4 amino acids, 1 to 3 amino acids, or 1 to 2 amino acids. In one embodiment, the modified uORF peptide comprises a deletion or modification of at least one amino acid in the uORF peptide. In another embodiment, the modified uORF peptide comprises a deletion or modification of at least two amino acids in the uORF peptide. In one embodiment, the modified uORF peptide comprises a deletion or modification of at least three amino acids in the uORF peptide. In one embodiment, the modified uORF peptide comprises a deletion or modification of at least four amino acids in the uORF peptide. In one embodiment, the modified uORF peptide comprises a deletion or modification of at least five amino acids in the uORF peptide. In one embodiment, the modified uORF peptide comprises a deletion or modification of one amino acid in the uORF peptide. In another embodiment, the modified uORF peptide comprises a deletion or substitution of two amino acids in the uORF peptide. In one embodiment, the modified uORF peptide comprises a deletion or modification of three amino acids in the uORF peptide. In one embodiment, the modified uORF peptide comprises a deletion or modification of 4 amino acids in the uORF peptide. In one embodiment, the modified uORF peptide comprises a deletion or modification of 5 amino acids in the uORF peptide. In one embodiment, the modified uORF peptide comprises a deletion or of 6 amino acids in the uORF peptide. In one embodiment, the modified uORF peptide comprises a deletion or modification of 7 amino acids in the uORF peptide. In one embodiment, the modified uORF peptide comprises a deletion or modification of 8 amino acids in the uORF peptide. In one embodiment, the modified uORF peptide comprises a deletion or modification of 9 amino acids in the uORF peptide. In one embodiment, the modified uORF peptide comprises a deletion or modification of 10 amino acids in the uORF peptide. In one embodiment, the modified uORF peptide comprises a deletion or modification of up to 15 amino acids in the uORF peptide. In one embodiment, the modified uORF peptide comprises a deletion or modification of up to 18 amino acids in the uORF peptide. In one embodiment, the modified uORF peptide comprises a deletion or modification of up to 20 amino acids in the uORF peptide. [0097] The modified uORF region may comprise a deletion or non-conservative modification of 1 to 200 nucleotides. In one embodiment, the modified uORF region may comprise a deletion or non-conservative modification of 1 to 200 nucleotides, 1 to 190 nucleotides, 1 to 180 nucleotides, 1 to 170 nucleotides, 1 to 160 nucleotides, 1 to 150 nucleotides, 1 to 140 nucleotides, 1 to 130 nucleotides, 1 to 120 nucleotides, 1 to 110 nucleotides, 1 to 100 nucleotides, 1 to 99 nucleotides, 1 to 98 nucleotides, 1 to 97 nucleotides, 1 to 96 nucleotides, 1 to 95 nucleotides, 1 to 94 nucleotides, 1 to 93 nucleotides, 1 to 92 nucleotides, 1 to 91 nucleotides, 1 to 90 nucleotides, 1 to 89 nucleotides, 1 to 88 nucleotides, 1 to 87 nucleotides, 1 to 86 nucleotides, 1 to 85 nucleotides, 1 to 84 nucleotides, 1 to 83 nucleotides, 1 to 82 nucleotides, 1 to 81 nucleotides, 1 to 80 nucleotides, 1 to 79 nucleotides, 1 to 78 nucleotides, 1 to 77 nucleotides, 1 to 76 nucleotides, 1 to 75 nucleotides, 1 to 74 nucleotides, 1 to 73 nucleotides, 1 to 72 nucleotides, 1 to 71 nucleotides, 1 to 70 nucleotides , 1 to 69 nucleotides, 1 to 68 nucleotides, 1 to 67 nucleotides, 1 to 66 nucleotides, 1 to 65 nucleotides, 1 to 64 nucleotides, 1 to 63 nucleotides, 1 to 62 nucleotides, 1 to 61 nucleotides, 1 to 60 nucleotides , 1 to 59 nucleotides, 1 to 58 nucleotides, 1 to 57 nucleotides, 1 to 56 nucleotides, 1 to 55 nucleotides, 1 to 54 nucleotides, 1 to 53 nucleotides, 1 to 52 nucleotides, 1 to 51 nucleotides, 1 to 50 nucleotides, 1 to 49 nucleotides, 1 to 48 nucleotides, 1 to 47 nucleotides, 1 to 46 nucleotides, 1 to 45 nucleotides, 1 to 44 nucleotides, 1 to 43 nucleotides, 1 to 42 nucleotides, 1 to 41 nucleotides, 1 to 40 nucleotides, 1 to 39 nucleotides, 1 to 38 nucleotides, 1 to 37 nucleotides, 1 to 36 nucleotides, 1 to 35 nucleotides, 1 to 34 nucleotides, 1 to 33 nucleotides, 1 to 32 nucleotides, 1 to 31 nucleotides, 1 to 30 nucleotides, 1 to 29 nucleotides, 1 to 28 nucleotides, 1 to 27 nucleotides, 1 to 26 nucleotides, 1 to 25 nucleotides, 1 to 24 nucleotides, 1 to 23 1 to 22 nucleotides, 1 to 21 nucleotides, 1 to 20 nucleotides, 1 to 19 nucleotides, 1 to 18 nucleotides, 1 to 17 nucleotides, 1 to 16 nucleotides, 1 to 15 nucleotides, 1 to 14 nucleotides, 1 to 13 nucleotides, 1 to 12 nucleotides, 1 to 11 nucleotides, 1 to 10 amino, 1 to 9 nucleotides, 1 to 8 nucleotides, 1 to 7 nucleotides, 1 to 6 nucleotides, 1 to 5 nucleotides, 1 to 4 nucleotides, 1 to 3 nucleotides, or 1 to 2 nucleotides. In one embodiment, the modified uORF region comprises a deletion or non- conservative substitution of at least one amino acid in the uORF region. In another embodiment, the modified uORF region comprises a deletion or non-conservative substitution of at least two nucleotides in the uORF region. In one embodiment, the modified uORF region comprises a deletion or non-conservative substitution of at least three nucleotides in the uORF region. In one embodiment, the modified uORF region comprises a deletion or non-conservative substitution of at least four nucleotides in the uORF region. In one embodiment, the modified uORF region comprises a deletion or non-conservative substitution of at least five nucleotides in the uORF region. In one embodiment, the modified uORF region comprises a deletion or non-conservative substitution of one nucleotide in the uORF region. In another embodiment, the modified uORF region comprises a deletion or non-conservative substitution of two nucleotides in the uORF region. In one embodiment, the modified uORF region comprises a deletion or non-conservative substitution of three nucleotides in the uORF region. In one embodiment, the modified uORF region comprises a deletion or non-conservative substitution of 4 nucleotides in the uORF region. In one embodiment, the modified uORF region comprises a deletion or non-conservative substitution of 5 nucleotides in the uORF region. In one embodiment, the modified uORF region comprises a deletion or non-conservative substitution of 6 nucleotides in the uORF region. In one embodiment, the modified uORF region comprises a deletion or non- conservative substitution of 7 nucleotides in the uORF region. In one embodiment, the modified uORF region comprises a deletion or non-conservative substitution of 8 nucleotides in the uORF region. In one embodiment, the modified uORF region comprises a deletion or non-conservative substitution of 9 nucleotides in the uORF region. In one embodiment, the modified uORF region comprises a deletion or non-conservative substitution of 10 nucleotides in the uORF region. In one embodiment, the modified uORF region comprises a deletion or non-conservative substitution of up to 15 nucleotides in the uORF region. In one embodiment, the modified uORF region comprises a deletion or non- conservative substitution of up to 20 nucleotides in the uORF region. In one embodiment, the modified uORF region comprises a or non-conservative substitution of up to 25 nucleotides in the uORF region. In one embodiment, the modified uORF region comprises a deletion or non-conservative substitution of up to 30 nucleotides in the uORF region. In one embodiment, the modified uORF region comprises a deletion or non-conservative substitution of up to 35 nucleotides in the uORF region. In one embodiment, the modified uORF region comprises a deletion or non-conservative substitution of up to 40 nucleotides in the uORF region. In one embodiment, the modified uORF region comprises a deletion or non-conservative substitution of up to 45 nucleotides in the uORF region. In one embodiment, the modified uORF region comprises a deletion or non-conservative substitution of up to 50 nucleotides in the uORF region. In one embodiment, the modified uORF region comprises a deletion or non-conservative substitution of up to 55 nucleotides in the uORF region. In one embodiment, the modified uORF region comprises a deletion or non-conservative substitution of up to 60 nucleotides in the uORF region. In one embodiment, the modified uORF region comprises a deletion or non-conservative substitution of up to 65 nucleotides in the uORF region. In one embodiment, the modified uORF region comprises a deletion or non-conservative substitution of up to 63 nucleotides in the uORF region. In one embodiment, the modified uORF region comprises a deletion or non-conservative substitution of up to 70 nucleotides in the uORF region. [0098] In one embodiment, the modified uORF of GGP comprises at least one deletion or non-conservative substitution of at least one base pair in the uORF. In another embodiment, the modified uORF of GGP comprises a deletion or non-conservative substitution of 1 to 100 base pairs. In another embodiment, the modified uORF of GGP comprises a deletion or non-conservative substitution of 2 to 80 base pairs. In another embodiment, the modified uORF of GGP comprises a deletion or non-conservative substitution of 5 to 70 base pairs in the uORF. [0099] In another embodiment, the modified uORF of GGP comprises a non-conservative substitution of 1 base pair in the uORF. In another embodiment, the modified uORF of GGP comprises a deletion of 5 base pairs in the uORF. In another embodiment, the modified uORF of GGP comprises deletion of 63 base pairs in the uORF. [0100] In another embodiment, the modified uORF of GGP comprises an amino acid substitution, deletion or insertion of one or more amino acid residues at amino acid positions corresponding to amino acid positions 30 to 61 of the GGP uORF peptide of SEQ ID NO: 10. In another embodiment, the uORF of GGP comprises an amino acid substitution, deletion or insertion of one or more amino acid residues at amino acid positions corresponding to amino acid positions 57 to 61 of the GGP uORF peptide of SEQ ID NO: 10. In another embodiment, the modified uORF of GGP an amino acid substitution, deletion or insertion of one or more amino acid residues at amino acid positions corresponding to amino acid positions 55 to 56 of the GGP uORF peptide of SEQ ID NO: 10. In another embodiment, the modified uORF of GGP comprises an amino acid substitution, deletion or insertion of one or more amino acid residues at amino acid positions corresponding to amino acid positions 37 to 57 of the GGP uORF peptide of SEQ ID NO: 10. [0101] The modified GGP uORF may comprise a deletion or modification of regions of high amino acid sequence conservation in GGP gene uORF (Laing et al.2015 Plant Cell; and Broad et al.2019 BMC Plant Biol.). In one embodiment, the modified GGP uORF comprises a deletion or modification within the amino acid sequence "GGGRL" (SEQ ID NO: 4); "LAGGG" (SEQ ID NO: 5); "GGGRTL" (SEQ ID NO: 6); GGGRLIL (SEQ ID NO: 7); GGGRLLL (SEQ ID NO: 8); GGGRILL (SEQ ID NO: 9); "GGRGALPSXGGSPSDLLFLAG", wherein X can be any amino acid (SEQ ID NO: 1); or "GGRGALPSAGGSPSDLLFLAGGG" wherein X can be any amino acid (SEQ ID NO: 106), at the C-terminal end of the GGP uORF peptide. [0102] In another embodiment, the modified uORF of GGP comprises an amino acid substitution, deletion or insertion of one or more amino acid residues at amino acid positions corresponding to amino acid positions 30 to 61 of the GGP uORF peptide of SEQ ID NO: 10, wherein the GGP uORF comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 12-14, 18-20, 24, 26-27, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48-49, and 52-53, and amino acid sequences having at least 70% sequence identity to any of the foregoing. In another embodiment, the modified uORF of GGP comprises an amino acid substitution, deletion or insertion of one or more amino acid residues at amino acid positions corresponding to amino acid positions 57 to 61 of the GGP uORF peptide of SEQ ID NO: 10, wherein the GGP uORF comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 12-14, 18-20, 24, 26-27, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48-49, and 52-53, and amino acid sequences having at least 70% sequence identity to any of the foregoing. In another embodiment, the modified uORF of GGP an amino acid substitution, deletion or insertion of one or more amino acid residues at amino acid positions corresponding to amino acid positions 56 of the GGP uORF peptide of SEQ ID NO: 10, wherein the GGP uORF comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 12-14, 18-20, 24, 26-27, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48-49, and 52-53, and amino acid sequences having at least 70% sequence identity to any of the foregoing. In another embodiment, the modified uORF of GGP comprises an amino acid substitution, deletion or insertion of one or more amino acid residues at amino acid positions corresponding to amino acid positions 37 to 57 of the GGP uORF peptide of SEQ ID NO: 10, wherein the GGP uORF comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 12-14, 18-20, 24, 26-27, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48-49, and 52-53, and amino acid sequences having at least 70% sequence identity to any of the foregoing. [0103] In another embodiment, the modified uORF of GGP comprises an amino acid substitution, deletion or insertion of one or more amino acid residues from position 30 to 61 of the GGP uORF peptide of SEQ ID NO: 10. In another embodiment, the modified uORF of GGP comprises an amino acid substitution, deletion or insertion of one or more amino acid residues from position 57 to 61 of the GGP uORF peptide of SEQ ID NO: 10. In another embodiment, the modified uORF of GGP comprises an amino acid substitution, deletion or insertion of one or more amino acid residues from position 55 to 56 of the GGP uORF peptide of SEQ ID NO: 10. In another embodiment, the modified uORF of GGP comprises an amino acid substitution, deletion or insertion of one or more amino acid residues from position 37 to 57 of the GGP uORF peptide of SEQ ID NO: 10. [0104] In another embodiment, the modified uORF of GGP comprises a substitution of one or more amino acids in the amino acid sequence from position 30 to 61 the GGP uORF peptide of SEQ ID NO: 10. In another embodiment, the modified uORF of GGP comprises a substitution of one or more amino acids in the amino acid sequence from position 57 to 61 of SEQ ID NO: 10. In another embodiment, the modified uORF of GGP comprises a substitution of one or more amino acids in the amino acid sequence from position 55 to 56 of SEQ ID NO: 10. In another embodiment, the modified uORF of GGP comprises a substitution of one or more amino acids in the amino acid sequence from position 37 to 57 of SEQ ID NO: 10. [0105] In another embodiment, the modified uORF of GGP comprises a substitution of the amino acid sequence from position 30 to 61 the GGP uORF peptide of SEQ ID NO: 10. In another embodiment, the modified comprises a substitution of the amino acid sequence from position 57 to 61 of SEQ ID NO: 10. In another embodiment, the modified uORF of GGP comprises a substitution of the amino acid sequence from position 55 to 56 of SEQ ID NO: 10. In another embodiment, the modified uORF of GGP comprises a substitution of the amino acid sequence from position 37 to 57 of SEQ ID NO: 10. [0106] In another embodiment, the modified uORF of GGP comprises a deletion of one or more amino acids at the amino acid sequence from position 30 to 61 the GGP uORF peptide of SEQ ID NO: 10. In another embodiment, the modified uORF of GGP comprises a deletion of one or more amino acids at the amino acid sequence from position 57 to 61 of SEQ ID NO: 10. In another embodiment, the modified uORF of GGP comprises a deletion of one or more amino acids at the amino acid sequence from position 55 to 56 of SEQ ID NO: 10. In another embodiment, the modified uORF of GGP comprises a deletion of one or more amino acids at the amino acid sequence from position 37 to 57 of SEQ ID NO: 10. [0107] In another embodiment, the modified uORF of GGP comprises a deletion of the amino acid sequence from position 30 to 61 the GGP uORF peptide of SEQ ID NO: 10. In another embodiment, the modified uORF of GGP comprises a deletion of the amino acid sequence from position 57 to 61 of SEQ ID NO: 10. In another embodiment, the modified uORF of GGP comprises a deletion of the amino acid sequence from position 55 to 56 of SEQ ID NO: 10. In another embodiment, the modified uORF of GGP comprises a deletion of the amino acid sequence from position 37 to 57 of SEQ ID NO: 10. [0108] In another embodiment, the modified uORF of GGP comprises an insertion of one or more amino acids at the amino acid sequence from position 30 to 61 the GGP uORF peptide of SEQ ID NO: 10. In another embodiment, the modified uORF of GGP comprises an insertion of one or more amino acids at the amino acid sequence from position 57 to 61 of SEQ ID NO: 10. In another embodiment, the modified uORF of GGP comprises an insertion of one or more amino acids at the amino acid sequence from position 55 to 56 of SEQ ID NO: 10. In another embodiment, the modified uORF of GGP an insertion of one or more amino acids at the amino acid sequence from position 37 to 57 of SEQ ID NO: 10. [0109] In some embodiments, the deletion or modification of the uORF of GGP results in disruption of the reading frame of the GGP uORF, wherein the GGP uORF comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 12-14, 18-20, 24, 26-27, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48-49, and 52-53, and amino acid sequences having at least 70% sequence identity of the foregoing. In some embodiments, the deletion or modification of the uORF of GGP results in disruption of the reading frame of the GGP uORF, wherein the GGP uORF comprises the nucleic acid sequence encoding any one of SEQ ID NOs: 10, 12-14, 18-20, 24, 26-27, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48-49, and 52-53 or the nucleic acid sequence of SEQ ID NO: 11, 15-17, 21-23, 25, 28, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 50-51, and 54-55. [0110] In an embodiment, the modified GGP uORF peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 56-58. In an embodiment, the modified GGP uORF comprises a nucleic acid sequence encoding any one of SEQ ID NOs: 56-58. Trait modifications and crop yield and [0111] 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 or of 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. [0112] “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 or unmodified 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. [0113] 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 useful numbers or amounts. In one embodiment, the crop plant is a cereal grass plant. [0114] In a plant or a crop plant a desirable “trait” may include, for example, one or more of the following, as compared to a control plant: increased vigour, increased “yield”, darker coloration, increased organ size, improved root growth, increased photosynthesis, increased SPAD, increased nutrient content, increased nutrient 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). [0115] In one embodiment, the plant having a modified genome comprising a polynucleotide comprising a modification to an upstream open reading frame (uORF) in a 5' untranslated region (UTR) of a GDP-L-galactose phosphorylase (GGP) gene or an ortholog thereof, wherein the modification comprises a deletion, addition, or substitution of at least one base pair of in the 5' UTR of the GGP gene, has increased or enhanced foliar ascorbate levels. In one embodiment, the plant having a modified genome comprising a polynucleotide comprising a modification to an upstream open reading frame (uORF) in a 5' untranslated region (UTR) of a GDP-L-galactose phosphorylase (GGP) gene or an ortholog thereof, wherein the modification comprises a deletion, addition, or substitution of at least one base pair of in the 5' UTR of the GGP gene, has increased or enhanced root ascorbate levels. In one embodiment, the plant having a modified genome comprising a polynucleotide comprising a modification to an upstream open reading frame (uORF) in a 5' untranslated region (UTR) of a GDP-L-galactose phosphorylase (GGP) gene or an ortholog thereof, wherein the modification comprises a deletion, addition, or substitution of at least one base pair of in the 5' UTR of the GGP gene, has increased or enhanced foliar ascorbate levels. In one embodiment, the plant having a modified genome comprising a polynucleotide comprising a modification to an upstream open reading frame (uORF) in a 5' untranslated region (UTR) of a GDP-L-galactose phosphorylase (GGP) gene or an ortholog thereof, wherein the modification comprises a deletion, addition, or substitution of at least one base pair of in the 5' UTR of the GGP gene, results in increased ascorbate levels in grain. In another embodiment, having a modified genome comprising a polynucleotide comprising a modification to an upstream open reading frame (uORF) in a 5' untranslated region (UTR) of a GDP-L-galactose phosphorylase (GGP) gene or an ortholog thereof, wherein the modification comprises a deletion, addition, or substitution of at least one base pair of in the 5' UTR of the GGP gene, results in increased ascorbate levels in germinated grain. [0116] The modification enhances the ascorbate foliar content in the plant or plant tissue, including foliar, root or germinated grain tissue 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. [0117] In one embodiment, the plant having a modified genome comprising a polynucleotide comprising a modification to an upstream open reading frame (uORF) in a 5' untranslated region (UTR) of a GDP-L-galactose phosphorylase (GGP) gene or an ortholog thereof, wherein the modification comprises a deletion, addition, or substitution of at least one base pair of in the 5' UTR of the GGP gene, has the same or improved salt tolerance, 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, wherein the improvement of salt tolerance is 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. [0118] As disclosed here in, a plant having a modified genome comprising a polynucleotide comprising a modification to an upstream open reading frame (uORF) in a 5' untranslated region (UTR) of a GDP-L-galactose phosphorylase (GGP) gene or an ortholog thereof, wherein the modification comprises a deletion, addition, or substitution of at least one base pair of in the 5' UTR of the GGP gene, is capable of increased yield. Also disclosed herein are methods of modifying the genome of a cereal grass plant, comprising introducing a modification to an reading frame (uORF) in a 5' untranslated region (UTR) of a GDP-L-galactose phosphorylase (GGP) gene or an ortholog thereof, wherein the modification comprises a deletion, addition, or substitution of at least one base pair of in the 5' UTR of the GGP gene, is capable of increased yield. [0119] The term “yield” typically refers to the portion of the plant that is harvestable such as grain, fruit, biomass; or part of the plant that generates the harvestable grain fruit or biomass such as flowers or panicles; or an edible vegetative part such as a stem, leaf or tuber. [0120] In one embodiment, the increased yield is increased grain crop yield. In another embodiment, the increase in grain crop yield is the result of increased grain weight per plant. In another embodiment, the increased yield is an increase in the number of panicles per plant. [0121] Therefore as disclosed herein, there is provided methods of increasing the grain crop yield of a cereal grass plant, comprising introducing a modification to an upstream open reading frame (uORF) in a 5' untranslated region (UTR) of a GDP-L-galactose phosphorylase (GGP) gene or an ortholog thereof, wherein the modification comprises a deletion, addition, or substitution of at least one base pair of in the 5' UTR of the GGP gene. In an embodiment, the increase in grain crop yield is the result of increased grain weight per plant. In an embodiment, the increase in grain crop yield is the result of an increase in the number of panicles per plant. [0122] In an embodiment, the modified uORF results in an increase in the grain crop yield of a plant or a cereal grass plant by at least about 1%, by at least 2%, by at least 3%, by at least 4%, by at least 5%, by at least 6%, by at least 7%, by at least 8%, by at least 9%, by at least 10%, by at least 15%, by at least 20%, by at least 21%, by at least 22%, by at least 23%, by at least 24%, by at least 25%, by at least 26%, by at least 27%, by at least 28%, by at least 29%, by at least 30%, by at least 31%, by at least 32%, by at least 33%, by at least 34%, by at least 35%, by at least 36%, by at least 37%, by at least 38%, by at least 39%, by at least 40%, by at least 41%, by at least 42%, by at least 43%, by at least 44%, by at least 45%, by at least 46%, by at least 47%, by at least 48%, by at least 49%, by at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, by at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, by at least 60%, by at least 61%, by at least 62%, by at least 63%, by at least 64%, by at least 65%, by at least 66%, by at least 67%, by at least 68%, by at least 69%, by at least 70%, by at least 75%, by at least 80%, by at least 85%, by at least 90%, by at least 95%, or by at least 100%, when compared to the grain crop yield of a plant or a cereal grass plant harbouring a corresponding uORF of the same plant species. In an embodiment, the modified uORF results in at least a 1.1 fold increase in the grain crop yield of a plant or a cereal grass plant, when compared to the grain crop yield of a plant or a cereal grass plant harbouring a corresponding wild-type uORF of the same plant species. In an embodiment, the modified uORF results in at least a 1.2 fold increase in the grain crop yield of a plant or a cereal grass plant, when compared to the grain crop yield of a plant or a cereal grass plant harbouring a corresponding wild-type uORF of the same plant species. In an embodiment, the modified uORF results in a 1.3 fold increase in the grain crop yield of a plant or a cereal grass plant, when compared to the grain crop yield of a plant or a cereal grass plant harbouring a corresponding wild-type uORF of the same plant species. In an embodiment, the modified uORF results in at least a 1.3 fold increase in the grain crop yield of a plant or a cereal grass plant, when compared to the grain crop yield of a plant or a cereal grass plant harbouring a corresponding wild-type uORF of the same plant species. In an embodiment, the modified uORF results in at least a 1.4 fold increase in the grain crop yield of a plant or a cereal grass plant, when compared to the grain crop yield of a plant or a cereal grass plant harbouring a corresponding wild-type uORF of the same plant species. In an embodiment, the modified uORF results in at least a 1.5 fold increase in the grain crop yield of a plant or a cereal grass plant, when compared to the grain crop yield of a plant or a cereal grass plant harbouring a corresponding wild-type uORF of the same plant species. In an embodiment, the modified uORF results in at least a 2 fold increase in the grain crop yield of a plant or a cereal grass plant, when compared to the grain crop yield of a plant or a cereal grass plant harbouring a corresponding wild-type uORF of the same plant species.. In an embodiment, the modified uORF results in at least a 3 fold increase in the grain crop yield of a plant or a cereal grass plant, when compared to the grain crop yield of a plant or a cereal grass plant harbouring a corresponding wild-type uORF of the same plant species.. In an embodiment, the modified uORF results in at least a 4 fold increase in the grain crop yield of a plant or a cereal grass plant, when compared to the grain crop yield of a plant or a cereal grass plant harbouring a corresponding wild-type uORF of the same plant species.. In an embodiment, the modified uORF results in at least a 5 fold increase in the grain crop yield of a plant or a cereal grass plant, when compared to the grain crop yield of a plant or a cereal grass plant harbouring a corresponding wild-type uORF of the same plant species. [0123] In an embodiment, the modified uORF results in an increase in the grain weight per plant of a plant or a cereal grass plant by at least about 1%, by at least 2%, by at least 3%, by at least 4%, by at least 5%, by at by at least 7%, by at least 8%, by at least 9%, by at least 10%, by at least 15%, by at least 20%, by at least 21%, by at least 22%, by at least 23%, by at least 24%, by at least 25%, by at least 26%, by at least 27%, by at least 28%, by at least 29%, by at least 30%, by at least 31%, by at least 32%, by at least 33%, by at least 34%, by at least 35%, by at least 36%, by at least 37%, by at least 38%, by at least 39%, by at least 40%, by at least 41%, by at least 42%, by at least 43%, by at least 44%, by at least 45%, by at least 46%, by at least 47%, by at least 48%, by at least 49%, by at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, by at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, by at least 60%, by at least 61%, by at least 62%, by at least 63%, by at least 64%, by at least 65%, by at least 66%, by at least 67%, by at least 68%, by at least 69%, by at least 70%, by at least 75%, by at least 80%, by at least 85%, by at least 90%, by at least 95%, or by at least 100%, when compared to the grain weight per plant of a plant or a cereal grass plant harbouring a corresponding wild-type uORF of the same plant species. In an embodiment, the modified uORF results in at least a 1.1 fold increase in the grain weight per plant of a plant or a cereal grass plant, when compared to the grain weight per plant of a plant or a cereal grass plant harbouring a corresponding wild-type uORF of the same plant species. In an embodiment, the modified uORF results in at least a 1.2 fold increase in the grain weight per plant of a plant or a cereal grass plant, when compared to the grain weight per plant of a plant or a cereal grass plant harbouring a corresponding wild- type uORF of the same plant species. In one embodiment, the modified uORF results in a 1.3 fold increase in the grain weight per plant of a plant or a cereal grass plant, when compared to the grain weight per plant of a plant or a cereal grass plant harbouring a corresponding wild-type uORF of the same plant species. In an embodiment, the modified uORF results in at least a 1.3 fold increase in the grain weight per plant of a plant or a cereal grass plant, when compared to the grain weight per plant of a plant or a cereal grass plant harbouring a corresponding wild-type uORF of the same plant species. In an embodiment, the modified uORF results in at least a 1.4 fold increase in the grain weight per plant of a plant or a cereal grass plant, when compared to the grain weight per plant of a plant or a cereal grass plant harbouring a corresponding wild-type uORF of the same plant species. In an embodiment, the modified uORF results in at least a 1.5 fold increase in the grain weight per plant of a plant or a cereal grass plant, when compared to the grain weight per plant of a plant or a cereal grass plant harbouring a corresponding wild-type uORF of the same plant species. In an embodiment, the modified uORF results in at least a 2 fold increase in the grain weight per plant of a plant or a plant, when compared to the grain weight per plant of a plant or a cereal grass plant harbouring a corresponding wild-type uORF of the same plant species. In an embodiment, the modified uORF results in at least a 3 fold increase in the grain weight per plant of a plant or a cereal grass plant, when compared to the grain weight per plant of a plant or a cereal grass plant harbouring a corresponding wild- type uORF of the same plant species. In an embodiment, the modified uORF results in at least a 4 fold increase in the grain weight per plant of a plant or a cereal grass plant, when compared to the grain weight per plant of a plant or a cereal grass plant harbouring a corresponding wild-type uORF of the same plant species. In an embodiment, the modified uORF results in at least a 5 fold increase in the grain weight per plant of a plant or a cereal grass plant, when compared to the grain weight per plant of a plant or a cereal grass plant harbouring a corresponding wild-type uORF of the same plant species. [0124] There is also provided, a method of increasing the number of panicles of a cereal grass plant, the method comprising introducing a modification to an upstream open reading frame (uORF) in a 5' untranslated region (UTR) of a GDP-L-galactose phosphorylase (GGP) gene or an ortholog thereof. In an embodiment, the modified uORF results in an increase in the number of panicles per plant of a plant or a cereal grass plant by at least about 1%, by at least 2%, by at least 3%, by at least 4%, by at least 5%, by at least 6%, by at least 7%, by at least 8%, by at least 9%, by at least 10%, by at least 15%, by at least 20%, by at least 21%, by at least 22%, by at least 23%, by at least 24%, by at least 25%, by at least 26%, by at least 27%, by at least 28%, by at least 29%, by at least 30%, by at least 31%, by at least 32%, by at least 33%, by at least 34%, by at least 35%, by at least 36%, by at least 37%, by at least 38%, by at least 39%, by at least 40%, by at least 41%, by at least 42%, by at least 43%, by at least 44%, by at least 45%, by at least 46%, by at least 47%, by at least 48%, by at least 49%, by at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, by at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, by at least 60%, by at least 61%, by at least 62%, by at least 63%, by at least 64%, by at least 65%, by at least 66%, by at least 67%, by at least 68%, by at least 69%, by at least 70%, by at least 75%, by at least 80%, by at least 85%, by at least 90%, by at least 95%, or by at least 100%, when compared to the number of panicles per plant of a plant or a cereal grass plant harbouring a corresponding wild-type uORF of the same plant species. In an embodiment, the modified uORF results in at least a 1.1 fold increase in the number of panicles per plant of a plant or a cereal grass plant, when compared to the number of panicles per plant of a plant or a cereal grass plant harbouring a corresponding wild-type uORF of the plant species. In an embodiment, the modified uORF results in at least a 1.2 fold increase in the number of panicles per plant of a plant or a cereal grass plant, when compared to the number of panicles per plant of a plant or a cereal grass plant harbouring a corresponding wild-type uORF of the same plant species. In an embodiment, the modified uORF results in a 1.3 fold increase in the number of panicles per plant of a plant or a cereal grass plant, when compared to the number of panicles per plant of a plant or a cereal grass plant harbouring a corresponding wild-type uORF of the same plant species. In an embodiment, the modified uORF results in at least a 1.3 fold increase in the number of panicles per plant of a plant or a cereal grass plant, when compared to the number of panicles per plant of a plant or a cereal grass plant harbouring a corresponding wild-type uORF of the same plant species. In an embodiment, the modified uORF results in at least a 1.4 fold increase in the number of panicles per plant of a plant or a cereal grass plant, when compared to the number of panicles per plant of a plant or a cereal grass plant harbouring a corresponding wild-type uORF of the same plant species. In an embodiment, the modified uORF results in at least a 1.5 fold increase in the number of panicles per plant of a plant or a cereal grass plant, when compared to the number of panicles per plant of a plant or a cereal grass plant harbouring a corresponding wild-type uORF of the same plant species. In an embodiment, the modified uORF results in at least a 2 fold increase in the number of panicles per plant of a plant or a cereal grass plant, when compared to the number of panicles per plant of a plant or a cereal grass plant harbouring a corresponding wild-type uORF of the same plant species. In an embodiment, the modified uORF results in at least a 3 fold increase in the number of panicles per plant of a plant or a cereal grass plant, when compared to the number of panicles per plant of a plant or a cereal grass plant harbouring a corresponding wild-type uORF of the same plant species. In an embodiment, the modified uORF results in at least a 4 fold increase in the number of panicles per plant of a plant or a cereal grass plant, when compared to the number of panicles per plant of a plant or a cereal grass plant harbouring a corresponding wild-type uORF of the same plant species. In an embodiment, the modified uORF results in at least a 5 fold increase in the number of panicles per plant of a plant or a cereal grass plant, when compared to the number of panicles per plant of a plant or a cereal grass plant harbouring a corresponding wild-type uORF of the same plant species. [0125] In an embodiment, the modified uORF results in an increase in the grain weight per plant of a plant or a cereal grass plant by at least about 1%, by at least 2%, by at least 3%, by at least 4%, by at least 5%, by at by at least 7%, by at least 8%, by at least 9%, by at least 10%, by at least 15%, by at least 20%, by at least 21%, by at least 22%, by at least 23%, by at least 24%, by at least 25%, by at least 26%, by at least 27%, by at least 28%, by at least 29%, by at least 30%, by at least 31%, by at least 32%, by at least 33%, by at least 34%, by at least 35%, by at least 36%, by at least 37%, by at least 38%, by at least 39%, by at least 40%, by at least 41%, by at least 42%, by at least 43%, by at least 44%, by at least 45%, by at least 46%, by at least 47%, by at least 48%, by at least 49%, by at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, by at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, by at least 60%, by at least 61%, by at least 62%, by at least 63%, by at least 64%, by at least 65%, by at least 66%, by at least 67%, by at least 68%, by at least 69%, by at least 70%, by at least 75%, by at least 80%, by at least 85%, by at least 90%, by at least 95%, or by at least 100%, when compared to the grain weight per plant of a plant or a cereal grass plant harbouring a corresponding wild-type uORF of the same plant species. Polynucleotides [0126] The present disclosure extends to polynucleotides encoding the polypeptides and uORFs disclosed herein. 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. The present disclosure also extends to polynucleotides encoding the polypeptides and uORFs disclosed herein. [0127] 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. [0128] Preferably, the term "polynucleotide" includes both the specified sequence and its complement. [0129] 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. [0130] The term "primer" refers to 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. [0131] 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. [0132] 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. [0133] 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]) In bl2seq (Tatiana A. Tatusova, Thomas L. Madden (1999), "Blast 2 sequences--a new tool for 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. [0134] The identity of polynucleotide sequences may be examined using the following Unix command line parameters: bl2seq -i nucleotideseq1 -j nucleotideseq2 -F F -p blastn [0135] 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=". [0136] 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 online at world-wide web.ebi.ac.uk/emboss/align/. [0137] 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. [0138] 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.) [0139] 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 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/). [0140] The similarity of polynucleotide sequences may be examined using the following Unix command line parameters: bl2seq -i nucleotideseq1 -j nucleotideseq2 -F F -p tblastx. [0141] 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. [0142] Variant polynucleotide sequences preferably exhibit an E value of less than 1x10-6 more 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 1x10-90 and most preferably less than 1x10-100 when compared with any one of the specifically identified sequences. [0143] Alternatively, variant polynucleotides of the present disclosure hybridize to the specified polynucleotide sequences, or complements thereof under stringent conditions. [0144] 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. [0145] 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 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 greater than 100 bases in length would be hybridization conditions such as prewashing in a solution of 6x Saline-Sodium Citrate (SSC) , 0.2% SDS; hybridizing at 65 °C., 6xSSC, 0.2% SDS overnight; followed by two washes of 30 minutes each in 1xSSC, 0.1% SDS at 65 °C. and two washes of 30 minutes each in 0.2xSSC, 0.1% SDS at 65 °C. [0146] 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. [0147] With respect to the DNA mimics known as peptide nucleic acids (PNAs) (Nielsen et al., 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. [0148] 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. [0149] 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). [0150] Variant polynucleotides due 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. [0151] 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 encoded protein as described in the Example section. Function of a variant may also be tested for its ability to alter the level of ascorbate synthesis, recycling and/or ascorbate content, a trait, or content of useful compounds, nutrients, or components in plants, also as described in the Examples section herein. Modified Genomes, Constructs, Vectors and Components Thereof [0152] The present disclosure also extends to modified genomes and polynucleotide constructs comprising the polynucleotides disclosed herein, and to expression vectors comprising the polynucleotides or the polynucleotide constructs disclosed herein. [0153] The term "modified genome" and the like refer to genomes that have been modified when compared to wild-type genomes of the corresponding plant species. [0154] 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. [0155] 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 from the host cell, or may be derived from 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. [0156] 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. [0157] The term "expression 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. [0158] 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" is capable of being expressed when it is operably linked to promoter and terminator sequences. [0159] "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. [0160] 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. [0161] The term "noncoding region" refers to untranslated sequences that are upstream of the translational start site and downstream of the translational stop site of a main 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. [0162] 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 regulatory functions. [0163] 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 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 the sequence of a polynucleotide and polypeptide produced. [0164] The term “native genomic locus” or "corresponding 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). [0165] 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. [0166] 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 rules. Or, there may be “complete” or between the nucleic acids. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands. [0167] 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-occurring 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. [0168] 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. [0169] 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 (i.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 sites, and locus control regions. In some embodiments the gene may comprise coding sequences and non-coding sequences. In other embodiments, 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. [0170] 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. [0171] 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 mRNA, rRNA, tRNA, miRNA or siRNA, or the gene product may be a polypeptide product. [0172] 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 sequence. [0173] 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 [0174] 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. [0175] 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. [0176] The term "isolated", as applied to the polynucleotide or polypeptide sequences disclosed herein, is used to refer to 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. [0177] 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. 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 its exogenous nucleic acid, typically exogenous DNA, such as a vector or other 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. [0178] A "recombinant" polypeptide sequence is produced by translation from a "recombinant" polynucleotide sequence. [0179] 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”, are taken to refer to a biological equivalent of the sequence from which it was derived. [0180] 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. [0181] 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 the entire length of a polypeptide of the disclosure. [0182] "Identity" or "similarity" refers to sequence similarity between two polynucleotide sequences 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 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 there between. 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. [0183] 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, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, 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 (i.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 optimally over 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 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). [0184] Polypeptide sequence identity can be determined in the following manner. The subject polypeptide sequence is compared to a candidate polypeptide sequence using BLASTP (from the BLAST suite of programs, version 2.2.5 [November 2002]) in bl2seq, which is publicly available from 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. [0185] 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. [0186] 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.) [0187] 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 transfer protocol ftp.ncbi.nih.gov/blast/). The similarity of polypeptide sequences may be examined using the following Unix command bl2seq -i peptideseq1 -j peptideseq2 -F F -p blastp [0188] Variant polypeptide sequences preferably exhibit an E value of less than 1x10-6 more 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 1x10-90 and most preferably 1x10-100 when compared with any one of the specifically identified sequences. [0189] 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. [0190] 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. [0191] Non-conservative substitutions will entail exchanging a member of one of these classes for a member of another class. [0192] 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 non- conservative 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]. The BLOSUM62 matrix is used to generate a score for each aligned amino acid pair found at the intersection of the corresponding 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. [0193] 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. [0194] 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. [0195] The function of a polypeptide variant, as a variant of a polypeptide encoded by a GGP gene, may be assessed by the methods described in the Example section herein. Methods for Isolating or Producing Polynucleotides [0196] 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. [0197] 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 5.0xSSC, 0.5% sodium dodecyl sulfate, 1xDenhardt's solution; washing (three washes of twenty minutes each at 55 °C) in 1.0xSSC, 1% (w/v) sodium dodecyl sulfate, 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 be conducted under conditions of 0.1xSSC, 1% (w/v) sodium dodecyl sulfate, at 60 °C. [0198] The polynucleotide 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. [0199] 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). [0200] 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 [0201] 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. [0202] Alternatively, library 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. [0203] 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. [0204] 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. [0205] 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 The BLAST programs may be used with default parameters or the parameters may be altered as required to refine the screen. [0206] 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). [0207] 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. [0208] 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. [0209] 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 weight 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). [0210] 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 same motifs in query sequences. The MAST results 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. [0211] 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 [0212] 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 431A Peptide Synthesizer (Foster City, Calif.). Mutated forms of the polypeptides may also be produced during such syntheses. [0213] 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). [0214] 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 [0215] 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 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, restriction endonucleases may be used to excise parts of existing sequences. Altered polynucleotide sequences may also be conveniently synthesized in a modified form. [0216] 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/Cas12 gene editing or so called “base editing”. Methods for Producing Constructs and Vectors [0217] 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. [0218] 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 [0219] 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, rye, oats, millet, sorghum, triticale, buckwheat, quinoa, spelt, einkorn, amaranth, grass, corn, 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, sweet potato, yam and family plant. [0220] Host cells comprising genetic constructs, such as expression constructs, of the invention 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 [0221] 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. [0222] 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-Verlag, 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 [0223] 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 the plant species to be transformed or may be derived from a different plant species. [0224] 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. [0225] 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. [0226] 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. [0227] Exemplary terminators that are commonly used in plant transformation genetic construct include, e.g., the cauliflower mosaic virus (CaMV) 35S terminator, the Agrobacterium tumefaciens nopaline synthase or octopine synthase terminators, the Zea mays zein gene terminator, the Oryza sativa ADP-glucose pyrophosphorylase terminator and the Solanum tuberosum PI-II terminator. [0228] Selectable markers commonly used in plant transformation include the neomycin phophotransferase II gene (NPT II) 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 the hygromycin phosphotransferase gene (hpt) for hygromycin resistance. [0229] 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), β-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 Verlag. Berline, pp.325-336. [0230] 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. [0231] 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. [0232] 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 [0233] 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., ^ 5'-GATCTA...TAGATC-3' ^ 3'-CTAGAT...ATCTAG-5' [0234] 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. [0235] Another silencing approach involves the use of a small antisense RNA targeted to the transcript equivalent to a miRNA (Llave et al., 2002, Science 297, 2053). Use of such small antisense RNA corresponding to polynucleotide of the invention is expressly contemplated. [0236] The term genetic construct as used herein also includes small antisense RNAs and other such polypeptides effecting gene silencing. [0237] 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 co- ordinately 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. [0238] 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. [0239] Other gene silencing strategies include dominant negative approaches and the use of ribozyme constructs (see, e.g., McIntyre, 1996, Transgenic Res, 5, 257). [0240] 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. [0241] 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 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(1):45-51); Prunus (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(1):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. [0242] 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 Plants [0243] Methods for modifying endogenous genomic DNA sequences in plants are known to those skilled in the art, illustrative of which may involve the use of sequence- specific 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" (Cermak et al., 2011, Nucleic Acids Res.39:e82; Mahfouz et al., 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 et al., 2012. Plant Biotechnol. J.10:373-389). [0244] 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 change the encoded amino acid, or at least partially, disable or render it inoperative, so it is no longer translatable. [0245] Alternatively, a codon for a highly conserved amino acid in the uORF can be changed to stop the uORF from functioning in downregulating transcription or translation of the downstream coding sequence. For example, a His residue in the conserved region of the uORF can be changed to a Leu. [0246] 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. In another embodiment, the uORF may be altered to remove a stop codon. [0247] 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/Cas12 gene editing or so called “base editing”. [0248] 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 modulate expression of a downstream coding sequence. Any such method is included within the scope of the present disclosure. Plants, plant parts and plant cells [0249] The present disclosure also extends to a plant, plant cell or plant part produced by the methods disclosed herein. [0250] In another aspect disclosed is provided a plant, plant cell, or plant part including a modified plant, plant cell or plant part, comprising a comprising a modification to an upstream open reading frame (uORF) in a 5' untranslated region (UTR) of a GDP-L- galactose phosphorylase (GGP) gene or an ortholog thereof, wherein the modification comprises a deletion, addition, or substitution of at least one base pair of in the 5' UTR of the GGP gene. In one embodiment, wherein the expression of GGP gene is not silenced. In one embodiment, the modification is capable of increasing foliar ascorbate levels. In one embodiment, the modification is capable of increased grain crop yield. In one embodiment, the increase in grain crop yield is increased grain weight per plant. In one embodiment, the modification is capable of generating increased number of panicles per plant. [0251] In an embodiment, the modified plant or plant cell exhibits a desirable trait selected from the group consisting of increased cellular ascorbate levels, increased foliar ascorbate content, increased root ascorbate content, increased fruit ascorbate content, increased grain ascorbate content, increased germinated grain ascorbate content, increased cellular iron content, increased photosynthesis, improved increased SPAD, increased vigour, increased tolerance to an abiotic stress, such as increased salt tolerance and increased tolerance to a pathogen. [0252] In an embodiment, the GGP gene 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: 59-81, and amino acid sequences having at least 70% sequence identity to any of the foregoing. In an embodiment, the 5' UTR of the GGP gene or ortholog thereof comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 11, 15-17, 21-23, 25, 28, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 50-51, and 54-55and nucleic acid sequences having at least 70% sequence identity to any of the foregoing. In an embodiment, the modified uORF of the GGP gene comprises at least one deletion or non- conservative substitution of at least one base pair in the uORF [0253] In an embodiment, the modified plant comprises a modified genome, comprises a modified GGP gene comprising a modified GGP uORF as disclosed herein. [0254] In one embodiment, the modified uORF of GGP comprises at least one deletion or non-conservative substitution of at least one base pair in the uORF. In one embodiment, the modified uORF of GGP comprises deletion or non-conservative substitution of 1 to 100 base pairs in the uORF. In one embodiment, the modified uORF of GGP comprises deletion or non-conservative substitution of 2 to pairs in the uORF. In another embodiment, the modified uORF comprises deletion or non-conservative substitution of 5 to 70 base pairs in the uORF. In one embodiment, the modified uORF of GGP comprises non-conservative substitution of 1 base pair in the uORF. In another embodiment the modified uORF of GGP comprises deletion of 5 base pairs in the uORF. In another embodiment the modified uORF of GGP comprises deletion of 63 base pairs in the uORF. In another embodiment the modified uORF of GGP results in disruption of the reading frame of the GGP uORF, wherein the GGP uORF comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 12-14, 18-20, 24, 26-27, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48-49, and 52- 53, and amino acid sequences having at least 70% sequence identity to any of the foregoing. [0255] In one embodiment, the modified uORF results in the disruption of the uORF reading frame results in amino acid substitution, deletion or insertion of one or more amino acid residues from position 30 to position 61 of SEQ ID NO: 10. In another embodiment the disruption of the uORF reading frame comprises substitution of an amino acid sequence comprising the amino acid residues at position 57-61 of SEQ ID NO: 10. In an embodiment, the disruption of the uORF reading frame comprises deletion of an amino acid sequence comprising the amino acid residues at position 55-56 of SEQ ID NO: 10. In another embodiment the disruption of the uORF reading frame comprises deletion of an amino acid sequence comprising the amino acid residues at position 37-57 of SEQ ID NO: 10. In some embodiments, the modified GGP uORF peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 56-58. In some embodiments, the entire GGP uORF has been modified or deleted. [0256] In one embodiment, the modified GGP uORF results in reduced ribosomal stalling in the uORF. [0257] In an embodiment, the modified uORF results in enhancing the expression of GGP by at least about 1%, by at least 2%, by at least 3%, by at least 4%, by at least 5%, by at least 6%, by at least 7%, by at least 8%, by at least 9%, by at least 10%, by at least 15%, by at least 20%, by at least 25%, by at least 30%, by at least 35%, by at least 40%, by at least 45%, by at least 50%, by at least 55%, by at least 60%, by at least 65%, by at least 70%, by at least 75%, by at least 80%, by at least 85%, by at least 90%, by at least 95%, or by at least 100%,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. [0258] The present disclosure also to plants comprising the plant cells, the polynucleotides, the polynucleotide constructs, or the expression vectors disclosed herein. [0259] The present disclosure also extends to plants, plant cells, transgenic plants or transgenic plant cells comprising a comprising a modification to an upstream open reading frame (uORF) in a 5' untranslated region (UTR) of a GDP-L-galactose phosphorylase (GGP) gene or an ortholog thereof, wherein the modification comprises a deletion, addition, or substitution of at least one base pair of in the 5' UTR of the GGP gene. 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 GGP gene or ortholog thereof when compared to the level of expression of the GGP gene or ortholog thereof in a control plant cell harbouring a corresponding native the GGP gene or ortholog thereof of the same plant species. The transgenic plant or plant cells may comprise the polynucleotides disclosed herein. [0260] In one embodiment, the plant, plant cell, transgenic plant or transgenic plant cell is a cereal grass plant. [0261] In an embodiment, the plant is selected from the group consisting of rice, barley, wheat, rye, oats, millet, sorghum, triticale, buckwheat, quinoa, spelt, einkorn, amaranth, grass, corn, 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, a leguminous plant, sweet potato, yam and a plant of the nightshade family. [0262] In an embodiment, the plant is a rice or wheat plant. [0263] The present disclosure also extends to seeds produced by the plants disclosed herein, to plants derived from such seeds, and to plant parts derived from the plants disclosed herein. [0264] 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 rice grain, wheat grain, rye grain, barley grain or oat grain. In an embodiment, the grain is rice grain. In an grain is wheat grain. [0265] 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. [0266] 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. [0267] 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. [0268] 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. [0269] In an embodiment, the plant is elected from the group consisting of rice, barley, wheat, rye, oats, millet, sorghum, triticale, buckwheat, quinoa, spelt, einkorn, amaranth, grass, corn, 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, 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. [0270] 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 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, ferns, 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. [0271] 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. In an embodiment, the plant part is a seed or a grain. [0272] 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, ferns, 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. [0273] In one embodiment, the plant is rice, barley (Hordeum vulgare), wheat (Triticum aestivum), rye (Secale cereal), oats (Avena sativa), millet, sorghum, triticale, buckwheat, quinoa, einkorn, amaranth, spelt (Triticum spelta), grass, corn, 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, pistachio, walnut, chestnut, a vine, a forage grass, alfalfa, a plant of the genus Prunus, 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 potatoes, eggplant, bell and chili peppers. [0274] 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 embodiment, the plant is a wheat, barley, oat, rye or spelt plant. [0275] 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. [0276] In one embodiment, the plant tissue is leaf tissue, stem tissue, root tissue, seed tissue or fruit tissue. [0277] The present invention is further described by reference to the following non- limiting examples. EXAMPLES Methods Plant growth conditions [0278] Oryza sativa cv. Nipponbare was used for all experiments. Rice grain were surface sterilized with 70% (v/v) ethanol (Chem-Supply, SA, Australia) for 1 min and 30% (v/v) bleach (White King, NSW, Australia) with a few drops of Tween-20 (Sigma-Aldrich, MO, USA) for 30 min and washed three times with sterile dH2O. Surface sterilized rice grain were germinated in a petri dish with moist sterile filter paper (Whatman, UK) for 7 to 9 days prior to transplanting to 1 L pots filled with potting mix in a glasshouse maintained at 26°C and 70% relative humidity at The University of Melbourne (Melbourne, VIC, Australia). The potting mix was prepared by mixing one part washed fine sand (Col Smith, VIC, Australia), one part propagating sand (Brunnings, VIC, Australia), two parts premium vermiculate (Exfoliators, VIC, Australia), and one part General Mix potting media (Australian Growing Solutions, VIC, Australia) fertilized with Osmocote Exact Standard 8- 9 M (ICL, NSW, Australia) at a rate of 6 g/L. Vector construction for constitutive OsGGP expression in rice [0279] The OsGGP (LOC_Os12g08810) CDS was PCR-amplified from rice cv. Nipponbare cDNA and recombined into the Gateway-compatible pMDC32 vector [197], which placed the OsGGP CDS under control of a dual CaMV 35S promoter and also contained the hygromycin phosphotransferase II (hptII) plant-selectable marker gene. Agrobacterium-mediated transformation of rice callus was carried out using established protocols (Sallaud et al.2003 Theor Appl Genet) Vector construction for mutated OsGGP uORF in rice [0280] Two CRISPR/Cas9 vectors, p35S-Cas9 and pUbi-Cas9, containing the human codon optimised Streptococcus pyogenes Cas9 (hSpCas9) gene under the transcriptional control of the dual CaMV 35S promoter and the maize ubiquitin (Ubi) promoter, respectively, a single guide RNA (sgRNA) targeting the most conserved region of the OsGGP uORF under the transcriptional control of the rice OsU3 promoter, and the hygromycin phosphotransferase II (hptII) plant selectable marker gene were assembled as previously described (Naim et al. 2018 Transgenic Res). The sgRNAs were inserted downstream of the OsU3 promoter using the tRNA-gRNA system and BsaI cloning strategy as previously described (Xie et al 2015 PNAS). The p35S-Cas9 sgRNA was assembled in two fragments with primers S1 (5’-GGTCTCATGGCAACAAAGCACCAGTGGTCTAG- 3’) (SEQ ID NO: 108) and AS1 (5’- TAGGTCTCAGGCTGGGCTGGATGCACCAGCCGGGAATC-3’) (SEQ ID NO: 109) and S2 (5’- TAGGTCTCCAGCCCCGCACGGGTTTTAGAGCTAGAAATAG-3’) (SEQ ID NO: 110) and AS2 (5’- TAGGCATGACTATGGTCTCTGTAGTAAAAAAAAAAGCACCGACTCGGTGCCAC 3’) (SEQ ID NO: 111), respectively. Similarly, the pUbi-Cas9 sgRNA was assembled in two fragments with primers S3 (5’-GGTCTCATGGCAACAAAGCACCAGTGGTCTAG- 3’) (SEQ ID NO: 112) and AS3 (5’- TAGGTCTCAGAAGAGGAGGTCTGCACCAGCCGGGAATC-3’) (SEQ ID NO: 113) and S4 (5’- TAGGTCTCCCTTCCTCGCCGGGTTTTAGAGCTAGAAATAG-3’) (SEQ ID NO: 114) and AS4 (5’TAGGCATGACTATGGTCTCTGTAGTAAAAAAAAAAGCACCGACTCGGTGCC AC-3’) (SEQ ID NO: 115), respectively. The resulting vectors were named p35S-Cas9- uORF and pUbi-Cas9-uORF, respectively, and were mobilised into Agrobacterium tumefaciens (strain AGL1) by electroporation prior to rice transformation. Rice transformation [0281] Transformation of rice callus was performed using established protocols [198]. Briefly, seed callus was induced on for 4 weeks (28°C, dark) followed by multiplication of embryonic units on fresh NB medium for a further 10-14 d (28°C, dark). Calli were then co-cultured with A. tumefaciens and placed on R2-CS medium for 3 d (25°C, dark). Transformed calli were then selected on R2-S medium for 14 d (28°C, dark), proliferated on NBS medium for 2-3 weeks (28°C, dark), and matured on PR-AG medium for 7-10 d (28°C, dark) prior to regeneration on RN medium for 2 d (28°C, dark) and then a further 4-6 weeks (28°C, 12 h light/12 h dark). Plantlets were then rooted on P medium for 3-4 weeks (28°C, 12 h light/12 h dark) prior to greenhouse acclimation in Jiffy peat pellets for 15 d, followed by transfer to soil pots. Preparation of GBR [0282] Following surface sterilization, BR samples were immediately snap-frozen in liquid nitrogen, lyophilized, and ground using a Tube Mill with a 40 mL grinding chamber (IKA), whereas GBR samples were imbibed in sterile dH2O for 12 h in the dark at 28°C, washed three times with sterile dH2O, and spread in a petri dish with moist sterile filter paper (Whatman) for 72 h in the dark at 28°C. The GBR was then washed three times with sterile dH2O, snap-frozen in liquid nitrogen, lyophilized, and ground using a Tube Mill with a 40 mL grinding chamber (IKA). Assessment of salt tolerance with automated imaging [0283] Surface sterilized rice grain were germinated in plastic boxes on moist paper towels (denoted DAP 0). At DAP 6, three uniformly germinated grain per line were transplanted to 2.8 L pots filled with 2.6 kg of UC Davis Mix, placed into square containers for water to collect, and grown on benches in the NW Smarthouse maintained at 26°C and 70% relative humidity at The Plant Accelerator, Adelaide, Australia under manual watering (not to weight). At DAP 14, plants were thinned to one seedling per pot and loaded onto the NW Smarthouse for automated imaging. A split-unit design was used to randomize pots (i.e. line and treatment combinations) to cart positions within the Smarthouse. The plants were automatically watered daily so that approximately 500 mL of water was maintained in the soil. The salt plants were treated with saline solution on DAP 20 and 23 (100 mL of 225mM NaCl) to reach a final concentration of 90 mM NaCl in the soil solution, while the control plants received equivalent water applications. Imaging was carried out daily from DAP 14 to 40 inclusive. From these images the PSA of the plant, as viewed using an RGB camera, was obtained as previously described. For analysis purposes, PSA was defined as the sum of the areas as measured (in six side/oblique camera views plus twice the shoot area (top view). The data was prepared for analysis using growthPheno [205], a package for the R statistical computing environment. Quantitative reverse-transcription-PCR analysis [0284] Total RNA from foliar tissue was isolated using the Direct-zol™ RNA MiniPrep Kit (Zymo Research, CA, USA) according to manufacturer’s instructions. To isolate total RNA from BR and GBR, sub-samples of snap-frozen BR and GBR were ground using a Tube Mill with a chilled 40 mL grinding chamber (IKA, Germany) and suspended in an extraction buffer containing 0.1M glycine-NaOH (Chem-Supply), 100 mM NaCl (Chem- Supply), 10 mM EDTA (Chem-Supply), 2% SDS (Sigma-Aldrich), and 1% sodium lauryl- sarcosine (Sigma-Aldrich) at pH 9 for 7 min at 1,400 RPM [200]. The solution was then mixed with an equal volume of phenol to chloroform to asoamyl alcohol at a ratio of 25:24:1 (Sigma-Aldrich) for 1 min at 1,400 RPM followed by incubation on ice for 5 min. After centrifugation for 20 min at 4°C, total RNA was then purified from the upper aqueous phase using the Direct-zol™ RNA MiniPrep Kit (Zymo Research) according to manufacturer’s instructions. Quantification, DNase treatment, and reverse 57 transcription of the RNA was performed using the QuantiFluor® RNA System (Promega, WI, USA), RQ1 RNase-Free DNase (Promega), and the Tetro cDNA Synthesis Kit (Bioline), respectively, according to manufacturer’s instructions. The quantitative reverse transcription-PCR (qRT-PCR) primers were designed to amplify the OsGGP, 35S-OsGGP, OsGME1, OsGME2, and OsGPP genes using Primer 3 software [201, 202]. Primer efficiency was ≥93% for all primer pairs. The qRT-PCR analysis was carried out on a CFX Connect™ Real-Time System (Bio-Rad, CA, USA) with Hard-Shell® 96-Well PCR Plates (Bio-Rad). The qRT-PCR amplification cycles consisted of 1 cycle = 3 min 95°C; 40 cycles = 20 s 95°C, 20 s 60°C, 20 s 72°C and were followed by a melt curve from 65°C to 95°C in 0.5°C steps for 5 s/step. The qRT-PCRs were performed in a final volume of 10 µL for KAPA SYBR® FAST (Kapa Biosystems, MA, USA) according to manufacturer’s instructions, each with four technical replicates. The absolute quantification of transcript copy number was determined using a 10-fold serial dilution of 108 –102 copies for each PCR product and a standard curve generated with the Bio-Rad CFX Manager 3.1 software (Bio-Rad). The PCR products were purified and quantified with a DNA Clean & Concentrator™-5 Kit (Zymo Research) and the QuantiFluor® ONE dsDNA System (Promega), respectively, prior to serial dilution. The geometric mean expression of three housekeeping genes selected from OsGAPDH, OsELF1, OsACT1 and OsUBQ5 using the NormFinder Excel add-in was used to normalize the expression of the 35S-OsGGP, OsGGP, OsGME1, OsGME2, and OsGPP genes. Quantification of total ascorbate [0285] Total ascorbate was extracted and measured as previously described with modifications [199]. Briefly, total ascorbate was extracted from ground, lyophilized tissue homogenised in extraction fluid containing 8% metaphosphoric acid, 2 mM EDTA, and 2 mM TCEP (Sigma-Aldrich) at 40°C for 2 h. The extract was centrifuged and 5 µL of supernatant was injected onto a C183 µm 33 x 7 mm Alltima Rocket column (Hichrom Limited, UK) maintained at 40°C with a flow rate of 1 mL/min. The concentration was determined by reverse phase chromatography on a Shimadzu Nexera UHPLC system (Shimadzu, Japan) with simultaneous UV and MS detection. Mobile phase A consisted of MS grade water with 0.1% formic acid (ThermoFisher Scientific, MA, USA). The elution procedure utilized a 1 min gradient from 0% to 90% of mobile phase B: MS grade acetonitrile with 0.1% formic acid (ThermoFisher Scientific). The UV absorption signal was acquired using a Shimadzu SPD-20A detector at 245 nm wavelength. The MS data was acquired on a Shimadzu LCMS-8050 triple quadrupole mass spectrometer equipped with a DUIS source. The MS instrument was operated in Multiple Reaction Monitoring mode monitoring ions in both positive (177>95, 177>85) and negative (175 >115, 175>71, 175>59) ionisation modes. The product ion 115 m/z was used as a quantifier. A calibration curve in the range of 1.95 to 250 ppm of L-ascorbic acid analytical standard (Sigma-Aldrich) was used to determine absolute concentration of analyte in the extracts. Example 1. Constitutive overexpression of OsGGP in rice [0286] Rice cv. Nipponbare transformants constitutively overexpressing the OsGGP CDS were generated through Agrobacterium tumefaciens-mediated transformation of a T-DNA containing the OsGGP CDS under transcriptional control of the dual CaMV 35S promoter. [0287] A total of 27 independent, hemizygous 35S-OsGGP transformation events were regenerated from tissue culture and ascorbate concentrations were measured in the T1 BR. Ascorbate concentrations were negligible in both wild-type (WT) and 35S-OsGGP brown rice (BR) but was significantly increased up to 5.0-fold in 35S-OsGGP germinated brown rice (GBR) relative to WT. [0288] Two events (35S-OsGGP-1 35S-OsGGP-2) with increased ascorbate concentrations in T1 GBR and Mendelian segregation rations of 3:1 for presence/absence of the T-DNA in T1 seedling were advanced and homozygous and null segregant (NS) progeny were identified (Table 3).
Table 3: Ascorbate concentrations of T135S-OsGGP GBR and the number of T1 35S-OsGGP seedlings with the 35SGGP T-DNA present or absent. Genotype Total ascorbate No. of seedlings No. of seedlings Chi-square p- (mg/100 g DW)a with T-DNA without T-DNA valueb WT 1.66 - - - Event 1 4.35 8 2 0.715 Event 2 2.88 10 2 0.505 Event 4 2.37 10 1 0.223 Event 5 2.94 11 1 0.182 Event 6 3.07 9 2 0.602 Event 7 5.64 10 2 0.505 Event 8 4.44 8 4 0.505 Event 10 3.82 11 1 0.182 Event 13 4.10 11 1 0.182 Event 16 2.31 11 1 0.182 Event 19 2.62 11 1 0.182 Event 20 (35S-OsGGP-2) 5.19 8 2 0.715 Event 22 2.17 9 1 0.273 Event 24 5.36 9 1 0.273 Event 25 6.29 10 1 0.223 Event 26 5.18 8 2 0.715 Event 27 6.26 10 1 0.223 Event 30 1.84 10 1 0.223 Event 33 0.95 8 0 0.103 Event 41 2.26 11 1 0.182 Event 42 4.07 7 1 0.414 Event 43 3.15 8 3 0.862 Event 45 2.52 10 1 0.223 Event 46 6.11 12 0 0.046* Event 49 4.22 10 2 0.505 Event 50 3.74 11 1 0.182 Event 51 (35S-OsGGP-1) 8.30 10 2 0.505 aTotal ascorbate values represent one replicate of four to eleven grain. bAsterisks indicate statistically significant differences between observed and expected segregation ratio of 3:1 for the presence to absence of the 35S-OsGGP T-DNA (chi-square test; * p-value ≤0.05). [0289] Ascorbate concentrations in homozygous NS and 35S-OsGGP BR and did not differ significantly (Figure 2b). Ascorbate concentrations were, however, significantly increased 8.7- and 5.1-fold in homozygous 35S-OsGGP-1 and 35S-OsGGP-2 GBR relative to NS-1 and NS-2, respectively (Figure 2b). [0290] At the vegetative growth phase, foliar ascorbate concentrations were significantly increased 1.8-fold in both homozygous 35S-OsGGP-1 and 35S-OsGGP-2 plants relative to NS-1 and NS-2, respectively (Figure 2c). Similarly, root ascorbate concentrations were significantly increased 4.2- and 4.5-fold in homozygous 35S-OsGGP-1 and 35S-OsGGP-2 plants relative to NS-1 and NS-2, respectively (Figure 2c). [0291] In contrast to the vegetative growth phase, ascorbate concentrations at the reproductive growth phase foliar were significantly reduced 2.0-fold in both homozygous 35S-OsGGP-1 and 35S-OsGGP-2 plants relative to NS-1 and NS-2, respectively (Figure 2e). The reduction in ascorbate concentrations in 35S-OsGGP plants relative to NS at the reproductive growth phase was only observed in homozygous 35S-OsGGP plants; hemizygous 35S-OsGGP plants had significantly increased ascorbate concentrations relative to NS at the reproductive growth phase (Figure 2). Transcript levels of the endogenous OsGGP gene were significantly reduced 2.0-fold in homozygous 35S-OsGGP-1 plants relative to NS-1 at the reproductive phase. [0292] Similarly, transcript levels of the endogenous OsGGP gene were reduced 2.4-fold in homozygous 35S-OsGGP-2 plants relative to NS-2 but did not differ significantly (Figure 2f). Even though foliar ascorbate concentrations and endogenous OsGGP transcript levels were significantly reduced, high transcript levels of the 35S-OsGGP transgene were detected in the 35S-OsGGP plants (Figure 2g). Transcript levels of the GDP-D-mannose-3′,5′- epimerase 1 and 2 (OsGME1 and OsGME2) genes, which encode the enzyme responsible for the fourth enzymatic step of the L-galactose pathway, and the L-galactose-1-phosphate phosphatase (OsGPP) gene, which encodes the sixth enzymatic step of the L-galactose pathway, did not differ significantly between the NS and 35S-OsGGP plants (Figure 2h-j). Example 2: Constitutive overexpression of OsGGP and salt tolerance [0293] Shoot growth measurements of control and salt-stressed 35S-OsGGP plants during the vegetative growth phase were carried out using automated imaging. Salt stress was applied in two steps to the plants at days after planting (DAP) 20 and 23 and the plants were imaged daily from DAP 14 to 40 inclusive. Under control conditions, foliar ascorbate concentrations were significantly fold in 35S-OsGGP-1 plants relative to NS- 1 at DAP 40, however foliar ascorbate concentrations did not differ significantly between NS-2 and 35S-OsGGP-2 plants (Figure 3a). Under salt conditions, foliar ascorbate concentrations were significantly increased 1.3-fold in both 35S-OsGGP-1 and 35S- OsGGP-2 plants relative to NS-1 and NS-2, respectively, at DAP 40 (Figure 3b). From smoothed projected shoot area (sPSA) values—a strong predictor of shoot biomass—the sPSA absolute growth rate (AGR) and sPSA relative growth rate (RGR) were calculated at the following DAP intervals: 16-20, 20-24, 24-30, 30-35, and 35-40 (Figure 3c). As expected, the sPSA RGR declined more rapidly in salt-stressed plants than control plants (Figure 3c-f). However, a similar trend in the sPSA RGR was observed for both control and salt-stressed 35S-OsGGP-1 plants relative to NS-1, with the 35S-OsGGP-1 plants not differing significantly relative to NS-1 at any of the calculated DAP intervals (Figure 3c & d). Likewise, a similar trend in the sPSA RGR was observed for both control and salt- stressed 35S-OsGGP-2 plants relative to NS-2, with the 35S-OsGGP-2 plants having a significantly lower sPSA RGR at the DAP interval 30-35 relative to NS-2 but otherwise did not differ significantly for any of the other calculated DAP intervals (Figure 3e-f). Example 3: Production of rice with CRISPR/Cas9-induced mutations in the OsGGP uORF [0294] Two CRISPR/Cas9 vectors, p35S-Cas9-uORF and pUbi-Cas9-uORF, were transformed into rice cv. Nipponbare via A. tumefaciens (Figure 4). No target mutations were detected in the p35S-Cas9-uORF transformation. From the pUbi-Cas9-uORF transformation, two plants with mutations in the OsGGP uORF were detected. Approximately five weeks later when the pUbi-Cas9-uORF transformation events were transferred to Jiffy peat pellets for greenhouse acclimation, DNA was once again extracted from shoots from a subsample of 49 independent pUbi-Cas9-uORF transformation events and the presence of CRISPR/Cas9-induced mutations in the OsGGP uORF screened via Sanger sequencing.29 plants with mutations in the OsGGP uORF were detected. [0295] Three independent T0 uorfOsGGP mutants recovered from CRISPR/Cas9-targeted mutagenesis with the pUbi-Cas9-uORF were chosen for analysis. One that was bi-allelic for a single adenine insertion and a single thymine insertion, one that was bi-allelic for a single thymine insertion and a 5-bp deletion, and one that was heterozygous for a 63-bp deletion (Figure 5). From these three parental T0 mutants, three independent homozygous, transgene- free uorfOsGGP mutants were subsequently isolated through PCR-based genotyping: one with a single adenine insertion 1), one with a 5-bp deletion (uorfOsGGP-2), and one with a 63-bp deletion (uorfOsGGP-3) (Figure 6a). [0296] At the peptide level, the uorfOsGGP-1 mutation disrupts the reading frame replacing the final 5 residues and introduces a delayed stop codon extending the peptide from 61 to 72 residues in length (Figure 6b). The uorfOsGGP-2 mutation deletes two residues and disrupts the reading frame replacing the final 5 residues and introduces a delayed stop codon extending the peptide from 61 to 70 residues in length (Figure 6b). The uorfOsGGP-3 mutation deletes 21 residues whilst maintaining the reading frame reducing the peptide from 61 to 40 residues in length (Figure 6b). An independent plant recovered from CRISPR/Cas9-targeted mutagenesis with the pUbi-Cas9- uORF with no mutations detected in the OsGGP uORF was used as a wild-type (WT) control. Example 4: Transcript levels of OsGGP in uorfOsGGP mutants [0297] Transcript levels of the OsGGP gene did not differ significantly between the T2 homozygous, transgene-free WT and uorfOsGGP plants (Figure 7). Similarly, transcript levels of the GDP-D-mannose-3′, 5′-epimerase 1 and 2 (OsGME1 and OsGME2) genes, which encode the enzyme responsible for the fourth enzymatic step of the L-galactose pathway, and the L-galactose-1- phosphate phosphatase (OsGPP) gene, which encodes the sixth enzymatic step of the L-galactose pathway, did not differ significantly between the WT and uorfOsGGP plants (Figure 7c-d). Example 5: Ascorbate levels in uorfOsGGP mutants [0298] Foliar ascorbate concentrations were significantly increased 1.2- and 1.3-fold in the T2 homozygous, transgene-free uorfOsGGP-2 and uorfOsGGP-3 mutants, respectively, relative to WT (Figure 8a). Similarly, foliar ascorbate concentrations were increased 1.2- fold in the T2 homozygous, transgene-free uorfOsGGP-1 mutant relative to WT but did not differ significantly (Figure 8a). Foliar ascorbate concentrations were also increased 1.2- to 1.7-fold in a range of T1 heterozygous, homozygous, and bi-allelic, transgene-free uorfOsGGP-2 and uorfOsGGP-3 mutants relative to WT (Figure 8b). Ascorbate concentrations were negligible in both T3 homozygous, transgene-free WT and uorfOsGGP- 1 brown rice and did not differ significantly (Figure 8c). Ascorbate concentrations were significantly decreased 1.4-fold in uorfOsGGP-1 brown rice germinated for 24 h relative to WT, but otherwise did not differ significantly in brown rice germinated for 48 and 72 h. Example 6: Shoot growth uorfOsGGP mutants [0299] Shoot growth measurements of T2 homozygous, transgene-free WT and uorfOsGGP-1 plants were carried out using automated imaging over the course of a full lifecycle (128 days). Imaging of the plants commenced at DAP 14 and was carried out approximately weekly (DAP 14 to 128 inclusive). The smoothed PSA (sPSA), a strong predictor of shoot biomass, was calculated for DAP 16, 30, 44, 58, 79, 101, and 128. The WT and uorfOsGGP-1 plants did not differ significantly with respect to sPSA values at any of the calculated timepoints (Figure 9a). From the sPSA values, the sPSA absolute growth rate (AGR) and sPSA relative growth rate (RGR) were calculated at the following DAP intervals: 16-30, 30-44, 44-58, 58-79, 79- 101, and 101-128. The WT and uorfOsGGP-1 plants did not differ significantly with respect to sPSA AGR values at any of the calculated DAP intervals (Figure 9b). The uorfOsGGP-1 mutant had a significantly higher sPSA RGR value for the DAP interval 16-30 relative to WT, but otherwise did not differ significantly at any of the other calculated DAP intervals (Figure 9c). Example 7: Salt stress tolerance assessment of the uorfOsGGP mutants [0300] Shoot growth measurements of control and salt-stressed T2 homozygous, transgene-free WT and uorfOsGGP-1 plants were carried out using automated imaging. Salt stress was applied in two steps to the plants at DAP 20 and 23 and the plants were imaged daily from DAP 14 to 40 inclusive. Foliar ascorbate concentrations were significantly increased 1.3-fold in both the control and salt-stressed uorfOsGGP-1 mutants relative to WT (Figure 10a, b). From the sPSA values, the sPSA AGR and sPSA RGR were calculated at the following DAP intervals: 16-20, 20-24, 24-30, 30-35, and 35-40 (Figure 10 and Figure 11). As expected, the sPSA RGR declined more rapidly in salt-stressed plants than control plants (Figure 10c, d). However, a similar trend in the sPSA RGR of both the control and salt-stressed uorfOsGGP-1 mutant relative to WT was observed, with the uorfOsGGP-1 mutant having a significantly higher sPSA RGR for the DAP intervals 16-20, 20-24, and 24- 30 relative to WT, but otherwise did not differ significantly for the DAP intervals 30-35 and 35-40. Example 8: Agro-morphological performance of the uorfOsGGP mutants [0301] The T2 homozygous, transgene-free uorfOsGGP-1 mutant had a significant 1.2- fold decrease in panicle length, 1.1-fold decrease in the number of primary branches per panicle, and 1.2-fold decrease in the number of spikelets per panicle relative to WT but had a significant 1.3-fold increase in both of panicles per plant and grain weight per plant (Table 4). The WT and uorfOsGGP-1 plants did not differ significantly with respect to straw biomass, the number of fertile spikelets per panicle, spikelet fertility, and thousand grain weight (Table 4). The uorfOsGGP-1 mutant had a minor significant increase in the grain length to width ratio relative to WT, but otherwise did not differ significantly with respect to the percentage of chalky grain, grain length, grain width, and grain area (Table 5). Table 4: Agro-morphological performance of T2 homozygous, transgene-free WT and uorfOsGGP-1 plants WT uorfOsGGP-1 Straw biomass (g DW) 36.72 ± 1.05 35.33 ± 0.71 No. of panicles per plant 20.33 ± 1.02 27.33 ± 0.76*** Panicle length (cm) 16.45 ± 0.57 13.49 ± 0.28** No. of primary branches per panicle 7.66 ± 0.26 6.91 ± 0.08* No. of spikelets per panicle 67.66 ± 4.29 53.46 ± 1.62* No. of fertile spikelets per panicle 46.13 ± 5.08 43.09 ± 3.21 Spikelet fertility (%) 61.61 ± 5.57 77.27 ± 5.18 Grain weight per plant (g) 16.73 ± 1.59 21.82 ± 1.35* Thousand grain weight (g) 29.37 ± 0.78 28.55 ± 1.13 Values represent mean ± SEM of six biological replicates. Asterisks indicate significant differences between the WT and uorfOsGGP plants (two-sample t-test, * p-value ≤0.05, ** p-value ≤0.01, *** p-value ≤0.001). Table 5: Quality parameters of T3 homozygous, transgene-free WT and uorfOsGGP-1grain. Genotype Chalky grains (%) WT 4.60 ± 1.32 uorfOsGGP-1 3.72 ± 1.23 Values represent mean ± SEM of six biological replicates. Asterisks indicate significant differences between the WT and uorfOsGGP plants (two-sample t-test, * p-value ≤0.05). Table 6: Sequence listing SEQ description sequence ID NO GSP GC TC GC TG LFL LFL unmodified uORF peptide AGGGRL of TaGGP1-D GCA ACG CTC GCA ACG CTC GCA ACG CTC L L L GCA GC CTC GCA GC CTC GCA GC CTC SD GC CCC CC LFL unmodified uORF AGGTGGAGCCGCACGGAGGACGCGGAGCACTCCCCTCGGCCGGGGG nucleotide of CAGCCCCTCCGACCTCCTCTTCCTCGCCGGCGGCGGTCGCATCCTTCT GCA ACG CTC LFL GCA ACG CTC L GC GG TCT LFL CA ACG CTT LFL GCA ACG CT LFL CA ACG CTT LLL GC GG nucleotide of AsGGP2-A CCTCCTCCTCTGA (oat) LL GC GG CCT LL GC GG CCT SD GC CC GC L FL GC GG TCT CA CG CTC L unmodified uORF peptide AGGGRL of AeGGP1 GCA GC CTC GCA ACG CTC GSP GSP SK TA KV LLI HF EGG ALG LSE VL FK IPG FQ HV LTV LAN LD VK FK IPG FQ HLP TVP AN LD VK Amino acid sequence of ANPTKLPLQEDAVPTDFFINLLLGQWEDRMTQGLFRYDVTACETKVIPG TaGGP1-D NLGFVAQLNEGRHLKKRPTEFRVDRVLQPFDSAKFNFTKVGQEEALFQ HLP TVP AN LD VK AD GEL FE PQQ FP VV TQ CIF AD GEL FG PQQ FP VV TQ CIF AD GEL FE PQ PF DV DT AC YAF IPG FQ LPQ PFP VVS TQ KA AD GEL FE QQI FPV VT QV IFQ PVY TK EEV LD YY DLANVVSSACIWLQDNNVPYNVLISDSGRKIFLFPQCYAEKQALGEVSQ ELLDTQVNPAVWEISGHIVLKRRDDYEEASEASAWRLLAEVSLSEARFE VY ETK EE RVL YY MN VSQ RFE AD GEL FE PQR FP VV TQV IFQ EL AC VG LIP FQ EG ALG LSE VRQ SEL AC VG LLIP FQ VN GE SE VRL EL AC VG IPR QA GNT GEV EAR Q AN GD RFE QQ FP VV TQ CI Amino acid sequence of PKKPVQEDGLPTEFFLNSLLLAQWEDRVARGLFRYDVTACETKVIPGDL AsGGP2-C GFVAQLNEGRHLKKRPTEFRVDCVLQPFDSAKFNFTKVGQEEVLFRFE PQQ FP VV TQ CIF AN GDL FE QQ FP VV TQ CIF YA VIP LF RL SV AN LL VK KA VIP LF RL SV LGD LD KA VY CET QE PR QA GN LG LSE CLV AD GEL FE PQ PF DV DT AC FK IPG FQ HLP (Aegilops tauschii) FPVEKAATQRIPLAEGGIKSGVKVSKLMNYPVRGLVFEEGNTLNDLAN LVSSACIWLQDNNVPYNVLISDSGRKIFLFPQCYAEKQALGEVSQELLD VK CA GC CCA CG GG TCA CA CC TC TG CT TA GA AA TT GC AG CC CTG GT CTT AG TG GA AC TT AC GT CC CT TT CCT AT GG GC CA GG TTC GT TC TT TCA TC GC GA TTT GT AT TG CA AAC CT AG GGAAACTGATGAGGCCATTTATGCACCTGTGCTGGTTGGCCCTTCAG CTGTTGGGGAGGGCTGCCTCGTAATTCCGTGA CC CT CTT CCT AT GG GC CA GG TTC GT TC TTC CAG CC CA AG TTT TGC TT AG GG CGT CC GCC GA TG CC CT TT CCT AT GG GC CA GG TTC GT TC TTC CAG CC CA AG TTT TGC TT AG GG CGT CC GC GG CT Nucleotide sequence of ACTACCAGGAAGAAGGAGGCGGGGGCTGCGGCAGGAACTGCCTCG TaGGP2-A GGGATTGCTGCTTGCCTGCTTCCAAGCTCCCCCTCTATGCTTTCAAGG TT CC AT CC CCA GGT TG GC AC TG GCT CT CT GA AG TT CT AA TG GA CT CA GG CC CA CG GG TT CC AT CC CCA GGT TG GC GC TG GCT CT CT GA AG TTG TG AG GA AG TC AT GA CT CA CG GG CTTCCTCAATTCTCTCCTCCTCGCACAGTGGGAGGACAGGGTGGCCC GAGGCCTATTCAGATATGATGTAACAGCCTGCGAGACCAAGGTGAT ACC CCA GGT TG GC GC TG GCT TTT TA AA GA TG TG AG GA AG TC AT GA CT CA GA AC TT GG AA GC TC GA TT TT AT CT TTC CT GG GG TAT AAT AT AG CA AA AC AG GA TC TG CA CGG GC TC CG CCTGGCGAGCTTGGGTTTGTTGCACAGCTTAACGAAGGCCGCCACCT CAAGAAGCGCCCAACCGAGTTCCGTGTTGACCGTGTGCTCCAGCCAT GTT GA CAA GT CT TTG TC AC AG AG GC GA AC AA GG CG TG AA TCT GT TGG TG AT AG GA TG AA TGG AC CC TC GG CC CA TCG AA GA AGT TG CT GA TAC TG AAG TG TTC GT TGG TG GAT GA GTG AT TC GGCCAAGAGGAGGTGCTCTTCCAATTTGAGAACAGTGGTAGTGATG ACAGCTACTTCCTGAGGAGTGCCGCAGTCACTGTTGCTGATCGTGCT TGT CCA GCC TC GTC AA GG TGG AC TGC GG GTA AT GA GG TCT CA TGG GC TCT GA TCC TCA TTT TCT AA ATC GT TG GGT CA CA GT AG ATT TC GG CC CG GG AT CC TC CA CGA CCG CA GGC CA CT CC TCA TTT GA GCCACATTCTTCTTATTCCTCGTGTTCTGGACCATCTACCTCAGAGGA TTGACCAAGAGAGCTTCTTGCTTGCACTGCACATGGCAGCTGAGGCA GC TT GGC CTG GCT CC TCC AA TC GC GCT AG GC AA CA CGG CTG CA GGC TAC TC CG CAC GT GA CG GG GGC CG CT GGG CCT GGC GC TTC GA GC AG CGC CA TG TG CA CGA CCG CA GGC CA CT CC TCA TTT GA CG GA GCTAGCCCATACTTCAGGCTTGGTTACAATAGCTTGGGTGCCTTCGC AACCATCAACCACCTCCACTTTCAGGCTTACTACCTGACAGCGCCTT GGC CTG GCT CC TCC AA TC GC GCT AG GC AA CA GG GC TC CG TTC CTC TT TTC AA AAT TG TT TGG CCA CA GT GAG TT TCT GC CA GG AG CTT CT TC CA CGG GC TC CG TTC CTC TT TTC CA AT TG TT GG CC CC TGTCAAAACTGACGAATTTCCCTGTGAGAGGGCTGGTGTTTGAGAGA GGCAACACACTGAAGGATTTGGCTGATGTGGTTTCCAACGCTTGCAT TTC GG CC CT GGA TCT AC CTC CA GG GGC TC CG TTC CTC TT TTC CA AAT TG TT TGG CCA CA GT GAG TT TCC GC CG GG AG CTT CT TC CA GAA CGC CA GAT GG GC CA AG TC GTT CC CT GG CA AG GTG GA CT TGACTGTGGAAAGAGGGTCTTCCTGTTCCCCCAGTGTTATGCAGAGA AGCAGGCTCTGGGTGAAGTGAGCCAGGAGCTGCTGGACACTCAGGT GG CC GC GG CC A CA CCT CAA CC AT AG CG TCC AG TT TTG TT TT TA TC GC GA ATT AT ATC GAG GG CG TT GG GAT GT CA CGG GG AG TGG ATG AA GA AG GAT GC ATG ACC AGC AT TGT TG AG GTG AA GTG TG TACTGAAACGGAGGGACGATTATGAGGAGGCATCAGAAGCTTCGGC ATGGCGGCTCCTTGCCGAGGTCTCTCTATCGGAGTCACGCTTCGAGG TG CTG AG CA CG GG TT CC AT ACC CCA GGT TG GC GC TG GCT TTT TA AA GA TG TG AG GA AG TC AT GA CT CC CT TT CCT AT GG GC CA GG TTC GT TC TTC CAG CC CA AG TTT TGC TT AG GG CGT CC CTACATCTTTGATGCTGCTTGTTTGGTTCAGTCCCAGGTGGAGGAGG AAACTGATGACGCCATTTATGCACCTGTGCCGGTTGGCCCTTCAGCT
BIBLIOGRAPHY Bulley, S. et al. Enhancing ascorbate in fruits and tubers through over‐expression of the L‐ galactose pathway gene GDP‐L‐galactose phosphorylase. Plant Biotechnol J 2012, 10, (4), 390-397. Bulley, S.; Laing, W., The regulation of ascorbate biosynthesis. Curr Opin Plant Biol 2016, 33, 15-22. Dowdle, J. et al. Two genes in Arabidopsis thaliana encoding GDP‐L‐galactose phosphorylase are required for ascorbate biosynthesis and seedling viability. Plant J 2007, 52, (4), 673-689. Juntawong et al.2014 Translational dynamics revealed by genome-wide profiling of ribosome footprints in Arabidopsis Proc Natl Acad Sci USA 111(1):E203-12. Laing, W. A.; et al. An upstream open reading frame is essential for feedback regulation of ascorbate biosynthesis in Arabidopsis. Plant Cell 2015, 27, (3), 772-786. Li, T. et al. Domestication of wild tomato is accelerated by genome editing. Nat Biotechnol 2018, 36, 1160-1163. Naim, F.; Dugdale, B.; Kleidon, J.; Brinin, A.; Shand, K.; Waterhouse, P.; Dale, J., Gene editing the phytoene desaturase alleles of Cavendish banana using CRISPR/Cas9. Transgenic Res 2018, 27, (5), 451-460. Sallaud, C. et al. Highly efficient production and characterization of T-DNA plants for rice (Oryza sativa L.) functional genomics. Theor Appl Genet 2003, 106, (8), 1396-1408. Starck, S. R.; Jiang, V; Pavon-Eternod, M; Prasad, S; McCarthy, B; Pan, T; Shastri, N (2012). "Leucine-tRNA initiates at CUG start codons for protein synthesis and presentation by MHC class I". Science.336 (6089): 1719–23. Xie, K. et al. Boosting CRISPR/Cas9 multiplex editing capability with the endogenous tRNA- processing system. Proc Natl Acad Sci U S A 2015, 112, (11), 3570-3575. Zhang, G.-Y. et al. Manipulation of the rice L-galactose pathway: evaluation of the effects of transgene overexpression on ascorbate accumulation and abiotic stress tolerance. PLoS One 2015, 10, (5), e0125870. Zhang, H. et al.; Genome editing of upstream open reading frames enables translational control in plants. Nat Biotechnol 2018, 36, 894-898.

Claims

CLAIMS: 1. A modified genome of a cereal grass plant comprising a polynucleotide comprising a modification to an upstream open reading frame (uORF) in a 5' untranslated region (UTR) of a GDP-L-galactose phosphorylase (GGP) gene or an ortholog thereof, wherein the modification comprises a deletion, addition, or substitution of at least one base pair in the 5' UTR of the GGP gene.
2. The modified genome of the cereal grass plant of claim 1, wherein the expression of the GGP gene is not silenced.
3. The modified genome of the cereal grass plant of claim 1 or claim 2, wherein the modified plant has increased grain crop yield.
4. The modified genome of the cereal grass plant of claim 3, wherein the increase in grain crop yield is increased grain weight per plant.
5. The modified genome of the cereal grass plant of claim any one of claims 1-4, wherein the modified plant has an increase in the number of panicles per plant.
6. The modified genome of the cereal grass plant of any one of claims 1-5, wherein the modified plant has increased foliar ascorbate levels.
7. The modified genome of the cereal grass plant of any one of claims 1 to 6, wherein the GGP gene 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: 59-81, and amino acid sequences having at least 70% sequence identity to any of the foregoing.
8. The modified genome of the cereal grass plant of any one of claims 1 to 7, wherein the 5' UTR of GGP gene or ortholog thereof comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 11, 15-17, 21-23, 25, 28, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 50-51, and 54-55 and nucleic acid sequences having at least 70% sequence identity to any of the foregoing.
9. The modified genome of the cereal grass plant of any one of claims 1 to 8, wherein the modified uORF of GGP comprises at least one deletion or non-conservative substitution of at least one base pair in the uORF.
10. The modified genome of the cereal grass plant of any one of claims 1 to 9, wherein the modified uORF of GGP comprises deletion or non-conservative substitution of 1 to 100 base pairs in the uORF.
11. The modified genome of the cereal grass plant of any one of claims 1 to 10, wherein the modified uORF of GGP comprises deletion or non-conservative substitution of 2 to 80 base pairs in the uORF.
12. The modified genome of the cereal grass plant of any one of claims 1 to 11, wherein the modified uORF of GGP comprises deletion or non-conservative substitution of 5 to 70 base pairs in the uORF.
13. The modified genome of the cereal grass plant of any one of claims 1 to 12, wherein the modified uORF of GGP comprises non-conservative substitution of 1 base pair in the uORF.
14. The modified genome of the cereal grass plant of any one of claims 1 to 12, wherein the modified uORF of GGP comprises deletion of 5 base pairs in the uORF.
15. The modified genome of the cereal grass plant of any one of claims 1 to 13, wherein the modified uORF of GGP comprises deletion of 63 base pairs in the uORF.
16. The modified genome of the cereal grass plant of any one of claims 1 to 15, wherein the modified uORF of GGP results in disruption of the reading frame of the GGP uORF, wherein the GGP uROF comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 12-14, 18-20, 24, 26-27, 30, 32, 34, 36-37, or 40-41.
17. The modified genome of the cereal of claim 16, wherein the disruption of the uORF reading frame results in amino acid substitution, deletion or insertion of one or more amino acid residues from position 30 to position 61 the GGP uORF peptide of SEQ ID NO: 10.
18. The modified genome of the cereal grass plant of claim 16 or claim 17, wherein the disruption of the uORF reading frame comprises substitution of an amino acid sequence comprising the amino acid residues at position 57-61 of SEQ ID NO:10.
19. The modified genome of the cereal grass plant of claim 16 or claim 17, wherein the disruption of the uORF reading frame comprises deletion of an amino acid sequence comprising the amino acid residues at positions 55-56 of SEQ ID NO :10.
20. The modified genome of the cereal grass plant of claim 16 or claim 17, wherein the disruption of the uORF reading frame comprises deletion of an amino acid sequence comprising the amino acid residues at positions 37-57 of SEQ ID NO:10.
21. The modified genome of the cereal grass plant of any one of claims 1-20, wherein the modified GGP uORF peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 56-58.
22. The modified genome of the cereal grass plant of any one of claims 1-21, wherein the modified GGP uORF results in reduced ribosomal stalling in the uORF.
23. The modified genome of the cereal grass plant of any one of claims 1-22, wherein modifying the uORF comprises site-directed nucleases or oligonucleotide-directed mutagenesis.
24. The modified genome of the cereal grass plant of claim 23, wherein said mutagenesis is achieved using SDN-1.
25. The modified genome of the plant of claim 23, wherein said mutagenesis is achieved using Zinc finger nucleases, TALENS and / or CRISPR/Cas gene editing and/or base editing.
26. The modified genome of the cereal grass plant of claim 25, wherein said mutagenesis is achieved using CRISPR/Cas9 or CRISPR/Cas12 gene editing.
27. The modified genome of the cereal grass plant of any one of claims 1-26, wherein the cereal grass plant is selected from the group consisting of rice, barley, wheat, rye, oats, millet, sorghum, triticale, buckwheat, quinoa, spelt, einkorn, amaranth, grass, corn, wild rice, teff; Aegilops tauschii, brachypodium, Miscanthus, switchgrass or a forage grass.
28. The modified genome of the cereal grass plant of any one of claims 1-27, wherein the cereal grass plant is a wheat plant or a rice plant.
29. The modified genome of the cereal grass plant of claim 28, wherein the cereal grass plant is a rice plant.
30. A polynucleotide construct comprising a modification to an upstream open reading frame (uORF) in a 5' untranslated region (UTR) of a GDP-L-galactose phosphorylase (GGP) gene or an ortholog thereof, wherein the modification comprises a deletion, addition, or substitution of at least one base pair of in the 5' UTR of the GGP gene.
31. The polynucleotide construct of claim 30, wherein the GGP gene 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: 59-81, and amino acid sequences having at least 70% sequence identity to any of the foregoing.
32. The polynucleotide construct of claim 30 or claim 31, wherein the 5' UTR of the GGP gene or ortholog thereof comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs:11, 15-17, 21-23, 25, 28, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 50-51, and 54-55 and nucleic acid sequences having at least 70% sequence identity to any of the foregoing.
33. A modified cereal grass plant comprising a polynucleotide comprising a modification to an upstream open reading frame (uORF) in a 5' untranslated region (UTR) of a GDP-L- galactose phosphorylase (GGP) gene or an ortholog thereof, wherein the modification comprises a deletion, addition, or substitution of at least one base pair of in the 5' UTR of the GGP gene.
34. A method of modifying the genome of a cereal grass plant, comprising introducing a modification to an upstream open reading frame (uORF) in a 5' untranslated region (UTR) of a GDP-L-galactose phosphorylase (GGP) gene or an ortholog thereof, wherein the modification comprises a deletion, addition, or substitution of at least one base pair of in the 5' UTR of the GGP gene.
35. A method of increasing the grain crop yield of a cereal grass plant, comprising introducing a modification to an upstream open reading frame (uORF) in a 5' untranslated region (UTR) of a GDP-L-galactose phosphorylase (GGP) gene or an ortholog thereof, wherein the modification comprises a deletion, addition, or substitution of at least one base pair of in the 5' UTR of the GGP gene.
36. The method of claim 35, wherein the increase in grain crop yield is increased grain weight per plant.
37. The method of claim 36, wherein the increase in grain crop yield is the result of an increase in the number of panicles per plant.
38. A method of increasing the number of panicles of a cereal grass plant, the method comprising introducing a modification to an upstream open reading frame (uORF) in a 5' untranslated region (UTR) of a GDP-L-galactose phosphorylase (GGP) gene or an ortholog thereof.
39. The method of any one of claims 34-38, wherein the GGP gene or ortholog thereof comprises a nucleic acid sequence that encodes a polypeptide having an amino acid sequence selected from the group of SEQ ID NOs: 59-81, and amino acid sequences having at least 70% sequence identity to any of the foregoing.
40. The method of any one of claims 34-39, wherein the 5' UTR of the GGP gene or ortholog thereof comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs:11, 15-17, 21-23, 25, 28, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 50-51, and 54-55 and nucleic acid sequences having at least 70% sequence identity to any of the foregoing.
41. The method of any one of claims 34-40, wherein modifying the uORF comprises at least one deletion or non-conservative substitution of at least one base pair in the uORF.
42. The method of claim 41, wherein modifying the uORF comprises deletion or non- conservative substitution of 1 to 100 base pairs in the uORF.
43. The method of claim 41 or claim 42, wherein modifying the uORF comprises deletion or non-conservative substitution of 2-80 base pairs in the uORF.
44. The method of any one of claims 41-43, wherein modifying the uORF comprises deletion or non-conservative substitution of 5-70 base pairs in the uORF.
45. The method of any one of claims 41-44, wherein modifying the uORF comprises a non- conservative substitution of 1 base pair in the uORF.
46. The method of any one of claims 41-44, wherein modifying the uORF comprises deletion of 5 base pairs in the uORF.
47. The method of any one of claims 41-44, wherein modifying the uORF comprises deletion of 63 base pairs in the uORF.
48. The method of any one of claims 34-47, wherein modifying the uORF results in disruption of the reading frame of the GGP uORF, wherein the GGP uROF comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 12-14, 18-20, 24, 26-27, 30, 32, 34, 36-37, or 40-41.
49. The method of claim 48, wherein the disruption of the uORF reading frame results in amino acid substitution, deletion or insertion of one or more amino acid residues from position 30 to position 61 the GGP uORF peptide of SEQ ID NO: 10.
50. The method of claim 49, wherein of the uORF reading frame comprises substitution of an amino acid sequence comprising the amino acid residues at positions 57-61 of SEQ ID NO: 10.
51. The method of claim 49, wherein the disruption of the uORF reading frame comprises deletion of an amino acid sequence comprising the amino acid residues at positions 55- 56 of SEQ ID NO: 10.
52. The method of claim 49, wherein the disruption of the uORF reading frame comprises deletion of an amino acid sequence comprising the amino acid residues at positions 37- 57 of SEQ ID NO: 10.
53. The method of any one of claims 34-52, wherein the modified GGP uORF peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 56-58.
54. The method of any one of claims 34-53, wherein the modified GGP uORF results in reduced ribosomal stalling in the uORF.
55. The method of any one of claims 34-54, wherein modifying the uORF comprises site- directed nucleases or oligonucleotide-directed mutagenesis.
56. The method of claim 55, wherein said mutagenesis is achieved using SDN-1.
57. The method of claim 56, wherein said mutagenesis is achieved using Zinc finger nucleases, TALENS and / or CRISPR/Cas gene editing and/or base editing.
58. The method of claim 57, wherein said mutagenesis is achieved using CRISPR/Cas9 or CRISPR/Cas12 gene editing.
59. The method of any claims 34-58, wherein the cereal grass plant is selected from the group consisting of rice, barley, wheat, rye, oats, millet, sorghum, triticale, buckwheat, quinoa, spelt, einkorn, amaranth, grass, corn, canola, wild rice, teff; Aegilops tauschii, brachypodium, Miscanthus, switchgrass or a forage grass.
60. The method of any one of claims 34-59, wherein the cereal grass plant is a wheat plant or a rice plant.
61. The method of claim 60, wherein grass plant is a rice plant.
62. A plant comprising the modified genome of any one of claims 1-29, or the polynucleotide of any one of claims 30-32.
63. A seed produced by the plant of claim 62.
64. A plant derived from the seed of claim 63.
65. A plant part derived from the plant of claim 62 or claim 64.
66. The plant part of claim 65, wherein the plant part is selected from the group consisting of a seed, grain, fruit, leaf, flower, tuber, stalk, rhizome, cutting, a panicle and root.
67. The plant part of claim 66, wherein the plant part is grain.
68. The plant part of claim 67, wherein the grain is a germinated grain.
69. The plant part of claim 66 or claim 67, wherein the grain is selected from the group consisting of rice grain, wheat grain, rye grain, barley grain or oat grain.
70. The plant part of claim 69, wherein the grain is rice.
71. A flour produced from the grain of any one of claims 67-70.
72. A food product comprising the plant of claim 62 or claim 64; the plant part of any one of claims 65-70, or the flour of claim 71.
PCT/AU2025/050512 2024-05-15 2025-05-15 Regulation of gene expression Pending WO2025236045A1 (en)

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