# Patent

## Title
Regulation of gene expression via uORF of GDP-L-galactose phosphorylase (GGP) gene

## Abstract

The invention provides isolated polynucleotides comprising sequences encoding a u ORF peptides and variants and fragments thereof. The invention also provides constructs and vectors containing the polynucleotides. The invention further provides cells, plant cells and plants transformed with the polynucleotides and constructs. The invention also provides methods of using the polynucleotides to control expression of operably linked polynucleotides. The invention also provides methods of manipulating GDP-L-Galactose phosphorylase (GGP) expression and ascorbate production in plants utilizing the polynucleotides of the invention.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a U.S. National Stage Application filed under 35 U.S.C. §371 of International Application No. PCT/IB2013/061166, filed Dec. 20, 2013, which claims the benefit of U.S. Provisional Application No. 61/740,751, filed Dec. 21, 2012. Both of these applications are hereby incorporated by reference in their entireties.


TECHNICAL FIELD
The present invention relates to control and manipulation of expression of GDP-L-Galactose phosphorylase (GGP) and ascorbate production. The invention also relates to sequence elements controlling such expression and production, and their use.
BACKGROUND ART
Ascorbate is the most abundant soluble antioxidant in plants and is also an essential nutrient for humans and a few other animals. Ascorbate contributes significantly to the overall intake of “free radical scavengers” or “anti-oxidative metabolites” in the human diet. Convincing evidence now shows that such metabolites either singly or in combination, benefit health and well-being, acting as anti-cancer forming agents and protecting against coronary heart disease.
Almost the entire dietary ascorbate intake in humans is derived from plant products. The ascorbate content of plant tissues however, is remarkably variable. Whilst leaf ascorbate content is generally high and relatively uniform in herbaceous and woody plants, a huge and unexplained variability in ascorbate content found is in non-green edible plant tissues. For example, in fruits, the levels vary from up to 30 mg gFW-1 AsA in the camu camu of Mirciaria dubia, to less than 3 μg gFW-1 AsA in the medlar of Mespilus germanica (Rodriguez et al. 1992, J Chromatogr Sci, 30:433-437). A range of values for ascorbate have been reported in kiwifruit (Ferguson, A. R., Botanical nominclature: Actinidia chinensis, Actinidia deliciosa, and Actinidia setosa. Kiwifruit: science and management, ed. I. J. Warrington and G. C. Weston. 1990, Palmerston North; New Zealand: New Zealand Society for Horticultural Science. 576. Beever, D. J. and G. Hopkirk, Fruit development and fruit physiology. Kiwifruit: science and management, ed. I. J. Warrington and G. C. Weston. 1990, Palmerston North; New Zealand: New Zealand Society for Horticultural Science. 576.) Ascorbate content of fruits from different vines range for A. deliciosa, 30-400 mg/100 g (Ferguson, A. R., 1991 Acta Hort. 290: p. 603-656, Spano, D., et al., 1997 Acta Hort., 444: p. 501-506.) while for the cultivar ‘Hayward’ the reported range is 80-120 mg/100 g (Beever, D. J. and G. Hopkirk, Fruit development and fruit physiology. Kiwifruit: science and management, ed. I. J. Warrington and G. C. Weston. 1990, Palmerston North; New Zealand: New Zealand Society for Horticultural Science. 576.). Higher concentrations of ascorbate are reported in fruit of, A. arguta, A. chinensis (Muggleston, S., et al., Orchardist, 1998. 71(8): p. 38-40, Chen, Q. and Q. Chen, Crop Genetic Resources, 1998(2): p. 3, Coggiatti, S., 1971 Ital Agr, October, 108(10): p. 935-941) A. chrysantha and A. polygama with very high levels in A. eriantha, and A. latifolia (>1% fresh weight) (Ferguson 1991 Acta Hort. 290: p. 603-656, and A. kolomikta (Kola, J. and J. Pavelka, 1988 Nahrung, 32(5): p. 513-515).
Three pathways of biosynthesis of ascorbic acid have been proposed in plants, one through L-Galactose (L-Gal) (Wheeler et al., 1998, Nature 393, 365-369), another from myo-Inositol (Loewus & Kelly, 1961, Arch. Biochem. Biophys. 95, 483-493; Lorence et al., (2004) Plant Physiol. 134, 1200-1205) and a third through Galacturonic acid (Agius et al., 2003, Nat Biotechnol 21, 177-81). The L-Gal pathway proceeds through L-Gal to galactono-1,4-lactone and thence to ascorbate (Wheeler et al., 1998, Nature 393, 365-369).
All the genes encoding enzymes, and their associated enzymatic activities, for the L-Galactose pathway have been identified and at least partially characterised.
The characterised genes and enzyme activities include the GDP-D-Mannose Pyrophosphorylase (Conklin, 1998, Trends Plant Sci 3: 329-330.; Conklin et al., 1999 Proc Natl Acad Sci USA 96: 4198-4203.; Keller et al., 1999 Plant J 19: 131-141.), the GDP-D-Mannose 3′,5′-Epimerase (Wolucka et al., 2001, Anal Biochem 294: 161-168; Wolucka and Van Montagu, 2003, J. Biol. Chem. 278: 47483-47490; Watanabe et al., 2006 Phytochemistry 67: 338-346.), the L-Galactose-1-P Phosphatase (Laing et al., 2004, Proceedings of the National Academy of Sciences (USA) 101: 16976-16981.; Conklin et al., 2006, J. Biol. Chem. 281: 15662-15670.), L-Galactose Dehydrogenase (Wheeler et al., 1998, Nature 393: 365-369.; Gatzek et al., 2002, Plant J. 30, 541 (2002; Laing et al., 2004 Proceedings of the National Academy of Sciences (USA) 101: 16976-16981), L-Galactono-1,4-lactone Dehydrogenase (Imai et al., 1998 Plant and Cell Physiology 39: 1350-1358.; Bartoli et al., 2005, Plant, Cell and Environment 28: 1073-1081.), and GDP-L galactose phosphorylase (GGP) (Laing et al., 2007, Proceedings of the National Academy of Sciences (USA) 104:9534-9). The applicants have previously shown that GDP-L galactose phosphorylase is central in determining ascorbate production Bulley S, et al 2012 Plant Biotechnol J 2012, 10:390-397.
Ascorbate concentrations are regulated according to demand. Under high light intensities when the need for high ascorbate is greatest, leaf ascorbate concentrations are raised (Bartoli et al., J. Exp. Bot. 57, 1621 (2006); Gatzek, et al., Plant J. 30, 541 (2002)). However little is known about the mechanism of regulation of ascorbate biosynthesis in plants (Bulley et al., Plant Biotechnol J 10, 390 (2012); Bulley et al., J. Exp. Bot. 60, 765 (2009).). Understanding how ascorbate biosynthesis is regulated may provide tools to manipulate biosynthesis in plants. Understanding the regulation of gene expression, and the factors/elements controlling such expression also provide valuable tools for genetic manipulation.
It is one object of the invention to provide improved compositions and methods for modulating GGP (also known as GDP-L-Galactose phosphorylase) activity; and/or ascorbate content in plants and/or to provide improved tools useful for genetic manipulation, or at least to provide the public with a useful choice.
SUMMARY OF THE INVENTION
In one aspect the invention provides an isolated polynucleotide comprising a sequence encoding a polypeptide with an amino acid sequence selected from SEQ ID NO:1 to 20 and 132 to 134 (uORF peptides) or a variant or fragment thereof.
In one embodiment the variant or fragment comprises a sequence with at least 70% Identity to an amino sequence selected from SEQ ID NO:21 to 40 and 135 to 137 (conserved region of uORF peptides).
In a further embodiment the variant or fragment comprises a sequence with an amino acid selected from SEQ ID NO:21 to 40 and 135 to 137 (conserved region of uORF peptides).
In one embodiment the variant or fragment comprises a sequence with at least 70% Identity to an amino sequence selected from SEQ ID NO: 21-30, 33-37 and 135 to 137 (conserved region of dicot uORF peptides).
In a further embodiment the variant or fragment comprises a sequence with an amino acid selected from SEQ ID NO: 21 to 30, 33 to 37 and 135 to 137 (conserved region of dicot uORF peptides).
In one embodiment the variant or fragment comprises a sequence with at least 70% Identity to the amino sequence of SEQ ID NO: 108 (consensus motif uORF peptides).
In a further embodiment the variant or fragment comprises the amino sequence of SEQ ID NO: 108 (consensus motif uORF peptides).
In a further embodiment the variant comprises a sequence with at least 70% identity to an amino acid sequence selected from SEQ ID NO:1 to 20 and 132 to 134 (uORF peptides).
In a further embodiment the variant comprises an amino acid sequence selected from SEQ ID NO:1 to 20 and 132 to 134 (uORF peptides).
In a further embodiment the variant comprises a sequence with at least 70% identity to an amino acid sequence selected from SEQ ID NO: 1 to 10, 13 to 17 and 132 to 134 (dicot uORF peptides).
In a further embodiment the variant comprises an amino acid sequence selected from SEQ ID NO: 1 to 10, 13 to 17 and 132 to 134 (dicot uORF peptides).
In a further embodiment the isolated polynucleotide comprises a sequence with at least 70% Identity to a sequence selected from SEQ ID NO:61 to 80 and 138 to 140 (conserved region of uORF DNA sequences).
In a further embodiment the isolated polynucleotide comprises a sequence selected from SEQ ID NO:61 to 80 and 138 to 140 (conserved region of uORF DNA sequences).
In a further embodiment the isolated polynucleotide comprises a sequence with at least 70% identity to a sequence selected from SEQ ID NO: 61 to 70, 73 to 77 and 138 to 140 (conserved region of dicot uORF DNA sequences).
In a further embodiment the isolated polynucleotide comprises a sequence selected from SEQ ID NO: 61 to 70, 73 to 77 and 138 to 140 (conserved region of dicot uORF DNA sequences).
In a further embodiment the isolated polynucleotide comprises a sequence with at least 70% Identity to a sequence selected from SEQ ID NO:41 to 60 and 129 to 131 (uORF DNA sequences).
In a further embodiment the isolated polynucleotide comprises a sequence selected from SEQ ID NO:41 to 60 and 129 to 131 (uORF DNA sequences).
In a further embodiment the isolated polynucleotide comprises a sequence with at least 70% Identity to a sequence selected from SEQ ID NO: 41 to 50, 53 to 57 and 129 to 131 (dicot uORF DNA sequences).
In a further embodiment the isolated polynucleotide comprises a sequence selected from SEQ ID NO: 41 to 50, 53 to 57 and 129 to 131 (dicot uORF DNA sequences).
In a further embodiment the isolated polynucleotide comprises a sequence with at least 70% Identity to a sequence selected from SEQ ID NO:111 to 125 (5′-UTR sub-sequences).
In a further embodiment the isolated polynucleotide comprises a sequence selected from SEQ ID NO:111 to 125 (5′-UTR sub-sequences).
In a further embodiment the isolated polynucleotide comprises a sequence with at least 70% Identity to a sequence selected from SEQ ID NO:81 to 100 and 126 to 128 (whole 5′-UTR sequences).
In a further embodiment the isolated polynucleotide comprises a sequence selected from SEQ ID NO:81 to 100 and 126 to 128 (whole 5′-UTR sequences).
In a further embodiment the isolated polynucleotide comprises a sequence with at least 70% Identity to a sequence selected from SEQ ID NO: 81 to 90, 93 to 97 and 125 to 128 (whole dicot 5′-UTR sequences).
In a further embodiment the isolated polynucleotide comprises a sequence selected from SEQ ID NO: 81 to 90, 93 to 97 and 125 to 128 (whole dicot 5′-UTR sequences).
In a further aspect the invention provides an isolated polynucleotide comprising a sequence selected from SEQ ID NO:41 to 60 and 129 to 131 (uORF DNA sequences) or a variant or fragment thereof.
In one embodiment the variant or fragment comprises a sequence with at least 70% identity to a sequence selected from SEQ ID NO:61 to 80 and 138 to 140 (conserved region of uORF DNA sequences).
In one embodiment the variant or fragment comprises a sequence with at least 70% identity to a sequence selected from SEQ ID NO: 61 to 70, 73 to 77 and 138 to 140 (conserved region of dicot uORF DNA sequences).
In a further embodiment the isolated polynucleotide comprises a sequence selected from SEQ ID NO: 41 to 60 and 129 to 131 (uORF DNA sequences).
In one embodiment the variant comprises a sequence with at least 70% identity to a sequence selected from SEQ ID NO:41 to 60 and 129 to 131 (uORF DNA sequences).
In one embodiment the variant comprises a sequence with at least 70% Identity to a sequence selected from SEQ ID NO: 41 to 50 and 53 to 57 and 129 to 131 (dicot uORF DNA sequences).
In a further embodiment the isolated polynucleotide comprises a sequence with at least 70% identity to a sequence selected from SEQ ID NO:111 to 125 (5′-UTR sub-sequences).
In a further embodiment the isolated polynucleotide comprises a sequence selected from SEQ ID NO: 111 to 125 (5′-UTR sub-sequences).
In a further embodiment the isolated polynucleotide comprises a sequence with at least 70% Identity to a sequence selected from SEQ ID NO:81 to 100 and 126 to 128 (whole 5′-UTR sequences).
In a further embodiment the isolated polynucleotide comprises a sequence selected from SEQ ID NO:81 to 100 and 126 to 128(whole 5′-UTR sequences).
In a further embodiment the isolated polynucleotide comprises a sequence with at least 70% identity to a sequence selected from SEQ ID NO: 81 to 90, 93 to 97 and 126 to 128 (whole dicot 5′-UTR sequences).
In a further embodiment the isolated polynucleotide comprises a sequence selected from SEQ ID NO: 81 to 90, 93 to 97 and 126 to 128 (whole dicot 5′-UTR sequences).
In a further aspect the invention provides an isolated polynucleotide with a sequence selected from SEQ ID NO 81 to 100 and 126 to 128 (whole 5′-UTR sequences) or a variant or fragment thereof.
In one embodiment the variant has at least 70% identity to a sequence selected from SEQ ID NO 81 to 100 and 126 to 128 (whole 5′-UTR sequences).
In a further aspect the invention provides an isolated polynucleotide with a sequence selected from SEQ ID NO 111 to 125 (5′-UTR sub-sequences) or a variant or fragment thereof.
In one embodiment the variant has at least 70% identity to a sequence selected from SEQ ID NO 111 to 125 (5′-UTR sub-sequences).
In a further embodiment the variant or fragment comprises a sequence with at least 70% Identity to a sequence selected from SEQ ID NO 41 to 60 and 129 to 131 (uORF DNA sequences).
In a further embodiment the variant or fragment comprises a sequence selected from SEQ ID NO 41 to 60 and 129 to 131 (uORF DNA sequences).
In a further embodiment the variant or fragment comprises a sequence with at least 70% identity to a sequence selected from SEQ ID NO 41 to 50, 53 to 57 and 129 to 131 (dicot uORF DNA sequences).
In a further embodiment the variant or fragment comprises a sequence selected from SEQ ID NO 41 to 50, 53 to 57 and 129 to 131 (dicot uORF DNA sequences).
In a further embodiment the variant or fragment comprises a sequence with at least 70% identity to a sequence selected from SEQ ID NO:61 to 80 and 138 to 140 (conserved region of uORF DNA sequences).
In a further embodiment the variant or fragment comprises a sequence selected from SEQ ID NO:61 to 80 and 138 to 140 (conserved region of uORF DNA sequences).
In a further embodiment the variant or fragment comprises a sequence with at least 70% Identity to a sequence selected from SEQ ID NO: 61 to 70, 73 to 77 and 138 to 140 (conserved region of dicot uORF DNA sequences).
In a further embodiment the variant or fragment comprises a sequence selected from SEQ ID NO: 61 to 70, 73 to 77 and 138 to 140 (conserved region of dicot uORF DNA sequences).
In a further embodiment the variant encodes a sequence with at least 70% identity to at least one of SEQ ID NO:21 to 40 and 135 to 137 (conserved region of uORF peptides).
In a further embodiment the variant encodes a sequence selected from at least one of SEQ ID NO:21 to 40 and 135 to 137 (conserved region of uORF peptides).
In a further embodiment the variant encodes a sequence with at least 70% identity to at least one of SEQ ID NO: 21 to 30, 33 to 37 and 135 to 137 (conserved region of dicot uORF peptides).
In a further embodiment the variant encodes a sequence selected from at least one of SEQ ID NO: 21 to 30, 33 to 37 and 135 to 137 (conserved region of dicot uORF peptides).
In one embodiment the variant or fragment comprises a sequence with at least 70% identity to the amino sequence of SEQ ID NO: 108 (consensus motif uORF peptides).
In a further embodiment the variant or fragment comprises the amino sequence of SEQ ID NO: 108 (consensus motif uORF peptides).
In a further embodiment the variant encodes a sequence with at least 70% Identity to at least one of SEQ ID NO:1 to 20 and 132 to 134 (uORF peptides).
In a further embodiment the variant or fragment encodes a sequence selected from at least one of SEQ ID NO:1 to 20 and 132 to 134 (uORF peptides).
In a further embodiment the variant encodes a sequence with at least 70% identity to at least one of SEQ ID NO: 1 to 10, 13 to 17 and 132 to 134 (dicot uORF peptides).
In a further embodiment the variant or fragment encodes a sequence selected from at least one of SEQ ID NO: 1 to 10, 13 to 17 and 132 to 134 (dicot uORF peptides).
In one embodiment the isolated polynucleotide is modified.
In one embodiment, the modification is at least one of a deletion, an addition, or a substitution of at least one nucleotide in the sequence encoding the 5′-UTR.
In one embodiment the modification, reduced, disrupts, or prevents translation of a uORF polypeptide with the sequence of any one of SEQ ID NO: 1 to 20 and 132 to 134 (uORF peptides) or a variant thereof.
In a further embodiment the modification reduces, disrupts or destroys the activity of a uORF polypeptide with the sequence of any one of SEQ ID NO: 1 to 20 and 132 to 134 (uORF peptides) or a variant thereof.
In one embodiment the variant comprises a sequence with at least 70% identity to any one of SEQ ID NO: 1 to 20 and 132 to 134 (uORF peptides).
In a further embodiment the variant comprises a sequence with at least 70% Identity to any one of SEQ ID NO: 1 to 10, 13 to 17 and 132 to 134 (dicot uORF peptides).
In a further embodiment the variant comprises a sequence with at least 70% identity to at least one of SEQ ID NO: 21 to 40 and 135 to 137 (uORF peptides conserved region).
In a further embodiment the variant comprises a sequence with at least one of SEQ ID NO: 21 to 40 and 135 to 137 (uORF peptides conserved region).
In a further embodiment the variant comprises a sequence with at least 70% identity to at least one of SEQ ID NO: 21 to 30, 33 to 37 and 135 to 137 (dicot uORF peptides conserved region).
In a further embodiment the variant comprises a sequence with at least one of SEQ ID NO: 21 to 30, 33 to 37 and 135 to 137 (dicot uORF peptides conserved region).
In one embodiment the variant or fragment comprises a sequence with at least 70% identity to the amino sequence of SEQ ID NO: 108 (consensus motif uORF peptides).
In a further embodiment the variant or fragment comprises the amino sequence of SEQ ID NO: 108 (consensus motif uORF peptides).
In one embodiment the polynucleotide, or variant, or fragment, is operably linked to a nucleic acid sequence of interest.
In a further embodiment the nucleic acid sequence of interest encodes a protein of interest.
In one embodiment the polynucleotide and nucleic acid sequence are not normally associated in nature.
When the polynucleotide is modified as discussed above, to disrupt expression or activity of the uORF polypeptide, the operably liked sequence may be a GGP sequence. In this embodiment the modification removes repression, via the uORF, by ascorbate. Expressing the GGP under the control of the modified polynucleotide may advantageously retain spatial and/or temporal expression of GGP similar to control by the native GGP promoter and 5′-UTR but stop the negative regulation of expression by ascorbate via the uORF polypeptide. In this embodiment the polynucleotide and nucleic acid sequence of interest may be normally associated in nature, except that the polynucleotide is in a modified form as discussed above.
Polypeptides
In a further aspect the invention provides an isolated polypeptide comprising a sequence selected from any one of SEQ ID NO:1 to 20 and 132 to 134 (uORF peptides) or a variant or fragment thereof.
In one embodiment the variant or fragment comprises a sequence with at least 70% identity to a sequence selected from SEQ ID NO:21 to 40 and 135 to 137 (conserved region of uORF peptides).
In a further embodiment the variant or fragment comprises a sequence selected from SEQ ID NO:21 to 40 and 135 to 137 (conserved region of uORF peptides).
In one embodiment the variant or fragment comprises a sequence with at least 70% Identity to a sequence selected from SEQ ID NO: 21 to 30, 33 to 37 and 135 to 137 (conserved region of dicot uORF peptides).
In a further embodiment the variant or fragment comprises a sequence selected from SEQ ID NO: 21 to 30, 33 to 37 and 135 to 137 (conserved region of dicot uORF peptides).
In one embodiment the variant or fragment comprises a sequence with at least 70% identity to the amino sequence of SEQ ID NO: 108 (consensus motif uORF peptides).
In a further embodiment the variant or fragment comprises the amino sequence of SEQ ID NO: 108 (consensus motif uORF peptides).
In a further embodiment the variant comprises a sequence with at least 70% identity to a sequence selected from SEQ ID NO:1 to 20 and 132 to 134 (uORF peptides).
In a further embodiment the variant comprises a sequence with at least 70% identity to a sequence selected from SEQ ID NO: 1 to 10, 13 to 17 and 132 to 134 (dicot uORF peptides).
Construct
In a further embodiment the invention provides a construct comprising a polynucleotide of the invention.
In one embodiment the polynucleotide is operably linked to a nucleic acid sequence of interest.
In a further embodiment the polynucleotide and nucleic acid sequence are not normally associated in nature.
In a further embodiment the nucleic acid sequence of interest encodes a protein of interest.
Activity of Polynucleotides
In one embodiment the polynucleotide of the invention is regulatable by a compound.
In this embodiment the expression of any nucleic acid sequence operably linked to the polynucleotide of the invention is regulated by the compound.
In a preferred embodiment regulation is post-transcriptional.
Preferably expression of the polypeptide encoded by the operably linked nucleic acid is regulated by the compound.
In one embodiment, expression of the operably linked nucleic acid is regulated by interaction between the compound and the uORF peptide expressed by the polynucleotide of the invention. In one embodiment interaction is direct. In a further embodiment the interaction is indirect. In a further embodiment the indirect interaction is via a further protein.
In one embodiment the compound is ascorbate, or a related metabolite. In a preferred embodiment the compound is ascorbate
When modified, as discussed above, the polynucleotide may no longer be regulatable by the compound. An application of this embodiment could be to express a GGP coding sequence under the control of the modified polynucleotide sequence. In this embodiment, the modification results in a reduction or removal of repression by the compound. When the compound is ascorbate, this results in a loss of a repression of GGP translation, and hence increased GGP production and increased ascorbate accumulation. In this embodiment the modification of the uORF, or uORF encoding sequence, may be in the context of the promoter and 5′-UTR sequence. Examples of the whole GGP promoter and 5-UTR sequence are provided in SEQ ID NO: 101 to 107 or a variant thereof. Use of the modified uORF in the context of the promoter and 5′-UTR sequence may retain some spatial or temporal expression of a native GGP sequence, but without repression of GGP translation by ascorbate via the uORF polypeptide.
Cell
In a further embodiment the invention provides a cell comprising a polynucleotide of the invention, or a construct of the invention.
Preferably the cell, or it precursor cell, has been genetically modified to comprise the polynucleotide of the invention, or a construct of the invention.
Preferably the cell, or it precursor cell, has been transformed to comprise the polynucleotide of the invention, or a construct of the invention.
Plant Cells and Plants
In a further embodiment the invention provides a plant cell or plant comprising a polynucleotide of the invention or a construct of the invention.
Preferably the plant cell or plant, or it precursor plant cell or plant, has been genetically modified to comprise the polynucleotide of the invention, or a construct of the invention.
Preferably the plant cell or plant, or it precursor plant cell or plant, has been transformed to comprise the polynucleotide of the invention, or a construct of the invention.
Also Express an Epimerase
In one embodiment the cell or plant is also genetically modified, or transformed, to express a polynucleotide encoding a GDP-D-Mannose epimerase.
Preferably the cell or plant also comprises an expression construct of the invention capable of expressing a GDP-D-Mannose epimerase.
Plant Part or Propagule
In a further embodiment the invention provides a plant part or propagule comprising a polynucleotide of the invention or a construct of the invention.
Preferably the plant part or propagule, or it precursor plant cell or plant, has been genetically modified to comprise the polynucleotide of the invention, or a construct of the invention.
Preferably the plant part or propagule, or it precursor plant cell or plant, has been transformed to comprise the polynucleotide of the invention, or a construct of the invention.
In a further aspect the invention provides a method for controlling or regulating expression of at least one nucleic acid sequence in a cell comprising transformation of the cell with a polynucleotide or construct of the invention.
In a further aspect the invention provides a method for controlling expression of at least one nucleic acid sequence in a plant cell or plant comprising transformation of the plant cell or plant with a polynucleotide or construct of the invention.
In a further aspect the invention provides a method for producing a cell with modified gene expression the method comprising transforming the cell with a polynucleotide or construct of the invention.
In a further aspect the invention provides a method for producing a plant cell or plant with modified gene expression the method comprising transforming plant cell or plant with a polynucleotide or construct of the invention.
In a further aspect of the invention provides a method for modifying the phenotype of a plant, the method including the stable incorporation into the genome of the plant, a polynucleotide or construct of the invention.
Those skilled in the art will understand that introduction of the polynucleotide of the invention into the cell, plant cell, or plant, may result in regulation or control of a nucleic acid sequence that is operably linked to the nucleic acid sequence before these sequences are introduced. In such an embodiment the polynucleotide of the invention and operably linked nucleic acid of interest will be introduced together, for example on a construct of the invention.
In an alternative embodiment, the polynucleotide of the invention may be inserted into the genome, and control or regulate expression of a nucleic acid sequence, such as a protein encoding nucleic acid sequence, adjacent to the site of insertion.
In a preferred embodiment, the cell, plant cell or plant produces a compound that regulates or controls expression via the introduced polynucleotide of the invention, or via a uORF polypeptide encoded by the introduced polynucleotide of the invention.
Alternatively the compound may be applied to the cell, plant cell or plant.
In a further aspect the invention provides a plant cell or plant produced by a method of the invention.
In one aspect the invention provides a method for producing a plant cell or plant with at least one of:
increased GGP translation,
increased GGP production,
increased GGP activity, and
increased ascorbate production,
the method comprising modification of the 5′-UTR of a GGP gene in the plant cell or plant.
In one embodiment, the 5′-UTR is in the context of a polynucleotide sequence selected from any one of SEQ ID NO: 101 to 107 (GGP genomic sequences with promoter a 5′-UTR) or a variant thereof.
Preferably the variant has at least 70% Identity to the sequence of any one of SEQ ID NO: 101 to 107 (GGP genomic sequences with promoter a 5′-UTR).
In a further embodiment the 5′-UTR has a polynucleotide sequence selected from any one of SEQ ID NO: 81 to 100 and 126 to 128 (whole 5′-UTR sequences) or a variant thereof.
Preferably the variant has at least 70% Identity to the sequence of any one of SEQ ID NO: 81 to 100 and 126 to 128 (whole 5′-UTR sequences).
In a preferred embodiment the modification is in a uORF sequence in the 5′-UTR.
In a preferred embodiment the uORF has a sequence selected from any one of SEQ ID NO: 41 to 60 and 129 to 131 (uORF DNA sequences) or a variant thereof.
In a preferred embodiment the variant has at least 70% Identity to any one of SEQ ID NO: 41 to 60 and 129 to 131 (uORF DNA sequences).
In a preferred embodiment the variant has at least 70% identity to any one of SEQ ID NO: 41-50, 53-57 and 129 to 131 (dicot uORF DNA sequences).
In a further embodiment the variant comprises a sequence with at least 70% identity to any one of SEQ ID NO: 61-80 and 138 to 140 (conserved region of uORF DNA sequences).
In a further embodiment the variant comprises the sequence of any one of SEQ ID NO: 61-80 and 138 to 140 (conserved region of uORF DNA sequences).
In a further embodiment the variant comprises a sequence with at least 70% identity to any one of SEQ ID NO: 61-70, 73-77 and 138 to 140 (conserved region of dicot uORF DNA sequences).
In a further embodiment the variant comprises the sequence of any one of SEQ ID NO: 61-70, 73-77 and 138 to 140 (conserved region of dicot uORF DNA sequences).
In a further embodiment the uORF has the sequence of any one of SEQ ID NO:1 to 20 and 132 to 134 (uORF polypeptide sequences) or a variant thereof.
In a further embodiment the variant has at least 70% identity the sequence of any one of SEQ ID NO: 1 to 20 and 132 to 134 (uORF polypeptide sequences).
In a further embodiment the variant has the sequence of any one of SEQ ID NO: 1 to 20 and 132 to 134 (uORF polypeptide sequences).
In a further embodiment the variant has at least 70% Identity the sequence of any one of SEQ ID NO: 1 to 10, 13 to 17 and 132 to 134 (dicot uORF polypeptide sequences).
In a further embodiment the variant has the sequence of any one of SEQ ID NO: 1 to 10, 13 to 17 and 132 to 134 (dicot uORF polypeptide sequences).
In a further embodiment the variant has at least 70% identity the sequence of any one of SEQ ID NO: 21 to 40 and 135 to 137 (conserved region of uORF polypeptide sequences).
In a further embodiment the variant has the sequence of any one of SEQ ID NO:21 to 40 and 135 to 137 (conserved region of uORF polypeptide sequences).
In a further embodiment the variant has at least 70% identity the sequence of any one of SEQ ID NO: 21 to 30, 33 to 37 and 135 to 137 (conserved region of dicot uORF polypeptide sequences).
In a further embodiment the variant has the sequence of any one of SEQ ID NO: 21 to 30, 33 to 37 and 135 to 137 (conserved region of dicot uORF polypeptide sequences).
In one embodiment the variant or fragment comprises a sequence with at least 70% identity to the amino sequence of SEQ ID NO: 108 (consensus motif uORF peptides).
In a further embodiment the variant or fragment comprises the amino sequence of SEQ ID NO: 108 (consensus motif uORF peptides).
Modification
In one embodiment, the modification is at least one of a deletion, an addition, or a substitution of at least one nucleotide in the sequence encoding the 5′-UTR.
In one embodiment the modification, reduced, disrupts, or prevents translation of a uORF polypeptide with the sequence of any one of SEQ ID NO: 1 to 20 and 132 to 134 (uORF peptides) or a variant thereof.
In a further embodiment the modification reduces, disrupts or destroys the activity of a uORF polypeptide with the sequence of any one of SEQ ID NO: 1 to 20 and 132 to 134 (uORF peptides) or a variant thereof.
In one embodiment the variant comprises a sequence with at least 70% identity to any one of SEQ ID NO: 1 to 20 and 132 to 134 (uORF peptides).
In a further embodiment the variant comprises a sequence with at least 70% identity to any one of SEQ ID NO: 1 to 10, 13 to 17 and 132 to 134 (dicot uORF peptides).
In a further embodiment the variant comprises a sequence with at least 70% identity to at least one of SEQ ID NO: 21 to 40 and 135 to 137 (uORF peptides conserved region).
In a further embodiment the variant comprises a sequence with at least one of SEQ ID NO: 21 to 40 and 135 to 137 (uORF peptides conserved region).
In a further embodiment the variant comprises a sequence with at least 70% identity to at least one of SEQ ID NO: 21 to 30, 33 to 37 and 135 to 137 (dicot uORF peptides conserved region).
In a further embodiment the variant comprises a sequence with at least one of SEQ ID NO: 21 to 30, 33 to 37 and 135 to 137 (dicot uORF peptides conserved region).
In one embodiment the variant or fragment comprises a sequence with at least 70% identity to the amino sequence of SEQ ID NO: 108 (consensus motif uORF peptides).
In a further embodiment the variant or fragment comprises the amino sequence of SEQ ID NO: 108 (consensus motif uORF peptides).
In a further aspect the invention provides a method for selecting a plant with at least one of:

    
    
        a) Increased GGP translation,
        b) increased GGP production,
        c) increased GGP activity, and
        d) increased ascorbate production,
    
    


the method comprising testing of a plant for the presence of a first polymorphism in a polynucleotide of the invention in the plant, or a further polymorphism linked to the first polymorphism.
In one embodiment presence of the first polymorphism, or the further polymorphism linked to the first polymorphism, Is indicative of at least one of a) to d).
In a further embodiment the further polymorphism is in linkage disequilibrium (LD) with the first polymorphism.
In a further embodiment the method includes the step of separating a selected plant from one of more non-selected plants.
In a further aspect the invention provides a plant selected by the method of the invention.
In a further aspect the invention provides a group of plants selected by the method of the invention. Preferably the group comprises at least 2, more preferably at least 3, more preferably at least 4, more preferably at least 5, more preferably at least 6, more preferably at least 7, more preferably at least 8, more preferably at least 9, more preferably at least 10, more preferably at least 11, more preferably at least 12, more preferably at least 13, more preferably at least 14, more preferably at least 15, more preferably at least 16, more preferably at least 17, more preferably at least 18, more preferably at least 19, more preferably at least 20 plants.
In a further aspect the invention provides a method of producing ascorbate, the method comprising extracting ascorbate from a plant cell or plant of the invention.
In a further aspect the invention provides an antibody raised against a polypeptide of the invention. In a further embodiment the invention provides an antibody specific for a polypeptide of the invention.
The polynucleotides, polypeptides, variants and fragments, of the invention may be derived from any species. The polynucleotides, polypeptides, variants and fragments may be naturally occurring or non-naturally occurring. The polynucleotides, variants and fragments may be recombinantly produced and also may be the products of “gene shuffling’ approaches.
In one embodiment the polynucleotide, polypeptide, variant or fragment, is derived from any plant species. The plant to be transformed or modified in the methods of the invention may be from any plant species. The plant cells to be transformed or modified in the methods of the invention may be from any plant species.
In a further embodiment the plant is from a gymnosperm plant species.
In a further embodiment the plant is from an angiosperm plant species.
In a further embodiment the plant is from a from dicotyledonuous plant species.
In a further embodiment the plant is from a fruit species selected from a group comprising but not limited to the following genera: Actinidia, Malus, Citrus, Fragaria and Vaccinium. 
Particularly preferred fruit plant species are: Actidinia deliciosa, A. chinensis, A. eriantha, A. arguta, hybrids of the four Actinidia species, Malus domestica and Malus sieboldii. 
In a further embodiment the plant is selected from the group consisting of Actinidia eriantha, Cucumis sativus, Glycine max, Solanum lycopersicum, Vitis vinifera, Arabidopsis thaliana, Malus×domesticus, Medicago truncatula, Populus trichocarpa, Actinidia arguta, Actinidia chinensis, Fragaria vulgaris, Solanum tuberosum, and Zea mays. 
In a further embodiment the plant is from a vegetable species selected from a group comprising but not limited to the following genera: Brassica, Lycopersicon and Solanum. 
Particularly preferred vegetable plant species are: Lycopersicon esculentum and Solanum tuberosum. 
In a further embodiment the plant is from monocotyledonous species.
In a further embodiment the plant is from a crop species selected from a group comprising but not limited to the following genera: Glycine, Zea, Hordeum and Oryza. 
Particularly preferred crop plant species are: Oryza sativa, Glycine max and Zea mays. 
In a further embodiment the plant is selected from the group consisting of Actinidia eriantha, Cucumis sativus, Glycine max, Solanum lycopersicum, Vitis vinifera, Arabidopsis thaliana, Malus×domesticus, Medicago truncatula, Populus trichocarpa, Actinidia arguta, Actinidia chinensis, Fragaria vulgaris, Solanum tuberosum, and Zea mays. 
DETAILED DESCRIPTION
In this specification where reference has been made to patent specifications, other external documents, or other sources of information, this is generally for the purpose of providing a context for discussing the features of the invention. Unless specifically stated otherwise, reference to such external documents is not to be construed as an admission that such documents, or such sources of information, in any jurisdiction, are prior art, or form part of the common general knowledge in the art.
The term “comprising” as used in this specification means “consisting at least in part of”. When interpreting each statement in this specification that includes the term “comprising”, features other than that or those prefaced by the term may also be present. Related terms such as “comprise” and “comprises” are to be interpreted in the same manner.
In certain embodiments the term “comprising” and related terms such as “comprise” and “comprises”, can be replaced with “consisting” and related terms, such as “consist” and “consists”.
Polynucleotides and Fragments
The term “polynucleotide(s),” as used herein, means a single or double-stranded deoxyribonucleotide or ribonucleotide polymer of any length but preferably at least 15 nucleotides, 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.
Preferably the term “polynucleotide” Includes both the specified sequence and its compliment.
A “fragment” of a polynucleotide sequence provided herein is a subsequence of contiguous nucleotides, e.g., a sequence that is at least 15 nucleotides in length.
The fragments of the invention comprise 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 invention.
The term “primer” refers to a short polynucleotide, usually having a free 3′OH group, that is hybridized to a template and used for priming polymerization of a polynucleotide complementary to the target.
Polypeptides and Fragments
The term “polypeptide”, as used herein, encompasses amino acid chains of any length but preferably at least 5 amino acids, including full-length proteins, in which amino acid residues are linked by covalent peptide bonds. Polypeptides of the present invention may be purified natural products, or may be produced partially or wholly using recombinant or synthetic techniques. The term may refer to a polypeptide, an aggregate of a polypeptide such as a dimer or other multimer, a fusion polypeptide, a polypeptide fragment, a polypeptide variant, or derivative thereof.
A “fragment” of a polypeptide is a subsequence of the polypeptide. Preferably the fragment performs a function that is required for the biological activity and/or provides three dimensional structure of the polypeptide.
The term “isolated” as applied to the polynucleotide or polypeptide sequences disclosed herein is used to refer to sequences that are removed from their natural cellular environment. In one embodiment the sequence is separated from its flanking sequences as found in nature. An isolated molecule may be obtained by any method or combination of methods including biochemical, recombinant, and synthetic techniques.
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.
A “recombinant” polypeptide sequence is produced by translation from a “recombinant” polynucleotide sequence.
The term “derived from” with respect to polynucleotides or polypeptides of the invention 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.
Variants
As used herein, the term “variant” refers to polynucleotide or polypeptide sequences different from the specifically identified sequences, wherein one or more nucleotides or amino acid residues is deleted, substituted, or added. Variants may be naturally occurring allelic variants, or non-naturally occurring variants. Variants may be from the same or from other species and may encompass homologues, paralogues and orthologues. In certain embodiments, variants of the inventive polypeptides and polypeptides possess biological activities that are the same or similar to those of the inventive polypeptides or polypeptides. The term “variant” with reference to polypeptides and polypeptides encompasses all forms of polypeptides and polypeptides as defined herein.
Polynucleotide Variants
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 invention. 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 invention.
Polynucleotide sequence identity can be determined in the following manner. The subject polynucleotide sequence is compared 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 comparing protein and nucleotide sequences”, FEMS Microbiol Lett. 174:247-250), which is publicly available from NCBI (ftp colon slash slash file transfer protocol.ncbi.nih.gov/blast/). The default parameters of bl2seq are utilized except that filtering of low complexity parts should be turned off.
The identity of polynucleotide sequences may be examined using the following unix command line parameters:

    
    
        bl2seq -i nucleotideseq1 -j nucleotideseq2 -F F -p blastn
    
    


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=”.
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 http colon slash slash world wide web.hgmp.mrc.ac.uk/Software/EMBOSS/. The European Bioinformatics Institute server also provides the facility to perform EMBOSS-needle global alignments between two sequences on line at http colon slash slash world wide web.ebi.ac.uk/emboss/align/.
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.
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 invention 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 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 1×10-6 more preferably less than 1×10-9, more preferably less than 1×10-12, more preferably less than 1×10-15, more preferably less than 1×10-18, more preferably less than 1×10-21, more preferably less than 1×10-30, more preferably less than 1×10-40, more preferably less than 1×10-50, more preferably less than 1×10-60, more preferably less than 1×10-70, more preferably less than 1×10-80, more preferably less than 1×10-90 and most preferably less than 1×10-100 when compared with any one of the specifically identified sequences.
Alternatively, variant polynucleotides of the present invention hybridize to the specified polynucleotide sequences, or complements thereof under stringent conditions.
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.
With respect to polynucleotide molecules greater than about 100 bases in length, typical stringent hybridization conditions are no more than 25 to 30° C. (for example, 10° C.) below the melting temperature (Tm) of the native duplex (see generally, Sambrook et al., Eds, 1987, Molecular Cloning, A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press; Ausubel et al., 1987, Current Protocols in Molecular Biology, Greene Publishing,). Tm for polynucleotide molecules greater than about 100 bases can be calculated by the formula Tm=81. 5+0. 41% (G+C-log (Na+). (Sambrook et al., Eds, 1987, Molecular Cloning, A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press; Bolton and McCarthy, 1962, PNAS 84:1390). Typical stringent conditions for polynucleotide of greater than 100 bases in length would be hybridization conditions such as prewashing in a solution of 6×SSC, 0.2% SDS; hybridizing at 65° C., 6×SSC, 0.2% SDS overnight; followed by two washes of 30 minutes each in 1×SSC, 0.1% SDS at 65° C. and two washes of 30 minutes each in 0.2×SSC, 0.1% SDS at 65° C.
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.
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.
Variant polynucleotides of the present invention also encompasses polynucleotides that differ from the sequences of the invention 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 invention. 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.
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 invention. 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).
Variant polynucleotides due to silent variations and conservative substitutions in the encoded polypeptide sequence may be determined using the publicly available bl2seq program from the BLAST suite of programs (version 2.2.5 [November 2002]) from NCBI (ftp colon slash slash file transfer protocol ftp.ncbi.nih.gov/blast/) via the tblastx algorithm as previously described.
The function of a variant polynucleotide of the invention as a GGP 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 it ability to alter GGP activity or ascorbate content in plants, also as describe in the Examples section herein.
The function of a variant polynucleotide of the invention as a GDP-D-Mannose epimerase 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 it ability to alter GDP-D-Mannose epimerase activity or ascorbate content in plants, also as describe in the Examples section herein.
Polypeptide Variants
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 invention. 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 invention.
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.
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 http colon slash slash 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.
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.)
Polypeptide variants of the present invention 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 line parameters:

    
    
        bl2seq -i peptideseq1 -j peptideseq2 -F F -p blastp
    
    


Variant polypeptide sequences preferably exhibit an E value of less than 1×10-6 more preferably less than 1×10-9, more preferably less than 1×10-12, more preferably less than 1×10-15, more preferably less than 1×10-18, more preferably less than 1×10-21, more preferably less than 1×10-30, more preferably less than 1×10-40, more preferably less than 1×10-50, more preferably less than 1×10-60, more preferably less than 1×10-70, more preferably less than 1×10-80, more preferably less than 1×10-90 and most preferably 1×10-100 when compared with any one of the specifically identified sequences.
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.
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; 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. 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 shown below (Table A).







TABLE A







The BLOSUM62 matrix containing 


all possible substitution scores 


[Henikoff and Henikoff, 1992].


















A
R
N
D
C
Q
E
G
H
I




















A
4
−1
−2
−2
0
−1
−1
0
−2
−1


R
−1
5
0
−2
−3
1
0
−2
0
−3


N
−2
0
6
1
−3
0
0
0
1
−3


D
−2
−2
1
6
−3
0
2
−1
−1
−3


C
0
−3
−3
−3
9
−3
−4
−3
−3
−1


Q
−1
1
0
0
−3
5
2
−2
0
−3


E
−1
0
0
2
−4
2
5
−2
0
−3


G
0
−2
0
−1
−3
−2
−2
6
−2
−4


H
−2
0
1
−1
−3
0
0
−2
8
−3


I
−1
−3
−3
−3
−1
−3
−3
−4
−3
4


L
−1
−2
−3
−4
−1
−2
−3
−4
−3
2


K
−1
2
0
−1
−3
1
1
−2
−1
−3


M
−1
−1
−2
−3
−1
0
−2
−3
−2
1


F
−2
−3
−3
−3
−2
−3
−3
−3
−1
0


P
−1
−2
−2
−1
−3
−1
−1
−2
−2
−3


S
1
−1
1
0
−1
0
0
0
−1
−2


T
0
−1
0
−1
−1
−1
−1
−2
−2
−1


W
−3
−3
−4
−4
−2
−2
−3
−2
−2
−3


Y
−2
−2
−2
−3
−2
−1
−2
−3
2
−1


V
0
−3
−3
−3
−1
−2
−2
−3
−3
3









The BLOSUM62 matrix shown is used to generate a score for each aligned amino acid pair found at the intersection of the corresponding column and row. For example, the substitution score from a glutamic acid residue (E) to an aspartic acid residue (D) is 2. The diagonal show scores for amino acids which have not changed. Most substitutions changes have a negative score. The matrix contains only whole numbers.
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 in table 1 is also used as the default matrix in BLAST searches, although not limited thereto.
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
The function of a polypeptide variant as a GGP may be assessed by the methods described in the Example section herein.
The function of a polypeptide variant as a GDP-D-Mannose epimerase may be assessed by the methods described in the Example section herein.
Constructs, Vectors and Components Thereof
The term “genetic 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 genetic 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.
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. 
The term “expression construct” refers to a genetic construct that includes the necessary elements that permit transcribing the insert polynucleotide molecule, and, optionally, translating the transcript into a polypeptide. An expression construct typically comprises in a 5′ to 3′ direction:

    
    
        a) a promoter functional in the host cell into which the construct will be transformed,
        b) the polynucleotide to be expressed, and
        c) a terminator functional in the host cell into which the construct will be transformed.
    
    


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.
“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.
In a preferred embodiment the regulatory elements include a polynucleotide sequence of the invention.
Preferably the sequence of the invention comprises a 5′-UTR sequence. Preferably the 5′-UTR sequence comprises a uORF.
The term “noncoding region” refers to untranslated sequences that are upstream of the translational start site and downstream of the translational stop site. 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.
A 5′-UTR sequence is the sequence between the transcription initiation site, and the translation start site.
The 5′-UTR sequence is an mRNA sequence encoded by the genomic DNA. However as used herein the term 5′-UTR sequence includes the genomic sequence encoding the 5′-UTR sequence, and the compliment of that genomic sequence, and the 5′-UTR mRNA sequence.
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 term “uORF” or “upstream open reading frame” is an mRNA element, defined by a start codon (any three base pair codon with at least two of the following bases in order: AUG) in the 5′-UTR, with an in frame stop codon (UAA, UAG, UGA), that is upstream (i.e. in a 5′ direction) and not overlapping with the main coding sequence.
The term “promoter” refers to cis-regulatory elements upstream of the coding region that regulate gene transcription. Promoters comprise cis-initiator elements which specify the transcription initiation site and conserved boxes such as the TATA box, and motifs that are bound by transcription factors.
A “transgene” is a polynucleotide that is taken from one organism and introduced into a different organism by transformation. The transgene may be derived from the same species or from a different species as the species of the organism into which the transgene is introduced.
A “transgenic plant” refers to a plant which contains new genetic material as a result of genetic manipulation or transformation. The new genetic material may be derived from a plant of the same species as the resulting transgenic plant or from a different species.
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′)






Read-through transcription will produce a transcript that undergoes complementary base-pairing to form a hairpin structure provided that there is a 3-5 bp spacer between the repeated regions.
The terms “to alter expression of” and “altered expression” of a polynucleotide or polypeptide of the invention, are intended to encompass the situation where genomic DNA corresponding to a polynucleotide of the invention is modified thus leading to altered expression of a polynucleotide or polypeptide of the invention. 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 and/or polypeptide produced and may also result in altered activity of a polypeptide due to alterations in the sequence of a polynucleotide and polypeptide produced.
Methods for Isolating or Producing Polynucleotides
The polynucleotide molecules of the invention can be isolated by using a variety of techniques known to those of ordinary skill in the art. By way of example, such polypeptides can be isolated through use of the polymerase chain reaction (PCR) described in Mullis et al., Eds. 1994 The Polymerase Chain Reaction, Birkhauser, incorporated herein by reference. The polypeptides of the invention can be amplified using primers, as defined herein, derived from the polynucleotide sequences of the invention.
Further methods for isolating polynucleotides of the invention 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. 0×SSC, 0. 5% sodium dodecyl sulfate, 1×Denhardt's solution; washing (three washes of twenty minutes each at 55° C.) in 1. 0×SSC, 1% (w/v) sodium dodecyl sulfate, and optionally one wash (for twenty minutes) in 0. 5×SSC, 1% (w/v) sodium dodecyl sulfate, at 60° C. An optional further wash (for twenty minutes) can be conducted under conditions of 0. 1×SSC, 1% (w/v) sodium dodecyl sulfate, at 60° C.
The polynucleotide fragments of the invention may be produced by techniques well-known in the art such as restriction endonuclease digestion, oligonucleotide synthesis and PCR amplification.
A partial polynucleotide sequence may be used, in methods well-known in the art to identify the corresponding full length polynucleotide sequence. Such methods include PCR-based methods, 5′RACE (Frohman M A, 1993, Methods Enzymol. 218: 340-56) and hybridization-based method, computer/database-based methods. Further, by way of example, inverse PCR permits acquisition of unknown sequences, flanking the polynucleotide sequences disclosed herein, starting with primers based on a known region (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 (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press, 1987).
It may be beneficial, when producing a transgenic 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 transgenic organisms. Additionally when down-regulation of a gene is the desired result, it may be necessary to utilise 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 orthologues of a particular gene in several different plant species.
Variants (including orthologues) may be identified by the methods described.
Methods for Identifying Variants
Physical Methods
Variant polypeptides may be identified using PCR-based methods (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 invention by PCR, may be based on a sequence encoding a conserved region of the corresponding amino acid sequence.
Alternatively library screening methods, well known to those skilled in the art, may be employed (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.
Polypeptide variants may also be identified by physical methods, for example by screening expression libraries using antibodies raised against polypeptides of the invention (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.
Computer Based Methods
The variant sequences of the invention, including both polynucleotide and polypeptide variants, may also be identified by computer-based methods well-known to those skilled in the art, 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.
An exemplary family of programs useful for identifying variants in sequence databases is the BLAST suite of programs (version 2.2.5 [November 2002]) including BLASTN, BLASTP, BLASTX, tBLASTN and tBLASTX, which are publicly available from (ftp colon slash slash file transfer protocol ftp.ncbi.nih.gov/blast/) or from the National Center for Biotechnology Information (NCBI), National Library of Medicine, Building 38A, Room 8N805, Bethesda, Md. 20894 USA. The NCBI server also provides the facility to use the programs to screen a number of publicly available sequence databases. BLASTN compares a nucleotide query sequence against a nucleotide sequence database. BLASTP compares an amino acid query sequence against a protein sequence database. BLASTX compares a nucleotide query sequence translated in all reading frames against a protein sequence database. tBLASTN compares a protein query sequence against a nucleotide sequence database dynamically translated in all reading frames. tBLASTX compares the six-frame translations of a nucleotide query sequence against the six-frame translations of a nucleotide sequence database. The BLAST programs may be used with default parameters or the parameters may be altered as required to refine the screen.
The use of the BLAST family of algorithms, Including BLASTN, BLASTP, and BLASTX, is described in the publication of Altschul et al., Nucleic Acids Res. 25: 3389-3402, 1997.
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. 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.
Multiple sequence alignments of a group of related sequences can be carried out with CLUSTALW (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, http colon slash slash world wide web -igbmc.u-strasbg.fr/BioInfo/ClustalW/Top.html) or T-COFFEE (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. (Feng and Doolittle, 1987, J. Mol. Evol. 25, 351).
Pattern recognition software applications are available for finding motifs or signature sequences. For example, MEME (Multiple Em for Motif Elicitation) finds motifs and signature sequences in a set of sequences, and MAST (Motif Alignment and Search Tool) uses these motifs to identify similar or the same motifs in query sequences. The 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 (Bairoch and Bucher, 1994, Nucleic Acids Res. 22, 3583; 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
The polypeptides of the invention, including variant polypeptides, may be prepared using peptide synthesis methods well known in the art such as direct peptide synthesis using solid phase techniques (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.
The polypeptides and variant polypeptides of the invention may also be purified from natural sources using a variety of techniques that are well known in the art (e.g. Deutscher, 1990, Ed, Methods in Enzymology, Vol. 182, Guide to Protein Purification,).
Alternatively the polypeptides and variant polypeptides of the invention may be expressed recombinantly in suitable host cells and separated from the cells as discussed below.
Methods for Modifying Sequences
Methods for modifying the sequence of proteins, or the polynucleotide sequences encoding them, are well known to those skilled in the art. The sequence of a protein may be conveniently be modified by altering/modifying the sequence encoding the protein and expressing the modified protein. Approaches such as site-directed mutagenesis may be applied to modify existing polynucleotide sequences. Alternatively restriction endonucleases may be used to excise parts of existing sequences. Altered polynucleotide sequences may also be conveniently synthesised in a modified form.
Methods for Producing Constructs and Vectors
The genetic constructs of the present invention comprise one or more polynucleotide sequences of the invention and/or polynucleotides encoding polypeptides of the invention, and may be useful for transforming, for example, bacterial, fungal, Insect, mammalian or plant organisms. The genetic constructs of the invention are intended to include expression constructs as herein defined.
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; Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing, 1987).
Methods for Producing Host Cells Comprising Polynucleotides, Constructs or Vectors
The invention provides a host cell which comprises a genetic construct or vector of the invention. Host cells may be derived from, for example, bacterial, fungal, insect, mammalian or plant organisms.
Host cells comprising genetic constructs, such as expression constructs, of the invention are useful in methods well known in the art (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 well known in the art (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
The invention further provides plant cells which comprise 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.
Methods for transforming plant cells, plants and portions thereof with polypeptides are 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
A number of plant transformation strategies are available (e.g. Birch, 1997, Ann Rev Plant Phys Plant Mol Biol, 48, 297, Hellens R P, et al (2000) Plant Mol Biol 42: 819-32, Hellens R et al (2005) Plant Meth 1: 13). For example, strategies may be designed to increase expression of a polynucleotide/polypeptide in a plant cell, organ and/or at a particular developmental stage where/when it is normally expressed or to ectopically express a polynucleotide/polypeptide in a cell, tissue, organ and/or at a particular developmental stage which/when it is not normally expressed. The expressed polynucleotide/polypeptide may be derived from the plant species to be transformed or may be derived from a different plant species.
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 transgenic 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 invention 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. Those 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 invention. 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.
Selectable markers commonly used in plant transformation include the neomycin phophotransferase II gene (NPT II) which confers kanamycin resistance, the aadA gene, which confers spectinomycin and streptomycin resistance, the phosphinothricin acetyl transferase (bar gene) for Ignite (AgrEvo) and Basta (Hoechst) resistance, and the hygromycin phosphotransferase gene (hpt) for hygromycin resistance.
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, GUS, 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.
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 invention may include an antisense copy of a polynucleotide of the invention. 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






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 an miRNA (Llave et al., 2002, Science 297, 2053). Use of such small antisense RNA corresponding to polynucleotide of the invention is expressly contemplated.
The term 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 (e.g. Napoli et al., 1990, Plant Cell 2, 279; de Carvalho Niebel et al., 1995, Plant Cell, 7, 347). In some cases sense suppression may involve over-expression of the whole or a partial coding sequence but may also involve expression of non-coding region of the gene, such as an intron or a 5′ or 3′ untranslated region (UTR). Chimeric partial sense constructs can be used to coordinately silence multiple genes (Abbott et al., 2002, Plant Physiol. 128(3): 844-53; Jones et al., 1998, Planta 204: 499-505). The use of such sense suppression strategies to silence the expression of a polynucleotide of the invention is also contemplated.
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.
Other gene silencing strategies include dominant negative approaches and the use of ribozyme constructs (McIntyre, 1996, Transgenic Res, 5, 257).
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.
The following are representative publications disclosing genetic transformation protocols that can be used to genetically transform the following plant species: Rice (Alam et al., 1999, Plant Cell Rep. 18, 572); apple (Yao et al., 1995, Plant Cell Reports 14, 407-412); maize (U.S. Pat. Nos. 5,177,010 and 5,981,840); wheat (Ortiz et al., 1996, Plant Cell Rep. 15, 1996, 877); tomato (U.S. Pat. No. 5,159,135); potato (Kumar et al., 1996 Plant J. 9, 821); cassava (Li et al., 1996 Nat. Biotechnology 14, 736); lettuce (Michelmore et al., 1987, Plant Cell Rep. 6, 439); tobacco (Horsch et al., 1985, Science 227, 1229); cotton (U.S. Pat. Nos. 5,846,797 and 5,004,863); grasses (U.S. Pat. Nos. 5,187,073 and 6,020,539); peppermint (Niu et al., 1998, Plant Cell Rep. 17, 165); citrus plants (Pena et al., 1995, Plant Sci. 104, 183); caraway (Krens et al., 1997, Plant Cell Rep, 17, 39); banana (U.S. Pat. No. 5,792,935); soybean (U.S. Pat. Nos. 5,416,011; 5,569,834; 5,824,877; 5,563,04455 and 5,968,830); pineapple (U.S. Pat. No. 5,952,543); poplar (U.S. Pat. No. 4,795,855); monocots in general (U.S. Pat. Nos. 5,591,616 and 6,037,522); brassica (U.S. Pat. Nos. 5,188,958; 5,463,174 and 5,750,871); cereals (U.S. Pat. No. 6,074,877); pear (Matsuda et al., 2005, Plant Cell Rep. 24(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 invention. 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 invention. 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 invention 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 invention. Use of each of the above approaches in alteration of expression of a nucleotide and/or polypeptide of the invention is specifically contemplated.
Methods for Modifying Endogenous DNA Sequences in Plant
Methods for modifying endogenous genomic DNA sequences in plants are known to those skilled in the art. Such methods may involve the use of sequence-specific nucleases that generate targeted double-stranded DNA breaks in genes of interest. 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).
In certain embodiments of the invention, one of these technologies (e.g. TALENs or a Zinc finger nuclease) can be used to modify one or more base pairs in the uORF in order to disable it, so it is no longer translatable.
In one embodiment the first base pair of the ACG start codon is changed to TCG to accomplish this. This would inactivate the ascorbate feed back regulation of GGP translation and allow increases of ascorbate concentration in the plant.
Alternatively, a codon for a highly conserved amino acid in the uORF can be changed to stop the uORF from functioning in down regulating translation of the GGP at high ascorbate. For example a His residue in the conserved region of the uORF can be changed to a Leu.
In a further embodiment an early base pair in the uORF is altered to introduce a stop codon, and cause early termination of the uORF which stops ascorbate feedback regulation of the translation of GGP.
Those skilled in the art will thus appreciate that there are numerous ways in which the uORF can be disrupted to remove negative regulation by ascorbate and to increase ascorbate production. Any such method is included within the scope of the invention.
Plants
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 invention 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 invention, provided they maintain the transgene or modification of the invention.
Methods for Extracting and Measuring Ascorbate from Plants
Methods are also provided for the production of ascorbate by extraction of ascorbate from a plant of the invention. Ascorbate may be extracted from plants as follows:
Frozen tissue samples are ground to a fine powder in a Cryomill at liquid nitrogen temperature. About 200 mg of frozen powdered tissue is then suspended in 5 volumes of 7% metaphosphoric acid containing 2 mM TCEP (Pierce), vortexed for 20 sec and incubated in a heating block for 2 h at 40° C. TCEP Is used in the extraction solution, because it is more effective reducing agent under acidic conditions than DTT, ensuring that all of vitamin C is in the ascorbic acid reduced form. The extract is centrifuged at 4° C. and twenty μL of the supernatant is injected into a Rocket Column And eluted using two solvents A (0.28% o-phosphoric acid, 0.1 mM EDTA and 0.25% methanol) and B (acetonitrile). Ascorbate and other compounds were eluted using a 5-min gradient to 90% B. Standards were run with every batch or 20 samples processed. Ascorbate was calculated from the area under the absorption at 240 nm curve at  ̃1 minute of elution.
This method may be up-scaled for larger scale ascorbate extraction using approaches well-known to those skilled in the art.
This invention 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 invention relates, such known equivalents are deemed to be incorporated herein as if individually set forth.



BRIEF DESCRIPTION OF THE DRAWINGS
The present invention will be better understood with reference to the accompanying drawings in which:
FIG. 1 shows the effect of ascorbate on reporter gene activity driven by the GGP promoter or by a control promoter. A, LUC/REN ratio as a function of leaf ascorbate concentration for GGP promoter B, LUC/REN ratio as a function of leaf ascorbate concentration for TT8 promoter. ▴low ascorbate (KO) leaves, ▪ control leaves, ◯ high ascorbate (GGP) leaves.
FIG. 2 shows the effect of ascorbate on the GGP promoter strength in various changed forms of the ACG uORFs present in the 5′-UTR of the GGP gene (FIG. S7B). A. LUC/REN ratios, B. Ascorbate concentrations in the same treatments. VTC2 is the wild type 5′-UTR and promoter of GGP. ACG1 had the initiating ACG of ACG1 changed to a TCG to no longer make it a start codon. ACG2 had the first His in a highly conserved region of the uORF (FIG. S5, S7B) changed to a Leu. GGP was added to manipulate the ascorbate concentrations. In A, the treatment VTC2+GGP had a significantly lower LUC/REN value than all other treatments (p=0.001), which were not significantly different. In B, The addition of GGP to the leaves increased ascorbate significantly (p=0.001), but differences between treatments were not significant at either high or low ascorbate. The bars are standard errors, n=4.
FIG. 3 shows the results of testing whether the non canonical uORF in the 5′-UTR of GGP acts in a cis or trans manner. VTC2 wt refers to the ascorbate repressed promoter and 5′-UTR, ACG1 refers to the mutated ACG codon of the uORF (non-responding), uORF is addition of the uORF driven by a 35S promoter and GGP is addition of the GGP coding sequence also driven by 35S to raise ascorbate.
FIG. 4 shows a comparison of the response of the reporter gene to ascorbate for GGP full promoter constructs from kiwifruit and Arabidopsis. GGP was used to raise ascorbate levels as in FIG. 1. ▪ Arabidopsis GGP (promoter and 5′-UTR), a standard kiwifruit GGP (promoter and 5′-UTR). Other details are found in the methods section.
FIG. 5 shows the effect of high ascorbate concentration of the amount of LUC protein as measured by an anti-LUC antibody in transiently transformed tobacco leaves driven by either the GGP promoter or the control TT8 promoter. Experiments were carried out as described in FIG. 1. The ascorbate concentration in the +319998 (GGP) lanes was 55 to 63 mg/100 g FW while in the lanes without GGP it was 21 mg/100 g FW. Other details are found in the methods section.
FIG. 6 shows the effect of manipulating ascorbate concentration independent of the GGP enzyme used to increase the leaf ascorbate by using GME to further enhance ascorbate at variable levels of GGP. A the effect of different combinations of GGP and GME on the LUC/REN ratio, B the effect of different combinations of GGP and GME on the ascorbate concentration. In A and B columns with the same letters are not significantly different at the 5% level. C the relationship between the individual leaf LUC/REN ratios and leaf ascorbate. Other details are found in the methods section. In C, ▪, control; •, GME; ♦, 0.33×GGP; ▴, 1×GGP; □, 0.33×GGP+1×GME; ▾, 1×GGP+GME. 1 and 0.33 refer to relative amounts of GGP and GME Injected into the leaf.
FIG. 7 shows an alignment of 5′-UTR sequences from a range of dicotyledonous species. Alignment was done using Clustal X as executed by Vector NTI. The suffix letters code species that the 5′-UTR sequences came from: Aa is Actinidia arguta, Ae is A. eriantha and Ac is A. Chinensis, MXd is Malus×domestica, Fv is Fragaria vesca, SI is Solanum lycospersicum, St, is Solanum tuberosum, Vv is Vitis vinifera, Gm is Glycine max, Mt is Medicago truncatula, Pt is Populus trichocarpa, Cs is Cucumis sativus. The prefix before each name is the GenBank accession number. The two genomic sequences came from published resources (T. P. G. S. Consortium, 2011 Nature 475, 189); Velasco et al., 2010 Nat Genet 42, 833). The short uORF beginning with ATG is in bold while the highly conserved non-canonical uORF starting with ACG is underlined in bold (ACG1). The bracketed number of each sequence refers to the total length of its 5′-UTR. The lower schematic shows the highly conserved region of the 5′-UTR and the three deletions used in testing their functions. The colored regions are the highly conserved regions that were deleted either individually (deletion 2 and 3 from left to right) or in their entirety (deletion 1). The yellow motif is a small conserved uORF that starts with ATG (ATG1), while the orange motifs are two non-canonical uORFs that start with ACG (ACG1, bottom, ACG2 top). All three uORFs are conserved between species although ACG2 is less conserved at the protein level ( ̃40 to  ̃60% Identical between families) compared to ACG1 ( ̃60 to  ̃80%)
FIG. 8 shows the effect of deletions in the 5′-UTR region of the GGP gene on the down regulation of the promoter by ascorbate. Deletions are as labeled FIG. S4. The bottom graph shows the corresponding ascorbate concentrations in the same leaf as the LUC/REN ratios were measured. Bars represent standard errors.
FIG. 9 shows a time course of the effect of the presence or absence of the 5′-UTR in front of the GGP CDS on leaf ascorbate. Both constructs were driven by the 35S promoter. Other details are found in the methods section. U, control (P19 only); •, -5′-UTR GGP; ▴, +5′-UTR GGP; ▾, -5′-UTR GGP+GME; ♦, +5′-UTR GGP; ◯, GME.
FIG. 10 shows an alignment of the predicted peptide sequences of the ACG uORF in the 5′-UTR of GGP over a wide range of species. Alignment was done using Clustal X (Thompson et al., Nucleic Acids Res 25, 4876 (1997).) as executed by Vector NTI. The suffixes are as listed in FIG. 7 with the addition of plants outside the dicotyledonous plants. Cr, Chlamydomonas rheinhardtii; Pp, Physcomitrella patens; Ps, Picea sitchensis; Sm, Selaginella moellendorffi; Zm, Zea mays. The prefix before each name is the GenBank accession number. Also shown is the consensus sequence for the whole uORF. Further highlighted underlined is a highly conserved consensus motif NPSPHGGRGALPSEGGSPSDLLFLAGGG (SEQ ID NO: 108).
FIG. 11 shows the effect of the small ATG uORF on the response of reporter gene activity driven by the GGP promoter and 5′-UTR to ascorbate concentration. The start codon in the small 30 bp ORF in the 5′-UTR was inactivated by converting the start ATG to a TTG. □, inactivated uORF; ▪, uORF start codon present. In this experiment and in a replicate experiment the reporter gene activity from the 5′-UTR without the start codon was more highly expressed at both low and high ascorbate than the gene with the uORF Intact. The solid lines are polynomial fits to the data. Other details are found in the methods section.
FIG. 12 shows the reduction state of ascorbate as a function of total leaf ascorbate. The data comes from three separate experiments. Ascorbate was manipulated using GGP and measured with and without reducing agent. When ascorbate concentrations were classified into low (27±0.7 mg/100 g FW), medium (59±2.7) and high (179±17.5) (mean±standard error) the values of redox potential were significantly different and decreased with increasing ascorbate from 74.8±1.0, 70.1±1.2 and 65.6±0.8 (p=0.05). The solid line is a hyperbolic fit to the % reduced data, and the dotted line is the linear fit to the redox potential.
FIG. 13 shows the effect of ascorbate on reporter gene activity driven by the GGP promoter or by a control promoter. A, LUC activity versus REN activity for the GGP promoter; B, LUC activity versus REN activity for TT8 promoter. The three slopes in A are significantly different (p<0.001) while only the low ascorbate slope in B is significantly lower (p<0.001) than the other two lines which do not differ in slope significantly).
FIG. 14 shows a schematic illustrating the positioning of the pGreen 0800 based reporter gene construct designed to test the 5′UTR from GGP from different species.
FIG. 15 shows sequences of 5′UTRs for GGP genes from potato, tomato and apple. The uORF is shown in bold.
FIG. 16 shows a plot of LUC values versus REN at high (+319998) and low ascorbate for the tomato 5′UTR.
FIG. 17 shows a plot of LUC values versus REN at high (+319998) and low ascorbate for the potato 5′UTR.
FIG. 18 shows a plot of LUC values versus REN at high (+319998) and low ascorbate for the apple 5′UTR.
FIG. 19 shows a plot of LUC values versus REN at high (+319998) and low ascorbate for a control promoter-5′UTR-LUC construct. Note that the slope is lower at low ascorbate than at high ascorbate
FIG. 20 shows a plot of LUC values versus REN at high (+319998) and low ascorbate for the wild type kiwifruit GGP promoter-5′UTR-LUC construct.



EXAMPLES
The invention will now be illustrated with reference to the following non-limiting example.
It is not the intention to limit the scope of the invention to the abovementioned example only. As would be appreciated by a skilled person in the art, many variations are possible without departing from the scope of the invention.
Example 1
Elucidation of the Control of GGP Expression
Summary
Ascorbate (vitamin C) Is an essential antioxidant and enzyme co-factor in both plants and animals. Ascorbate concentration is tightly regulated in plants, partly to respond to stress. The applicants have shown that ascorbate levels are controlled via the post-transcriptional repression of GDP-L-galactose phosphorylase (GGP), the rate-limiting enzyme in the ascorbate biosynthesis pathway. This regulation requires the translation of a Cis-acting uORF (upstream open reading frame), which initiates from a non-canonical start codon and represses the translation of the downstream GGP ORF under high ascorbate. Removal of this uORF allows plants to produce high levels of ascorbate. The uORF is present in the GGP gene from both lower and higher plants indicating it is an ancient mechanism to control ascorbate levels.
Ascorbate (vitamin C) is an essential biochemical found in most living organisms with a central role of controlling the redox potential of the cell (Asensi-Fabado et al., 2010, Trends Plant Sci. 15, 582; Foyer et al., Plant Physiol. 155, 2 (2011)) as well as serving as an enzyme cofactor (Mandl et al., 2009, Br. J. Pharmacol. 157, 1097). Ascorbate concentrations are regulated according to demand; for example, leaf ascorbate concentrations increase under high light intensities when the need for ascorbate is greatest (Bartoli et al., 2006, J. Exp. Bot. 57, 1621; Gatzek et al., 2002, Plant J. 30, 541). However the mechanism by which ascorbate biosynthesis is regulated is not known. The applicants have shown previously that the enzyme GDP L-Galactose phosphorylase (GGP) is central to determining ascorbate in plants (Bulley et al., 2012, Plant Biotechnol J 10, 390; Bulley et al., 2009, J. Exp. Bot. 60, 765), suggesting it may serve a regulatory role.
Results and Discussion
To investigate if the GGP gene is regulated by ascorbate levels, the applicants fused the kiwifruit GGP promoter with its 5′-UTR (SEQ ID NO: 101) to the luciferase (LUC) reporter gene and expressed the construct transiently in Nicotiana benthamiana leaves. (Hellens et al., 2005, Plant Methods 1, 13.). The applicants manipulated ascorbate by also expressing just the coding sequence of GGP under a strong constitutive promoter. A doubling of ascorbate concentration from  ̃2 mM (20 mg/100 g FW) to 4 mM was sufficient to reduce the relative LUC activity by 50%, and when ascorbate was increased close to 10 mM, >90% of LUC activity was abolished (FIG. 1). Similarly, the Arabidopsis GGP (VTC2; At4g26850) promoter and 5′-UTR (SEQ ID NO: 102) also conferred ascorbate dependent repression on a LUC reporter gene (FIG. 4).
In contrast, high ascorbate had no affect on relative LUC activity using a control promoter for a gene unrelated to ascorbate metabolism (T-8: an Arabidopsis bHLH transcription factor controlling polyphenolic biosynthesis) (FIG. 1). Additional controls demonstrate that this regulation was specific to GGP sequences, independent of the level of expression of the transgenes and was reflected in LUC protein changes (Tables 1-3, FIG. 5).







TABLE 1







The absolute value of LUC or REN does not significantly influence the LUC/REN


ratio. The relationship between LUC and REN was linear over a 200 fold range.


The subscript P refers to the whole promoter including any 5′-UTR from


that gene. Other details are found in the methods section.














Slope of

Mean LUC
Mean REN




relationship

Luminescent
Luminescent



Agrobacterium
between

units +/−
units +/−



culture
LUC and

Standard
Standard


Construct
dilution as a %
REN
LUC/REN
Error
Error















GGPP
100
0.07
0.11
7442 ± 785
67908 ± 7429


GGPP
10
0.12
0.11
2777 ± 277
25001 ± 1684


TT8P
100
2.66
2.96
122573 ± 15748
41478 ± 4557


TT8P
10
2.64
2.91
25560 ± 2255
8786 ± 710


TT8P
8
2.97
2.69
23621 ± 2672
8785 ± 552


TT8P
5
1.56
2.24
10176 ± 547 
4537 ± 210


TT8P
3
1.72
2.40
6412 ± 450
2676 ± 172


TT8P
1
2.39
2.49
1739 ± 274
 699 ± 101


TT8P
0.5
2.53
2.79
1482 ± 175
531 ± 43
















TABLE 2







Comparison of the effect of ascorbate on a range of control genes. 














ascorbate








mg/100 g




Slope



FW

Std


of



Ascorbate

error


LUC



mg/100
LUC/REN
Std
N

REN


Treatment
g FW
LUC/REN
error
N

slope
















TT8P
22
1.95
0.24
6
a
1.89


TT8P + GGP
78
4.09
0.28
12
b
4.71


EF1aP
23
0.767
0.086
6
a
0.780


ET1aP + GGP
82
0.733
0.065
12
a
0.775


ACt2P
24
0.385
0.042
6
a
0.327


ACt2P + GGP
82
0.550
0.074
12
a
0.586


ACt7P
22
0.815
0.037
6
a
0.757


ACt7P + GGP
78
0.843
0.036
12
a
0.834





+ GGP refers to the cotransformation of the CDS of the GGP from kiwifruit (Genbank accession FG528585) under the control of the 35S promoter in order to raise ascorbate. The slope of LUC REN is the slope of the plot of LUC values against REN values forced through the origin as a comparison to the LUC/REN ratios. N is the number of independent LUC/REN ratios measured. The subscript P refers to the whole promoter including any 51-UTR from that gene. Within a block of two rows, LUC/REN means with the same letter do not significantly differ at p < 0.01. Other details are found in the methods section.













TABLE 3





Effect of adding a control gene to the gene 


used to manipulate ascorbate concentration.























GGPP
22
0.251
0.027
12
a
0.230



GGPP + GGP
65
0.028
0.008
12
b
0.022



GGPP + FG429343
22
0.226
0.044
12
a
0.183



TT8P
25
3.87
0.44
12
a
3.12



TT8P + GGP
50
2.42
0.21
12
b
2.25



TT8P + FG429343
25
2.73
0.16
12
b
3.06







To check that expression of an extra gene (GGP) to increase ascorbate did not directly affect the LUC/REN ratio, we substituted another control gene, GenBank accession FG429343, an Actinidia deliciosa methyl transferase which had no direct affect of the ascorbate concentration. The subscript P refers to the whole promoter. Within a block of three rows, LUC/REN means with the same letter do not significantly differ at p < 0.01. Other details are found in the methods section.






In the experiments described, leaf ascorbate was manipulated through changes in expression of the GGP coding sequence. In order to separate the effects of ascorbate from possible effect of the GGP protein (Müller-Moulé, P., 2008, Plant Mol. Biol. 68, 31), the applicants expressed both GGP and GME separately and together. The applicants have previously shown (Bulley et al., 2009, J. Exp. Bot. 60, 765.) that GGP expressed alone in tobacco has a moderate effect, GME very little effect, whereas when expressed together there is a strong synergistic stimulation of ascorbate concentration. Thus by varying the ratios of these two genes, ascorbate can be manipulated independently of the amount of GGP protein (FIG. 6). The response of the ratio to ascorbate followed a smooth curve in spite of different levels of GGP protein associated with different ascorbate concentrations (FIG. 6), showing ascorbate or a related metabolite is the factor reducing the LUC activity.
To test whether the effect of ascorbate was mediated by the untranscribed promoter or the 5′-UTR the applicants undertook two experiments. Firstly the applicants swapped the 5′-UTR regions between the GGP TT8 promoters. We transiently expressed these in leaves and measured the relative LUC activity. Increased ascorbate reduced the LUC activity only when the 5′-UTR from GGP was present. (Table 4). Secondly the applicants deleted two regions within the 5′-UTR that were especially strongly conserved at the DNA level between species. The first was from  ̃387 to  ̃432 bp and the second between  ̃514b and  ̃597 bp (FIG. 7) with the rest of the GGP promoter intact, and tested them using the reporter assay. All deletions caused the loss of the ability of ascorbate to down regulate the reporter gene expression (FIG. 8). These experiments show that the 5′-UTR is necessary and sufficient for down regulation by ascorbate.













TABLE 4






ascorbate




Slope



mg/100 g
LUC/
Std


of LUC


Treatment
FW
REN
Error
N

REN





















TT8P′ − GGPUTR
22
0.057
0.008
12
a
0.044


TT8P′ − GGPUTR + GGP
66
0.007
0.001
12
b
0.0063


TT8P′ − GGPUTR + FG429343
24
0.041
0.002
12
a
0.041


GGPP′ − TT8UTR
18
0.861
0.112
12
a
0.753


GGPP′ − TT8UTR + GGP
72
0.479
0.027
12
b
0.441


GGPP′ − TT8UTR + FG429343
22
0.553
0.085
12
b
0.492





The ascorbate down regulation of the GGP promoter is expressed through the 5′-UTR region of the gene. Subscript UTR refers to the 5′-UTR of the gene and subscript P′ refers to just the untranscribed promoter of the respective gene. GGP refers to the cotransformation of the CDS of GGP under the control of the 355 promoter in order to raise ascorbate levels while FG429343, is a methyl transferase control gene. The slope of LUC REN is the slope of the plot of LUC values against REN values forced through the origin. N is the number of independent LUC/REN ratios measured. Within a block of three rows, LUC/REN means with the same letter do not significantly differ at p < 0.01. Other details are found in the methods section.






To investigate whether the ascorbate control is at the transcriptional or post-transcriptional level, the applicants measured transcript levels of the reporter gene construct. Our data show little effect of ascorbate on the levels of LUC mRNA (Table 5), indicating that ascorbate, directly or indirectly, acts through the 5′-UTR to control the translation of GGP.







TABLE 5







Effect of ascorbate level on the RNA levels for LUC driven


either by the GGP promoter or TT8, a control gene. Gene


expression was measured relative to the expression of REN


in the same RNA preparation. Values are the mean of three


biological replicates, each involving three combined leaves.


Standard errors are shown brackets. Within each promoter


pair, there was no significant difference in gene expression


or the LUC activity for the TT8 promoter. The change in LUC


activity for the GGP promoter was significant (p < 0.001) as


were the changes in ascorbate for both promoters (p < 0.003).













Ascorbate


Promoter/Treatment
Gene expression
LUC/REN
mg/100 g FW





GGP/low ascorbate
6.0E−02 ± 8.8E−03
1.08 ± 0.05
 25.3 ± 0.93


GGP/high ascorbate
5.3E−02 ± 7.4E−03
0.15 ± 0.03
92.4 ± 7.1


TT8/low ascorbate
8.5E−01 ± 5.7E−02
19.9 ± 0.59
26.1 ± 6.3


TT8/high ascorbate
9.3E−01 ± 1.1E−01
16.3 ± 0.83
84.6 ± 4.7









To verify that the 5′-UTR acts directly to affect leaf ascorbate concentrations, we constructed a 35S driven GGP coding sequence with and without the GGP 5′-UTR in front of the coding sequence. Both constructs enhanced leaf ascorbate in the transient system, but the construct without the 5′-UTR had about 30% more ascorbate than the construct with the 5′-UTR (FIG. 9). Furthermore, co-infiltrating GME into the leaf so to drive the ascorbate even higher than GGP alone (Bulley et al., 2009, J. Exp. Bot. 60, 765.) resulted in over two-fold higher ascorbate in the construct without the 5′-UTR than the construct containing the 5′-UTR. Thus, in high ascorbate conditions the GGP 5′-UTR limits both GGP production and ascorbate synthesis. Removal of this regulation provides a way of generate plants with high ascorbate levels.
Given that the effect of ascorbate was mediated through the 5′-UTR region of GGP, we examined the properties of the 5′-UTR. GGP is unusual in having a long 5′-UTR, over 500 bp long in many species with strongly conserved elements (FIG. 7). Aligned GGP 5′-UTRs from different species including an algae and two mosses revealed the presence of a highly conserved uORF with the potential to encode a 60 to 65 amino acid peptide (FIG. 10). Interestingly, for this peptide to be made, translation would need to initiate at a non-canonical ACG initiation codon. A few examples of non-canonical translation initiation have been described (Ivanov et al., 2008, Proc. Natl. Acad. Sci. USA 105, 10079). Efficient translation requires Kozak sequences which is the case for this ORF (FIG. 8). To test if this uORF is required for ascorbate dependent regulation of the GGP gene, the applicants mutated the potential ACG initiation codon to TCG. LUC activity from the mutated construct remained high, even in the presence of high ascorbate (FIG. 2). To further examine the requirement of the uORF, the applicants mutated a highly conserved His (CGG codon) at residue 36 to Leu (CTG). Again, this abolished ascorbate dependent regulation (FIG. 2). Mutating an internal ATG codon with potential to encode a short uORF of 10 amino acids, increased the relative LUC activity by over two fold, probably because it removed a competing start codon, but did not change the relative sensitivity of the promoter to ascorbate concentration (FIG. 11).
The applicants then used the ACG uORF mutant that did not respond to ascorbate to test whether the predicted uORF worked in a Cis or Trans configuration. The applicants tested whether expressing the ACG uORF separately could recover ascorbate repression of LUC activity in this mutated vector. In FIG. 3 the applicants show that the presence of the ACG uORF had no effect on any treatment and did not complement the mutant form of the ACG uORF. This is consistent with the uORF working in a Cis conformation with the GGP CDS.
In this work the applicants provide evidence that ascorbate, or a precursor of ascorbate, interacts either directly or indirectly through an intermediate with a peptide produced by a non canonical uORF in the 5′-UTR of GGP, the key control gene of ascorbate biosynthesis, resulting in inhibition of the translation of the GGP enzyme. Reports of the control of protein expression in eukaryotes by products of a biosynthesis pathway are rare. Often gene expression is controlled by signaling cascades via a separate receptor to a transcription factor or through posttranslational modification of the target proteins (Smeekens et al., 2010, Curr. Opin. Plant Biol. 13, 273). While it has been reported that 5′-UTR sequences are important in controlling protein expression (Hulzink et al., 2003, Plant Physiol. 132, 75), reports on the control by a small molecule of gene expression through the 5′-UTR of the mRNA In eukaryotes are uncommon (Rahmani et al., 2009, Plant Physiol. 150, 1356) and control through a non-canonical start codon uORF are extremely rare.
A simple model of action may be that the ACG uORF is translated but in the presence of high ascorbate, the ribosome is stalled on the uORF. At low ascorbate, the translation terminates at the stop codon and immediately restarts downstream at the start ATG of GGP. There is no obvious Kozak sequence associated with the GGP primary start codon, but a reasonably strong Kozak sequence associated with ACG1. This effectively primes the ribosome at high ascorbate on the GGP mRNA ready for translation to respond rapidly to any reduction in ascorbate due to stress.
It appears that another factor may be required for the action of this feed-back loop. This is because the ascorbate regulation of the 5′-UTR for GGP from A. eriantha, a kiwifruit species with very high fruit ascorbate (Bulley et al., 2009, J. Exp. Bot. 60, 765.), functions in N. benthamiana. For A. eriantha to have high ascorbate suggests a mutation has occurred disrupting the ascorbate feedback of GGP translation in A. eriantha. However, that control of the A. eriantha GGP by ascorbate is functional in N. benthamiana suggests a factor mediating between ascorbate and the ACG1 uORF Is functional in N. benthamiana. This factor is likely to be a protein.
There are two types of uORF (Tran et al., 2008. BMC Genomics 9, 361): sequence-independent uORFs, where translation of the uORF influences the reinitiation efficiency of a downstream ORF and thus affects overall translation (Calvo et al., 2009. Proc. Natl. Acad. Sci. USA 106, 7507) but the uORF-encoded peptide sequence is not important (the short ATG 10 amino acid uORF In the GGP 5′-UTR appears to fit into the class (FIG. 7), and sequence-dependent ORFs, where the nascent uORF peptide causes ribosome stalling during translational elongation and termination. The fact that the GGP uORF encodes a highly conserved peptide over a wide range of plant taxonomies and that ascorbate repression is abolished by a single amino acid mutation in the uORF, indicates the latter type. Two examples in plants, polyamine and sucrose regulation (Rahmani et al., 2009, Plant Physiol. 150, 1356; Gong and Pua, 2005, Plant Physiol. 138, 276) involve sequence dependent uORFs. Our new example is different in that it initiates translation with a highly conserved non-canonical codon.
In conclusion we have shown that the level of ascorbate in a leaf can be controlled through ascorbate feedback through a non canonical uORF in the long 5′-UTR of the controlling gene of ascorbate biosynthesis, GGP. We show evidence that this feedback acts post-transcriptionally by controlling the level of the GGP enzyme. We propose that this is a major mechanism that ascorbate concentrations are controlled in the L-galactose pathway of ascorbate biosynthesis.
Materials and Methods:
Plant Materials and Chemical Assays
The Nicotiana benthamiana leaf transient reporter gene system using luciferase (LUC) as the promoter specific reporter and renilla (REN) as the transformation reporter was as described previously (Hellens et al., 2005, Plant Methods 1, 13). Ascorbate concentration in the leaf was manipulated by co-injecting either the coding sequence for Actinidia chinensis GGP in pGreen (Hellens et al., 2000, Plant Mol. Biol. 42, 819) under the 35S promoter transformed into Agrobacterium tumifaciens (Bulley et al., 2009, J. Exp. Bot. 60, 765.) or a KO vector constructed using as a template the GGP sequence from N. benthamiana assembled from seven ESTs in GenBank as described by (Snowden et al., The Plant Cell 17, 746). In addition, the version of the CDS of A. eriantha GME (GenBank accession FG424114) described earlier (Bulley et al., 2009, J. Exp. Bot. 60, 765.) was used to synergistically enhance ascorbate with GGP. Ascorbate was measured in extracts of the same leaf using an HPLC based assay also as previously described (Rassam et al., 2005, J. Agric. Food Chem. 53, 2322). Ascorbate was measured as total ascorbate by reducing extracts before HPLC (Rassam et al., 2005, J. Agric. Food Chem. 53, 2322). Measurement of the redox state of the ascorbate found the redox potential decreased significantly with increased concentration of ascorbate (FIG. 12). This raises the possibility that the effect of ascorbate may be either through ascorbate itself or through the decreased redox potential at increased ascorbate.
FIG. 13 shows a typical experiment where each point (mean of two to three measurements) represents a different leaf of N. benthamiana showing how ascorbate affects the LUC activity but has little effect on the REN activity. This data is also plotted in FIG. 1 where the mean LUC/REN ratio for each leaf is plotted against the ascorbate concentration.
To test whether the effect of ascorbate was mediated by the promoter or the 5′-UTR we constructed two vectors where the 5′-UTR regions were swapped between the GGP promoter and the TT8 promoter. The resulting constructs consisted of the TT8 core promoter (TT8P′) followed by the GGP 5′-UTR (GGPUTR) and vice versa. We transiently expressed these in leaves and measured the relative LUC activity.
To test the effect of the 5′-UTR in front of the GGP coding sequence on ascorbate concentration, a different GGP gene (GenBank accession FG460629) was used instead of the GGP used in other experiments. At the protein level it was 96% identical to the standard GGP and in the absence of the 5′-UTR, raised ascorbate concentrations to similar levels seen for the usual GGP. The version with the 5′-UTR had the full 5′-UTR, while the version labeled without the UTR had all but 37 bp upstream of the start ATG deleted using the XhoI restriction enzyme. In this region at the 3′ end of the 5′-UTR, there is little homology between GGPs (FIG. 8). Both versions were ligated into the pART277 vector (Gleave, A., 1992, Plant Mol. Biol. 20, 1203 (1992).
The amount of LUC protein was measured using an antibody to LUC (Promega) using a Western blot of 50 μg soluble cellular protein per lane (extracted in 40 mM phosphate buffer, pH 7.4, 150 mM NaCl) from various constructs transiently expressed in Nicotiana benthamiana leaves. The large subunit of RubisCO as stained by SYPRO Tangerine protein gel stain is shown as a loading control.
To separate the effects of GGP protein from ascorbate, we initially attempted to use ascorbate or its precursors injected directly into the leaf by syringe. However we could not get sustained changes in leaf ascorbate. We also tried allowing detached leaves or discs previously injected with Agrobacterium LUC/REN constructs to take up ascorbate precursors. While these leaves did have very significant increases in ascorbate, the leaves deteriorated before LUC/REN values could rise enough to be measurable. We then tried lowering ascorbate without lowering GGP concentrations by knocking out two genes involved in ascorbate biosynthesis (encoding galactose dehydrogenase and GDP mannose epimerase). However, again we failed to have significant changes in leaf ascorbate, suggesting that their expressed enzymes may be stable or in excess over the seven day extent of the experiment.
Experiments were repeated at least twice with similar results, and although in some cases the high ascorbate reduced REN expression as well as LUC, this did not alter the effect of ascorbate on reducing the slope of the relationship (i.e. the LUC/REN ratio) for the GGP promoter but not the TT8.
Gene Cloning and Plasmids
The GGP promoter from Actinidia eriantha (SEQ ID NO: 101) was cloned by genome walking and has been deposited in GenBank as accession number JX486682. A. eriantha gDNA (2.0 μg) was digested using seven blunt cutting restriction enzymes; DraI, EclII 136, EcoRV, HpaI, MScI, ScaI, SspI and StuI. Digests were purified and eluted in 10 μL using PCR Clean and Concentrate spin columns (Zymogen). Double stranded adapter sequences (Clontech) containing nested PCR primer sites were ligated onto cut fragments overnight at 16 C using T4 Rapid Ligase (Roche). Ligations were column purified a second time and eluted in 30 μL. First round PCR was performed using 1 uL of each digest with primers 319998NRWLK1, RPH-149 and Ex Taq polymerase (Takara) using the following two step cycling conditions. For the first high stringency step, one cycle of denaturation was performed at 94 C for two min, followed by seven cycles of 94 C for 25 sec and elongation/annealing at 72 C for three min. The second step consisted of 32 cycles of 94 C for 25 sec and 67 C for three min before a final 67 C extension for three min. The first round products were run on a 1% agarose gel and 1 μL of a 1:50 dilution was used as template for second round PCR with 319998NRWLK2 and RPH-150. Second round PCR was also performed as a two step PCR with an initial denaturation of 94 C for two min followed by five cycles of 94 C for 25 sec and 72 C for 3 m. This was followed by a further 20 cycles of 94 C for 25 s and 67 C for three min before a final 67 C extension. Gel electrophoresis was used to identify PCR products between 500-2 kb in size for cloning into pGem T Easy vector (Promega) according to the manufacturer's instructions. Clones were DNA sequenced confirmed for overlap with the known 5′-UTR. A second set of nested primers was designed to the end of the first promoter walk to extend the known A. eriantha promoter sequence to 2 kb. This 2 kb promoter sequence was then PCR amplified from A. eriantha gDNA using primers Eriantha gDNA PCR 5′ and 319998NRWLK2 and sub-cloned into pGreen0800-5′_LUC (Hellens et al., 2000, Plant Mol. Biol. 42, 819) using EcoRV and NcoI restriction enzymes. The final construct is called the GGP-promoter-pGreenII 0800-5 LUC vector.
The promoter for GGP from Arabidopsis thaliana (At4g26850) (SEQ ID NO: 102) and control promoters from a range of sources were cloned by PCR. The control promoters were TT8 (AT4G09820), EF1α (AT1G07940), Act2P (AT3G18780) and Act7P (AT5G09810).
Generation of the inactivated start codons or other deletions and mutations in the uORFs of the 5′-UTR was done by chemical synthesis (GenScript, www.genscript.com) of mutated and control sequences. In the inactivated versions, the ATG or ACG start codons of the uORFs were changed to a TTG. Other changes were done by site-specific mutagenesis. There is a StuI site on the 5′ side of the UTR 28 bp into the 5′-UTR which was used as the 5′ border of the synthesized fragment. We added an extra CC to the 3′ end of the synthesized genes to create a NcoI site (ccatgg) and removed the sequence equivalent to the synthesized fragment from the GGP-promoter-pGreenII 0800-5 LUC by digesting it with StuI and NcoI. Then the synthesized fragments were separately cloned into the vector to create the two versions with and without a uORF.
RNA Isolation and cDNA Synthesis:
Total RNA was isolated from 100 mg leaf tissue using a RNeasy Plant Mini kit (Qiagen) and concentrations were measured using a Nanodrop 1000 spectrophotometer (Thermo Fisher Scientific Inc.). Complementary DNA was then synthesized from 1 μg total RNA and random hexamers in a 10 μl total volume using a BluePrint Reagent Kit for Real Time PCR (Takara Bio Company) following manufacturer instructions. Following cDNA synthesis the preparation was diluted 75 times in preparation for quantitative real time PCR.
Quantitative PCR
Quantitative PCR was performed in 5 μl total volume using a LightCycler® 480 Real-Time PCR System (Roche Diagnostics) and the following primer pair: LUC1/2: 5′-TATCCGCTGGAAGATGGAAC-3′ (SEQ ID NO:109); 5′-TCCACCTCGATATGTGCATC-3′. (SEQ ID NO:110) Primers were designed with annealing temperatures of 60 C using Primer3 (Rozen and Skaletsky, 2000, Methods Mol Biol. 132, 365. The luciferase primer pair amplifies regions from the 5′-end of the luciferase open reading frame. Reaction components (using LightCycler 480® SYBR Green I Master Mix) were as follows: 2 μM each primer, 1.25 μl diluted cDNA preparation. The standard cycling protocol with a Tm of 60° C. was used and relative quantification analysis normalized to Renilla transcripts was performed using The LightCycler® 480 Software (Roche Diagnostics).
Control Tests of the System Used
As the control gene promoter TT8 expressed about 10 times higher than the GGT promoter we considered the possibility that the TT8 promoter might have saturated the ability of the tobacco cell to express the transcript or translate the LUC and thus any inhibition would not be seen. To check this, we titrated the amount of Agrobacterium containing the TT8-LUC construct over a 200 fold range. The ratio of LUC/REN and the slope of the relationship between LUC and REN was unchanged by the amount of Agrobacterium injected (Table 1) showing little sign of any saturation of expression of the reporter genes. The TT8 promoter driven LUC values overlapped with the LUC values expressed using the GGP promoter. We also tested whether several different alternative promoters were inhibited by ascorbate. These included EF1α, Act2P, and Act7P. None of these promoters were negatively affected by ascorbate (Table 2) although in this experiment, the TT8 promoter strength was actual increased by ascorbate. In a third test we verified that the effect of ascorbate on the GGP promoter strength was not restricted to the kiwifruit GGP gene promoter by testing the same promoter from Arabidopsis GGP (At4g26850). As shown in FIG. 4, the response of the LUC/REN ratio to ascorbate was essentially identical for the two promoters from different species. In a final test, we checked whether expressing a gene to raise ascorbate (kiwifruit GGP) might in itself affect the results. Consequently we added an extra control gene in the form of a methyl transferase. While we got a small reduction in this experiment in the LUC/REN ratio of the added control gene (Table 3), this did not change the conclusion that increased ascorbate reduced the strength of the GGP promoter but had little effect of other control promoters.
Examples 2
Testing the Effect of Ascorbate on Other 5′UTR Sequences in the LUC/REN Reporter Assay
Methods
A 35S driven-LUC construct was derived from pGreen 0800 LUC (Hellens et al., 2000, Plant Mol. Biol. 42, 819), where a second copy of the 35S promoter without its 5′UTR was cloned into the multiple cloning site in front of the LUC coding sequence, and 5′UTRs from apple, potato and tomato (SEQ ID NO: 126, 127 and 128 respectively) were inserted between this 35S promoter and the beginning of the LUC coding sequence replacing the 35S 5′UTR (FIG. 14). The 5′UTR sequences are shown in FIG. 15.
These constructs were tested as described for other GGP promoter constructs in Example 1 above.
Results
The LUC genes, each driven by different GGP 5′UTR inserts, were all down regulated by ascorbate (FIGS. 16, 17 and 18, Table 6). In each case both the LUC/REN ratios and the slope of the relationship between LUC and REN were reduced by ascorbate. In these experiments, the ascorbate increased less than shown in Example 1 (Table 6), possibly due to varying growth conditions (lower light and higher temperature) but the increased ascorbate still reduced the LUC values significantly. Also shown are corresponding data for a typical non-responsive promoter-5′UTR (EF1alpha: FIG. 19) and for the standard GGP promoter-5′UTR (FIG. 20). The further variant 5′UTR sequences disclosed can of course be tested in the same way.














TABLE 6











Mean




Mean




ascorbate




LUC/
Std



mg/100 g
Std


5′UTR
REN
error
p
Slope
R2
FW
error






















Tomato low
1.88
0.21
0.000
0.77
0.30
19.1
1.7


ascorbate









Tomato
0.69
0.22

0.04
0.00
36.6
1.5


high









ascorbate









Potato low
2.03
0.22
0.000
1.55
0.57
21.5
1.4


ascorbate









Potato high
0.30
0.04

0.13
0.63
40.3
3.4


ascorbate









Apple low
1.77
0.24
0.024
2.31
0.78
18.4
1.1


ascorbate









Apple high
0.89
0.26

0.41
0.34
33.6
2.9


ascorbate









EF1alpha
1.821
0.15
0.028
1.51
0.71
19.1
0.6


promoter









low









ascorbate









EF1alpha
2.278
0.13

2.21
0.85
34.9
2.0


promoter









high









ascorbate









Kiwifruit
0.236
0.02
0.000
0.27
0.77
20.5
0.8


GGP-5′UTR









low









ascorbate









Kiwifruit
0.079
0.01

−0.03
0.08
40.3
1.9


GGP-5′UTR.









high









ascorbate





Tabulation of LUC/REN ratios and slopes for plots of LUC against REN for various 5′UTR from GGPs from three different species. We also include a control promoter and its own 5′UTR and the original GGP promoter and 5′UTR used previously as controls. The p value is the statistical significance for the difference between high and low ascorbate for each construct.






CONCLUSION
The GGP 5′UTR constructs from these three different species tested all showed significant down regulation by increased ascorbate. Along with the 5′UTRs from GGPs from Arabidopsis and kiwifruit (Example 1), this makes a total of five diverse GGP 5′UTRs that have been tested and all have been shown to be down regulated by increased ascorbate, strongly suggesting that this is a widespread phenomenon.










SUMMARY OF SEQUENCES











SEQ ID
Molecule





NO.
type
Species
Classification
Reference














1
polypeptide
Actinidia eriantha
Dicot
uORF peptide


2
polypeptide
Cucumis sativus
Dicot
uORF peptide


3
polypeptide
Glycine max
Dicot
uORF peptide


4
polypeptide
Solanum
Dicot
uORF peptide




lycopersicum


5
polypeptide
Vitis vinifera
Dicot
uORF peptide


6
polypeptide
Arabidopsis
Dicot
uORF peptide




thaliana


7
polypeptide
Arabidopsis
Dicot
uORF peptide




thaliana

at5g55120


8
polypeptide
Malus x
Dicot
uORF peptide




domesticus


9
polypeptide
Medicago
Dicot
uORF peptide




truncatula


10
polypeptide
Populus
Dicot
uORF peptide




trichocarpa


11
polypeptide
Picea sitchensis
Pinophyta
uORF peptide


12
polypeptide
Physcomitrella
Bryophyta
uORF peptide




patens


13
polypeptide
Actinidia arguta
Dicot
uORF peptide


14
polypeptide
Actinidia eriantha
Dicot
uORF peptide


15
polypeptide
Actinidia chinensis
Dicot
uORF peptide


16
polypeptide
Fragaria vulgaris
Dicot
uORF peptide


17
polypeptide
Solanum
Dicot
uORF peptide




tuberosum


18
polypeptide
Chlamydomonas
Protista
uORF peptide




reinhardtii


19
polypeptide
Zea mays
Monocot
uORF peptide


20
polypeptide
Selaginella
Lycopodiophyta
uORF peptide




moellendorffii


21
polypeptide
Actinidia eriantha
Dicot
uORF peptide






conserved region


22
polypeptide
Cucumis sativus
Dicot
uORF peptide






conserved region


23
polypeptide
Glycine max
Dicot
uORF peptide






conserved region


24
polypeptide
Solanum
Dicot
uORF peptide




lycopersicum

conserved region


25
polypeptide
Vitis vinifera
Dicot
uORF peptide






conserved region


26
polypeptide
Arabidopsis
Dicot
uORF peptide




thaliana

conserved region






at4g26850


27
polypeptide
Arabidopsis
Dicot
uORF peptide




thaliana

conserved region






at5g55120


28
polypeptide
Malus x
Dicot
uORF peptide




domesticus

conserved region


29
polypeptide
Medicago
Dicot
uORF peptide




truncatula

conserved region


30
polypeptide
Populus
Dicot
uORF peptide




trichocarpa

conserved region


31
polypeptide
Picea sitchensis
Pinophyta
uORF peptide






conserved region


32
polypeptide
Physcomitrella
Bryophyta
uORF peptide




patens

conserved region


33
polypeptide
Actinidia arguta
Dicot
uORF peptide






conserved region


34
polypeptide
Actinidia eriantha
Dicot
uORF peptide






conserved region


35
polypeptide
Actinidia chinensis
Dicot
uORF peptide






conserved region


36
polypeptide
Fragaria vulgaris
Dicot
uORF peptide






conserved region


37
polypeptide
Solanum
Dicot
uORF peptide




tuberosum

conserved region


38
polypeptide
Chlamydomonas
Protista
uORF peptide




reinhardtii

conserved region


39
polypeptide
Zea mays
Monocot
uORF peptide






conserved region


40
polypeptide
Selaginella
Lycopodiophyta
uORF peptide




moellendorffii

conserved region


41
polynucleotide
Actinidia eriantha
Dicot
uORF DNA


42
polynucleotide
Cucumis sativus
Dicot
uORF DNA


43
polynucleotide
Glycine max
Dicot
uORF DNA


44
polynucleotide
Solanum
Dicot
uORF DNA




lycopersicum


45
polynucleotide
Vitis vinifera
Dicot
uORF DNA


46
polynucleotide
Arabidopsis
Dicot
uORF DNA




thaliana

at4g26850


47
polynucleotide
Arabidopsis
Dicot
uORF DNA




thaliana

at5g55120


48
polynucleotide
Malus x
Dicot
uORF DNA




domesticus


49
polynucleotide
Medicago
Dicot
uORF DNA




truncatula


50
polynucleotide
Populus
Dicot
uORF DNA




trichocarpa


51
polynucleotide
Picea sitchensis
Pinophyta
uORF DNA


52
polynucleotide
Physcomitrella
Bryophyta
uORF DNA




patens


53
polynucleotide
Actinidia arguta
Dicot
uORF DNA


54
polynucleotide
Actinidia eriantha
Dicot
uORF DNA


55
polynucleotide
Actinidia chinensis
Dicot
uORF DNA


56
polynucleotide
Fragaria vulgaris
Dicot
uORF DNA


57
polynucleotide
Solanum
Dicot
uORF DNA




tuberosum


58
polynucleotide
Chlamydomonas
Protista
uORF DNA




reinhardtii


59
polynucleotide
Zea mays
Monocot
uORF DNA


60
polynucleotide
Selaginella
Lycopodiaphyta
uORF DNA




moellendorffii


61
polynucleotide
Actinidia eriantha
Dicot
uORF DNA






conserved region


62
polynucleotide
Cucumis sativus
Dicot
uORF DNA






conserved region


63
polynucleotide
Glycine max
Dicot
uORF DNA






conserved region


64
polynucleotide
Solanum
Dicot
uORF DNA




lycopersicum

conserved region


65
polynucleotide
Vitis vinifera
Dicot
uORF DNA






conserved region


66
polynucleotide
Arabidopsis
Dicot
uORF DNA




thaliana

conserved region






at4g26850


67
polynucleotide
Arabidopsis
Dicot
uORF DNA




thaliana

conserved region






at5g55120


68
polynucleotide
Malus x
Dicot
uORF DNA




domesticus

conserved region


69
polynucleotide
Medicago
Dicot
uORF DNA




truncatula

conserved region


70
polynucleotide
Populus
Dicot
uORF DNA




trichocarpa

conserved region


71
polynucleotide
Picea sitchensis
Pinophyta
uORF DNA






conserved region


72
polynucleotide
Physcomitrella
Bryophyta
uORF DNA




patens

conserved region


73
polynucleotide
Actinidia arguta
Dicot
uORF DNA






conserved region


74
polynucleotide
Actinidia eriantha
Dicot
uORF DNA






conserved region


75
polynucleotide
Actinidia chinensis
Dicot
uORF DNA






conserved region


76
polynucleotide
Fragaria vulgaris
Dicot
uORF DNA






conserved region


77
polynucleotide
Solanum
Dicot
uORF DNA




tuberosum

conserved region


78
polynucleotide
Chlamydomonas
Protista
uORF DNA




reinhardtii

conserved region


79
polynucleotide
Zea mays
Monocot
uORF DNA






conserved region


80
polynucleotide
Selaginella
Lycopodiophyta
uORF DNA




moellendorffii

conserved region


81
polynucleotide
Actinidia eriantha
Dicot
Whole 5′-UTR


82
polynucleotide
Cucumis sativus
Dicot
Whole 5′-UTR


83
polynucleotide
Glycine max
Dicot
Whole 5′-UTR


84
polynucleotide
Solanum
Dicot
Whole 5′-UTR




lycopersicum


85
polynucleotide
Vitis vinifera
Dicot
Whole 5′-UTR


86
polynucleotide
Arabidopsis
Dicot
Whole 5′-UTR




thaliana

at4g26850


87
polynucleotide
Arabidopsis
Dicot
Whole 5′-UTR




thaliana

at5g55120


88
polynucleotide
Malus x
Dicot
Whole 5′-UTR




domesticus


89
polynucleotide
Medicago
Dicot
Whole 5′-UTR




truncatula


90
polynucleotide
Populus
Dicot
Whole 5′-UTR




trichocarpa


91
polynucleotide
Picea sitchensis
Pinophyta
Whole 5′-UTR


92
polynucleotide
Physcomitrella
Bryophyta
Whole 5′-UTR




patens


93
polynucleotide
Actinidia arguta
Dicot
Whole 5′-UTR


94
polynucleotide
Actinidia eriantha
Dicot
Whole 5′-UTR


95
polynucleotide
Actinidia chinensis
Dicot
Whole 5′-UTR


96
polynucleotide
Fragaria vulgaris
Dicot
Whole 5′-UTR


97
polynucleotide
Solanum
Dicot
Whole 5′-UTR




tuberosum


98
polynucleotide
Chlamydomonas
Protista
Whole 5′-UTR




reinhardtii


99
polynucleotide
Zea mays
Monocot
Whole 5′-UTR


100
polynucleotide
Selaginella
Lycopodiophyta
Whole 5′-UTR




moellendorffii


101
polynucleotide
Actinidia eriantha
Dicot
GGP genomic






promoter






sequence


102
polynucleotide
Arabidopsis
Dicot
GGP genomic




thaliana

promoter






sequence






at4g26850


103
polynucleotide
Arabidopsis
Dicot
GGP genomic




thaliana

promoter






sequence






at5g55120


104
polynucleotide
Malus x
Dicot
GGP2 genomic




domesticus

promoter






sequence






MDP0000288088


105
polynucleotide
Malus x
Dicot
GGP1 genomic




domesticus

promoter






sequence






MDP0000172222


106
polynucleotide
Malus x
Dicot
GGP3 genomic




domesticus

promoter






sequence






MDP0000191488


107
polynucleotide
Actinidia chinensis
Dicot
GGP genomic






promoter






sequence


108
polypeptide
Artificial
Consensus
Consensus motif


109
polynucleotide
Artificial
Primer
LUC1


110
polynucleotide
Artificial
Primer
LUC2


111
polynucleotide
Cucumis sativus
Dicot
Sub-sequence of






5′-UTR


112
polynucleotide
Actinidia eriantha
Dicot
Sub-sequence of






5′-UTR


113
polynucleotide
Actinidia arguta
Dicot
Sub-sequence of






5′-UTR


114
polynucleotide
Actinidia eriantha
Dicot
Sub-sequence of






5′-UTR


115
polynucleotide
Actinidia chinensis
Dicot
Sub-sequence of






5′-UTR


116
polynucleotide
Malus x
Dicot
Sub-sequence of




domesticus

5′-UTR


117
polynucleotide
Fragaria vulgaris
Dicot
Sub-sequence of






5′-UTR


118
polynucleotide
Arabidopsis
Dicot
Sub-sequence of




thaliana

5′-UTR


119
polynucleotide
Arabidopsis
Dicot
Sub-sequence of




thaliana

5′-UTR


120
polynucleotide
Populus
Dicot
Sub-sequence of




trichocarpa

5′-UTR


121
polynucleotide
Glycine max
Dicot
Sub-sequence of






5′-UTR


122
polynucleotide
Medicago
Dicot
Sub-sequence of




truncatula

5′-UTR


123
polynucleotide
Solanum
Dicot
Sub-sequence of




lycopersicum

5′-UTR


124
polynucleotide
Solanum
Dicot
Sub-sequence of




tuberosum

5′-UTR


125
polynucleotide
Vitis vinifera
Dicot
Sub-sequence of






5′-UTR


126
polynucleotide
Solanum
Dicot
Whole 5′-UTR




lycopersicum


127
polynucleotide
Solanum
Dicot
Whole 5′-UTR




tuberosum


128
polynucleotide
Malus x
Dicot
Whole 5′-UTR




domesticus


129
polynucleotide
Solanum
Dicot
uORF DNA




lycopersicum


130
polynucleotide
Solanum
Dicot
uORF DNA




tuberosum


131
polynucleotide
Malus x
Dicot
uORF DNA




domesticus


132
polypeptide
Solanum
Dicot
uORF peptide




lycopersicum


133
polypeptide
Solanum
Dicot
uORF peptide




tuberosum


134
polypeptide
Malus x
Dicot
uORF peptide




domesticus


135
polypeptide
Solanum
Dicot
uORF peptide




lycopersicum

conserved region


136
polypeptide
Solanum
Dicot
uORF peptide




tuberosum

conserved region


137
polypeptide
Malus x
Dicot
uORF peptide




domesticus

conserved region


138
polynucleotide
Solanum
Dicot
uORF DNA




lycopersicum

conserved region


139
polynucleotide
Solanum
Dicot
uORF DNA




tuberosum

conserved region


140
polynucleotide
Malus x
Dicot
uORF DNA




domesticus

conserved region

## Claims

1. A method for producing a plant cell or plant with at least one of:
a) increased GDP-L-Galactose phosphorylase (GGP) translation,
b) increased GGP production,
c) increased GGP activity, and
d) increased ascorbate production,
the method comprising modification of the 5′-UTR of a GGP gene in the plant cell or plant, wherein the 5′UTR comprises at least one of:
i) a sequence with at least 70% identity to any one of SEQ ID NOs: 41-100, and 111-131, and
ii) a sequence encoding a polypeptide with at least 70% identity to any one of SEQ ID NOs: 1-40, 108, and 132 to 140;
wherein the modification disrupts the function of an upstream open reading frame (uORF) encoded by the 5′UTR; and wherein said modification is at least one of a deletion, an addition, or a substitution of at least one nucleotide in the 5′UTR.

2. The method of claim 1 wherein the modification is in the uORF sequence in the 5′UTR.

3. The method of claim 2 wherein the uORF has a sequence selected from any one of SEQ ID NO: 41 to 60 and 129 to 131 or a variant thereof with at least 70% identity to any one of SEQ ID NO: 41 to 60 and 129 and 131.

4. A method for selecting a plant with at least one of:
a) increased GGP translation,
b) increased GGP production,
c) increased GGP activity, and
d) increased ascorbate production,

the method comprising testing of a plant for the presence of a first polymorphism in a polynucleotide comprising a sequence encoding a polypeptide with at least 70% identity to an amino acid sequence selected from SEQ ID NO:1 to 20, and 132 to 134 in the plant, or a further polymorphism linked to the first polymorphism, wherein the first polymorphism disrupts expression of the polypeptide.

5. The method of claim 4 which includes the step of separating the selected plant from one or more of non-selected plants.

6. A method of producing ascorbate, the method comprising extracting ascorbate from a plant cell or plant produced by the method of claim 1 or selected by the method of claim 4.

7. A plant cell or plant produced by the method of claim 1.

8. The plant cell or plant of claim 7 with at least one of:
a) increased GGP translation,
b) increased GGP production,
c) increased GGP activity, and
d) increased ascorbate production,

wherein the plant cell or plant comprises a modification in the 5′-UTR of a GGP gene in the plant cell or plant, wherein the 5′-UTR comprises at least one of:
i) a sequence with at least 80% identity to any one of 41-100, and 111-131, and
ii) a sequence encoding a polypeptide with at least 80% identity to any one of SEQ ID NO: 1-40, 108, and 132 to 140

wherein the modification disrupts the function of a uORF encoded by the 5′-UTR, and wherein said modification is at least one of a deletion, an addition, or a substitution of at least one nucleotide in the 5′-UTR.

9. A method of producing ascorbate, the method comprising extracting ascorbate from the plant cell or plant of claim 7.