WO2006011815A2 - Phosphatases, polynucleotides encoding these and uses thereof - Google Patents

Phosphatases, polynucleotides encoding these and uses thereof Download PDF

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Publication number
WO2006011815A2
WO2006011815A2 PCT/NZ2005/000188 NZ2005000188W WO2006011815A2 WO 2006011815 A2 WO2006011815 A2 WO 2006011815A2 NZ 2005000188 W NZ2005000188 W NZ 2005000188W WO 2006011815 A2 WO2006011815 A2 WO 2006011815A2
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Prior art keywords
polynucleotide
polypeptide
seq
plant
galactose
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PCT/NZ2005/000188
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French (fr)
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WO2006011815A3 (en
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William Laing
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The Horticulture And Food Research Institute Of New Zealand Limited
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Publication of WO2006011815A2 publication Critical patent/WO2006011815A2/en
Publication of WO2006011815A3 publication Critical patent/WO2006011815A3/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine

Definitions

  • the present invention relates to compositions and methods for producing plants with altered ascorbic acid content
  • 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.
  • L-GaI pathway proceeds through L-GaI to galactono-l,4-lactone and thence to ascorbate ( Wheeler et al, 1998, Nature 393, 365- 369).
  • Mutant and biochemical studies show that it is likely that the L-GaI is derived from GDP-D-Man through GDP-L-GaI to L-GaI-I -P, which has been proposed to be converted to L-GaI through the action of a specific unidentified phosphatase.
  • GaI-I -phosphate in a specific manner.
  • the invention provides plant cell or plant with altered L-Galactose-1 - phosphate phosphatase expression, the method comprising transformation of a plant cell or plant with a genetic construct including: a) a polynucleotide encoding of a polypeptide with the amino acid sequence of any one of SEQ ID NO:67 to 118, 120, 122, 124, 126, 130 to 132, or a variant of the polypeptide from a plant species, wherein the variant has the activity of an L-Galactose- 1 -phosphate phosphatase; b) a polynucleotide comprising a fragment, of at at least 15 nucleotides in length, of the polynucleotide of a), or c) a polynucleotide comprising a compliment, of at at least 15 nucleotides in length, of the polynucleotide of a); or d) a polynucleot
  • the variant has at least 50% sequence identity to a polypeptide with the amino acid sequence of any one of SEQ ID NO: 67 to 118, 120, 122, 124, 126, 130 to 132.
  • the variant comprises the amino acid sequence: X 1 X2X 3 X 4 X 5 SX6X 7 X8X9LX 1O X 11 X 12 LX 1 3X 14 X 1 5Xl6Xl7Xl8Xl9X2 ⁇ RDX2lX22X23X24X2S X2 6 X 27 TX28X29lX3 ⁇ X3lX32X33X34X35X36X37X38X39X4 ⁇ X4lX42X43X44X45X46X47X48X49X5
  • X 1 is N 5 K 5 T 5 A 5 D or V;
  • X 2 is P 5 R or S;
  • X 3 is I or M;
  • X 4 is K 5 R or H;
  • X 5 is V or A;
  • X 6 is S, A or T;
  • X 7 is Q 5 K 5 E or H;
  • X 8 is S 5 T 5 N 5 A or D;
  • X 9 is E or Q;
  • X 10 is V 5 1 5 L or M;
  • X 11 is K 5 T 5 N or S;
  • X 12 is S 5 A or C;
  • X 13 is L or V;
  • X 14 is A 5 G 5 S 5 V 5 L or M;
  • X 15 is T or A;
  • X 16 is V 5 A 5 D or I;
  • X 17 is G or I;
  • X 18 is T 5 V or P;
  • X 19 is K 5 N 5 T 5 E or M;
  • X 20 is K 5 N or Q;
  • polynucleotide of a) encodes a polypeptide with the amino acid sequence of any one of SEQ ID NO: 67 to 118, 120, 122, 124, 126, 130 to 132.
  • polynucleotide of a) encodes a polypeptide with the amino acid sequence of SEQ ID NO:67.
  • the invention provides a method of producing a plant cell or plant with altered L-Galactose-1 -phosphate phosphatase expression, the method comprising transformation of a plant cell or plant with a genetic construct including: a) a polynucleotide comprising a nucleotide sequence selected from any one the sequences of SEQ ID NO: 1, 5 to 54, 55 to 65, 119, 121, 123, 125 and 127 to 129, or a variant thereof from a plant species, wherein the variant encodes a polypeptide which has the activity of an L-Galactose-1 -phosphate phosphatase; b) a polynucleotide comprising a fragment, of at at least 15 nucleotides in length, of the polynucleotide of a), or c) a polynucleotide comprising a complement, of at at least 15 nucleotides in length, of the polynucleotide of
  • polynucleotide of a) comprises any one the sequences of SEQ ID NO: 1, 5 to 54, 55 to 65, 119, 121, 123, 125 and 127 to 129.
  • polynucleotide of a comprises the sequence of SEQ ID NO: 1.
  • the plant or plant cell produced by the methods of the invention have increased L-Galactose- 1 -phosphate phosphatase activity.
  • the plant or plant cell produced by the methods of the invention have altered ascorbate content, preferably increased ascorbate content.
  • the invention provides an isolated polynucleotide having at least 82% sequence identity to a nucleotide sequence that encodes a polypeptide comprising an amino acid sequence selected from any one of SEQ ID NO: 67 to 118, 120, 122, 124, 126, 130 to 132, wherein the polynucleotide encodes an L-Galactose- 1 -phosphate phosphatase.
  • said nucleotide sequence comprises a sequence selected from any one of SEQ ID NO: 1, 5 to 54, 55 to 65, 119, 121, 123, 125 and 127 to 129.
  • said said nucleotide sequence comprises a full-length coding sequence selected from any one of SEQ ID NO: 1, 5 to 54, 55 to 65, 119, 121, 123, 125 and 127 to 129.
  • nucleotide sequence comprises the nucleotide sequence of SEQ ID NO: 1. In a further embodiment said nucleotide sequence comprises the full-length coding sequence of SEQ ID NO: 1.
  • the invention provides an isolated polynucleotide that encodes a polypeptide comprising an amino acid sequence selected from any one of SEQ ID NO: 67 to 118, 120, 122, 124, 126, 130 to 132.
  • polynucleotide encodes a polypeptide comprising the amino acid sequence of SEQ ID NO : 67.
  • polynucleotide comprises a sequence selected from any one of SEQ ID NO: 1, 5 to 54, 55 to 65, 119, 121, 123, 125 and 127 to 129.
  • polynucleotide comprises the full-length coding sequence from any one of SEQ ID NO: 1, 5 to 54, 55 to 65, 119, 121, 123, 125 and 127 to 129.
  • polynucleotide comprises the sequence of SEQ ID NO: 1
  • polynucleotide comprises the full-length coding sequence from within the sequence of SEQ ID NO: 1.
  • the invention provides an isolated polynucleotide comprising a sequence selected from any one of SEQ ID NO: I 5 5 to 54, 55 to 65, 119, 121, 123, 125 and 127 to 129 or a variant thereof, wherein the variant is from an apple, kiwifruit or avocado species, and encodes a polypeptide which has the activity of an L-Galactose-1- phosphate phosphatase.
  • the isolated polynucleotide comprises the sequence of SEQ ID NO: 1 or a variant thereof, wherein the variant is from an apple, kiwifruit or avocado species, and encodes a polypeptide which has the activity of an L-Galactose-1 -phosphate phosphatase.
  • the isolated polynucleotide comprises the sequence of SEQ ID NO: 1
  • the invention provides an expression construct which includes a polynucleotide comprising the sequence selected from any one of SEQ ID NO: 1, 5 to 45, 47 to 54, 55 to 65, 119, 121, 123, 125 and 127 to 129 or a variant thereof from a plant species, wherein the variant encodes a polypeptide which has the activity of an L- Galactose-1 -phosphate phosphatase, and wherein the variant does not encode the polypeptide of Accession No. AAB19030.
  • the expression construct includes a polynucleotide comprising the sequence of SEQ ID NO: 1, or a variant thereof from a plant species, wherein the variant encodes a polypeptide which has the activity of an L-Galactose-1 -phosphate phosphatase, and wherein the variant does not encode the polypeptide of Accession No. AAB19030.
  • the expression construct includes a polynucleotide comprising the sequence of SEQ ID NO: 1.
  • the invention provides an isolated L-Galactose-1 -phosphate phosphatase polypeptide from a plant species excluding the polypeptide of Accession No. AAB19030.
  • the invention provides an isolated polypeptide having at least 81 % sequence identity to an amino acid sequence selected from any one of of SEQ ID NO: 67 to 118, 120, 122, 124, 126, 130 to 132, wherein the polypeptide has the activity of an L-Galactose-1 -phosphate phosphatase
  • the isolated polypeptide has at least 81 % sequence identity to the amino acid sequence of SEQ ID NO: 67.
  • the isolated polypeptide of comprises the amino acid sequence of SEQ ID NO: 67 to 118, 120, 122, 124, 126, 130 to 132.
  • the isolated polypeptide comprises the amino acid sequence of SEQ ID NO: 67.
  • the invention provides an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 67 to 105, 107 to 118, 120, 122, 124, 126, 130 to 132, or a variant thereof from a plant species, wherein the variant has the activity of an L- Galactose- 1 -phosphate phosphatase, and wherein the variant is not the polypeptide of Accession No. AAB19030.
  • the isolated polypeptide comprises the amino acid sequence of SEQ ID NO: 67, or a variant thereof from a plant species, wherein the variant has the activity of an L-Galactose-1 -phosphate phosphatase, and wherein the variant is not polypeptide of Accession No. AAB19030.
  • the invention provides an isolated polynucleotide encoding a polypeptide of the invention.
  • the invention provides an isolated polynucleotide comprising: a) a polynucleotide comprising a fragment, of at at least 15 nucleotides in length, of a polynucleotide of the invention; b) a polynucleotide comprising a complement, of at at least 15 nucleotides in length, of the polynucleotide of the invention; or d) a polynucleotide comprising a sequence, of at at least 15 nucleotides in length, capable of hybridising to the polynucleotide of the invention.
  • the invention provides a genetic construct which comprises a polynucleotide of the invention.
  • the invention provides a vector comprising an expression construct or genetic construct of the invention.
  • the invention provides a host cell genetically modified to express a polynucleotide of the invention, or a polypeptide of the invention. In a further aspect the invention provides a host cell comprising an expression construct or genetic construct of the invention.
  • the invention provides a method for producing an L-Galactose-1 - phosphate phosphatase polypeptide comprising culturing a host cell comprising an expression construct of the invention or a genetic construct of the invention, capable of expressing an L-Galactose-1 -phosphate phosphatase polypeptide.
  • the invention provides a method for producing the enzymic product of an L-Galactose-1 -phosphate phosphatase comprising culturing a host cell including an expression construct of the invention or an genetic construct of the invention, capable of expressing an L-Galactose-1 -phosphate phosphatase polypeptide, in the presence of enzymic substrate which may be supplied to, or may be naturally present within the host cell.
  • the invention provides a method for the biosynthesis of ascorbate comprising the steps of culturing a host cell comprising an expression construct of the invention or the genetic construct of the invention, capable of expressing an L- Galactose-1 -phosphate phosphatase polypeptide, in the presence of an ascorbate precursor which may be supplied to, or may be naturally present within the host cell.
  • the invention provides a plant cell genetically modified to express a polynucleotide of the invention, or a polypeptide of the invention.
  • the invention provides a plant cell which comprises an expression construct of the invention or the genetic construct of the invention.
  • the invention provides a plant which comprises a plant cell of the inenetion.
  • the invention provides a method for selecting a plant altered in L- Galactose-1 -phosphate phosphatase activity, the method comprising testing of a plant for altered expression of a polynucleotide of the invention.
  • the invention provides a method for selecting a plant with altered ascorbic acid content; the method comprising testing of a plant for altered expression of a polynucleotide of the invention.
  • the invention provides a method for selecting a plant altered in L- Galactose-1 -phosphate phosphatase activity, the method comprising testing of a plant for altered expression of a polypeptide of the invention.
  • the invention provides a method for selecting a plant altered in ascorbic acid content, the method comprising testing of a plant for altered expression of a polypeptide of the invention.
  • the invention provides a plant cell or plant produced by the method of the invention.
  • the invention provides a plant selected by the method of the invention.
  • the invention provides a method of producing ascorbate, the method comprising extracting ascorbate from a plant cell or plant of the invention.
  • the invention provides a method for identifying a compound as a candidate for a herbicide, comprising: a) contacting said compound with a polypeptide comprising a sequence selected from any one of SEQ ID NO : 67 to 118, 120, 122, 124, 126, 130 to 132, or a variant thereof which has the activity of an L-Galactose-1 -phosphate phosphatase, and b) detecting the presence and/or absence of binding between said compound and said polypeptide; wherein binding indicates that said compound is a candidate for a herbicide.
  • the invention provides a method for identifying a compound as a candidate for a herbicide, comprising: a) contacting said compound with a polypeptide comprising a sequence selected from any one of SEQ ID NO : 67 to 118, 120, 122, 124, 126, 130 to 132, or a variant thereof which has the activity of an L-Galactose-1 -phosphate phosphatase, and b) assessing the effect of the compound the an L-Galactose-1 -phosphate phosphatase activity of the polypeptide; wherein a decrease in activity indicates that said compound is a candidate for a herbicide.
  • the variant has at least 50% sequence identity to an amino acid sequence selected from any one of SEQ ID NO: 67 to 118, 120, 122, 124, 126, 130 to 132.
  • polypeptide comprises an amino acid sequence selected from any one of SEQ ID NO: 67 to 118, 120, 122, 124, 126, 130 to 132.
  • the variant has at least 50% sequence identity to the amino acid sequence of SEQ ID NO: 67.
  • polypeptide comprises an amino acid sequence of SEQ ID NO: 67.
  • the invention provides a compound identified by a method of the invention.
  • the invention provides a method for determining whether the compound of the invention has herbicidal activity, comprising: contacting a plant or plant cells with said herbicide candidate and detecting the presence or absence of a decrease in growth or viability of said plant or plant cells.
  • the invention provides an antibody raised against a polypeptide of the invention.
  • the invention provides method of converting galactose- 1 -phosphate to galactose, the method comprising contacting galactose- 1 -phosphate with the expression product of an expression construct comprising a polynucleotide of the invention to obtain galactose.
  • polynucleotides and variants of polynucleotides, of the invention may be derived from any species.
  • polynucleotide or variant is derived from a plant species.
  • polynucleotide or variant is derived from a gymnosperm plant species.
  • polynucleotide or variant is derived from a angiosperm plant species.
  • polynucleotide or variant is derived from a from dicotyledonuous plant species.
  • polynucleotide or variant is derived from a fruit plant species selected from a group comprising but not limited to the following genera: Actinidia, Malus, Citrus, Fragaria and Vaccinium
  • polynucleotide or variant is derived from a vegetable plant 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
  • polynucleotide or variant is derived from a monocotylenouous plant species.
  • polynucleotide or valiant is derived from a crop plant 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
  • OX 51 X 52 X 53 (SEQ ID NO:2) wherein X 1 is N, K, S, T, A, D or V; X 2 is P, R or S; X 3 is I or M; X 4 is K, R, H or S; X 5 is V, A or T; X 6 is S, P, A or T; X 7 is Q, K, E or H; X 8 is S, N, T, D or A; X 9 is E or Q;
  • X 10 is V, A, G, I, L or M;
  • X 11 is K, N, T or S;
  • X 12 is S, A or C;
  • X 13 is L, M or V;
  • X 14 is
  • X 15 is T or A;
  • X 16 is E or D;
  • X 17 is V, A, I or D;
  • X 18 is G or I;
  • X 19 is
  • X 20 is K, N 5 T, E or M;
  • X 21 is K, N or Q;
  • X 22 is L 5 A, S, E, P, T or V;
  • X 23 is
  • X 24 is V, L or I
  • X 25 is D, G or E
  • X 26 is A, D, V, I or T
  • X 27 is T, S, A, C or V
  • X 28 is N 5 D, G or R
  • X 29 is R, K or T
  • X 30 is N or K
  • X 31 is S, R, K, N or G
  • X 32 is L or V
  • X 33 is L or I
  • X 34 is F, Y or T
  • X 35 is K or E
  • X 36 is V, I or G
  • X 37 is R or A
  • X 38 is
  • X 39 is L, I, P or V;
  • X 40 is R or E;
  • X 41 is M or D;
  • X 42 is S, C, G, T or A;
  • X 43 is
  • X 44 is S or L;
  • X 45 is C 5 L or V;
  • X 46 is A or R;
  • X 47 is L or T;
  • X 48 is N 5 D 5 A or G;
  • X 49 is L, M or T; X 50 is C or L; X 51 is G 5 W or R; X 52 is I 5 V or C; X 53 is A or R
  • X 1 is N 5 K, T, A, D or V;
  • X 2 is P, R or S;
  • X 3 is I or M;
  • X 4 is K, R or H;
  • X 5 is V or A;
  • X 6 is S 3 A or T;
  • X 7 is Q, K, E or H;
  • X 8 is S, T, N, A or D;
  • X 9 is E or Q;
  • Xi 0 is V, I, L or M;
  • X 11 is K, T, N or S;
  • Xi 2 is S, A or C;
  • Xi 3 is L or V;
  • X !4 is A, G, S, V, L or M;
  • X 15 is T or A;
  • X 16 is V, A, D or I;
  • X 17 is G or I;
  • X 15 is T or A;
  • X 16 is V, A, D or I;
  • X 17 is G or I;
  • Variants of SEQ ID NO:1 which are derived from a plant species and encode polypeptides comprising the amino acid sequence:
  • X 1 is N, K, S, T, A, D or V
  • X 2 is P, R or S
  • X 3 is I or M
  • X 4 is K, R, H or S
  • X 5 is V, A or T
  • X 6 is S, P, A or T
  • X 7 is Q, K, E or H
  • X 8 is S, N, T, D or A
  • X 9 is E or Q
  • X 10 is V 5 A, G, I, L or M
  • X 11 is K, N, T or S
  • X 12 is S, A or C
  • X 13 is L, M or V
  • X 14 is A, G, V, S, L or M
  • X 15 is T or A
  • X 16 is E or D
  • X 17 is V, A 5 1 or D
  • X 18 is G or I
  • Xi 9 is T 5 V or P
  • X 20 is K 5 N 5 T 5 E or M
  • X 21 is K
  • SEQ ID NO: 5 to 66 are disclosed herein and listed as SEQ ID NO: 5 to 66.
  • Variants of SEQ ID NO:1 which are derived from a dicotyledonous plant species and encode polypeptides comprising the amino acid sequence:
  • SEQ ID NO:1 which are derived from a monocotyledonous plant species and encode polypeptides comprising the amino acid sequence: S PIXIX 2 SX 3 QX 4 ELX 5 KALX 5 VTX 7 X 8 GTX 9 RDKX IO TX I IDDTTNRINX I2 LLX I3 KI RS I R MCGSLALNMCGVA ( SEQ ID NO : 4 )
  • polypeptides and variants of polypeptides of the invention are disclosed herein and listed as SEQ ID NO: 55 to 65.
  • the polypeptides and variants of polypeptides of the invention may be derived from any species.
  • polypeptides or variants of the invention are derived from plant species.
  • polypeptides or variants of the invention are derived from gymnosperm plant species.
  • polypeptides or variants of the invention are derived from angiosperm plant , species.
  • polypeptides or variants of the invention are derived from dicotyledonuous plant species.
  • polypeptide or variant is derived from a fruit plant 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 and hybrids of the four species and Malus domestica
  • polypeptide or variant is derived from a vegetable plant 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
  • polypeptide or variant is derived from a monocotylenouous plant species. In a further embodiment polypeptide or variant is derived from a crop plant 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
  • X 51 X 52 X 53 (SEQ ID NO :2) wherein X 1 is N, K, S 5 T, A, D or V; X 2 is P, R or S; X 3 is I or M; X 4 is K, R, H or S; X 5 is V, A or T; X 6 is S, P, A or T; X 7 is Q, K, E or H; X 8 is S, N, T, D or A; X 9 is E or Q; X 10 is V, A, G, I, L or M; X 11 is K, N, T or S; Xj 2 is S, A or C; X 13 is L 5 M or V; X 14 is A, G, V, S, L or M; X 15 is T or A; X 16 is E or D; X 17 is V, A, I or D; X 18 is G or I; X 19 is T, V or P; X 20 is K 5 N, T 5 E or M; X 21 is K,
  • X 1 is N 5 K 5 T 5 A, D or V
  • X 2 is P, R or S
  • X 3 is I or M
  • X 4 is K 5 R or H
  • X 5 is V or A
  • X 6 is S 5 A or T
  • X 7 is Q 5 K 5 E or H
  • X 8 is S, T 5 N, A or D
  • X 9 is E or Q
  • X 10 is V, I 5
  • X 11 is K 5 T, N or S
  • X 12 is S 5 A or C
  • X 13 is L or V
  • X 14 is A 5 G 5 S 5 V 5 L or M
  • polypeptides and variants are disclosed herein and listed as SEQ ID NO: 68 to 112.
  • polypeptide or variants are disclosed herein and listed as SEQ ID NO: 113 to 117.
  • Consensus sequences (both DNA and polypeptide) for L-Galactose-1 -phosphate phosphatase for the species Actinidia Arguta, Actinidia deliciosa and Malus domestica axe disclosed herein and listed as SEQ ID NO: 119 to 126. These sequences are also preferred sequences for use in the invention.
  • the plant cells and plants of the invention may be from any species.
  • plants cells and plants of the invention are from gymnosperm species.
  • plants cells and plants of the invention are from angiosperm species. In a further embodiment the plants cells and plants of the invention are from dicotyledonuous species.
  • plants cells and plants 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 and hybrids of the four species and Malus domestica
  • plants cells and plants are 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
  • plants cells and plants of the invention are from monocotylenouous species.
  • the variant plants cells and plants are 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
  • plant is intended to include a whole plant, any part of a plant, propagules and progeny of a plant.
  • 'propagule' means any part of a plant that may be used in reproduction or propagation, either sexual or asexual, including seeds and cuttings.
  • polynucleotide(s), 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.
  • a "fragment" of a polynucleotide sequence provided herein is a subsequence of contiguous nucleotides that is capable of specific hybridization to a target of interest, 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.
  • a fragment of a polynucleotide sequence can be used in antisense, gene silencing, triple helix or ribozyme technology, or as a primer, a probe, included in a microarray, or used in polynucleotide-based selection methods of the invention.
  • 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.
  • probe refers to a short polynucleotide that is used to detect a polynucleotide sequence, that is complementary to the probe, in a hybridization-based assay.
  • the probe may consist of a "fragment" of a polynucleotide as defined herein.
  • polypeptide 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 that performs a function that is required for the biological activity and/or provides three dimensional structure of the polypeptide.
  • 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 capable of performing the above enzymatic activity.
  • 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.
  • An isolated molecule may be obtained by any method or combination of methods including biochemical, recombinant, and synthetic techniques.
  • 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.
  • 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, paralogies 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.
  • variants of the inventive polypeptides and polypeptides possess biological activities that are the same or similar to those of the inventive polypeptides or polypeptides.
  • variant with reference to polypeptides and polypeptides encompasses all forms of polypeptides and polypeptides as defined herein.
  • Variant polynucleotide sequences preferably exhibit at least 50%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, 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 [Nov 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 .-//ftp .ncbi.nih. go v/blast/) .
  • the default parameters of bl2seq are utilized except that filtering of low complexity parts should be turned off.
  • the parameter -F F turns off filtering of low complexity sections.
  • the parameter -p selects the appropriate algorithm for the pair of sequences.
  • Polynucleotide sequence identity may also be calculated over the entire length of the overlap between a candidate and subject polynucleotide sequences using global sequence alignment programs (e.g. Needleman, S. B. and Wunsch, C. D. (1970) J. MoI. Biol. 48, 443-453).
  • Needleman- Wunsch global alignment algorithm is found in the needle program in the EMBOSS package (Rice,P.
  • 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://www.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:/www.ebi.ac.uk/emboss/align/.
  • the GAP program may be used which computes an optimal global alignment of two sequences without penalizing terminal gaps. GAP is described in the following paper: Huang, X. (1994) On Global Sequence Alignment. Computer Applications in the Biosciences 10, 227-235.
  • BLASTN as described above is preferred for use in the determination of sequence identity for polynucleotide variants according to the present invention.
  • 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.
  • 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 [Nov 2002]) from NCBI (ftp://ftp.ncbi.nih.gov/blast/).
  • 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 x 10 '6 more preferably less than 1 x 10 "9 , more preferably less than 1 x 10 ⁇ n , more preferably less than 1 x 10 "15 , more preferably less than 1 x 10 "18 and most preferably less than 1 x 10 "21 when compared with any one of the specifically identified sequences.
  • variant polynucleotides of the present invention hybridize to the polynucleotide sequences recited in SEQ ID NO: 1 and 5 to 67, 119, 121 and 123, or complements thereof under stringent conditions.
  • hybridize under stringent conditions refers to the ability of a polynucleotide molecule to hybridize to a target polynucleotide molecule (such as a target polynucleotide molecule immobilized on a DNA or RNA blot, such as a Southern blot or Northern blot) under defined conditions of temperature and salt concentration.
  • the ability to hybridize under stringent hybridization conditions can be determined by initially hybridizing under less stringent conditions then increasing the stringency to the desired stringency.
  • Tm melting temperature
  • Typical stringent conditions for polynucleotide of greater than 100 bases in length would be hybridization conditions such as prewashing in a solution of 6X SSC, 0.2% SDS; hybridizing at 65 0 C, 6X SSC, 0.2% SDS overnight; followed by two washes of 30 minutes each in IX SSC 5 0.1% SDS at 65° C and two washes of 30 minutes each in 0.2X SSC, 0.1% SDS at 65 0 C.
  • exemplary stringent hybridization conditions are 5 to 10° C below Tm.
  • Tm of a polynucleotide molecule of length less than 100 bp is reduced by approximately (500/oligonucleotide length) 0 C.
  • Tm values are higher than those for DNA- DNA or DNA-RNA hybrids, and can be calculated using the formula described in Giesen et ah, Nucleic Acids Res. 1998 Nov l;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 [Nov 2002]) from NCBI (ftp://ftp.ncbi.nih.gov/blast/ r ) via the tblastx algorithm as previously described.
  • variant polypeptide sequences preferably exhibit at least 50%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, 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 [Nov 2002]) in bl2seq, which is publicly available from NCBI (ftp ://ftp.ncbi.nih. gov/blast/).
  • BLASTP from the BLAST suite of programs, version 2.2.5 [Nov 2002]
  • bl2seq which is publicly available from NCBI (ftp ://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.
  • EMBOS S-needle available at http:/www.ebi.ac.uk/emboss/align/
  • GAP Human, X. (1994) On Global Sequence Alignment. Computer Applications in the Biosciences 10, 227-235.) as discussed above are also suitable global sequence alignment programs for calculating polypeptide sequence identity.
  • BLASTP as described above is preferred for use in the determination of polypeptide variants according to the present invention.
  • 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.
  • 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 [Nov 2002]) from NCBI (ftp://ftp.ncbi.nih. gov/blasfrO.
  • the similarity of polypeptide sequences may be examined using the following unix command line parameters:
  • Variant polypeptide sequences preferably exhibit an E value of less than 1 x 10 "6 more preferably less than 1 x 10 '9 , more preferably less than 1 x 10 ⁇ n , more preferably less than 1 x 10 "15 , more preferably less than 1 x 10 "18 and most preferably less than 1 x 10 "21 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.
  • 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.
  • vector refers to a polynucleotide molecule, usually double stranded DNA, which is used to transport the genetic construct into a host cell.
  • the vector may be capable of replication in at least one additional host system, such as E. coli.
  • expression 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.
  • 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.
  • coding sequence is capable of being expressed when it is operably linked to promoter and terminator sequences.
  • “Operably-linked” means that the sequenced to be expressed is placed under the control of regulatory elements that include promoters, tissue-specific regulatory elements, temporal regulatory elements, enhancers, repressors and terminators.
  • 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.
  • 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.
  • promoter refers to nontranscribed 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.
  • 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 TAGATQT)
  • 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.
  • SEQ ID NO: 1 which encodes a polypeptide (SEQ ID NO: 67) with activity as L-Galactose-1 -phosphate phosphatase.
  • SEQ ID NO: 67 polypeptide with activity as L-Galactose-1 -phosphate phosphatase.
  • the applicants have identified a distinct group of polypeptides which are variants of SEQ ID NO: 67 which were not previously known to be L-Galactose-1 -phosphate phosphatases, and further identified a consensus sequence (SEQ ID NO: 2) present in all of such variants.
  • the applicants have identified sub groups of those polypeptides from dicocotyledonous and monotyledonous plant species and identified a consensus sequence (SEQ ID NO: 3) specific to the dicotyledonous polypeptides and a consensus sequence (SEQ ID NO: 4) specific to the monotyledonous polypeptides.
  • the applicants also identified polynucleotide sequences encoding all of the polypeptides disclosed.
  • the invention provides genetic constructs, vectors and plants confirming the polynucleotide sequences.
  • the invention also provides plants comprising the genetic construct and vectors of the invention.
  • the invention provides plants altered relative to wild-type plants in L-Galactose-1 - phosphate phosphatase activity and plants altered to wild-type plants in ascorbic acid content.
  • the invention provides both plants with increased and decreased L-Galactose- 1 -phosphate phosphatase activity and ascorbic content.
  • the invention also provides methods for the production of such plants and methods of selection of such plants.
  • the invention also provides methods for identifying herbicidal compounds which inhibit the activity of the L-Galactose-1 -phosphate phosphatase polypeptides of the invention.
  • polypeptides of the invention can be isolated by using a variety of techniques known to those of ordinary skill in the art.
  • such polypeptides can be isolated through use of the polymerase chain reaction (PCR) described in Mullis et ah, Eds. 1994 The Polymerase Chain Reaction, Birkhauser, incorporated herein by reference.
  • PCR polymerase chain reaction
  • 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 in SEQ ID NO: 1 and 5 to 67, 119, 121, 123 and 125 as hybridization probes.
  • the technique of hybridizing labeled 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 X SSC, 0. 5% sodium dodecyl sulfate, 1 X Denhardt's solution ; washing (three washes of twenty minutes each at 55°C) in 1.
  • polynucleotide fragments of the invention may be produced by techniques well- known in the art such as restriction endonuclease digestion and oligonucleotide synthesis.
  • 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 MA, 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 ah, 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.
  • standard molecular biology approaches can be utilized (Sambrook et al. , Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press, 1987).
  • transgenic plant from a particular species, 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.
  • 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.
  • Variant polypeptides may be identified using PCR-based methods (Mullis et al. , Eds. 1994 The Polymerase Chain Reaction, Birkhauser).
  • 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.
  • valiant sequences of the invention 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 [Nov 2002]) including BLASTN, BLASTP, BLASTX, tBLASTN and tBLASTX, which are publicly available from (ftp ://ftp.ncbi.nih. gov/blast ⁇ or from the National Center for Biotechnology Information (NCBI), National Library of Medicine, Building 38 A, Room 8N805, Bethesda, MD 20894 USA.
  • NCBI National Center for Biotechnology Information
  • the NCBI server also provides the facility to use the programs to screen a number of publicly available sequence databases.
  • BLASTN compares a nucleotide query sequence against a nucleotide sequence database.
  • BLASTP compares an amino acid query sequence against a protein sequence database.
  • BLASTX compares a nucleotide query sequence translated in all reading frames against a protein sequence database.
  • tBLASTN compares a protein query sequence against a nucleotide sequence database dynamically translated in all reading frames.
  • tBLASTX compares the six- frame translations of a nucleotide query sequence against the six-frame translations of a nucleotide sequence database.
  • the BLAST programs may be used with default parameters or the parameters may be altered as required to refine the screen.
  • BLAST family of algorithms including BLASTN, BLASTP 5 and BLASTX, is described in the publication of Altschul et ah, Nucleic Acids Res. 25: 3389-3402, 1997.
  • the "hits" to one or more database sequences by a queried sequence produced by BLASTN, BLASTP, BLASTX 5 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 5 BLASTP 5 BLASTX, tBLASTN and tBLASTX algorithms also produce "Expect" values for alignments.
  • the Expect value (E) indicates the number of hits one can "expect” to see by chance when searching a database of the same size containing random contiguous sequences.
  • the Expect value is used as a significance threshold for determining whether the hit to a database indicates true similarity. For example, an E value of 0.1 assigned to a polynucleotide hit is interpreted as meaning that in a database of the size of the database screened, one might expect to see 0.1 matches over the aligned portion of the sequence with a similar score simply by chance.
  • the probability of finding a match by chance in that database is 1% or less using the BLASTN, BLASTP, BLASTX, tBLASTN or tBLASTX algorithm.
  • Pattern recognition software applications are available for finding motifs or signature sequences.
  • MEME Multiple Em for Motif Elicitation
  • MAST Motif Alignment and Search Tool
  • the MAST results are provided as a series of alignments with appropriate statistical data and a visual overview of the motifs found.
  • MEME and MAST were developed at the University of California, San Diego.
  • PROSITE (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 www.expasy.org/prosite
  • Prosearch is a tool that can search SWISS-PROT and EMBL databases with a given sequence pattern or signature.
  • polypeptide variants of the invention may 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.
  • variant polynucleotide of the invention as an L-Galactose-1 -phosphate phosphatase may be assessed for example by expressing such a sequence in bacteria and testing activity of the encoded protein as described in Example 2.
  • 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 ah, 1969, in Solid-Phase Peptide Synthesis, WH Freeman Co, San Francisco California, or automated synthesis, for example using an Applied Biosystems 43 IA Peptide Synthesizer (Foster City, California). Mutated forms of the polypeptides may also be produced during such syntheses.
  • 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
  • polypeptides and variant polypeptides of the invention may be expressed recombinantly in suitable host cells and separated from the cells as discussed below.
  • the genetic constructs of the present invention comprise one or more polynucleotide sequences of the invention and/or polynycleotides 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.
  • 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
  • polypeptides of the invention 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, VoI 182, Guide to Protein Purification).
  • Host cells of the invention may also be useful in methods for production of an enzymatic product generated by an expressed polypeptide of the invention. Such methods may involve culturing the host cells of the invention in a medium suitable for expression of a recombinant polypeptide of the invention, optionally in the presence of additional enzymatic substrate for the expressed polypeptide of the invention. The enzymatic product produced may then be separated from the host cells or medium by a variety of art standard methods.
  • 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.
  • Alteration of L-Galactose-1 -phosphate phosphatase activity and/or ascorbic acid content may also be altered in a plant through methods of the invention. Such methods may involve the transformation of plant cells and plants, with a construct of the invention designed to alter expression of a polynucleotide or polypeptide which modulates L- Galactose-1 -phosphate phosphatase activity and/or ascorbic acid content in such plant cells and plants.
  • Such methods also include the transformation of plant cells and plants with a combination of the construct of the invention and one or more other constructs designed to alter expression of one or more polypeptides or polypeptides which modulate L-Galactose-1 -phosphate phosphatase activity and/or ascorbic acid content in such plant cells and plants.
  • a number of plant transformation strategies are available (e.g. Birch, 1997, Ann Rev Plant Phys Plant MoI Biol, 48, 297).
  • 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.
  • promoters that are suitable for use in modifying and modulating plant traits using genetic constructs comprising the polynucleotide sequences of the invention.
  • 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 zin gene terminator, the Oryza sativa ADP-glucose pyrophosphorylase terminator and the Solarium tuberosum PI-II terminator.
  • CaMV cauliflower mosaic virus
  • Agrobacterium tumefaciens nopaline synthase or octopine synthase terminators the Zea mays zin gene terminator
  • the Oryza sativa ADP-glucose pyrophosphorylase terminator the Solarium tuberosum PI-II terminator.
  • NPT II neomycin phophotransferase II gene
  • aadA gene which confers spectinomycin and streptomycin resistance
  • phosphinothricin acetyl transferase ⁇ bar gene for Ignite (AgrEvo) and Basta (Hoechst) resistance
  • hpt hygromycin phosphotransferase gene
  • reporter genes coding sequences which express an activity that is foreign to the host, usually an enzymatic activity and/or a visible signal (e.g., luciferase, GUS, GFP) which may be used for promoter expression analysis in plants and plant tissues are also contemplated.
  • a visible signal e.g., luciferase, GUS, GFP
  • 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., Spangenbert. 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.
  • 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.
  • 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
  • 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.
  • 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.
  • nucleotide and/or polypeptide of the invention 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 MoI 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.
  • 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.
  • Ascorbate may be extracted from plants as follows:
  • Each frozen tissue sample was ground to a fine powder in a Cryomill at liquid nitrogen temperature. About 200 mg of frozen powdered tissue was suspended in 4 volumes of 0.5 N HCl containing 4 mM TCEP (Pierce), vortexed for 20 sec and incubated in a heating block for 2 h at 40 0 C. TCEP was 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 was centrifuged at 4 0 C and twenty ⁇ L of the supernatant was injected into a 7.8 x 300 mm Aminex HPX-87H HPLC column (BioRad).
  • the column was run with 2.8 mM H 2 SO 4 , at a flow rate of 0.6 mL/min and the amount of ascorbic acid was calculated from absorbance at 245 nm (retention time 9.6 min), using ascorbic acid (Sigma St Louis) as a standard.
  • the peak was authenticated as ascorbic acid by showing that it was completely degraded by ascorbate oxidase at pH 5.5.
  • Methods are also provided for selecting plants with altered L-Galactose-1 -phosphate phosphatase activity and/or ascorbic acid content. Such methods involve testing of plants for altered for the expression of a polynucleotide or polypeptide of the invention. Such methods may be applied at a young age or early developmental stage to accelerate breeding programs directed toward improving L-Galactose-1 -phosphate phosphatase activity and/or ascorbic acid content.
  • a polynucleotide such as a messenger RNA
  • exemplary methods for measuring the expression of a polynucleotide include but are not limited to Northern analysis, RT-PCR and dot-blot analysis (Sambrook et al, Molecular Cloning : A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press, 1987).
  • Polypeptides or portions of the polypeptides of the invention are thus useful as probes or primers, as herein defined, in methods for the identification of plants with altered levels of polypeptides involved in modulation of L-Galactose-1 -phosphate phosphatase activity and/or ascorbic acid content.
  • an altered level in a plant of a polypeptide involved in L-Galactose-1 -phosphate phosphatase activity and/or ascorbic acid content may be used as an indicator of eventual altered L-Galactose-1 -phosphate phosphatase activity and/or ascorbic acid content in such a plant.
  • the polypeptides of the invention may be used as probes in hybridization experiments, or as primers in PCR based experiments, designed to identify such plants.
  • antibodies may be raised against polypeptides of the invention.
  • Methods for raising and using antibodies are standard in the art (see for example: Antibodies, A Laboratory Manual, Harlow A Lane, Eds, Cold Spring Harbour Laboratory, 1998).
  • Such antibodies may be used in methods to detect altered expression of polypeptides which modulate flower size in plants.
  • Such methods may include ELISA (Kemeny, 1991, A Practical Guide to ELISA, NY Pergamon Press) and Western analysis (Towbin & Gordon, 1994, J Immunol Methods, 72, 313).
  • the plants of the invention may be grown and either selfed or crossed with a different plant strain and the resulting hybrids, with the desired phenotypic characteristics, may be identified. Two or more generations may be grown to ensure that the subject phenotypic characteristics are stably maintained and inherited. Plants resulting from such standard breeding approaches also form an aspect of the present invention.
  • the invention provides methods for identifying herbicidal compounds which bind to and/or inhibit the activity of the polypeptides of the invention.
  • Any compound may be screened as a candidate herbicide using the methods of the invention.
  • compounds that could be screened include inorganic and organic compounds such as, but not limited to, amino acids, peptides, proteins, nucleotides, nucleic acids, glyco-conjugates, oligosaccharides, lipids, alcohols, thiols, aldehydes, alkylators, carbonic ethers, hydrazides, hydrazines, ketons, nitrils, amines, sulfochlorides, triazines, piperizines, sulphonamides and the like.
  • compound libraries are screened in the methods of the invention.
  • 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.
  • Figure 1 shows purification profiles of kiwifruit L-GaI-I -P ase.
  • A G75 chromatography of a crude extract of kiwifruit fruit. Fractions were 6 mL and 20 ⁇ L aliquots were assayed per fraction.
  • B Hitrap Q chromatography of L-GaI- 1-Pase. Fractions 22 to 40 from Fig IA were applied to the column. A gradient from 0 to 0.5 M NaCl was run from fraction 1 to fraction 90. Fractions were 2 mL and 20 ⁇ L were assayed for enzyme activity.
  • C Hitrap Butyl FF chromatography of the phosphatase activity. A gradient from 1 to 0 M (NH 4 ) 2 S ⁇ 4 was run from fraction 1 to fraction 90. Continuous line, OD at
  • Figure 2 shows a gel filtration chromatography profile of L-GaI-I -Phosphate phosphatase activity. Details are given in the methods section and in Fig 1. Fractions were 1 mL and 20 ⁇ L were assayed. Symbols are as for Figure 1, with down triangles at the top of the figure representing the peak elution volumes of standard proteins. These had masses, from left to right, of 136, 68, 34, 17 and 12.4 KD.
  • Figure 3 shows the pH response of Gal -1 -Phosphate phosphatase. Assays were run in 0.2M BTP buffer, pH as specified on the graph, in 2 mM MgCl 2 , 0.5 niM L-GaI-I -P for 30 minutes at 30 C. Data is the mean + standard error of three replicates. Separate experiments, showed very similar responses
  • Figure 4 shows the response of L-Gal-Pase activity to MgCl 2 .
  • the reaction mix contained either 0.4 (triangles) or 0.2 mM (squares) EDTA in 0.2 M Bis Tris Propane buffer, pH 7.0 and 0.5mM Gal 1-P.
  • Assays (30 minutes, 30C) were initiated with enzyme.
  • Free Mg +"1" was calculated using the CHEAQS programme (a program for calculating CHemical Equilibria in AQuatic Systems) written by Wilko Verweij
  • Figure 5 shows the response of L-GaI-I -Pase activity to L-GaI 1-P concentration.
  • Figure 6 shows SDS PAGE of partially purified Arabidopsis L-GaI-I -Pase preparation. Arrows are at the position of the indicated standards.
  • Figure 7 shows a cluster analysis of purported my ⁇ -inositol phosphate phosphatases. The alignment was based on translated Genbank sequences or Arabidopsis (At) TAIR peptide sequences. Tomato sequences (Le) represent isoforms 1 (LeIMPl; SLU39444), 2 (LeIMP2; SLU39443) and 3 (LeIMP3; SLU39059). Rice sequences are coded Os, potato St, The ESTs from the HortResearch fruit database are coded A (apple) or K (kiwifruit), after their EST number. est233909 is the EST shown to posseses L-GaI-I- Pase activity of the invention.
  • Figure 8 shows typical Sypro Ruby stained SDS Gel electrophoresis of a selection of E coli expressed L-galactose-1-P phosphatases. Expressed enzymes were partially purified using a HisTrap column as described in Laing et al, (2004) except for At3g02870, which was purified through gelfiltration and Anion exchange chromatography. Lane 1, standards; Lane 2, 233909; Lane 3, At3g02870; Lane 4, 158264; Lane 5, 159754 (Group 3 MlPase); Lane 6, 169480 (Group 2 MlPase); lane 7, 180144; Lane 8, Atlg31190 (Group 2 MlPase); Lane 9, blank, Lane 10 standards.
  • Figure 9 shows a Clustal analysis of MlPase protein sequences derived from cloned genes.
  • Aa Actinidia aguta; Ad, A. deliciosa; At, Arabidopsis thaliana; Md, Mains domestica; Pa Persea americana; Le, Lycopersicon esculentum (Solanum lycopersicum); Os, Oryza sativa; St, Solanum tuberosum; Ta, Triticum aestivum.
  • the Group 1 genes high lit in yellow have been expressed and specific L-Galactose-1-P phosphatase activity measured.
  • Figure 10 shows the response of Tomato L-Galactose-1-P phosphatase to L- Galactose-1-P, and inhibition by D-Mannose-1-P.
  • Tomato SLU39059 was assayed as a function of L-Galactose concentration in the absence (squares) and presence (triangles) of 4.5 niM D-mannose-1-P.
  • MgC12 was
  • Tomato SLU39059 was assayed as a function of MgC12 concentration in the absence (squares) and presence (triangles) of 4.5 mM D-mannose-1-P.
  • L-Galactose-1-P was 4.5 mM.
  • Hydrolysis of Mannose-1-P was taken into account in the backgrounds. Inhibition at saturating Mg was 28%, while at saturating L-Galactose-1-P it was 48%.
  • FIG 11 shows the response of Tomato L-Galactose-1-P phosphatase to L-Galactose- 1-P, and inhibition by D-Galactose-1-P.
  • Tomato SLU39059 was assayed as a function of MgC12 concentration in the absence (squares) and presence (triangles) of 4.5 mM D- galactose-1-P.
  • L-Galactose-1-P was 4.5 mM. Hydrolysis of D-galactose-1-P was taken into account in the backgrounds. Inhibition at saturating Mg was 42%.
  • FIG 12 shows the response of the three Tomato L-Galactose-1-P phosphatases to L- MgC12. Tomato phosphatases was assayed as a function of MgC12 concentration. L- Galactose-1-P was 4.5 mM. The calculated Mg Ka values were 0.80 ⁇ 0.08, 0.79 ⁇ 0.08 and 0.76 ⁇ 0.07 mM for SLU39443, SLU39444 and SLU39059 respectively. There is no apparent substrate inhibition.
  • FIG 13 shows the response of the three Tomato L-Galactose-1-P phosphatases to L- L-Galactose-1-P.
  • Tomato phosphatases were assayed as a function of L-Galactose-1-P concentration.
  • MgC12 was 4.5 mM.
  • the calculated Mg Km values were 0.169 ⁇ 0.03, 0.296 ⁇ 0.06 and 0.244 + 0.04 mM for SLU39443, SLU39444 and SLU39059 respectively. There is no apparent substrate inhibition.
  • Figure 14 shows an alignment of wheat L-Galactose- 1 -phosphate phosphatase polypeptides discussed in Example 3.
  • Kiwifruit Actinidia deliciosa (A. Chev.) C. F. Liang et A.R. Ferguson var. deliciosa
  • Tissues were stored frozen at -80C until use.
  • the first assay measured the phosphate formed by the hydrolysis of L-GaI-I -P using a microplate based colorimetric phosphate assay (Chifflet et ah, (1988) Anal. Biochem. 168, 1-4) and was used during purification and the characterise the enzyme.
  • the standard conditions for this assay consisted of 0.1 M Bis Tris Propane pH 7.0, 2 mM MgC12 , 0.2 mM
  • the basic assay in 100 ⁇ L volume was the same as the first assay, but
  • GaIDH was assayed in 0.1 M Tris, pH 8, 0.25 mM NAD and L-GaI in a volume of 100
  • Either kiwifruit fruit or Arabidopsis thaliana (CoI-I) leaves were ground to a fine powder under liquid nitrogen and ⁇ 70 grams of frozen tissue was extracted in 100 mL (kiwifruit) or 200 mL ⁇ A. thaliana) of 0.18 M MOPS buffer adjusted to pH 7.0,
  • the active fractions from the butyl column were concentrated using a 10,000 MW cut off centrifugal concentrator to 1 mL, and the sample was applied to a 40 cm x 1.6 cm G75 superdex column equilibrated with the same buffer as the large G75 superdex column. Active fractions were combined and used to characterise the enzyme.
  • Tryptic peptides were separated at a flow rate of 400nL/min with a linear gradient from 2 to 80% B (acetonitrile + 0.1% formic acid) over 50 mins.
  • Solvent A was 0.1% aqueous formic acid.
  • the column flow rate was produced by splitting the primary flow
  • the mass spectrometer was operated in the positive ion mode and the mass range acquired was
  • MS/MS data were analysed using TurboSEQUEST, a computer programme that allows the correlation of experimental data with theoretical spectra generated from known protein sequences (Yates, JR III; Eng, JK; McCormack, AL: Schieltz, D. Anal. Chem. 1995, 67, 1426-1436. Eng, J; McCormack, AL; Yates, JR III. J.Am. Mass Spectrom. 1994, 5, 976-989.). Spectra were searched against the latest version of the public non-redundant protein database of the National Center for Biotechnology Information (NCBI) and with an A. thaliana protein database.
  • NCBI National Center for Biotechnology Information
  • the criteria used for a positive peptide identification for a doubly-charged peptide were a correlation factor (XCorr) greater than 2.0, a delta cross- correlation factor (dCn) greater than 0.1 (indicating a significant difference between the best match reported and the next best match) and a high preliminary scoring (Sp).
  • XCorr correlation factor
  • dCn delta cross- correlation factor
  • Sp high preliminary scoring
  • the correlation factor threshold was set at 2.5. All matched peptides were confirmed by visual examination of the spectra.
  • the full length cDNA was obtained from the HortResearch kiwifruit EST resource cloned into Bluescript SK(-).
  • the 880 bp fragment was cut out using BAMHl and HindIII and cloned in frame into PET30c(+) (Novagen, San Diego, CA) and this construct was transformed into E. coli BL21.
  • the construct was verified by PCR amplification form PET primers and by restriction enzyme digestion. This construct contains an N terminal His 6 tag.
  • E coli was grown to an OD600 of 0.6 at 37 C, then the protein was expressed under IPTG control (0.3mM) for 20 hours at 2OC.
  • Fractions containing protein were exhaustively dialysed against 10 mM BTP, pH 7.1, 1 mM EDTA, 5 mM B mercaptoethanol and 30 mM NaCl.
  • the first major peak of phosphatase activity was unaffected by MgCl 2 , while the second peak was stimulated nine fold by MgCl 2 (Table 1).
  • the first peak which had a optimum below pH 6 (data not shown), was most likely a non-specific acid phosphatase with activity against L-GaI-I -P.
  • the L-GaI-I -P ase was linear with the amount of enzyme until at least 10 nmoles of phosphate had been generated in the assay, which represented about 20% of the substrate present in the assay.
  • the activity was also linear with time until over 30 minutes.
  • the pH optimum showed a well-defined maximum at pH 7 with a fall off to half the maximum volume within 0.6 of a pH unit to either side of the maximum (Fig 3).
  • the K M (Gal-l-P) was dependent on MgCl 2 , being 0.041 ⁇ 0.002 mM at 1.8 mM
  • the enzyme was also prepared from A. thaliana seedlings and the the protein from this source was specifically identified.
  • the A. thaliana shoot preparation of the L-GaI-I- Pase showed similar properties to the kiwifruit enzyme, showing absolute Mg +"1"
  • the K M (L-Gal-1-P) was 0.044 ⁇ 0.003 mM and the pH optimum was between 6.8
  • the putative phosphatase protein was Arabidopsis protein AT3g02870, a protein annotated in TAIR as a putative inositol- 1 (or 4)-monophosphatase with a predicted MW of 29121 kD and a predicted pi of 5.01.
  • the report of myo inositol- 1-Pase activity in the tomato genes was based on screening a phage library for 5-bromo-4-chloro-3-indoyl phosphate phosphatase activity, and using the one positive clone (LeIMP) to screen a young fruit cDNA library for two other clones ( Gillaspy et al, 1995, Plant Cell 7, 2175-85).
  • EXAMPLE 2 CLONING AND BACTERIAL EXPRESSION OF A POLYNUCLEOTIDE FROM KIWIFRUIT ENCODING GAL-I -PHOSPHATE PHOSPHATASE
  • Gal-l-P of 0.15 ⁇ 0.02 mM
  • K M Myo inositol-1-P
  • Peak 1 refers to the average of fractions 29 to 32 in figure 2 while
  • peak 2 refers to fractions 70 to 73. Assays of 20 ⁇ L for each fraction were carried out
  • Actinidia arguta start of translated EST sequence 180844 Malus domestica variety Fruit stored for 24 hours under low Initial M in original
  • Actinidia deliciosa after hydrogen cyanamide UTR of the EST in treatment KALA (already in front of the N terminal patent) 260086 Persea americana, variety Avocado fruit (QAAB) Identical to EST
  • At3g02870 Arabidopsis thaliana Group 1 enzyme cDNA clone Identical to EST U09692 from TAIR sequence
  • Lycopersicon esculentum sequence (Solarium lycopersiciim)
  • Lycopersicon esculentum sequence (Solarium lycopersicum)
  • Inhibitors of L-Galactose-1-P phosphatase (233909). Inhibitors were included in the assay at 2.3 mM and assayed at 1 mM L-Galactose-1-P and 2 mM MgC12.
  • D-galactose- 1 -P was more inhibitory than D- mannose-1-P at saturating Mg (42%) as shown in figure 11.
  • the tomato phosphatases showed considerable differences in their specifity for D-galactose-1-P compared to 7?r ⁇ o-inositol-l-P (ratio of GaI-I -Pase to MIPase ranging from 11 to 56). To see if this reflected changes in the kinetic properties of the enzyme, we measured the response to Mg and L-Galactose-1-P for these three enzymes ( Figures 12 and 13).
  • the enzymes showed similar constants for Mg (0.76 to 0.8 mM) and L-Galactose-1-P (0.169 to 0.296 mM). This suggests that the differences in the specifity ratios were either due to differences in the Km(myo-inositol-l-P) (not measured, but unlikely as 1 mM myo-inositol-1-P was used in the assay, which should be near saturating (see Laing et al, 2004 PNAS 101, 16976-81) or due to intrinsic differences in the catalytic hydrolysis rate for the two substrates.
  • EXAMPLE 4 PLANT TRANSFORMATION WITH A POLYNUCLEOTIDE ENCODING GAL-1-PHOSPHATE PHOSPHATASE
  • ⁇ SAK778 is a derivative of pART7 and ⁇ ART27 (Gleave, et al. 1992, Plant MoI Biol. 20(6): 1203-7). To generate ⁇ SAK778 the following steps were carried out: The 49 bp Sall-Xbal fragment from the polylinker of pBK-CMV (Stratagene) was cloned into the Xhol-Xbal sites of pART7, generating ⁇ SAK7.
  • the 2187 bp SacI-NheI fragment of pSAK7 was then cloned into the Sacl-Spel sites of pART7, replacing the pART7 CaMV35S promoter-multiple cloning site-ocs 3' transcriptional terminator cassette with the corresponding pSAK7 cassette carrying the alternative multiple cloning site and generating pSAK8.
  • the 2171 bp 35S-mcs-ocs3' cassette of was then cloned as aNotl fragment into pART77, generating pSAK778.
  • pART77 was generated by replacing the 1578 bp Nhel-Aflll fragment of pART27 with the 963 bp Nhel-Aflll fragment of pGreen0029SK62, generating pART67. pART67 was then cleaved with Spel and ligated with the oligonucleotide 5'-CTAGGGCGCGCC-3', to destroy the Spel site of pART67, generating pART77.
  • pSAK778_233909 was introduced into Agrobacterium using standard techniques.
  • Agrobacterium, GV3101(MP90) were cultured on Lennox agar (Invitrogen) supplemented with 50ug.ml-l kanamycin (Sigma) and incubated at 28C.
  • Several lO ⁇ l loops of confluent bacterium were re-suspended in 1 OmI of infiltration media ( 1 OmM Mg C12, 0.5 ⁇ M acetosyringone (Sigma)), to an OD600 of -0.2, and allowed to incubate at room temperature for 2 hours before infiltration.
  • Pl 9 containing Agrobacterium were treated in a similar way.
  • Nicotiana benthamiana were grown in a glass house at an average temperature of 22C using natural light with daylight extension to 16hrs. Plants were grown until they had 4 leaves and the three youngest leaves over 1 cm long were infiltrated with Agrobacterium (Voinnet, et al 2003, Plant J. 33 (5): 949-56) and maintained in the glasshouse for the next 10 days of co- cultivation. Leaves were infiltrated with a mixture of Agrobacterium containing the gene of interest and a separate Agrobacterium containing the Pl 9 gene using a ImL syringe without a needle. Controls were infiltrated with Agrobacterium containing the Pl 9 gene. At harvest the leaves were immediately frozen and stored in liquid nitrogen.
  • Leaves were ground to a powder and then extracted four to six volumes of 200 mM Bis Tris Propane, pH 7.0, 0.5 mM EDTA, 2 mM DTT and one proteinase inhibitor tablet per 25 mL. Extracts were centrifuged and desalted using NAP columns (GE Healthcare) equilibrated with 100 mM Bis Tris Propane pH 7.0, and 0.5 mM EDTA.
  • Extracts were assayed in 10OmM Bis Tris Propane, pH 7.0 with 0.5 mM Gal-l-P and 0.5 mM EDTA with the addition of 2.5 mM MgCl 2 (Mg dependent phosphatase activity) or without the addition OfMgCl 2 (Mg independent phosphatase, or background).
  • MgCl 2 Mg dependent phosphatase activity
  • Mg independent phosphatase Mg independent phosphatase
  • Transient expression in tobacco leaves is a rapid powerful technique to express transgenic protein in a plant environment, in a native format. We used this technique to verify the identity of EST clone 233909.
  • the transiently expressed 233909 was tested for substrate specifity (Table 8).
  • Table 8 Ability of 233909 transiently transformed tobacco extracts to hydrolyze different sugar phosphates. Assays were carried out as described in the methods except with 1 rnM of the selected substrate per assay. Assays were carried out with and without MgCl 2 and the percentages were calculated on the rates corrected for hydrolysis in the absence of Mg. The rate in the absence of Mg and the P19 control rate are expressed as a percentage of the rate with Mg and the same substrate.

Abstract

The invention relates to L-galactose-1-phosphate phosphatases in plants and plants with altered expression of L-galactose-1- phosphate phosphatases. The invention also provides polynucleotides encoding L-galactose-1-phosphate phosphatases from apple, kiwifruit and avocado species and variants of these sequences. The polynucleotides are useful for transforming plants to provide altered expression of L-galactose-1- phosphate phosphatases. The invention also provides isolated L-galactose-1-phosphate phosphatases, host cells, plant selection methods, herbicide identification methods and antibodies.

Description

PHOSPHATASES, POLYNUCLEOTIDES ENCODING THESE AND USES THEREOF
TECHNICAL FIELD
The present invention relates to compositions and methods for producing plants with altered ascorbic acid content
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 all of the 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 duhia, 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. IJ. Warrington and G.C. Weston. 1990, Palmerston North; New Zealand: New Zealand Society for Horticultural Science. 576. Beever, DJ. and G. Hopkirk, Fruit development and fruit physiology. Kiwifruit: science and management, ed. IJ. 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, 30-400mg/l 0Og (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/100g (Beever, DJ. and G. Hopkirk, Fruit development and fruit physiology. Kiwifruit: science and management, ed. IJ. 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, Oct,. 108(10): p. 935-941) ,4. 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-GaI (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-GaI pathway proceeds through L-GaI to galactono-l,4-lactone and thence to ascorbate ( Wheeler et al, 1998, Nature 393, 365- 369). Mutant and biochemical studies show that it is likely that the L-GaI is derived from GDP-D-Man through GDP-L-GaI to L-GaI-I -P, which has been proposed to be converted to L-GaI through the action of a specific unidentified phosphatase.
The enzyme activities and genes for several specific plant phosphatases have been recently identified, for example phosphoglycolate phosphatase Mamedov, T. G.,
Suzuki, K., Miura, K., Kucho Ki, K. & Fukuzawa, H. (2001) J. Biol Chem. 276, 45573-
9. There is also a wide range of information on non-specific acid phosphatases, for example, Turner, W. L. & Plaxton, W. C. (2001) Planta 214, 243-9, Schenk, G.,
Guddat, L. W., Ge, Y., Carrington, L. E., Hume, D. A., Hamilton, S. & de Jersey, J. (2000) Gene 250, 117-25 and Bozzo, G. G., Raghothama, K. G. & Plaxton, W. C.
(2002) Eur. J. Biochem. 269, 6278-86. which would be expected to dephosphorylate L-
GaI-I -phosphate among other substrates, but there appears to be no published information on a plant phosphatase that would catalyse the dephosphorylation of L-
GaI-I -phosphate in a specific manner.
Identification of genes encoding enzymes in the biosynthetic pathway for ascorbate production provides the opportunity for gene-based approaches to manipulation of ascorbate content in plants.
It is an object of the invention to provide improved compositions and methods for modulating L-Galactose-1 -phosphate phosphatase activity and/or ascorbate content in plants or at least to provide the public with a useful choice.
SUMMARY OF THE INVENTION
In a first aspect the invention provides plant cell or plant with altered L-Galactose-1 - phosphate phosphatase expression, the method comprising transformation of a plant cell or plant with a genetic construct including: a) a polynucleotide encoding of a polypeptide with the amino acid sequence of any one of SEQ ID NO:67 to 118, 120, 122, 124, 126, 130 to 132, or a variant of the polypeptide from a plant species, wherein the variant has the activity of an L-Galactose- 1 -phosphate phosphatase; b) a polynucleotide comprising a fragment, of at at least 15 nucleotides in length, of the polynucleotide of a), or c) a polynucleotide comprising a compliment, of at at least 15 nucleotides in length, of the polynucleotide of a); or d) a polynucleotide comprising a sequence, of at at least 15 nucleotides in length, capable of hybridising to the polynucleotide of a) under stringent conditions
In one embodiment the variant has at least 50% sequence identity to a polypeptide with the amino acid sequence of any one of SEQ ID NO: 67 to 118, 120, 122, 124, 126, 130 to 132.
In a further embodiment the variant comprises the amino acid sequence: X1X2X3X4X5SX6X7X8X9LX1OX11X12LX13X14X15Xl6Xl7Xl8Xl9X2θRDX2lX22X23X24X2S X26X27TX28X29lX3θX3lX32X33X34X35X36X37X38X39X4θX4lX42X43X44X45X46X47X48X49X5
C)X51X52X53 (SEQ ID NO:2) wherein X1 is N5 K, S5 T, A, D or V; X2 is P5 R or S; X3 is I or M; X4 is K, R5 H or S; X5 is V5 A or T; X6 is S5 P5 A or T; X7 is Q5 K5 E or H; X8 is S, N5 T5 D or A; X9 is E or Q; X10 is V5 A5 G, I5 L or M; X11 is K, N5 T or S; X12 is S5 A or C; X13 is L5 M or V; X14 is A, G5 V5 S5 L or M; X15 is T or A; X16 is E or D; X17 is V5 A5 1 or D; X18 is G or I; X19 is T5 V or P; X20 is K5 N5 T, E or M; X21 is K5 N or Q; X22 is L5 A5 S5 E5 P, T or V; X23 is T5 1 or V; X24 is V5 L or I; X25 is D5 G or E; X26 is A5 D5 V5 1 or T; X27 is T5 S5 A5 C or V; X28 is N5 D5 G or R; X29 is R5 K or T; X30 is N or K; X31 is S, R5 K, N or G; X32 is L or V; X33 is L or I; X34 is F5 Y or T; X35 is K or E; X36 is V5 1 or G; X37 is R or A; X38 is S, A or V; X39 is L, I5 P or V; X40 is R or E; X41 is M or D; X42 is S, C5 G5 T or A; X43 is G or R; X44 is S or L; X45 is C, L or V; X46 is A or R; X47 is L or T; X48 is N5 D, A or G; X49 is L5 M or T; X50 is C or L; X51 is G5 W or R; X52 is I5 V or C; X53 is A or R.
In a further embodiment the the variant comprises the amino acid sequence:
X1X2X3LTX4X5X6TWIVDPX7DGTTNFVHGX8PX9VCVSIGLTIX10KX11PX12VX13V VYY14PrX15X16ELFTX17X18X19GX2OGAX21LNGX22 (SEQ ID NO: 133) wherein X1 is T5V5 N5 D or I5 X2 is T5 D5 F or A5 X3 is E or D, X4 is D or Y5 X5 is E5 D or Q5 X6 is P or H5 X7 is L or V5 X8 is F or Y5 X9 is F or S, X10 is G, E or A5 X11 is I5 V or K5 X12 is T or V5 X13 is G or A5 X14 is N or D5 X15 is M or I, X16 is D5 N or E5 X17 is G or A, X18 is I or V5 X19 is R5 H5 D5 L5 N5 Q or Y5 X20 is K5 Q5 G or R5 X21 is F or Y and X22 is K, N or S
In a further embodiment the variant is from a dicotyledonous species and comprises the amino acid sequence:
X1X2X3X4X5SX6X7X8X9LX1oX11X12LX13X14X15EX16X17X18X19RDX2oX21X22X23X24X2 5X26TX27X2sIX29X3θX3lX32X33X34X35X36X37X38X39X4θX4lX42X43X44X45X46X47X48X49X
S0X51 (SEQ ID NO:3)
wherein X1 is N5 K5 T5 A5 D or V; X2 is P5 R or S; X3 is I or M; X4 is K5 R or H; X5 is V or A; X6 is S, A or T; X7 is Q5 K5 E or H; X8 is S5 T5 N5 A or D; X9 is E or Q; X10 is V5 15 L or M; X11 is K5 T5 N or S; X12 is S5 A or C; X13 is L or V; X14 is A5 G5 S5 V5 L or M; X15 is T or A; X16 is V5 A5 D or I; X17 is G or I; X18 is T5 V or P; X19 is K5 N5 T5 E or M; X20 is K5 N or Q; X21 is L5 A5 S5 E5 P or V; X22 is T5 1 or V; X23 is V5 L or I; X24 is D5 G or E; X25 is A5 D5 V or T; X26 is T5 S5 A5 C or V; X27 is N5 D5 G or R; X28 is R5 K or T; X29 is N or K; X30 is S5 R5 N or G; X31 is L or V; X32 is L or I; X33 is F5 T or Y; X34 is K or E; X35 is V or G; X36 is R or A; X37 is S or V; X38 is L5 15 P or V; X39 is R or E; X40 is M or D; X41 is S, C, G, T or A; X42 is G or R; X43 is S or L; X44 is C or V; X45 is A or R; X46 is L or T; X47 is N, D, A or G; X48 is L or T; X49 is C or L; X50 is G, W or R; X51 is I, V or C.
In a further embodiment the variant is from a dicotyledonous species and comprises the amino acid sequence:
X1X2X3LTDX4X5TWrVDPX6DGTTNFVHGX7PX8VCVSIGLTIX9KX1OPX11VGVVY X12PrX13X14ELFTXi5X16Xi7GX18GAXi9LNGX2O (SEQ ID NO: 134) wherein X1 is V, N, D, I or T, X2 is T, F or A, X3 is E or D, X4 is E, D or Q, X5 is P or H, X6 is L or V, X7 is F or Y, X8 is F or S, X9 is G, E or A, X10 is V, I or K, Xn is T or V, Xi2 is N or D, Xi3 is I or M, X14 is D, E or N, Xi5 is G or A, X16 is I or V, Xi7 is H, D, L5 N, Q, R or Y, X18 is K, Q, G or R, Xi9 is F or Y and X20 is K or TST.
In a further embodiment the variant is from a monocotyledonous species and comprises the amino acid sequence:
SPIX1X2SX3QX4ELX5KALX6VTX7X8GTX9RDKX1OTX11DDTTNRINXJ2LLX13KIRSI RMCGSLALNMCGVA (SEQ ID NO:4) wherein X1 is K or R; X2 is T or A; X3 is S or P; X4 is N or D; X5 is V or A; X6 is L or M; X7 is E or D; X8 is V or A; X9 is K or N; Xi0 is A, S or T; Xn is L or V; Xi2 is K or R; X13 is F or Y.
In one embodiment the polynucleotide of a) encodes a polypeptide with the amino acid sequence of any one of SEQ ID NO: 67 to 118, 120, 122, 124, 126, 130 to 132.
In a further embodiment the polynucleotide of a) encodes a polypeptide with the amino acid sequence of SEQ ID NO:67.
In a further aspect the invention provides a method of producing a plant cell or plant with altered L-Galactose-1 -phosphate phosphatase expression, the method comprising transformation of a plant cell or plant with a genetic construct including: a) a polynucleotide comprising a nucleotide sequence selected from any one the sequences of SEQ ID NO: 1, 5 to 54, 55 to 65, 119, 121, 123, 125 and 127 to 129, or a variant thereof from a plant species, wherein the variant encodes a polypeptide which has the activity of an L-Galactose-1 -phosphate phosphatase; b) a polynucleotide comprising a fragment, of at at least 15 nucleotides in length, of the polynucleotide of a), or c) a polynucleotide comprising a complement, of at at least 15 nucleotides in length, of the polynucleotide of a); or d) a polynucleotide comprising a sequence, of at at least 15 nucleotides in length, capable of hybridising to the polynucleotide of a) under stringent conditions
In one embodiment the polynucleotide of a) comprises any one the sequences of SEQ ID NO: 1, 5 to 54, 55 to 65, 119, 121, 123, 125 and 127 to 129.
In a further embodiment the polynucleotide of a) comprises the sequence of SEQ ID NO: 1.
Preferably the plant or plant cell produced by the methods of the invention have increased L-Galactose- 1 -phosphate phosphatase activity.
Preferably the plant or plant cell produced by the methods of the invention have altered ascorbate content, preferably increased ascorbate content.
In a further aspect the invention provides an isolated polynucleotide having at least 82% sequence identity to a nucleotide sequence that encodes a polypeptide comprising an amino acid sequence selected from any one of SEQ ID NO: 67 to 118, 120, 122, 124, 126, 130 to 132, wherein the polynucleotide encodes an L-Galactose- 1 -phosphate phosphatase.
In one embodiment said nucleotide sequence comprises a sequence selected from any one of SEQ ID NO: 1, 5 to 54, 55 to 65, 119, 121, 123, 125 and 127 to 129.
In a further embodiment said said nucleotide sequence comprises a full-length coding sequence selected from any one of SEQ ID NO: 1, 5 to 54, 55 to 65, 119, 121, 123, 125 and 127 to 129.
In a further embodiment said nucleotide sequence comprises the nucleotide sequence of SEQ ID NO: 1. In a further embodiment said nucleotide sequence comprises the full-length coding sequence of SEQ ID NO: 1.
In a further aspect the invention provides an isolated polynucleotide that encodes a polypeptide comprising an amino acid sequence selected from any one of SEQ ID NO: 67 to 118, 120, 122, 124, 126, 130 to 132.
In one embodiment the polynucleotide encodes a polypeptide comprising the amino acid sequence of SEQ ID NO : 67.
In a further embodiment the polynucleotide comprises a sequence selected from any one of SEQ ID NO: 1, 5 to 54, 55 to 65, 119, 121, 123, 125 and 127 to 129.
In a further embodiment the polynucleotide comprises the full-length coding sequence from any one of SEQ ID NO: 1, 5 to 54, 55 to 65, 119, 121, 123, 125 and 127 to 129.
In a further embodiment the polynucleotide comprises the sequence of SEQ ID NO: 1
In a further embodiment the polynucleotide comprises the full-length coding sequence from within the sequence of SEQ ID NO: 1.
In a further aspect the invention provides an isolated polynucleotide comprising a sequence selected from any one of SEQ ID NO: I5 5 to 54, 55 to 65, 119, 121, 123, 125 and 127 to 129 or a variant thereof, wherein the variant is from an apple, kiwifruit or avocado species, and encodes a polypeptide which has the activity of an L-Galactose-1- phosphate phosphatase.
In one embodiment the isolated polynucleotide comprises the sequence of SEQ ID NO: 1 or a variant thereof, wherein the variant is from an apple, kiwifruit or avocado species, and encodes a polypeptide which has the activity of an L-Galactose-1 -phosphate phosphatase.
In one embodiment the isolated polynucleotide comprises the sequence of SEQ ID NO: 1 In a further aspect the invention provides an expression construct which includes a polynucleotide comprising the sequence selected from any one of SEQ ID NO: 1, 5 to 45, 47 to 54, 55 to 65, 119, 121, 123, 125 and 127 to 129 or a variant thereof from a plant species, wherein the variant encodes a polypeptide which has the activity of an L- Galactose-1 -phosphate phosphatase, and wherein the variant does not encode the polypeptide of Accession No. AAB19030.
In one embodiment the expression construct includes a polynucleotide comprising the sequence of SEQ ID NO: 1, or a variant thereof from a plant species, wherein the variant encodes a polypeptide which has the activity of an L-Galactose-1 -phosphate phosphatase, and wherein the variant does not encode the polypeptide of Accession No. AAB19030.
In a further embodiment the expression construct includes a polynucleotide comprising the sequence of SEQ ID NO: 1.
In a farther aspect the invention provides an isolated L-Galactose-1 -phosphate phosphatase polypeptide from a plant species excluding the polypeptide of Accession No. AAB19030.
In a further aspect the invention provides an isolated polypeptide having at least 81 % sequence identity to an amino acid sequence selected from any one of of SEQ ID NO: 67 to 118, 120, 122, 124, 126, 130 to 132, wherein the polypeptide has the activity of an L-Galactose-1 -phosphate phosphatase
In one embodiment the isolated polypeptide has at least 81 % sequence identity to the amino acid sequence of SEQ ID NO: 67.
In a further embodiment the isolated polypeptide of comprises the amino acid sequence of SEQ ID NO: 67 to 118, 120, 122, 124, 126, 130 to 132.
In a further embodiment the isolated polypeptide comprises the amino acid sequence of SEQ ID NO: 67. In a farther aspect the invention provides an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 67 to 105, 107 to 118, 120, 122, 124, 126, 130 to 132, or a variant thereof from a plant species, wherein the variant has the activity of an L- Galactose- 1 -phosphate phosphatase, and wherein the variant is not the polypeptide of Accession No. AAB19030.
In one embodiment the isolated polypeptide comprises the amino acid sequence of SEQ ID NO: 67, or a variant thereof from a plant species, wherein the variant has the activity of an L-Galactose-1 -phosphate phosphatase, and wherein the variant is not polypeptide of Accession No. AAB19030.
In a farther aspect the invention provides an isolated polynucleotide encoding a polypeptide of the invention.
In a farther aspect the invention provides an isolated polynucleotide comprising: a) a polynucleotide comprising a fragment, of at at least 15 nucleotides in length, of a polynucleotide of the invention; b) a polynucleotide comprising a complement, of at at least 15 nucleotides in length, of the polynucleotide of the invention; or d) a polynucleotide comprising a sequence, of at at least 15 nucleotides in length, capable of hybridising to the polynucleotide of the invention.
In a farther aspect the invention provides a genetic construct which comprises a polynucleotide of the invention.
In a farther aspect the invention provides a vector comprising an expression construct or genetic construct of the invention.
In a farther aspect the invention provides a host cell genetically modified to express a polynucleotide of the invention, or a polypeptide of the invention. In a further aspect the invention provides a host cell comprising an expression construct or genetic construct of the invention.
In a further aspect the invention provides a method for producing an L-Galactose-1 - phosphate phosphatase polypeptide comprising culturing a host cell comprising an expression construct of the invention or a genetic construct of the invention, capable of expressing an L-Galactose-1 -phosphate phosphatase polypeptide.
In a further aspect the invention provides a method for producing the enzymic product of an L-Galactose-1 -phosphate phosphatase comprising culturing a host cell including an expression construct of the invention or an genetic construct of the invention, capable of expressing an L-Galactose-1 -phosphate phosphatase polypeptide, in the presence of enzymic substrate which may be supplied to, or may be naturally present within the host cell.
In a further aspect the invention provides a method for the biosynthesis of ascorbate comprising the steps of culturing a host cell comprising an expression construct of the invention or the genetic construct of the invention, capable of expressing an L- Galactose-1 -phosphate phosphatase polypeptide, in the presence of an ascorbate precursor which may be supplied to, or may be naturally present within the host cell.
In a further aspect the invention provides a plant cell genetically modified to express a polynucleotide of the invention, or a polypeptide of the invention.
In a further aspect the invention provides a plant cell which comprises an expression construct of the invention or the genetic construct of the invention.
In a further aspect the invention provides a plant which comprises a plant cell of the inenetion.
In a further aspect the invention provides a method for selecting a plant altered in L- Galactose-1 -phosphate phosphatase activity, the method comprising testing of a plant for altered expression of a polynucleotide of the invention. In a further aspect the invention provides a method for selecting a plant with altered ascorbic acid content; the method comprising testing of a plant for altered expression of a polynucleotide of the invention.
In a further aspect the invention provides a method for selecting a plant altered in L- Galactose-1 -phosphate phosphatase activity, the method comprising testing of a plant for altered expression of a polypeptide of the invention.
In a further aspect the invention provides a method for selecting a plant altered in ascorbic acid content, the method comprising testing of a plant for altered expression of a polypeptide of the invention.
In a further aspect the invention provides a plant cell or plant produced by the method of the invention.
In a further aspect the invention provides a plant selected by the method of the invention.
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 a method for identifying a compound as a candidate for a herbicide, comprising: a) contacting said compound with a polypeptide comprising a sequence selected from any one of SEQ ID NO : 67 to 118, 120, 122, 124, 126, 130 to 132, or a variant thereof which has the activity of an L-Galactose-1 -phosphate phosphatase, and b) detecting the presence and/or absence of binding between said compound and said polypeptide; wherein binding indicates that said compound is a candidate for a herbicide.
In a further aspect the invention provides a method for identifying a compound as a candidate for a herbicide, comprising: a) contacting said compound with a polypeptide comprising a sequence selected from any one of SEQ ID NO : 67 to 118, 120, 122, 124, 126, 130 to 132, or a variant thereof which has the activity of an L-Galactose-1 -phosphate phosphatase, and b) assessing the effect of the compound the an L-Galactose-1 -phosphate phosphatase activity of the polypeptide; wherein a decrease in activity indicates that said compound is a candidate for a herbicide.
In one embodiment the variant has at least 50% sequence identity to an amino acid sequence selected from any one of SEQ ID NO: 67 to 118, 120, 122, 124, 126, 130 to 132.
In a further embodiment the polypeptide comprises an amino acid sequence selected from any one of SEQ ID NO: 67 to 118, 120, 122, 124, 126, 130 to 132.
In a further embodiment the variant has at least 50% sequence identity to the amino acid sequence of SEQ ID NO: 67.
In a further embodiment the polypeptide comprises an amino acid sequence of SEQ ID NO: 67.
In a further aspect the invention provides a compound identified by a method of the invention.
In a further aspect the invention provides a method for determining whether the compound of the invention has herbicidal activity, comprising: contacting a plant or plant cells with said herbicide candidate and detecting the presence or absence of a decrease in growth or viability of said plant or plant cells.
In a further aspect the invention provides an antibody raised against a polypeptide of the invention.
In a further aspect the invention provides method of converting galactose- 1 -phosphate to galactose, the method comprising contacting galactose- 1 -phosphate with the expression product of an expression construct comprising a polynucleotide of the invention to obtain galactose.
The polynucleotides and variants of polynucleotides, of the invention may be derived from any species.
In one embodiment the polynucleotide or variant, is derived from a plant species.
In a further embodiment the polynucleotide or variant, is derived from a gymnosperm plant species.
In a further embodiment the polynucleotide or variant, is derived from a angiosperm plant species.
In a further embodiment the polynucleotide or variant, is derived from a from dicotyledonuous plant species.
In a further embodiment the polynucleotide or variant, is derived from a fruit plant 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 deϊiciosa, A. chinensis, A. eriantha, A. arguta and hybrids of the four species and Malus domestica In a further embodiment the polynucleotide or variant, is derived from a vegetable plant 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 polynucleotide or variant, is derived from a monocotylenouous plant species.
In a further embodiment the polynucleotide or valiant, is derived from a crop plant 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 one embodiment the polynucleotide or variant is derived from a plant species and encodes polypeptide comprising the amino acid sequence:
X1X2X3X4X5SX6X7X8XgLX1OXnX12LX13X14X1SX16X17X18X19X2ORDX21X22X23X24X25
X26X27TX28X29lX3θX3lX32X33^34X35X36X37X38X39X4θX4lX42X43X44X45X46X47X48X49X5
OX51X52X53 (SEQ ID NO:2) wherein X1 is N, K, S, T, A, D or V; X2 is P, R or S; X3 is I or M; X4 is K, R, H or S; X5 is V, A or T; X6 is S, P, A or T; X7 is Q, K, E or H; X8 is S, N, T, D or A; X9 is E or Q;
X10 is V, A, G, I, L or M; X11 is K, N, T or S; X12 is S, A or C; X13 is L, M or V; X14 is
A, G, V, S3 L or M; X15 is T or A; X16 is E or D; X17 is V, A, I or D; X18 is G or I; X19 is
T, V or P; X20 is K, N5 T, E or M; X21 is K, N or Q; X22 is L5 A, S, E, P, T or V; X23 is
T, I or V; X24 is V, L or I; X25 is D, G or E; X26 is A, D, V, I or T; X27 is T, S, A, C or V; X28 is N5 D, G or R; X29 is R, K or T; X30 is N or K; X31 is S, R, K, N or G; X32 is L or V; X33 is L or I; X34 is F, Y or T; X35 is K or E; X36 is V, I or G; X37 is R or A; X38 is
S, A or V; X39 is L, I, P or V; X40 is R or E; X41 is M or D; X42 is S, C, G, T or A; X43 is
G or R; X44 is S or L; X45 is C5 L or V; X46 is A or R; X47 is L or T; X48 is N5 D5 A or G;
X49 is L, M or T; X50 is C or L; X51 is G5 W or R; X52 is I5 V or C; X53 is A or R
In a further embodiment the polynucleotide or variant is derived from a dicotyledonous plant species and encodes a polypeptide comprising the amino acid sequence:
Figure imgf000016_0001
5X26TX27X28IX29X3θX3lX32X33X34X35X36X37X38X39X4θX4lX42X43X44X45X46X47X48X49X
Figure imgf000016_0002
wherein X1 is N5 K, T, A, D or V; X2 is P, R or S; X3 is I or M; X4 is K, R or H; X5 is V or A; X6 is S3 A or T; X7 is Q, K, E or H; X8 is S, T, N, A or D; X9 is E or Q; Xi0 is V, I, L or M; X11 is K, T, N or S; Xi2 is S, A or C; Xi3 is L or V; X!4 is A, G, S, V, L or M; X15 is T or A; X16 is V, A, D or I; X17 is G or I; X18 is T, V or P; X19 is K, N, T, E or M; X20 is K, N or Q; X21 is L, A, S, E, P or V; X22 is T, I or V; X23 is V, L or I; X24 is D, G or E; X25 is A, D, V or T; X26 is T, S, A, C or V; X27 is N5 D, G or R; X28 is R, K or T; X29 is N or K; X30 is S, R, N or G; X31 is L or V; X32 is L or I; X33 is F, T or Y; X34 is K or E; X35 is V or G; X36 is R or A; X37 is S or V; X38 is L, I, P or V; X39 is R or E; X40 is M or D; X41 is S, C, G, T or A; X42 is G or R; X43 is S or L; X44 is C or V; X45 is A or R; X46 is L or T; X47 is N5 D, A or G; X48 is L or T; X49 is C or L; X50 is G, W or R; X51 is I, V or C;
In a further embodiment the polynucleotide or variant is derived from a monocotyledonous plant species and encodes a polypeptide comprising the amino acid sequence:
S P IXIX2SX3QX4ELX5KALX6VTX7X8GTX9RDKX1OTXIIDDTTNRINX12LLXI3KIRS IR MCGSLALNMCGVA ( SEQ ID NO : 4 )
X1 is K or R; X2 is T or A; X3 is S or P; X4 is N or D; X5 is V or A; X6 is L or M; X7 is E or D; X8 is V or A; X9 is K or N; Xi0 is A, S or T; Xn is L or V; Xi2 is K or R; X13 is F or Y;
Variants of SEQ ID NO:1 which are derived from a plant species and encode polypeptides comprising the amino acid sequence:
X1X2X3X4X5SX6X7X8X9LX1oX1iX12LX13X14X15X16X17X18Xi9X2θRDX21X22X23X24X25 X26X27TX28X29IX30X3lX32X33X34X35X36X37X38X39X40X4lX42X43X44X45X46X47X48X49X5
0X51X52X53 (SEQ ID NO:2) wherein X1 is N, K, S, T, A, D or V; X2 is P, R or S; X3 is I or M; X4 is K, R, H or S; X5 is V, A or T; X6 is S, P, A or T; X7 is Q, K, E or H; X8 is S, N, T, D or A; X9 is E or Q; X10 is V5 A, G, I, L or M; X11 is K, N, T or S; X12 is S, A or C; X13 is L, M or V; X14 is A, G, V, S, L or M; X15 is T or A; X16 is E or D; X17 is V, A5 1 or D; X18 is G or I; Xi9 is T5 V or P; X20 is K5 N5 T5 E or M; X21 is K5 N or Q; X22 is L5 A5 S, E5 P5 T or V; X23 is T5 1 or V; X24 is V5 L or I; X25 is D5 G or E; X26 is A5 D5 V, I or T; X27 is T5 S5 A5 C or V; X28 is N5 D5 G or R; X29 is R, K or T; X30 is N or K; X31 is S5 R5 K5 N or G; X32 is L or V; X33 is L or I; X34 is F5 Y or T; X35 is K or E; X36 is V5 1 or G; X37 is R or A; X38 is S, A or V; X39 is L, I, P or V; X40 is R or E; X41 is M or D; X42 is S, C, G, T or A;
X43 is G or R; X44 is S or L; X45 is C, L or V; X46 is A or R; X47 is L or T; X48 is N, D, A or G; X49 is L, M or T; X50 is C or L; X51 is G, W or R; X52 is I, V or C; X53 is A or R
are disclosed herein and listed as SEQ ID NO: 5 to 66.
Variants of SEQ ID NO:1 which are derived from a dicotyledonous plant species and encode polypeptides comprising the amino acid sequence:
X1X2X3X^X5SXeX7X8X9LX1OX11Xi2LXIaXuXi5EX15X17X18X19RDX2θX21X22X23X24X2 5X26TX27X28lX29X3θX3lX32X33X34X35X36X37X38X39X4θX4lX42X43X44X45X46X47X48X49X
Figure imgf000017_0001
are disclosed herein and listed as SEQ ID NO: 5 to 54.
Variants of SEQ ID NO:1 which are derived from a monocotyledonous plant species and encode polypeptides comprising the amino acid sequence: S PIXIX2SX3QX4ELX5KALX5VTX7X8GTX9RDKXIOTXIIDDTTNRINXI2LLXI3KI RS I R MCGSLALNMCGVA ( SEQ ID NO : 4 )
Xi is K or R; X2 is T or A; X3 is S or P; X4 is N or D; X5 is V or A; X6 is L or M; X7 is E or D; X8 is V or A; X9 is K or N; X10 is A, S or T; X11 is L or V; X12 is K or R; X13 is F or Y;
are disclosed herein and listed as SEQ ID NO: 55 to 65. The polypeptides and variants of polypeptides of the invention may be derived from any species.
In one embodiment the polypeptides or variants of the invention are derived from plant species.
In a further embodiment the polypeptides or variants of the invention are derived from gymnosperm plant species.
In a further embodiment the polypeptides or variants of the invention are derived from angiosperm plant , species.
In a further embodiment the polypeptides or variants of the invention are derived from dicotyledonuous plant species.
In a further embodiment polypeptide or variant is derived from a fruit plant 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 and hybrids of the four species and Malus domestica
In a further embodiment polypeptide or variant is derived from a vegetable plant 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 polypeptide or variant is derived from a monocotylenouous plant species. In a further embodiment polypeptide or variant is derived from a crop plant 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 one embodiment the polypeptide or variant is derived from a plant species and comprises the amino acid sequence:
X1X2XsX4X5SX6X7XsXgLXi0Xl IXl2LX13X14X15X16X17X18X19X2ORDX21X22X23X24X25 X26X27TX28X29lX30X3 !X32X33X34X35X36X37X38X39X40X41X42X43X44X45X46X47X48X49X5
OX51X52X53 (SEQ ID NO :2) wherein X1 is N, K, S5 T, A, D or V; X2 is P, R or S; X3 is I or M; X4 is K, R, H or S; X5 is V, A or T; X6 is S, P, A or T; X7 is Q, K, E or H; X8 is S, N, T, D or A; X9 is E or Q; X10 is V, A, G, I, L or M; X11 is K, N, T or S; Xj2 is S, A or C; X13 is L5 M or V; X14 is A, G, V, S, L or M; X15 is T or A; X16 is E or D; X17 is V, A, I or D; X18 is G or I; X19 is T, V or P; X20 is K5 N, T5 E or M; X21 is K, N or Q; X22 is L, A, S, E5 P, T or V; X23 is T, I or V; X24 is V, L or I; X25 is D5 G or E; X26 is A5 D5 V5 1 or T; X27 is T5 S5 A5 C or V; X28 is N5 D5 G or R; X29 is R5 K or T; X30 is N or K; X31 is S5 R5 K5 N or G; X32 is L or V; X33 is L or I; X34 is F5 Y or T; X35 is K or E; X36 is V5 1 or G; X37 is R or A; X38 is S5 A or V; X39 is L5 1, P or V; X40 is R or E; X41 is M or D; X42 is S5 C5 G5 T or A; X43 is G or R; X44 is S or L; X45 is C, L or V; X46 is A or R; X47 is L or T; X48 is N5 D5 A or G; X49 is L5 M or T; X50 is C or L; X51 is G5 W or R; X52 is I5 V or C; X53 is A or R
Such variants are disclosed herein and listed as SEQ ID NO: 68 to 118.
In a further embodiment polypeptide or variant is derived from a dicotyledonous plant species and comprises the amino acid sequence:
X1X2XSX4X5SX6X7X8X9LX1OX11X12LX13X14X15EX16X17X18X19RDX2oX21X22X23X24X2
5X26TX27X28IX29X3oX3lX32X33X34X35X36X37X38X39X4θX4lX42X43X44X45X46X47X48X49X
50X51 (SEQ ID NO:3) wherein X1 is N5 K5 T5 A, D or V; X2 is P, R or S; X3 is I or M; X4 is K5 R or H; X5 is V or A; X6 is S5 A or T; X7 is Q5 K5 E or H; X8 is S, T5 N, A or D; X9 is E or Q; X10 is V, I5
L or M; X11 is K5 T, N or S; X12 is S5 A or C; X13 is L or V; X14 is A5 G5 S5 V5 L or M;
X15 is T or A; Xj6 is V5 A, D or I; Xn is G or I; X18 is T5 V or P; X19 is K5 N, T5 E or M;
Figure imgf000020_0001
Such polypeptides and variants are disclosed herein and listed as SEQ ID NO: 68 to 112.
In a further embodiment polypeptide or variant is derived from a monocotyledonous plant species and comprises the amino acid sequence:
S PIXIX2SX3QX4ELX5KALX6VTX7X8GTX9RDKXIOTX11DDTTNRINXI2LLX13KIRS IR MCGSLALNMCGVA ( SEQ ID NO : 4 )
X1 is K or R; X2 is T or A; X3 is S or P; X4 is N or D; X5 is V or A; X6 is L or M; X7 is E or D; X8 is V or A; X9 is K or N; X10 is A, S or T; X11 is L or V; X12 is K or R; X13 is F or Y;
Such polypeptide or variants are disclosed herein and listed as SEQ ID NO: 113 to 117.
Consensus sequences (both DNA and polypeptide) for L-Galactose-1 -phosphate phosphatase for the species Actinidia Arguta, Actinidia deliciosa and Malus domestica axe disclosed herein and listed as SEQ ID NO: 119 to 126. These sequences are also preferred sequences for use in the invention.
The plant cells and plants of the invention may be from any species.
In one embodiment the plants cells and plants of the invention are from gymnosperm species.
In a further embodiment the plants cells and plants of the invention are from angiosperm species. In a further embodiment the plants cells and plants of the invention are from dicotyledonuous species.
In a further embodiment the plants cells and plants 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 and hybrids of the four species and Malus domestica
In a further embodiment the plants cells and plants are 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 plants cells and plants of the invention are from monocotylenouous species.
In a further embodiment the variant plants cells and plants are 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
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. DETAILED DESCRIPTION
Definitions
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.
A "fragment" of a polynucleotide sequence provided herein is a subsequence of contiguous nucleotides that is capable of specific hybridization to a target of interest, 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. A fragment of a polynucleotide sequence can be used in antisense, gene silencing, triple helix or ribozyme technology, or as a primer, a probe, included in a microarray, or used in polynucleotide-based selection methods 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.
The term "probe" refers to a short polynucleotide that is used to detect a polynucleotide sequence, that is complementary to the probe, in a hybridization-based assay. The probe may consist of a "fragment" of a polynucleotide as defined herein.
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 that performs a function that is required for the biological activity and/or provides three dimensional structure of the polypeptide. 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 capable of performing the above enzymatic activity.
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. 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.
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, paralogies 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.
Variant polynucleotide sequences preferably exhibit at least 50%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, 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 [Nov 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 .-//ftp .ncbi.nih. go v/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 nucleotideseql -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. MoI. 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,! 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://www.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:/www.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.
Use of BLASTN as described above is preferred for use in the determination of sequence identity for polynucleotide variants according to the present invention.
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 [Nov 2002]) from NCBI (ftp://ftp.ncbi.nih.gov/blast/).
The similarity of polynucleotide sequences may be examined using the following unix command line parameters:
bl2seq -i nucleotideseql -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 x 10 '6 more preferably less than 1 x 10 "9, more preferably less than 1 x 10 ~n, more preferably less than 1 x 10 "15, more preferably less than 1 x 10 "18 and most preferably less than 1 x 10 "21 when compared with any one of the specifically identified sequences.
Alternatively, variant polynucleotides of the present invention hybridize to the polynucleotide sequences recited in SEQ ID NO: 1 and 5 to 67, 119, 121 and 123, 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 6X SSC, 0.2% SDS; hybridizing at 650C, 6X SSC, 0.2% SDS overnight; followed by two washes of 30 minutes each in IX SSC5 0.1% SDS at 65° C and two washes of 30 minutes each in 0.2X SSC, 0.1% SDS at 650C.
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)0 C.
With respect to the DNA mimics known as peptide nucleic acids (PNAs) (Nielsen et ah, 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 ah, Nucleic Acids Res. 1998 Nov l;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 [Nov 2002]) from NCBI (ftp://ftp.ncbi.nih.gov/blast/r) via the tblastx algorithm as previously described.
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 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, 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 [Nov 2002]) in bl2seq, which is publicly available from NCBI (ftp ://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. EMBOS S-needle (available at http:/www.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.
Use of BLASTP as described above is preferred for use in the determination of polypeptide variants according to the present invention.
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 [Nov 2002]) from NCBI (ftp://ftp.ncbi.nih. gov/blasfrO. The similarity of polypeptide sequences may be examined using the following unix command line parameters:
bl2seq -i peptideseql -j peptideseq2 -F F -p blastp Variant polypeptide sequences preferably exhibit an E value of less than 1 x 10 "6 more preferably less than 1 x 10 '9, more preferably less than 1 x 10 ~n, more preferably less than 1 x 10 "15, more preferably less than 1 x 10 "18 and most preferably less than 1 x 10 "21 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.
Conservative substitutions of one or several amino acids of a described 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).
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 sequenced to be expressed is placed under the control of regulatory elements that include promoters, tissue-specific regulatory elements, temporal regulatory elements, enhancers, repressors and terminators.
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.
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 "promoter" refers to nontranscribed 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 TAGATQT)
(3 OCTAGAT 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.
The applicants have identified a polynucleotide (SEQ ID NO: 1) which encodes a polypeptide (SEQ ID NO: 67) with activity as L-Galactose-1 -phosphate phosphatase. The applicants have identified a distinct group of polypeptides which are variants of SEQ ID NO: 67 which were not previously known to be L-Galactose-1 -phosphate phosphatases, and further identified a consensus sequence (SEQ ID NO: 2) present in all of such variants. The applicants have identified sub groups of those polypeptides from dicocotyledonous and monotyledonous plant species and identified a consensus sequence (SEQ ID NO: 3) specific to the dicotyledonous polypeptides and a consensus sequence (SEQ ID NO: 4) specific to the monotyledonous polypeptides.
The applicants also identified polynucleotide sequences encoding all of the polypeptides disclosed.
The invention provides genetic constructs, vectors and plants confirming the polynucleotide sequences. The invention also provides plants comprising the genetic construct and vectors of the invention.
The invention provides plants altered relative to wild-type plants in L-Galactose-1 - phosphate phosphatase activity and plants altered to wild-type plants in ascorbic acid content. The invention provides both plants with increased and decreased L-Galactose- 1 -phosphate phosphatase activity and ascorbic content. The invention also provides methods for the production of such plants and methods of selection of such plants. The invention also provides methods for identifying herbicidal compounds which inhibit the activity of the L-Galactose-1 -phosphate phosphatase polypeptides of the invention.
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 ah, 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 in SEQ ID NO: 1 and 5 to 67, 119, 121, 123 and 125 as hybridization probes. The technique of hybridizing labeled 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 X SSC, 0. 5% sodium dodecyl sulfate, 1 X Denhardt's solution ; washing (three washes of twenty minutes each at 55°C) in 1. 0 X SSC, 1% (w/v) sodium dodecyl sulfate, and optionally one wash (for twenty minutes) in 0. 5 X SSC, 1% (w/v) sodium dodecyl sulfate, at 6O0C. An optional further wash (for twenty minutes) can be conducted under conditions of 0. 1 X 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 and oligonucleotide synthesis.
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 MA, 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 ah, 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.
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.
The valiant 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 [Nov 2002]) including BLASTN, BLASTP, BLASTX, tBLASTN and tBLASTX, which are publicly available from (ftp ://ftp.ncbi.nih. gov/blastΛ or from the National Center for Biotechnology Information (NCBI), National Library of Medicine, Building 38 A, 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, BLASTP5 and BLASTX, is described in the publication of Altschul et ah, Nucleic Acids Res. 25: 3389-3402, 1997.
The "hits" to one or more database sequences by a queried sequence produced by BLASTN, BLASTP, BLASTX5 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 BLASTN5 BLASTP5 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, TJ. (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^/www-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. MoI. Biol. (2000) 302: 205-217))or PILEUP, which uses progressive, pairwise alignments. (Feng and Doolittle, 1987, J. MoI. 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 (www.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.
In addition to the computer/database methods described above, polypeptide variants of the invention may 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.
The function of a variant polynucleotide of the invention as an L-Galactose-1 -phosphate phosphatase may be assessed for example by expressing such a sequence in bacteria and testing activity of the encoded protein as described in Example 2. 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 ah, 1969, in Solid-Phase Peptide Synthesis, WH Freeman Co, San Francisco California, or automated synthesis, for example using an Applied Biosystems 43 IA Peptide Synthesizer (Foster City, California). 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
Figure imgf000037_0001
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.
The genetic constructs of the present invention comprise one or more polynucleotide sequences of the invention and/or polynycleotides 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 ah, Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press, 1987 ; Ausubel et al, Current Protocols in Molecular Biology, Greene Publishing, 1987).
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, VoI 182, Guide to Protein Purification).
Host cells of the invention may also be useful in methods for production of an enzymatic product generated by an expressed polypeptide of the invention. Such methods may involve culturing the host cells of the invention in a medium suitable for expression of a recombinant polypeptide of the invention, optionally in the presence of additional enzymatic substrate for the expressed polypeptide of the invention. The enzymatic product produced may then be separated from the host cells or medium by a variety of art standard methods.
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.
Alteration of L-Galactose-1 -phosphate phosphatase activity and/or ascorbic acid content may also be altered in a plant through methods of the invention. Such methods may involve the transformation of plant cells and plants, with a construct of the invention designed to alter expression of a polynucleotide or polypeptide which modulates L- Galactose-1 -phosphate phosphatase activity and/or ascorbic acid content in such plant cells and plants. Such methods also include the transformation of plant cells and plants with a combination of the construct of the invention and one or more other constructs designed to alter expression of one or more polypeptides or polypeptides which modulate L-Galactose-1 -phosphate phosphatase activity and/or ascorbic acid content in such plant cells and plants.
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.
A number of plant transformation strategies are available (e.g. Birch, 1997, Ann Rev Plant Phys Plant MoI Biol, 48, 297). 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 zin gene terminator, the Oryza sativa ADP-glucose pyrophosphorylase terminator and the Solarium 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., Spangenbert. 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 (Mclntyre, 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); maize (US Patent Serial Nos. 5, 177, 010 and 5, 981, 840); wheat (Ortiz et al, 1996, Plant Cell Rep. 15, 1996, 877); tomato (US Patent Serial 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 (US Patent Serial Nos. 5, 846, 797 and 5, 004, 863); grasses (US Patent 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 ScL 104, 183); caraway (Krens et al, 1997, Plant Cell Rep, 17, 39); banana (US Patent Serial No. 5, 792, 935); soybean (US Patent Nos. 5, 416, 011 ; 5, 569, 834 ; 5, 824, 877 ; 5, 563, 04455 and 5, 968, 830); pineapple (US Patent Serial No. 5, 952,
543); poplar (US Patent No. 4, 795, 855); monocots in general (US Patent Nos. 5, 591, 616 and 6, 037, 522); brassica (US Patent Nos. 5, 188, 958 ; 5, 463, 174 and 5, 750, 871); and cereals (US Patent No. 6, 074, 877).
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 MoI 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 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:
Each frozen tissue sample was ground to a fine powder in a Cryomill at liquid nitrogen temperature. About 200 mg of frozen powdered tissue was suspended in 4 volumes of 0.5 N HCl containing 4 mM TCEP (Pierce), vortexed for 20 sec and incubated in a heating block for 2 h at 40 0C. TCEP was 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 was centrifuged at 4 0C and twenty μL of the supernatant was injected into a 7.8 x 300 mm Aminex HPX-87H HPLC column (BioRad). The column was run with 2.8 mM H2SO4, at a flow rate of 0.6 mL/min and the amount of ascorbic acid was calculated from absorbance at 245 nm (retention time 9.6 min), using ascorbic acid (Sigma St Louis) as a standard. The peak was authenticated as ascorbic acid by showing that it was completely degraded by ascorbate oxidase at pH 5.5.
Methods are also provided for selecting plants with altered L-Galactose-1 -phosphate phosphatase activity and/or ascorbic acid content. Such methods involve testing of plants for altered for the expression of a polynucleotide or polypeptide of the invention. Such methods may be applied at a young age or early developmental stage to accelerate breeding programs directed toward improving L-Galactose-1 -phosphate phosphatase activity and/or ascorbic acid content.
The expression of a polynucleotide, such as a messenger RNA, is often used as an indicator of expression of a corresponding polypeptide. Exemplary methods for measuring the expression of a polynucleotide include but are not limited to Northern analysis, RT-PCR and dot-blot analysis (Sambrook et al, Molecular Cloning : A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press, 1987). Polypeptides or portions of the polypeptides of the invention are thus useful as probes or primers, as herein defined, in methods for the identification of plants with altered levels of polypeptides involved in modulation of L-Galactose-1 -phosphate phosphatase activity and/or ascorbic acid content. For example an altered level in a plant, of a polypeptide involved in L-Galactose-1 -phosphate phosphatase activity and/or ascorbic acid content may be used as an indicator of eventual altered L-Galactose-1 -phosphate phosphatase activity and/or ascorbic acid content in such a plant. The polypeptides of the invention may be used as probes in hybridization experiments, or as primers in PCR based experiments, designed to identify such plants.
Alternatively antibodies may be raised against polypeptides of the invention. Methods for raising and using antibodies are standard in the art (see for example: Antibodies, A Laboratory Manual, Harlow A Lane, Eds, Cold Spring Harbour Laboratory, 1998). Such antibodies may be used in methods to detect altered expression of polypeptides which modulate flower size in plants. Such methods may include ELISA (Kemeny, 1991, A Practical Guide to ELISA, NY Pergamon Press) and Western analysis (Towbin & Gordon, 1994, J Immunol Methods, 72, 313). These approaches for analysis of polynucleotide or polypeptide expression and the selection of plants with altered expression are useful in conventional breeding programs designed to produce varieties with altered L-Galactose-1 -phosphate phosphatase activity and/or ascorbic acid content.
The plants of the invention may be grown and either selfed or crossed with a different plant strain and the resulting hybrids, with the desired phenotypic characteristics, may be identified. Two or more generations may be grown to ensure that the subject phenotypic characteristics are stably maintained and inherited. Plants resulting from such standard breeding approaches also form an aspect of the present invention.
The invention provides methods for identifying herbicidal compounds which bind to and/or inhibit the activity of the polypeptides of the invention.
Methods for identifying compounds which bind to such polypeptides are known and described for example in WO 03/077648. Methods for measuring the activity of polypeptides of the invention are described in the Examples provided herein.
Any compound may be screened as a candidate herbicide using the methods of the invention. Examples of compounds that could be screened include inorganic and organic compounds such as, but not limited to, amino acids, peptides, proteins, nucleotides, nucleic acids, glyco-conjugates, oligosaccharides, lipids, alcohols, thiols, aldehydes, alkylators, carbonic ethers, hydrazides, hydrazines, ketons, nitrils, amines, sulfochlorides, triazines, piperizines, sulphonamides and the like. Preferably compound libraries are screened in the methods of the invention. Methods for synthesising and screening compound libraries are known to those skilled in the art. See for example, U. S. Patent Nos. 5,463, 564; 5,574, 656; 5,684, 711; and 5,901, 069, the contents of which are incorporated by reference.
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:
Figure 1 shows purification profiles of kiwifruit L-GaI-I -P ase. A. G75 chromatography of a crude extract of kiwifruit fruit. Fractions were 6 mL and 20 μL aliquots were assayed per fraction. B. Hitrap Q chromatography of L-GaI- 1-Pase. Fractions 22 to 40 from Fig IA were applied to the column. A gradient from 0 to 0.5 M NaCl was run from fraction 1 to fraction 90. Fractions were 2 mL and 20 μL were assayed for enzyme activity. C. Hitrap Butyl FF chromatography of the phosphatase activity. A gradient from 1 to 0 M (NH4)2Sθ4 was run from fraction 1 to fraction 90. Continuous line, OD at
280 nm; triangles, Phosphatase activity; squares Gal Dehydrogenase (GDH) activity.
Figure 2 shows a gel filtration chromatography profile of L-GaI-I -Phosphate phosphatase activity. Details are given in the methods section and in Fig 1. Fractions were 1 mL and 20 μL were assayed. Symbols are as for Figure 1, with down triangles at the top of the figure representing the peak elution volumes of standard proteins. These had masses, from left to right, of 136, 68, 34, 17 and 12.4 KD.
Figure 3 shows the pH response of Gal -1 -Phosphate phosphatase. Assays were run in 0.2M BTP buffer, pH as specified on the graph, in 2 mM MgCl2, 0.5 niM L-GaI-I -P for 30 minutes at 30 C. Data is the mean + standard error of three replicates. Separate experiments, showed very similar responses
Figure 4 shows the response of L-Gal-Pase activity to MgCl2. The reaction mix contained either 0.4 (triangles) or 0.2 mM (squares) EDTA in 0.2 M Bis Tris Propane buffer, pH 7.0 and 0.5mM Gal 1-P. Assays (30 minutes, 30C) were initiated with enzyme. Free Mg+"1" was calculated using the CHEAQS programme (a program for calculating CHemical Equilibria in AQuatic Systems) written by Wilko Verweij Figure 5 shows the response of L-GaI-I -Pase activity to L-GaI 1-P concentration. Reactions were carried out in 100 niM Bis Tris Propane, pH 7.0, at 30C for 30 minutes. Reactions were run at either 1.9 (squares) or 4.8 (triangles) mM MgCl2.. The curves are the best fits of a Menton Michaelis equation to the data.
Figure 6 shows SDS PAGE of partially purified Arabidopsis L-GaI-I -Pase preparation. Arrows are at the position of the indicated standards.
Figure 7 shows a cluster analysis of purported myø-inositol phosphate phosphatases. The alignment was based on translated Genbank sequences or Arabidopsis (At) TAIR peptide sequences. Tomato sequences (Le) represent isoforms 1 (LeIMPl; SLU39444), 2 (LeIMP2; SLU39443) and 3 (LeIMP3; SLU39059). Rice sequences are coded Os, potato St, The ESTs from the HortResearch fruit database are coded A (apple) or K (kiwifruit), after their EST number. est233909 is the EST shown to posseses L-GaI-I- Pase activity of the invention.
Figure 8 shows typical Sypro Ruby stained SDS Gel electrophoresis of a selection of E coli expressed L-galactose-1-P phosphatases. Expressed enzymes were partially purified using a HisTrap column as described in Laing et al, (2004) except for At3g02870, which was purified through gelfiltration and Anion exchange chromatography. Lane 1, standards; Lane 2, 233909; Lane 3, At3g02870; Lane 4, 158264; Lane 5, 159754 (Group 3 MlPase); Lane 6, 169480 (Group 2 MlPase); lane 7, 180144; Lane 8, Atlg31190 (Group 2 MlPase); Lane 9, blank, Lane 10 standards.
Figure 9 shows a Clustal analysis of MlPase protein sequences derived from cloned genes. Aa, Actinidia aguta; Ad, A. deliciosa; At, Arabidopsis thaliana; Md, Mains domestica; Pa Persea americana; Le, Lycopersicon esculentum (Solanum lycopersicum); Os, Oryza sativa; St, Solanum tuberosum; Ta, Triticum aestivum. The Group 1 genes high lit in yellow have been expressed and specific L-Galactose-1-P phosphatase activity measured. Figure 10 shows the response of Tomato L-Galactose-1-P phosphatase to L- Galactose-1-P, and inhibition by D-Mannose-1-P.
A. Tomato SLU39059 was assayed as a function of L-Galactose concentration in the absence (squares) and presence (triangles) of 4.5 niM D-mannose-1-P. MgC12 was
1.8mM.
B. Tomato SLU39059 was assayed as a function of MgC12 concentration in the absence (squares) and presence (triangles) of 4.5 mM D-mannose-1-P. L-Galactose-1-P was 4.5 mM. Hydrolysis of Mannose-1-P was taken into account in the backgrounds. Inhibition at saturating Mg was 28%, while at saturating L-Galactose-1-P it was 48%.
Figure 11 shows the response of Tomato L-Galactose-1-P phosphatase to L-Galactose- 1-P, and inhibition by D-Galactose-1-P. Tomato SLU39059 was assayed as a function of MgC12 concentration in the absence (squares) and presence (triangles) of 4.5 mM D- galactose-1-P. L-Galactose-1-P was 4.5 mM. Hydrolysis of D-galactose-1-P was taken into account in the backgrounds. Inhibition at saturating Mg was 42%.
Figure 12 shows the response of the three Tomato L-Galactose-1-P phosphatases to L- MgC12. Tomato phosphatases was assayed as a function of MgC12 concentration. L- Galactose-1-P was 4.5 mM. The calculated Mg Ka values were 0.80 ± 0.08, 0.79 ± 0.08 and 0.76 ± 0.07 mM for SLU39443, SLU39444 and SLU39059 respectively. There is no apparent substrate inhibition.
Figure 13 shows the response of the three Tomato L-Galactose-1-P phosphatases to L- L-Galactose-1-P. Tomato phosphatases were assayed as a function of L-Galactose-1-P concentration. MgC12 was 4.5 mM. The calculated Mg Km values were 0.169 ± 0.03, 0.296 ± 0.06 and 0.244 + 0.04 mM for SLU39443, SLU39444 and SLU39059 respectively. There is no apparent substrate inhibition.
Figure 14 shows an alignment of wheat L-Galactose- 1 -phosphate phosphatase polypeptides discussed in Example 3. EXAMPLES
The invention will now be illustrated with reference to the following non-limiting example.
EXAMPLE 1: PURIFICATION OF L-GALACTOSE-I -PHOSPHATE
PHOSPHATASE FROM ACTINIDIA DELICIOSA Am)ARABIDOPSIS THALIANA
Materials and methods
Materials
Kiwifruit (Actinidia deliciosa (A. Chev.) C. F. Liang et A.R. Ferguson var. deliciosa
Ηayward') fruit were picked at Te Puke, New Zealand. For biochemical studies, small one cm diameter fruit were used, picked four weeks after anthesis. Arahidopsis plants were grown for approximately five weeks at 20 to 25C and 200 umole.m- .sec" PAR.
Tissues were stored frozen at -80C until use.
Most chemicals were obtained from Sigma, (St. Louis, MO), except the proteinase inhibitor tablets (Roche, Basel, Switzerland), and L-GaI-I -P (Glycoteam, Hamburg, Germany). The L-GaI-I -P appeared as a single peak by HPLC with a mass of 260 D.
Column chromatography media were obtained from Amersham Biosciences (Sweden).
Bradford's reagent was provided by Bio-Rad (Hercules, CA.).
Enzyme assays
Two assays were used to measure the activity of the Gal-1-Pase enzyme. The first assay measured the phosphate formed by the hydrolysis of L-GaI-I -P using a microplate based colorimetric phosphate assay (Chifflet et ah, (1988) Anal. Biochem. 168, 1-4) and was used during purification and the characterise the enzyme. The standard conditions for this assay consisted of 0.1 M Bis Tris Propane pH 7.0, 2 mM MgC12 , 0.2 mM
L-GaI-I -P in 100 μL. Between 1 to 20 μL of extract was added to this reaction mix to
start the reaction, incubated for 15 to 30 minutes at 30C and terminated by the addition
of 100 μL of 3% ascorbate and 0.5% (both w/v) ammonium molybdate in 0.5M HCl,
made up fresh each day. Backgrounds were run by adding the enzyme after the reaction was terminated. The second assay, used during purification and to verify products, coupled the production of L-GaI to L-GaIDH (Wheeler et al., 1998, Nature 393, 365- 369; Gatzek et al., 2002, Plant Journal 30, 541-53 and measured the reduction of
NAD. The basic assay in 100 μL volume was the same as the first assay, but
additionally contained 0.2 mM NAD as well as excess of L-GaIDH either partially purified from kiwifruit fruit (Laing et al., unpublished) or using the cloned enzyme expressed in Escherichia coli (Bulley et al. , unpublished). Time courses were followed up to 30 minutes at 2OC using either a Victor fluorescence plate reader (Wallac, Finland) to measure NADH fluorescence (excitation 340 nm, emission 460 nm) or by absorbance spectroscopy.
GaIDH was assayed in 0.1 M Tris, pH 8, 0.25 mM NAD and L-GaI in a volume of 100
μL at 2OC using the Victor fluorimeter to measure NADH fluorescence.
L-Gal-1-Phosphate phosphatase Purification
Either kiwifruit fruit or Arabidopsis thaliana (CoI-I) leaves were ground to a fine powder under liquid nitrogen and ~70 grams of frozen tissue was extracted in 100 mL (kiwifruit) or 200 mL {A. thaliana) of 0.18 M MOPS buffer adjusted to pH 7.0,
containing 1.0% Triton X-100, 4% PVPP, 2.5 mM DTT, 10% (v/v) glycerol, 10 μM
E64 (kiwifruit) and a proteinase inhibitor tablet per 50 mL. This was then filtered through 20 micron nylon mesh, centrifuged (20 minutes at 20,000 g) and the supernatant decanted. This was applied to a 5 x 60 cm G75 Superdex™ column run
at 5 niL min'1 equilibrated with 0.1 M Tris pH 8.0, 2 mM EDTA, 5 mM 2- mercaptoethanol and 10% glycerol. Active fractions were pooled, diluted with water
and glycerol to 50 mM TrisCl, 10% glycerol and applied to two 5 mL Hitrap™ Q FF
columns in series equilibrated with 20 mM TrisCl, pH 8.0 with 2 mM EDTA, 5 mM 2-
mercaptoethanol and 10% glycerol. The Hitrap™ Q column was eluted using a gradient
of 0 to 0.5 M NaCl in 20 mM TrisCl, pH 8.0 containing EDTA, 2-mercaptoethanol and glycerol.
Active fractions were combined and ammonium sulphate was added to 1 M before application of the sample to a 5 mL Hitrap Butyl FF column. Proteins bound to the butyl sepharose column were eluted with a reverse gradient of ammonium sulphate from 1 M to O M.
The active fractions from the butyl column were concentrated using a 10,000 MW cut off centrifugal concentrator to 1 mL, and the sample was applied to a 40 cm x 1.6 cm G75 superdex column equilibrated with the same buffer as the large G75 superdex column. Active fractions were combined and used to characterise the enzyme.
SDS-PAGE was performed using the Bio-Rad Mini-PROTEAN II™ system with a 4%
(w/v) acrylamide stacking gel and a 10% (w/v) acrylamide separating gel using the Tricine-based buffer (Schagger et ah, 1987, Anal. Biochem. 166, 368-79). A broad molecular mass range of Bio-Rad prestained marker proteins were used to determine
molecular mass. Gels were stained with Sypro Ruby™ (Molecular Probes, Eugene, OR,
USA) followed by coomassie using standard techniques.
Kinetic experiments Enzyme kinetic analyses for Km and Vmax measurements, Mg+* response, alternative substrates, and pH optimum were replicated three times within the experiment in a standard manner. Data are presented as means and standard errors as appropriate. Experiments were repeated at least twice and a representative experiment presented.
Liquid Chromatography-Mass Spectrometry Analysis of Peptides
A partially purified L-GaI- 1-Pase preparation was digested with trypsin (0.5 μg trypsin
(modified sequencing grade from Roche) per sample in 0.2 M NH4HCO3 with 0.5 mM CaC12) and gel slices containing proteins from an SDS PAGE separation of the same preparation were subjected to in gel digestion (27). Digested peptides were analysed by LC-MS using an LCQ Deca ion trap mass spectrometer fitted with a nanospray ESI interface (ThermoQuest, Finnigan, San Jose, CA, USA) and coupled to a SurveyorTM
HPLC. 5 μL of the digested protein sample was injected onto a reversed phase column
(PepMap Cl 8, 75 μm ID x 15cm, 3μm, LC Packings, San Francisco, CA, USA).
Tryptic peptides were separated at a flow rate of 400nL/min with a linear gradient from 2 to 80% B (acetonitrile + 0.1% formic acid) over 50 mins. Solvent A was 0.1% aqueous formic acid. The column flow rate was produced by splitting the primary flow
rate of lOOμL/min from the SurveyorTM HPLC system via an AcurateTM flow splitter
(LC Packings, San Francisco, CA, USA). The nanospray interface was used with a 30
μm ID fused-silica standard coated PicoTipTM (New Objective, Woburn, MA5USA)
and the spray voltage was supplied directly to the coated needle tip at 1.9 kV. The mass spectrometer was operated in the positive ion mode and the mass range acquired was
m/z 300-1800. The heated capillary temperature was set at 210 °C. Data was acquired
using a top 2 experiment in data-dependent mode with dynamic exclusion enabled. MS/MS data were analysed using TurboSEQUEST, a computer programme that allows the correlation of experimental data with theoretical spectra generated from known protein sequences (Yates, JR III; Eng, JK; McCormack, AL: Schieltz, D. Anal. Chem. 1995, 67, 1426-1436. Eng, J; McCormack, AL; Yates, JR III. J.Am. Mass Spectrom. 1994, 5, 976-989.). Spectra were searched against the latest version of the public non-redundant protein database of the National Center for Biotechnology Information (NCBI) and with an A. thaliana protein database. The criteria used for a positive peptide identification for a doubly-charged peptide were a correlation factor (XCorr) greater than 2.0, a delta cross- correlation factor (dCn) greater than 0.1 (indicating a significant difference between the best match reported and the next best match) and a high preliminary scoring (Sp). For triply-charged peptides the correlation factor threshold was set at 2.5. All matched peptides were confirmed by visual examination of the spectra.
Gene Cloning
The full length cDNA was obtained from the HortResearch kiwifruit EST resource cloned into Bluescript SK(-). The 880 bp fragment was cut out using BAMHl and HindIII and cloned in frame into PET30c(+) (Novagen, San Diego, CA) and this construct was transformed into E. coli BL21. The construct was verified by PCR amplification form PET primers and by restriction enzyme digestion. This construct contains an N terminal His6 tag. E coli was grown to an OD600 of 0.6 at 37 C, then the protein was expressed under IPTG control (0.3mM) for 20 hours at 2OC. Cells were harvested, resuspended in the His column binding buffer (Novagen) containing one proteinase inhibitor tablet per 50 niL and broken by a high-pressure emulsifier. The centrifuged and filtered supernatent was purified on a 5 mL Hitrap Nickel column.
Fractions containing protein were exhaustively dialysed against 10 mM BTP, pH 7.1, 1 mM EDTA, 5 mM B mercaptoethanol and 30 mM NaCl.
RESULTS
Purification of a specific L-Gal-1-Pcιse from kiwifruit
A large peak of kiwifruit L-GaI- 1-Pase activity was eluted from the large G75 superdex column after the void volume (Figure IA). Further analysis of this peak showed that the earlier eluting fractions consisted of a neutral pH, Mg+4" and L-GaI dependent phosphatase activity while the later eluting fractions had a more acid pH optimum and activity was independent Of MgCl2 (data not shown). Consequently, only the earlier fractions were applied to the anion exchange column where a clear separation of the remaining acid phosphatase and the Mg dependent phosphatase was achieved (Figure IB). The first major peak of phosphatase activity was unaffected by MgCl2, while the second peak was stimulated nine fold by MgCl2 (Table 1). The first peak, which had a optimum below pH 6 (data not shown), was most likely a non-specific acid phosphatase with activity against L-GaI-I -P.
Further purification of the second Hitrap Q peak was gained through fractionation on a hydrophobic interaction column (Fig 1C) and through re-chromatography on a G75 superdex column (Fig 2). The peak activity on this latter column eluted the fraction after BSA, suggesting a native molecular weight close to 65,000.
Further purification (eg chromatafocusing on a MonoP™ column, native gel electrophoresis etc.) resulted in increases in the protein purity, but catastrophic losses in activity. The enzyme would not bind to a Hitrap SP column at pH 5.2. Consequently, characterisation of the enzyme was carried out on combined fractions from the small G75 superdex. At this stage of the purification, at least 10 peaks were apparent by SDS PAGE, with molecular weights between 30,000 and 100,000 (data not shown).
Characterisation of the kiwifruit Phosphatase activity.
The L-GaI-I -P ase was linear with the amount of enzyme until at least 10 nmoles of phosphate had been generated in the assay, which represented about 20% of the substrate present in the assay. The activity was also linear with time until over 30 minutes.
Substrate speciβty of the kiwifruit enzyme
We verified that the product of the reaction was L-GaI by coupling the phosphatase activity to the reduction of NAD using semi purified L-GaIDH from kiwifruit. This enzyme has been shown to be specific to L-GaI with some activity with L-Sorbosone (Arabidopsis), L-Fucose (Arabidopsis, but not kiwifruit) and L-gulose but no activity against a wide range of other sugars Gatzek et al, Plant Journal 30,541-53 (2002). In all steps of purification the coupled reaction paralleled the direct phosphate assay (data not shown). As the substrate L-Gal-1-P is prepared from L-galactono-1, 4-lactone, it would be expected to form L-Gal-1-P.
The ability of the phosphatase activity to hydrolyse a range of phosphorylated compounds was measured using the standard phosphate assay. The results are the means of two experiments, and no substrate other than L-Gal-1-P was significantly and reproducibly hydrolysed by the partially purified enzyme (Table 2). Several substrates (fructose 2,6-BP, Ribulose bisP and Dihydroxyacetone phosphate) were partially hydrolysed (about 20% of the available substrate) by the acid stop mix and showed high backgrounds, explaining an apparent ability of the enzyme to slightly hydrolyse, fructose 2,6-BP. However, this substrate showed a near zero rate in other assays.
The pH optimum showed a well-defined maximum at pH 7 with a fall off to half the maximum volume within 0.6 of a pH unit to either side of the maximum (Fig 3).
The response of the phosphatase to MgCl2 showed a KM(Mg++) of 0.2 mM (±0.04) but
Mg++ was inhibitory at concentrations above 2 mM (Fig 4). The apparent Ki(Mg++) for
substrate inhibition was 0.46 mM (±0.07). No activity was found in the presence of 0.2
mM EDTA without added Mg++.
The KM(Gal-l-P) was dependent on MgCl2 , being 0.041 ± 0.002 mM at 1.8 mM
MgCl2 and 0.022 ± 0.001 mM at 4.8 mM MgCl2 (Fig 5). In some experiments, there
was evidence of mild substrate inhibition, but the Ki was in the range of 3 to 9 mM. As is the case for the Mg4+ response, the Vmax was halved when the Mg++ was raised from close to the optimum to 4.8 mM.
Purification and specific identification of a polypeptide sequence with L-Gal-1-Pase activity from Arabidopsis thalinua
The enzyme was also prepared from A. thaliana seedlings and the the protein from this source was specifically identified. The A. thaliana shoot preparation of the L-GaI-I- Pase showed similar properties to the kiwifruit enzyme, showing absolute Mg+"1"
dependency with a KM (Mg++) of 0.016 ± 0.004 mM and a Ki(Mg4+) of 0.620 ± 0.17
mM. The KM(L-Gal-1-P) was 0.044 ± 0.003 mM and the pH optimum was between 6.8
and 7 at 2 mM MgCl2 and 0.5 mM L-GaI-I -P (data not shown). For those substrates assayed, the Arabidopsis enzyme showed a similar spectrum of activity to the kiwifruit enzyme (Table T). The enzyme preparation was then subjected to trypsin digestion in order to identify the proteins in the mixture using liquid chromatography-mass spectroscopy (LCMS) utilizing the complete Arabidopsis predicted protein databases. In parallel, we separated the mixture using SDS PAGE (Figure 6) and excised nine protein stained gel bands, in gel trypsin digested these and analysed the mixture also using LCMS. We identified 24 proteins based on mass spectrometry analysis, one of which was annotated as myo inositol phosphatase. Nineteen proteins were identified from the analysis of the partially pure preparation and five extras were identified from the SDS bands. The protein annotated as myo inositol phosphatase was found both from the mixture and from a gel band. None of the other proteins appeared to be likely to be a phosphatase. The putative phosphatase protein was Arabidopsis protein AT3g02870, a protein annotated in TAIR as a putative inositol- 1 (or 4)-monophosphatase with a predicted MW of 29121 kD and a predicted pi of 5.01.
Identification of polynucleotides encoding L-Gal-1-Pase in plant species
We identified a range of sequences from public databases, as well as kiwifruit and apple full length ESTs from the HortResearch EST database resource, that showed homology to AT3g02870, and used clustalX (Jeanmougin et al., 1998, Trends Biochem. Sci. 23, 403-5.) to align the encoded polypeptide sequences. The polypeptides formed into three main clusters (Fig. 7), with the tomato myo inositol phosphatases ( Gillaspy et al, 1995, Plant Cell 7, 2175-85) all in one group with AT3g02870, and a kiwifruit and apple EST. The two other clusters also included kiwifruit apple and Arabidopsis sequences.
The report of myo inositol- 1-Pase activity in the tomato genes was based on screening a phage library for 5-bromo-4-chloro-3-indoyl phosphate phosphatase activity, and using the one positive clone (LeIMP) to screen a young fruit cDNA library for two other clones ( Gillaspy et al, 1995, Plant Cell 7, 2175-85).
The activity reported for the tomato clones was a very sensitive assay ( Gillaspy et al, 1995, Plant Cell 7, 2175-85), and as we show, the expressed L-Gal-1-Pase can hydrolyse myo inositol at about 7% the rate of L-GaI-I -P hydrolysis. These authors ( Gillaspy et al., 1995, Plant Cell 7, 2175-85) did not test specifity of activity against other substrates and did not characterise the enzyme as to its properties except for a suggestion it was a dimer, Mg dependent and Li4+ sensitive ( Gillaspy et al, 1995, Plant Cell 7, 2175-85).
Two pathways of ascorbic acid biosynthesis involve a phosphatase step; the L-GaI pathway ( Wheeler et al, 1998, Nature 393, 365-369) and the myo inositol pathway ( Lorence et al, 2004, Plant Physiol. 134, 1200-1205; Gillaspy et al, 1995, Plant Cell 7, 2175-85). The enzyme we describe shows activity against both of these substrates, albeit this enzyme is much more effective against L-GaI-I -P than myo inositol- 1 -P.
EXAMPLE 2: CLONING AND BACTERIAL EXPRESSION OF A POLYNUCLEOTIDE FROM KIWIFRUIT ENCODING GAL-I -PHOSPHATE PHOSPHATASE
We cloned and expressed EST 233909, the putative kiwifruit myo inositol- 1-Pase, in E. coli, and obtained high yields of protein after purification on a His column (up to 3 mg from 50 mL of culture). This protein showed some myoinositol activity, but had ~14 times more Gal- IP phosphatase activity and very little activity against a wide range of phosphorylated compounds (Table 2). The expressed enzyme had a maximum specific
activity against L-Gal-1-P of 16.4 ± 1.0 μmoles Pi generated min"1 (mg protein)"1 (calculated from the L-GaI-I -P response curve) while extract from E. coli expressing the empty vector after His column purification showed no phosphatase activity against L-GaI-I -P, myo inositol- 1 -P or other substrates tested. Coupled assays using E. coli expressed kiwifruit L-GDH, which uses L-GaI to reduce NAD, showed that the products of the reaction were L-galactose as well as inorganic phosphate (data not shown).
Kinetic analysis of the cloned and expressed kiwifruit enzyme showed it had a KM(L-
Gal-l-P) of 0.15 ± 0.02 mM, a KM(Myo inositol-1-P) of 0.33 ± 0.02 mM and a
KM(Mg++) of 0.47 ± 0.16 mM with a Ki(Mg+4) of 13.4 ± 5.9 mM (data not shown). The
high standard errors for Mg inhibition reflect the lesser degree of inhibition by Mg++ seen with this enzyme compared to the enzymes extracted from plants. The enzyme showed a pH optimum of 7.0 (data not shown), very similar to the Arabidopsis and kiwifruit enzymes. The enzyme also appeared to be a dimer by gel filtration. The predicted subunit MW of the expressed enzyme, including the His Tag, is ~34800 D. On a G75 Sephadex column, the enzyme eluted (data not shown) just before a BSA standard (MW 68,000 D), as would be expected for a dimer (MW -70,000 D).
Table 1
Effect Of MgCl2 on L-GaI-I -Phosphate phosphatase activity of the two peaks off the Hitrap Q column. Peak 1 refers to the average of fractions 29 to 32 in figure 2 while
peak 2 refers to fractions 70 to 73. Assays of 20 μL for each fraction were carried out
in the absence or presence of 5 niM MgCl2. No EDTA was present in the reaction mix. See text for assay details.
5 mM 0 mM Ratio
Mg4+ Mg+-1"
nmoles.min"
peak l 0.75 0.72 1.0
peak 2 0.51 0.05 9.5
Table 2. Substrate specifity for the partially purified phosphatase activity.
Reactions were run in 100 mM BisTris Propane buffer, pH 7.0 with 2 MgCl2, and 0.5 mM phosphorylated substrate for 30 minutes (kiwifruit and Arabidopsis) or 11 minutes (E. coli) at 3OC. Standard errors were around 2% of the mean for these assays. Empty positions indicate that the substrate was not assayed with that enzyme.
Kiwifruit enzyme Arabidopsis enzyme E. coli expressed
At3g02870 kiwifruit enzyme EST
233909
Substrate nmoles/min % nmoles/min % nmoles/min %
L Gal 1-P 0.220 100.0 0.163 100.0 1.61 100.0
D Gal 1-P 0.011 4.6 0.066 4.1
D Mannose 1-P 0.008 3.3 0.059 3.7
Dihydroxyacetone -0.001 -0.3 phosphate
Fructose 1, 6-BP -0.001 -0.4
Fructose 1-P -0.003 -1.3
Fructose 6-P 0.000 -0.2
Fructose 2, 6-BisP 0.020 9.8
Guanosine Diphosphate 0.007 3.6
Glucose 6-P 0.000 -0.1 -0.001 0.0
Glucose 1-P 0.016 6.7
Glucose 1-6BP 0.002 1.0 Guanosine Triphosphate -0.004 -2.1
Mannose 6-P -0.001 -0.3
p-Nitrophenyl Phosphate 0.009 4.2 0.001 0.4 -0.014 -0.9
Phosphoglyceric acid 0.001 0.3 0.000 0.0
Phosphoglycolate 0.008 3.6 0.009 5.9
Ribose 5-P 0.003 1.5 0.014 0.9
Ribulose 1,5-bisP -0.003 -1.2
Sucrose 6-P 0.006 2.7 0.003 1.9 -0.001 -0.1
Trehalose 6-P -0.001 -0.6 0.003 1.7 -0.003 -0.2
Myo inositol- 1 -P 0.353 21.9
• The test for alternative substrates used excess E. coli expressed enzyme in this assay to maximize sensitivity for alternative substrates, and the control rate was beyond the linear portion of the activity versus amount of enzyme curve (data not shown). The true level of L-GaI- 1-Pase activity was approximately three times this, and in separate measurements, we established that under standard substrate concentrations (0.5 mM sugar phosphate and 2 mM MgCl2 ) and pH 7.0, myo inositol-1-P was hydrolysed at 7.1% the rate of L-Gal-1-P. EXAMPLE 3: CLONING AND BACTERIAL EXPRESSION OF
POLYNUCLEOTIDES FROM SEVERAL SPECIES ENCODING GAL-I- PHOSPHATE PHOSPHATASE
The genes for group 1 MIPases (Figure 9) from a range of species were PCR amplified 5 and cloned into the pGEM vector, cut out with the corresponding restriction enzymes and re-cloned into the pET30 vector. Genes were sequenced before they were expressed as described in Laing et al, 2004 (PNAS 101, 16976-81). Full length Actinidia, Malus and Persea clones were obtained from the HortResearch EST resource, while the Arabidopsis clone was obtained from TAIR (http://arabidopsis.org/). Tomato
10 clones were PCR amplified from mRNA isolated from young micro torn tomatoes. Wheat clones were PCR amplified from cDNA prepared from wheat using primers designed based on genbank deposited EST sequences. Three variants were obtained (see alignment in figure 14). Primers were designed to have a Eco Rl and BAM Hl restriction site at the 5' and 3' ends (except for EST 260086, which had Sal I and Xho I
15 restriction sites) to facilitate cloning. Full details of clones, tissues and any sequencing deviations are listed in Table 3. Table 3. List of Group 1 genes used to test for L-galactose-1-P phosphatase activity.
Est # Species Tissue (Library) Comments
Group 1 MIPases
158264 Ripe fruit (KHFA) T to P at 71 AA (from
Actinidia arguta start of translated EST sequence) 180844 Malus domestica variety Fruit stored for 24 hours under low Initial M in original
Royal Gala oxygen/high CO2 (ABDA) sequence changed to T in pET30 vector
233909 Dormant kiwifruit buds three days 14 residues from the 5'
Actinidia deliciosa after hydrogen cyanamide UTR of the EST in treatment (KALA) (already in front of the N terminal patent) 260086 Persea americana, variety Avocado fruit (QAAB) Identical to EST
Haas sequence
At3g02870 Arabidopsis thaliana Group 1 enzyme cDNA clone Identical to EST U09692 from TAIR sequence
39059 MicroTom young fruit Identical to published
Lycopersicon esculentum sequence (Solarium lycopersiciim)
39443 MicroTom young fruit Identical to published
Lycopersicon esculentum sequence (Solarium lycopersicum)
39444 MicroTom young fruit Two changes from
Lycopersicon esculentum published sequence, (Solanum lycopersicum) Residue 12 G to A change
Residue 225 A to T
BT009424
Triticum aestivum Young seedlings Three genes identified. Clone #1 residue 28 S to T, 30 K to N and 118 G to A. Clone #3, Initial M to L. Clone #4, Initial M to L, plus significant C terminal changes (see Figure 14).
Genes from a wide range of group 1 dicotyledonous and one monocotyledonous species (Table 3) were expressed in E coli, partially purified on a His chelating column and activity measured. High levels of expression were obtained for most clones showing a major band on SDS PAGE at around 35000 D (Figure 8). In all cases much greater hydrolysis of L-Galactose-1-P phosphatase activity than myø-inositol-1-P was measured (Table 4).
10 Table 4. Activity of E coli expressed group 1 L-Galactose-1-P phosphatases. Enzymes were expressed as described in the methods and assayed at near saturating substrate concentrations (1 to 2 mM).
Figure imgf000064_0001
Figure imgf000065_0001
*minimal protein after dialysis (<0.3 mg/niL), no ~35000 Da band on SDS PAGE and no activity observed. All the three tomato clones, originally described as MIPases (Gillaspy et al, 1995
Plant Cell 7, 2175-85), expressed well and also showed much more L-Galactose-1-Pase than myo-inositol-1-Pase activity (Table 4). Over all enzymes tested, the ratio of L-GaI- 1-Pase activity to MIPase activity ranged from 5.4 to 88. Some of the enzymes expressed extremely high levels of protein (eg over 3 mg from 50 mL of E coli culture) and only one expressed poorly (SLU39059), but even that gene expressed for more protein than was needed. The specific activity of the various genes ranged from 0.27 to 25.6 umoles Pi released/mg protein/min (Table 5). We have no explanation for the range in specific activity.
Table 5. Specific activity of selected L-Galactose-1-P phosphatases. Enzymes were assayed at pH 7.0 at 1 mM L-galactose-1-P and 2 mM MgC12.
Figure imgf000066_0001
A clustal diagram summarizing the genes that were tested for specific L-galactose-1-P phosphatase activity is shown in Figure 9.
A range of compounds were tested as inhibitors of the phosphatase (Table 6).
Table 6. Inhibitors of L-Galactose-1-P phosphatase (233909). Inhibitors were included in the assay at 2.3 mM and assayed at 1 mM L-Galactose-1-P and 2 mM MgC12.
% residue
Inhibitor activity
None 100
D-Glucose-1-P 106
D-GaM P 73
D-Fructose-1-P 79
Pglycolate 73
L-Galactose 112
D-Ribose-5P 71
D-Myo inositol-1-P 113
D-mannose-1-P 72
Some of these compounds showed inhibition, especially those that are similar to the preferred substrate, L-galactose- 1 -P . This included D-mannose- 1 -P and D-galactose- 1 - P. These compounds were tested further, and it appeared some of the inhibition was due to Mg chelation by the phosphorylated substrate and inhibitor. When the phosphatase activity was measured as a function of L-Galactose-1-P concentration (Figure 10), inhibition by D-mannose-1-P appeared to increase at higher substrate concentrations (48%). However, when the enzyme activity was titrated against Mg concentration, the inhibition was reduced by higher Mg concentrations (maximum inhibition 28% at saturating Mg). D-galactose- 1 -P was more inhibitory than D- mannose-1-P at saturating Mg (42%) as shown in figure 11. The tomato phosphatases showed considerable differences in their specifity for D-galactose-1-P compared to 7?rμo-inositol-l-P (ratio of GaI-I -Pase to MIPase ranging from 11 to 56). To see if this reflected changes in the kinetic properties of the enzyme, we measured the response to Mg and L-Galactose-1-P for these three enzymes (Figures 12 and 13). However, the enzymes showed similar constants for Mg (0.76 to 0.8 mM) and L-Galactose-1-P (0.169 to 0.296 mM). This suggests that the differences in the specifity ratios were either due to differences in the Km(myo-inositol-l-P) (not measured, but unlikely as 1 mM myo-inositol-1-P was used in the assay, which should be near saturating (see Laing et al, 2004 PNAS 101, 16976-81) or due to intrinsic differences in the catalytic hydrolysis rate for the two substrates.
EXAMPLE 4: PLANT TRANSFORMATION WITH A POLYNUCLEOTIDE ENCODING GAL-1-PHOSPHATE PHOSPHATASE
Materials and methods
Full length ESTs were cloned into a vector for Agrobacterium tumefacians as follows. The 233909 cDNA clone was excised from pBluescript SK- as a 1168 bp Spel-Xhol fragment. This was then cloned into the Spel-Xhol sites of pSAK778, placing the cDNA between the CaMV35S promoter and ocs3' transcriptional terminator. A gene encoding the Pl 9 silencing suppressor gene was also cloned into another vector (Voinnet, et al 2003, Plant J. 33(5):949-56).
ρSAK778 is a derivative of pART7 and ρART27 (Gleave, et al. 1992, Plant MoI Biol. 20(6): 1203-7). To generate ρSAK778 the following steps were carried out: The 49 bp Sall-Xbal fragment from the polylinker of pBK-CMV (Stratagene) was cloned into the Xhol-Xbal sites of pART7, generating ρSAK7. The 2187 bp SacI-NheI fragment of pSAK7 was then cloned into the Sacl-Spel sites of pART7, replacing the pART7 CaMV35S promoter-multiple cloning site-ocs 3' transcriptional terminator cassette with the corresponding pSAK7 cassette carrying the alternative multiple cloning site and generating pSAK8. The 2171 bp 35S-mcs-ocs3' cassette of was then cloned as aNotl fragment into pART77, generating pSAK778. pART77 was generated by replacing the 1578 bp Nhel-Aflll fragment of pART27 with the 963 bp Nhel-Aflll fragment of pGreen0029SK62, generating pART67. pART67 was then cleaved with Spel and ligated with the oligonucleotide 5'-CTAGGGCGCGCC-3', to destroy the Spel site of pART67, generating pART77.
pSAK778_233909 was introduced into Agrobacterium using standard techniques. Agrobacterium, GV3101(MP90) were cultured on Lennox agar (Invitrogen) supplemented with 50ug.ml-l kanamycin (Sigma) and incubated at 28C. Several lOμl loops of confluent bacterium were re-suspended in 1 OmI of infiltration media ( 1 OmM Mg C12, 0.5μM acetosyringone (Sigma)), to an OD600 of -0.2, and allowed to incubate at room temperature for 2 hours before infiltration. Pl 9 containing Agrobacterium were treated in a similar way.
All other techniques (eg cloning into pET30 vectors) are as described in Example 2.
Nicotiana benthamiana were grown in a glass house at an average temperature of 22C using natural light with daylight extension to 16hrs. Plants were grown until they had 4 leaves and the three youngest leaves over 1 cm long were infiltrated with Agrobacterium (Voinnet, et al 2003, Plant J. 33 (5): 949-56) and maintained in the glasshouse for the next 10 days of co- cultivation. Leaves were infiltrated with a mixture of Agrobacterium containing the gene of interest and a separate Agrobacterium containing the Pl 9 gene using a ImL syringe without a needle. Controls were infiltrated with Agrobacterium containing the Pl 9 gene. At harvest the leaves were immediately frozen and stored in liquid nitrogen.
Extraction and Assay of Gal-1-Pase activity in tobacco leaves
Leaves were ground to a powder and then extracted four to six volumes of 200 mM Bis Tris Propane, pH 7.0, 0.5 mM EDTA, 2 mM DTT and one proteinase inhibitor tablet per 25 mL. Extracts were centrifuged and desalted using NAP columns (GE Healthcare) equilibrated with 100 mM Bis Tris Propane pH 7.0, and 0.5 mM EDTA. Extracts were assayed in 10OmM Bis Tris Propane, pH 7.0 with 0.5 mM Gal-l-P and 0.5 mM EDTA with the addition of 2.5 mM MgCl2 (Mg dependent phosphatase activity) or without the addition OfMgCl2 (Mg independent phosphatase, or background). Previously, we have established that the GaI-I -Phosphatase activity is completely dependent on Mg (Laing et al, 2004, PNAS 101, 16976-81). Assays were carried out at 2OC for 30 minutes and terminated as described earlier.
Results
Transient expression in tobacco leaves is a rapid powerful technique to express transgenic protein in a plant environment, in a native format. We used this technique to verify the identity of EST clone 233909.
Transient transformation of tobacco leaves with Agrobacterium tumefaciens containing a vector designed to express 233909 resulted in high levels of Mg dependent Gal-l-P phosphatase activity (Table 7). ++
Table 7. Mg dependent L-gal-1-Pase activity in tobacco leaves transiently transformed with 233909 or P19. Leaf 1 is the oldest leaf, leaf 3 the youngest. See methods for details.
Pase activity protein nmoles/min/ug
Clone Plant Leaf ug/uL protein
233909 3 1 0.705 0.0995
233909 3 2 0.812 0.1143
233909 3 3 0.654 0.1422
233909 1 1 0.508 0.10437
233909 1 2 0.380 0.14087
233909 1 3 0.407 0.2171
P19 2 1 0.503 0.0012
P19 2 2 0.646 0.0025
P19 2 3 0.627 0.0013
P19 1 1 0.466 0.0005
P19 1 2 0.405 0.0008
P19 1 3 0.458 0.0026
Arabidopsis 0.754 -0.0021
Virtually no Mg dependent activity was seen in the Pl 9 control while activity was readily measured in the leaves infiltrated with 233909. The activity responded to Mg
with a K3(Mg) of 0.096 ± 0.045 rnM but was inhibited at high Mg with an inhibition
constant of 7.69 ± 3.11 rnM. The response to Gal-l-P followed a typical Menton-
Michaelis curve with a K1n(GaI-I-P) of 0.25 ± 0.03 rnM.
The transiently expressed 233909 was tested for substrate specifity (Table 8).
Table 8. Ability of 233909 transiently transformed tobacco extracts to hydrolyze different sugar phosphates. Assays were carried out as described in the methods except with 1 rnM of the selected substrate per assay. Assays were carried out with and without MgCl2 and the percentages were calculated on the rates corrected for hydrolysis in the absence of Mg. The rate in the absence of Mg and the P19 control rate are expressed as a percentage of the rate with Mg and the same substrate.
233909 233909 P19 Rate
Rate in Rate in in presence absence presence of Mg of Mg of Mg substrate %
Gal-1-P 104 12 -7
Pglycolate 157 3 74
Ribose-5-P 9 73 4 pDNP 6 82 1
Fructose-1-P 2 97 5
Sucrose-6-P 42 6 35 trehalose-6-P 3 0 4
G!ucose-1-P 2 88 0
Dgal-1-P 5 79 3
D man-1-P 3 70 2
Myoinositol-1-P 4 76 2
In the case of Sucrose- 1 -Phosphate and PGlycolate phosphatases, the rate of hydrolysis by the Pl 9 transiently transformed plants were high, as expected for these important photosynthetically aligned enzymes. However, for the other substrates, little hydrolysis was seen in both the 233909 and P19 transformed plants in spite of a high rate of hydrolysis of L-Gal-1-P in the 233909 transformed plants. This is a very similar spectrum of hydrolysis as seen in the purified and E coli expressed L-GaI- 1-Pase
It is not the intention to limit the scope of the invention to the abovementioned examples only. As would be appreciated by a skilled person in the art, many variations are possible without departing from the scope of the invention.

Claims

CLAIMS:
1. A method of producing a plant cell or plant with altered L-Galactose-1- phosphate phosphatase expression, the method comprising transformation of a plant cell or plant with a genetic construct including: a) a polynucleotide encoding of a polypeptide with the amino acid sequence of any one of SEQ ID NO:67 to 118, 120, 122, 124, 126, 130 to 132, or a variant of the polypeptide from a plant species, wherein the variant has the activity of an L-Galactose- 1 -phosphate phosphatase; b) a polynucleotide comprising a fragment, of at least 15 nucleotides in length, of the polynucleotide of a), or c) a polynucleotide comprising a compliment, of at least 15 nucleotides in length, of the polynucleotide of a); or d) a polynucleotide comprising a sequence, of at least 15 nucleotides in length, capable of hybridising to the polynucleotide of a) under stringent conditions
2. The method of claim 1 wherein the variant has at least 50% sequence identity to a polypeptide with the amino acid sequence of any one of SEQ ID NO: 67 to 118, 120, 122, 124, 126, 130 to 132.
3. The method of claim 1 wherein the variant comprises the amino acid sequence:
X1X2X3X4X5SX6X7X8X9LX1OX1IX12LX13X14X15X16X17X18X19X2ORDX21X22X23X24X25 X26X27TX28X29IX3OX31X32X33X34X35X36X37X38X39X40X41X42X43X44X45X46X47X48X49X5 0X51X52X53 (SEQ ID NO:2) wherein X1 is N5 K, S, T, A, D or V; X2 is P, R or S; X3 is I or M; X4 is K, R, H or S; X5 is V, A or T; X6 is S, P, A or T; X7 is Q, K, E or H; X8 is S, N, T, D or A; X9 is E or Q; X10 is V, A, G, I, L or M; X11 is K, N, T or S; X12 is S, A or C; X13 is L, M or V; X14 is A5 G5 V5 S, L or M; X15 is T or A; Xi6 is E or D; X17 is V5 A5 1 or D; X18 is G or I; X19 is T5 V or P; X20 is K, N5 T, E or M; X21 is K, N or Q; X22 is L5 A, S5 E5 P5 T or V; X23 is T5 1 or V; X24 is V5 L or I; X25 is D5 G or E; X26 is A5 D5 V5 1 or T; X27 is T5 S5 A5 C or V; X28 is N5 D5 G or R; X29 is R5 K or T; X30 is N or K; X31 is S5 R5 K5 N or G; X32 is L or V; X33 is L or I; X34 is F5 Y or T; X35 is K or E; X36 is V5 1 or G; X37 is R or A; X38 is S5 A or V; X39 is L5 15 P or V; X40 is R or E; X41 is M or D; X42 is S, C5 G5 T or A; X43 is G or R; X44 is S or L; X45 is C, L or V; X46 is A or R; X47 is L or T; X48 is N, D, A or
G; X49 is L, M or T; X50 is C or L; X51 is G, W or R; X52 is I, V or C; X53 is A or R.
4. The method of claim 1 wherein the variant comprises the amino acid sequence: XIX2X3LTX4X5X6TWIVDPX7DGTTNFVHGX8PX9VCVSIGLTIX1OKX1 JPX12VXI3V VYY14PIX15X16ELFTX17X18X19GX2OGAX21LNGX22 (SEQ ID NO:133) wherein X1 is T,V, N5 D or I, X2 is T, D, F or A, X3 is E or D, X4 is D or Y, X5 is E, D or Q5 X6 is P or H, X7 is L or V, X8 is F or Y, X9 is F or S, X10 is G, E or A, X11 is I, V or K, X12 is T or V, X13 is G or A, X14 is N or D, Xi5 is M or I, X16 is D5 N or E5 Xi7 is G or A5 X18 is I or V5 X19 is R, H, D5 L, N, Q or Y5 X20 is K5 Q5 G or R5 X21 is F or Y and X22 is K, N or S
5. The method of claim 1 wherein the variant is from a dicotyledonous species and comprises the amino acid sequence:
X1X2X3X4X5SX6X7X8X9LXiOXi IXI2LXI3XI4XI5EXI6X17X1gX19RDX20X21X22X23X24X2
5X26TX27X28IX29X3oX31X32X33X34X35X36X37X38X39X4θX4lX42X43X44X45X46X47X48X49X
Figure imgf000074_0001
6. The method of claim 1 wherein the variant is from a dicotyledonous species and comprises the amino acid sequence:
X1X2X3LTDX4X5TWrVDPX6DGTTNFVHGX7PX8VCVSIGLTIX9KX1OPX11VGVVY X12PIXI3XI4ELFTX15X16X17GX18GAX19LNGX20 (SEQ ID NO:134) wherein X1 is V5 N5 D, I or T5 X2 is T5 F or A5 X3 is E or D5 X4 is E5 D or Q, X5 is P or H5 X6 is L or V5 X7 is F or Y5 X8 is F or S5 X9 is G, E or A5 Xi0 is V5 1 or K, X11 is T or V5 Xi2 is N or D5 XB is I or M5 XM is D5 E or N5 Xi5 is G or A5 Xi6 is I or V5 Xi7 is H5 D5 L5 N5 Q5 R or Y5 Xi8 is K5 Q5 G or R5 X19 is F or Y and X20 is K or N.
7. The method of claim 1 wherein the variant is from a monocotyledonous species and comprises the amino acid sequence: SPIX1X2SX3QX4ELX5KALX6VTX7X8GTX9RDKX1OTXnDDTlNRINX12LLX13KIRSI RMCGSLALNMCGVA (SEQ ID NO:4)
X1 is K or R; X2 is T or A; X3 is S or P; X4 is N or D; X5 is V or A; X6 is L or M; X7 is E or D; X8 is V or A; X9 is K or N; X10 is A, S or T; X11 is L or V; X12 is K or R;
X13 is F or Y.
8. The method of claim 1 wherein the polynucleotide of a) encodes a polypeptide with the amino acid sequence of any one of SEQ ID NO: 67 to 118, 120, 122, 124, 126, 130 to 132.
9. The method of claim 1 wherein the polynucleotide of a) encodes a polypeptide with the amino acid sequence of SEQ ID NO: 67.
10. A method of producing a plant cell or plant with altered L-Galactose-1- phosphate phosphatase expression, the method comprising transformation of a plant cell or plant with a genetic construct including: a) a polynucleotide comprising a nucleotide sequence selected from any one the sequences of SEQ ID NO: 1, 5 to 54, 55 to 65, 119, 121, 123, 125 and 127 to 129, or a variant thereof from a plant species, wherein the variant encodes a polypeptide which has the activity of an L-Galactose-1 -phosphate phosphatase; b) a polynucleotide comprising a fragment, of at least 15 nucleotides in length, of the polynucleotide of a), or c) a polynucleotide comprising a complement, of at least 15 nucleotides in length, of the polynucleotide of a); or d) a polynucleotide comprising a sequence, of at least 15 nucleotides in length, capable of hybridising to the polynucleotide of a) under stringent conditions
11. The method of claim 10 wherein the polynucleotide of a) comprises any one the sequences of SEQ ID NO: 1, 5 to 54, 55 to 65, 119, 121, 123, 125 and 127 to 129.
12. The method of claim 10 wherein the polynucleotide of a) comprises the sequence of SEQ ID NO: 1.
13. The method of any one of claims 1 to 12 in which the plant or plant cell produced as increased L-Galactose-1 -phosphate phosphatase activity.
14. The method of any one of claims 1 to 12 in which the plant are plant cell produced has altered ascorbate content.
15. An isolated polynucleotide having at least 82% sequence identity to a nucleotide sequence that encodes a polypeptide comprising an amino acid sequence selected from any one of SEQ ID NO: 67 to 118, 120, 122, 124, 126, 130 to 132, wherein the polynucleotide encodes an L-Galactose-1 -phosphate phosphatase.
16. The polynucleotide of claim 15, wherein said nucleotide sequence comprises a nucleotide sequence selected from any one of SEQ ID NO: 1, 5 to 54, 55 to 65, 119, 121, 123, 125 and 127 to 129.
17. The polynucleotide of claim 15 or 16, wherein said nucleotide sequence comprises a full-length coding sequence selected from any one of SEQ ID NO: 1, 5 to 54, 55 to 65, 119, 121, 123, 125 and 127 to 129.
18. The polynucleotide of claim 15 or 16, wherein said nucleotide sequence comprises the nucleotide sequence of SEQ ID NO: 1.
19. The polynucleotide of claim 15 or 16, wherein said nucleotide sequence comprises the full-length coding sequence of SEQ ID NO: 1.
20. An isolated polynucleotide that encodes a polypeptide comprising an amino acid sequence selected from any one of SEQ ID NO: 67 to 118, 120, 122, 124, 126, 130 to 132.
21. The polynucleotide of claim 20 that encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 67.
22. The polynucleotide of claim 20 or 21 comprising a sequence selected from any one of SEQ ID NO: 1, 5 to 54, 55 to 65, 119, 121, 123, 125 and 127 to 129.
23. The polynucleotide of claim 20 or 21 comprising the full-length coding sequence from any one of SEQ ID NO: 1, 5 to 54, 55 to 65, 119, 121, 123, 125 and 127 to 129.
24. The polynucleotide of claim 20 or 21 comprising the sequence of SEQ ID NO: 1
25. The polynucleotide of claim 20 or 21 comprising the full-length coding sequence from within the sequence of SEQ ID NO:1.
26. An isolated polynucleotide comprising a sequence selected from any one of SEQ ID NO: 1, 5 to 54, 55 to 65, 119, 121, 123, 125 and 127 to 129 or a variant thereof, wherein the variant is from an apple, kiwifruit or avocado species, and encodes a polypeptide which has the activity of an L-Galactose-1 -phosphate phosphatase.
27. The isolated polynucleotide of claim 26 comprising the sequence of SEQ ID NO: 1 or a variant thereof, wherein the variant is from an apple, kiwifruit or avocado species, and encodes a polypeptide which has the activity of an L-Galactose-1- phosphate phosphatase.
28. The isolated polynucleotide of claim 26 comprising the sequence of SEQ ID NO: 1
29. An expression construct which includes a polynucleotide comprising the sequence selected from any one of SEQ ID NO: 1, 5 to 45, 47 to 54, 55 to 65, 119, 121, 123, 125 and 127 to 129 or a variant thereof from a plant species, wherein the variant encodes a polypeptide which has the activity of an L-Galactose-1 -phosphate phosphatase, and wherein the variant does not encode the polypeptide of Accession No. AAB19030.
30. The expression construct of claim 29 which includes a polynucleotide comprising the sequence of SEQ ID NO: 1, or a variant thereof from a plant species, wherein the variant encodes a polypeptide which has the activity of an L-Galactose-
1 -phosphate phosphatase, and wherein the variant does not encode the polypeptide of Accession No. AAB19030.
31. The expression construct of claim 29 which includes a polynucleotide comprising the sequence of SEQ ID NO: 1.
32. An isolated L-Galactose-1 -phosphate phosphatase polypeptide from a plant species excluding the polypeptide of Accession No. AAB 19030.
33. An isolated polypeptide having at least 81 % sequence identity to an amino acid sequence selected from any one of SEQ ID NO: 67 to 118, 120, 122, 124, 126, 130 to 132, wherein the polypeptide has the activity of an L-Galactose-1 -phosphate phosphatase
34. The isolated polypeptide of claim 33 having at least 81 % sequence identity to the amino acid sequence of SEQ ID NO: 67.
35. The isolated polypeptide of claim 33 comprising to the amino acid sequence of SEQ ID NO: 67 to 118, 120, 122, 124, 126, 130 to 132.
36. The isolated polypeptide of claim 33 comprising the amino acid sequence of SEQ ID NO: 67.
37. An isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 67 to 105, 107 to 118, 120, 122, 124, 126, 130 to 132, or a variant thereof from a plant species, wherein the variant has the activity of an L-Galactose-1 -phosphate phosphatase, and wherein the variant is not the polypeptide of Accession No. AAB19030.
38. The isolated polypeptide of claim 37 comprising the amino acid sequence of SEQ ID NO: 67, or a variant thereof from a plant species, wherein the variant has the activity of an L-Galactose-1 -phosphate phosphatase, and wherein the variant is not polypeptide of Accession No. AAB19030.
39. An isolated polynucleotide encoding a polypeptide of any one of claims 32 to 38.
40. An isolated polynucleotide comprising: a) a polynucleotide comprising a fragment, of at least 15 nucleotides in length, of the polynucleotide of any one of claims 15 to 28 or 39; b) a polynucleotide comprising a complement, of at least 15 nucleotides in length, of the polynucleotide of any one of claims 15 to 28 or 39; or d) a polynucleotide comprising a sequence, of at least 15 nucleotides in length, capable of hybridising to the polynucleotide of any one of claims 15 to 28 or 39.
41. A genetic construct which comprises a polynucleotide of any one of claims 15 to 28, 39 or 40.
42. A vector comprising the expression construct of any one of claims 29 to 31 or the genetic construct of claim 41.
43. A host cell genetically modified to express a polynucleotide of any one of claims 15 to 28 or 39, or a polypeptide of any one of claims 32 to 38.
44. A host cell comprising the expression construct of any one of claims 29 to 31 or the genetic construct of claim 41.
45. A method for producing an L-Galactose-1 -phosphate phosphatase polypeptide comprising culturing a host cell comprising an expression construct of any one of claims 29 to 31 or the genetic construct of claim 41, capable of expressing an L- Galactose- 1 -phosphate phosphatase polypeptide.
46. A method for producing the enzymic product of an L-Galactose-1 -phosphate phosphatase comprising culturing a host cell including an expression construct of any one of claims 29 to 31 or the genetic construct of claim 41, capable of expressing an L-Galactose-1 -phosphate phosphatase polypeptide, in the presence of enzymic substrate which may be supplied to, or may be naturally present within the host cell.
47. A method for the biosynthesis of ascorbate comprising the steps of culturing a host cell comprising an expression construct of any one of claims 29 to 31 or the genetic construct of claim 41, capable of expressing an L-Galactose-1 -phosphate phosphatase polypeptide, in the presence of an ascorbate precursor which may be supplied to, or may be naturally present within the host cell.
48. A plant cell genetically modified to express a polynucleotide of any one of claims 15 to 28, or 39, or a polypeptide of any one of claims 32 to 38.
49. A plant cell which comprises an expression construct of any one of claims 29 to 31 or the genetic construct of claim 41.
50. A plant which comprises a plant cell of claim 48 or 49.
51. A method for selecting a plant altered in L-Galactose-1 -phosphate phosphatase activity, the method comprising testing of a plant for altered expression of a polynucleotide of any one of claims 15 to 28 or 39.
52. A method for selecting a plant with altered ascorbic acid content; the method comprising testing of a plant for altered expression of a polynucleotide of any one of claims 15 to 28 or 39.
53. A method for selecting a plant altered in L-Galactose-1 -phosphate phosphatase activity, the method comprising testing of a plant for altered expression of a polypeptide any one of claims 32 to 38.
54. A method for selecting a plant altered in ascorbic acid content, the method comprising testing of a plant for altered expression of a polypeptide of any one of claims 32 to 38.
55. A plant cell or plant produced by the method of any one of claims 1 to 14.
56. A plant selected by the method of any one of claims 51 to 54.
57. A method of producing ascorbate, the method comprising extracting ascorbate from a plant cell or plant of claim 55 or plant of claim 56.
58. A method for identifying a compound as a candidate for a herbicide, comprising: a) contacting said compound with a polypeptide comprising a sequence selected from any one of SEQ ID NO : 67 to 118, 120, 122, 124, 126, 130 to 132, or a variant thereof which has the activity of an L-Galactose-1 -phosphate phosphatase, and b) detecting the presence and/or absence of binding between said compound and said polypeptide; wherein binding indicates that said compound is a candidate for a herbicide.
59. A method for identifying a compound as a candidate for a herbicide, comprising: a) contacting said compound with a polypeptide comprising a sequence selected from any one of SEQ ID NO : 67 to 118, 120, 122, 124, 126, 130 to 132, or a variant thereof which has the activity of an L-Galactose-1 -phosphate phosphatase, and b) assessing the effect of the compound the an L-Galactose-1 -phosphate phosphatase activity of the polypeptide; wherein a decrease in activity indicates that said compound is a candidate for a herbicide.
60. The method of claim 58 or 59 wherein the variant has at least 50% sequence identity to an amino acid sequence selected from any one of SEQ ID NO: 67 to 118, 120, 122, 124, 126, 130 to 132.
61. The method of claim 58 or 59 in which polypeptide comprises an amino acid sequence selected from any one of SEQ ID NO: 67 to 118, 120, 122, 124, 126, 130 to 132.
62. The method of claim 58 or 59 wherein the variant has at least 50% sequence identity to the amino acid sequence of SEQ ID NO: 67.
63. The method of claim 58 or 59 in which polypeptide comprises the amino acid sequence of SEQ ID NO: 67.
64. A compound identified by a method of any one of claims 58 to 63.
65. A method for determining whether the compound of claim 64 has herbicidal activity, comprising: contacting a plant or plant cells with said herbicide candidate and detecting the presence or absence of a decrease in growth or viability of said plant or plant cells.
66. An antibody raised against a polypeptide of any one of claims 32 to 38.
67. A method of converting galactose- 1 -phosphate to galactose, the method comprising contacting galactose- 1 -phosphate with the expression product of an expression construct comprising a polynucleotide as defined in claim 1, part a) to obtain galactose.
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