Modulation of gene expression

Abstract

The present invention provides a method of producing a host cell, plant cell or plant having increased L-ascorbic acid (AsA) content or increased translation, production and / or activity of GDP-L-galactose phosphorylase (GGP), the method comprising transforming the host cell or plant cell with a polynucleotide encoding a polypeptide that modulates GDP-L-galactose phosphorylase (GGP). The invention also provides host cells, plant cells and plants genetically modified to contain and / or express the polynucleotides.

Description

Technical Field

[0001] The present invention relates to polynucleotides, gene constructs and methods for producing plants with altered GDP-L-galactose phosphorylase 3 (GGP) translation, production and / or activity; and / or altered L-ascorbic acid (AsA) content; and plants and plant cells produced by the method. Background Art

[0002] L-Ascorbic acid (AsA) is the most abundant soluble antioxidant in plants and is an essential nutrient for humans and a few other animals. AsA contributes significantly to the total intake of "free radical scavengers" or "antioxidant metabolites" in the human diet. There is now convincing evidence that these metabolites, either alone or in combination, have beneficial health effects, act as anticancer agents and prevent coronary heart disease. For people in both developed and developing countries, consuming sufficient amounts of fruits and vegetables to continuously maintain optimal AsA concentrations remains a challenge.

[0003] Almost all dietary AsA intake in humans comes from plant products. However, the AsA content of plant tissues is significantly variable. Although leaf AsA content is generally high and relatively uniform in herbaceous and woody plants, a large and unexplained variability in AsA content is found in non-green edible plant tissues.

[0004] Understanding how AsA biosynthesis is regulated may provide tools for manipulating biosynthesis in plants. Understanding the regulation of gene expression and the factors / elements that control this expression also provides valuable tools for genetic manipulation. The enzyme GDP-L-galactose phosphorylase (GGP) is known to play a key role in AsA biosynthesis. The regulation of GGP expression and activity has not been fully resolved.

[0005] It is desirable to increase the AsA content of plants.

[0006] It is an object of the present invention to provide new, improved and / or alternative tools for manipulating the expression and content of AsA in plants and / or to at least provide the public with a useful choice.

[0007] In this specification, where reference has been made to external sources of information (including patent specifications and other literature), this is generally for the purpose of providing a context for discussing the features of the invention. Unless otherwise stated, reference to such sources of information is not to be construed as an admission that such sources are prior art or form part of the common general knowledge in the art, in any jurisdiction. Summary of the invention

[0008] In a first aspect, the present invention relates to a method for producing a plant cell or plant having an increased L-ascorbic acid (AsA) content, the method comprising transforming a plant cell with a polynucleotide encoding a polypeptide having an amino acid sequence of any one of SEQ ID NOs: 1-102 and 348-356, or a variant of a polypeptide having at least about 70% sequence identity with a polypeptide having an amino acid sequence of any one of SEQ ID NOs: 1-102 and 348-356.

[0009] In a second aspect, the present invention relates to a method for producing a plant cell or plant having increased translation, production and / or activity of GDP-L-galactose phosphorylase 3 (GGP), the method comprising transforming a plant cell with a polynucleotide encoding a polypeptide having an amino acid sequence of any one of SEQ ID NOs: 1-102 and 348-356, or a polypeptide variant having at least about 70% sequence identity to a polypeptide having an amino acid sequence of any one of SEQ ID NOs: 1-102 and 348-356.

[0010] In a third aspect, the present invention relates to a method for producing a plant cell or plant having an increased L-ascorbic acid (AsA) content, the method comprising transforming a plant cell with a polynucleotide comprising a nucleotide sequence of any one of SEQ ID NOs: 103-108, or a variant thereof having at least about 70% sequence identity with a nucleotide having a nucleotide sequence of any one of SEQ ID NOs: 103-108.

[0011] In a fourth aspect, the present invention relates to a method for producing a plant cell or plant having increased translation, production and / or activity of GDP-L-galactose phosphorylase 3 (GGP), the method comprising transforming the plant cell with a polynucleotide comprising the nucleotide sequence of any one of SEQ ID NOs: 103-108, or a variant thereof having at least about 70% sequence identity with a nucleotide having the nucleotide sequence of any one of SEQ ID NOs: 103-108.

[0012] In a fifth aspect, the present invention relates to a method for producing a plant cell or plant having increased L-ascorbic acid (AsA) content or increased GDP-L-galactose phosphorylase 3 (GGP) translation, production and / or activity, the method comprising upregulating in a plant cell or expressing in a plant a polypeptide having an amino acid sequence of any one of SEQ ID NOs: 1-102 and 348-356, or a variant of a polypeptide having at least about 70% sequence identity with an amino acid sequence of any one of SEQ ID NOs: 1-102 and 348-356.

[0013] In a sixth aspect, the present invention relates to a method for producing a plant cell or plant having increased L-ascorbic acid (AsA) content or increased GDP-L-galactose phosphorylase 3 (GGP) translation, production and / or activity, the method comprising upregulating in a plant cell or expressing in a plant a polynucleotide comprising a nucleotide sequence selected from any one of SEQ ID NOs: 103-108 or a nucleotide sequence of a variant having at least about 70% sequence identity with the nucleotide sequence of any one of SEQ ID NOs: 103-108.

[0014] In the seventh aspect, the present invention relates to an isolated polynucleotide encoding a polypeptide having an amino acid sequence of any one of SEQ ID NOs: 1-4 and 102, or a variant of a polypeptide having at least about 70% sequence identity, preferably at least about 75% sequence identity, to the amino acid sequence of any one of SEQ ID NOs: 1-4 and 102.

[0015] In an eighth aspect, the invention relates to an isolated polynucleotide encoding a polypeptide having an amino acid sequence of any one of SEQ ID NOs: 109-153, 155-157, 159, 162-164, 166-174, 176, 178, 180-183, 185, 186, 188-190, 192-200, 202-204 and 206-209, or a variant of a polypeptide having at least about 70% sequence identity, preferably at least about 75% sequence identity, to the amino acid sequence of any one of SEQ ID NOs: 109-153, 155-157, 159, 162-164, 166-174, 176, 178, 180-183, 185, 186, 188-190, 192-200, 202-204 and 206-209.

[0016] In the ninth aspect, the present invention relates to a genetic construct comprising a polynucleotide encoding a polypeptide having an amino acid sequence of any one of SEQ ID NOs: 1-4 and 102, or a variant of a polypeptide having at least about 70% sequence identity, preferably at least about 75% sequence identity, to the amino acid sequence of any one of SEQ ID NOs: 1-4 and 102.

[0017] In a tenth aspect, the present invention relates to a gene construct comprising a polynucleotide encoding a polypeptide having an amino acid sequence of any one of SEQ ID NOs: 109-153, 155-157, 159, 162-164, 166-174, 176, 178, 180-183, 185, 186, 188-190, 192-200, 202-204 and 206-209, or a polypeptide having an amino acid sequence of any one of SEQ ID NOs: A variant of a polypeptide having at least about 70% sequence identity, preferably at least about 75% sequence identity, to the amino acid sequence of any one of NOs: 109-153, 155-157, 159, 162-164, 166-174, 176, 178, 180-183, 185, 186, 188-190, 192-200, 202-204 and 206-209.

[0018] In various embodiments, the gene construct may further include a second polynucleotide

[0019] a) which encodes a polypeptide having an amino acid sequence of any one of SEQ ID NOs: 109-209, or a variant of a polypeptide having at least about 70% sequence identity, preferably at least about 75% sequence identity, to an amino acid sequence of any one of SEQ ID NOs: 109-209; and / or

[0020] b) having the nucleotide sequence of SEQ ID NO: 210, or a nucleotide variant thereof having at least about 70% sequence identity with the nucleotide sequence of SEQ ID NO: 210.

[0021] In the eleventh aspect, the present invention relates to a genetic construct comprising a polynucleotide, the polynucleotide comprising the nucleotide sequence of any one of SEQ ID NOs: 103-108, or a polypeptide variant having at least about 70% sequence identity, preferably at least about 75% sequence identity, with the nucleotide sequence of any one of SEQ ID NOs: 103-108.

[0022] In a twelfth aspect, the present invention relates to a gene construct comprising a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 210, or a polypeptide variant having at least about 70% sequence identity with the nucleotide sequence of SEQ ID NO: 210.

[0023] In a thirteenth aspect, the present invention relates to a kit comprising the first gene construct of the ninth or eleventh aspect described herein; and the second gene construct of the tenth or twelfth aspect described herein.

[0024] In a fourteenth aspect, the present invention provides a host cell comprising one or more gene constructs described herein.

[0025] In a fifteenth aspect, the present invention provides a host cell genetically modified to express a polynucleotide encoding a polypeptide having an amino acid sequence of any one of SEQ ID NOs: 1-102 and 348-356, or a variant of a polypeptide having at least about 70% sequence identity with an amino acid sequence of any one of SEQ ID NOs: 1-102 and 348-356.

[0026] In a sixteenth aspect, the present invention provides a host cell genetically modified to express a polynucleotide comprising a nucleotide sequence of any one of SEQ ID NOs: 103-108, or a variant thereof having at least about 70% sequence identity with a nucleotide sequence of any one of SEQ ID NOs: 103-108.

[0027] In the seventeenth aspect, the present invention provides a plant cell genetically modified to express a polynucleotide encoding a polypeptide having an amino acid sequence of any one of SEQ ID NOs: 1-102 and 348-356 or a variant of a polypeptide having at least 70% sequence identity with a polypeptide having an amino acid sequence of any one of SEQ ID NOs: 1-102 and 348-356.

[0028] In an eighteenth aspect, the present invention provides a plant cell genetically modified to express a polynucleotide comprising a nucleotide sequence selected from any one of SEQ ID NOs: 103-108 or a variant thereof having at least 70% sequence identity with the nucleotide sequence of any one of SEQ ID NOs: 103-108.

[0029] In a nineteenth aspect, the present invention provides a plant comprising the plant cell described herein.

[0030] In a twentieth aspect, the present invention provides a method for selecting plants with altered GDP-L-galactose phosphorylase 3 (GGP) activity, the method comprising testing the plant for altered expression of a polynucleotide encoding a polypeptide having an amino acid sequence of any one of SEQ ID NOs: 1-102 and 348-356 or a variant of a polypeptide having at least 70% sequence identity with a polypeptide having an amino acid sequence of any one of SEQ ID NOs: 1-102 and 348-356.

[0031] In another aspect, the present invention provides a method for selecting a plant with altered GDP-L-galactose phosphorylase 3 (GGP) activity, the method comprising testing the plant for altered expression of a polynucleotide comprising a nucleotide sequence selected from any one of SEQ ID NOs: 103-108 or a variant thereof, wherein the variant comprises a sequence having at least 70% sequence identity with the nucleotide sequence of any one of SEQ ID NOs: 103-108.

[0032] In another aspect, the present invention provides a method for selecting plants having altered L-ascorbic acid (AsA) content, the method comprising testing a plant for altered expression of a polynucleotide encoding a polypeptide having an amino acid sequence of any one of SEQ ID NOs: 1-102 and 348-356 or a variant of a polypeptide having at least 70% sequence identity with a polypeptide having an amino acid sequence of any one of SEQ ID NOs: 1-102 and 348-356.

[0033] In another aspect, the present invention provides a method for selecting plants with altered L-ascorbic acid (AsA) content, the method comprising testing the plant for altered expression of a polynucleotide comprising a nucleotide sequence selected from any one of SEQ ID NOs: 103-108 or a variant thereof comprising a sequence having at least 70% sequence identity with the nucleotide sequence of any one of SEQ ID NOs: 103-108.

[0034] In another aspect, the invention provides a plant cell or plant produced by any of the methods described herein.

[0035] In another aspect, the invention provides a plant cell or plant selected by any of the methods described herein.

[0036] In another aspect, the invention provides a group or population of plants produced by any of the methods described herein.

[0037] In another aspect, the present invention provides a method for producing AsA, comprising extracting AsA from the host cell described herein.

[0038] In another aspect, the present invention provides a method for producing AsA, the method comprising extracting AsA from the plant cell or plant described herein.

[0039] The following embodiments may relate to any or all of the above aspects.

[0040] In various embodiments, the method can further include transforming or co-transforming a second polynucleotide with the polynucleotide encoding a polypeptide having an amino acid sequence of any one of SEQ ID NOs: 109-209 or a variant of a polypeptide having at least about 70% sequence identity to an amino acid sequence of any one of SEQ ID NOs: 109-209.

[0041] In various embodiments, the method can further include transforming or co-transforming a second polynucleotide with the polynucleotide, the second polynucleotide having the nucleotide sequence of SEQ ID NO: 210, or a variant thereof having at least about 70% sequence identity to the nucleotide sequence of SEQ ID NO: 210. In various embodiments, the variant has at least about 75, 80, 85, 90, 95, 96, 97, 98, 99%, or 100% sequence identity to the nucleotide sequence of SEQ ID NO: 210.

[0042] In various embodiments, the host cell or plant cell can be genetically modified to express a second polynucleotide encoding a polypeptide having an amino acid sequence of any one of SEQ ID NOs: 109-209, or a variant of a polypeptide having at least about 70% sequence identity to an amino acid sequence of any one of SEQ ID NOs: 109-209.

[0043] In various embodiments, the host cell or plant cell can be genetically modified to express a second polynucleotide having the nucleotide sequence of SEQ ID NO:210, or a variant thereof having at least about 70% sequence identity thereto.

[0044] In various embodiments, the polynucleotide may encode a polypeptide having the following amino acid sequence, or may encode a polypeptide variant having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the following amino acid sequence:

[0045] a) any one of SEQ ID Nos: 1-102 and 348-356;

[0046] b) any one of SEQ ID Nos: 1-50, 52-60, 62-83, 85-95, 97-102, 348-350 and 352-354;

[0047] c) any one of SEQ ID Nos: 1-49, 53-56, 58-60, 62, 64-66, 70-79, 82, 83, 85-87, 89-91, 93-95, 98, 102, 348-350, 352 and 353;

[0048] d) any one of SEQ ID No: 1-49, 53-56, 58, 60, 62, 64, 66, 70, 71, 74-76, 78, 79, 83, 85-87, 89-91, 93-95, 98, 102, 348-350, 352 and 353;

[0049] e) any one of SEQ ID No: 1-49, 53-56, 58, 60, 62, 64, 66, 70, 71, 74-76, 78, 79, 83, 85-87, 89-90, 93, 94, 98, 102, 348-350, 352 and 353;

[0050] f) any one of SEQ ID No: 1-5, 7-10, 12-19, 21-24, 28-31, 33, 35, 36, 38-45, 48, 49, 53, 55, 56, 58, 60, 62, 64, 66, 71, 74-76, 79, 83, 85, 87, 89, 90, 93, 94, 98, 102, 350, 352;

[0051] g) any one of SEQ ID Nos: 1-4, 38, 41-45, 48, 49, 51, 52, 56, 60, 63, 66, 79, 80, 85, 89, 90, 92, 96, 97, 100-102, and 348-356;

[0052] h) any one of SEQ ID Nos: 1-4, 49, 51, 66, 80, 85, 90, 92, 96, 102, and 348-356;

[0053] i) any one of SEQ ID Nos: 1-4 and 102;

[0054] j) any one of SEQ ID Nos: 1-4;

[0055] k) SEQ ID No: 1 or 102;

[0056] 1) SEQ ID No: 1; or

[0057] m) SEQ ID No: 102.

[0058] In various embodiments, the polynucleotide may have the following nucleotide sequence or may encode a polynucleotide variant having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the following nucleotide sequence:

[0059] a) any one of SEQ ID Nos: 103-108,

[0060] b) SEQ ID No: 103 or 105, or

[0061] c) SEQ ID No: 103.

[0062] In various embodiments, the polynucleotide or the second polynucleotide may encode a polypeptide having the following amino acid sequence, or may encode a polypeptide variant having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the following amino acid sequence:

[0063] a) any one of SEQ ID Nos: 109-209;

[0064] b) any one of SEQ ID Nos: 109-174, 176-178 and 180-209;

[0065] c) any one of SEQ ID Nos: 109-159, 162-164, 166-174, 176, 178, 180-186, 188-190, 192-204 and 206-209;

[0066] d) any one of SEQ ID Nos: 109-153, 155-157, 159, 162-164, 166-174, 176, 178, 180-186, 188-190, 192-204 and 206-209;

[0067] e) any one of SEQ ID Nos: 109-153, 155-157, 159, 162-164, 166-174, 176, 178, 180-183, 185, 186, 188-190, 192-200, 202-204 and 206-209;

[0068] f) any one of SEQ ID Nos: 109-114, 125, 126, 142, 177, 179, 187, 191 and 205;

[0069] g) any one of SEQ ID Nos: 109-114 and 179;

[0070] h) any one of SEQ ID Nos: 109-114; or

[0071] i) SEQ ID NO:109.

[0072] In various embodiments, the polynucleotide or the second polynucleotide can have a nucleotide sequence that is a variant of a polynucleotide that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleotide sequence of SEQ ID NO:210, or can encode a variant of a polynucleotide that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleotide sequence of SEQ ID NO:210.

[0073] In various embodiments, a polypeptide or polypeptide variant may include one or more SANT / MYB domains. In various embodiments, a polypeptide or polypeptide variant may include two SANT / MYB domains.

[0074] In various embodiments, upregulation may include genetic engineering.

[0075] In various embodiments, transformation comprises stable transformation. In various embodiments, the method comprises stable transformation of plant cells with polynucleotides or variants thereof. In various embodiments, the host cell or plant cell is stably transformed and / or stably genetically modified.

[0076] In various embodiments, the methods may include transforming a plant cell with a genetic construct described herein. In various embodiments, the methods may include stably transforming a plant cell with a genetic construct described herein.

[0077] In various embodiments, the host cell or plant cell includes an endogenous GGP gene, preferably a functional GGP gene. For example, in some embodiments, the host cell or plant cell has been previously transformed with a functional GGP gene. In some embodiments, the method includes transforming the host cell or plant cell with a GGP gene.

[0078] In some embodiments, the GGP gene has a promoter comprising one or more MYBS1 binding sites, preferably two or more MYBS1 binding sites. In some embodiments, the method comprises gene editing the promoter of the endogenous GGP gene to provide a promoter comprising one or more MYBS1 binding sites. In some embodiments, the method comprises modifying the endogenous GGP promoter to provide a GGP promoter comprising one or more MYBS1 binding sites. In some embodiments, the method comprises transforming a plant cell or a host cell with the GGP gene. In some embodiments, the method comprises transforming a plant cell or a host cell with a GGP gene having a promoter, the promoter comprising one or more MYBS1 binding sites, preferably two or more MYBS1 binding sites.

[0079] In various embodiments, upregulation can include crossing with a plant expressing a polypeptide comprising an amino acid sequence having at least 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to a polypeptide having an amino acid sequence of any one of SEQ ID NOs: 1-102 and 348-356 and / or SEQ ID NOs: 109-209.

[0080] According to certain embodiments, upregulating comprises hybridizing with a plant expressing a polynucleotide comprising a nucleotide sequence selected from any one of SEQ ID NOs: 103 to 108 and 210.

[0081] In various embodiments, the gene construct may include one or more polynucleotides operably linked to a promoter. In various embodiments, the promoter is at least one of:

[0082] i) a promoter not normally associated with a polynucleotide in nature,

[0083] ii) Promoters derived from bacteria, fungi, insects, mammals or viruses.

[0084] iii) a bacterial promoter,

[0085] iv) a fungal promoter,

[0086] v) insect promoters,

[0087] vi) a mammalian promoter, and

[0088] vii) Viral promoters.

[0089] In various embodiments, the promoter can be of bacterial, fungal, insect, mammalian, or viral origin.

[0090] In various embodiments, the promoter can be a constitutive promoter or a tissue-specific promoter. In various embodiments, the promoter causes expression in leaves and / or roots. Preferably, the promoter causes expression in leaves. More preferably, the promoter is a tissue-specific promoter that causes targeted expression in leaves and / or roots.

[0091] In various embodiments, one or more polynucleotides can be regulated by a compound. In some embodiments, the compound is L-ascorbic acid (AsA) or a related metabolite.

[0092] In various embodiments, the host cell can be a plant cell.

[0093] The term "comprising" as used in this specification and claims means "consisting at least in part of..." When interpreting statements in this specification and claims that include the term "comprising", other features may be present in addition to the features preceded by the term in each statement. Related terms such as "comprise" and "comprised" are to be interpreted in a similar manner.

[0094] As used herein, the term "and / or" means "and" or "or", or both.

[0095] As used herein, the term "(s)" following a noun is intended to refer to the plural and / or singular forms of that noun.

[0096] Reference to numerical ranges disclosed herein (e.g., 1 to 10) is intended to also include reference to all rational numbers within the range (e.g., 1, 1.1, 2, 3, 3.9, 4, 5, 6, 6.5, 7, 8, 9, and 10) and any range of rational numbers within the range (e.g., 2 to 8, 1.5 to 5.5, and 3.1 to 4.7), and thus, all subranges of all ranges explicitly disclosed herein are hereby explicitly disclosed. These are only examples of what is specifically intended, and all possible combinations of numerical values ​​between the lowest and highest values ​​recited are considered to be explicitly stated in this application in a similar manner.

[0097] The present invention may also be broadly said to include the parts, elements and features referred to or indicated individually or collectively in the specification of the present application, as well as any or all combinations of any two or more of the parts, elements or features, and where specific integers having known equivalents in the art to which the present invention relates are referred to herein, such known equivalents are deemed to be incorporated herein as if individually set forth.

[0098] Although the present invention is broadly defined above, it will be appreciated by those skilled in the art that the present invention is not limited thereto and that the present invention also includes embodiments for which the following description gives examples. BRIEF DESCRIPTION OF THE DRAWINGS

[0099] The present invention will now be described by way of example only and with reference to the accompanying drawings, in which:

[0100] Figure 1 Provided are line graphs showing AsA content in six Actinidia species over days after flowering (DAF).

[0101] Figure 2 Graphs showing the expression (lines) and AsA content (bars) of GGPs (GGP1, GGP2 and GGP3) in (A) A. rufa and (B) A. eriantha over days after flowering (DAF) are provided. Data are shown as mean + / - standard deviation (n=3).

[0102] Figure 3 Bar graphs showing that overexpression or knockdown of AceGGP3 alters AsA concentration in Actinidia chinensis fruit are provided. (A) shows the expression abundance of AceGGP3, (B) shows the AsA content in Actinidia chinensis fruit 7 days after infiltration; (C) shows the expression abundance of AceGGP3, (D) shows the AsA content in Actinidia chinensis callus 6 days after infiltration with the following substances: EV (empty vector); OX-AceGGP3 (AceGGP3 overexpression) or TRV-AceGGP3 (AceGGP3-antisense expression). Data are shown as mean + / - standard deviation (n = 3). Significant differences were detected by t-test using GraphPad Prism8 (*, p < 0.05; **, p < 0.01; ***, p < 0.001).

[0103] Figure 4 Provided are bar graphs showing (A) RT-qPCR analysis of AceGGP3 and (B) relative AsA content in five Actinidia chinensis transgenic lines overexpressing AceGGP3. WT: wild type; OE-GGP3#10, OE-GGP3#14, OE-GGP3#20, OE-GGP3#21, OE-GGP3#23 represent five different transgenic lines, respectively. Data are shown as mean + / - standard deviation (n=3). Significant differences were detected by t-test using GraphPad Prism 8 (*, p<0.05; **, p<0.01; ***, p<0.001).

[0104] Figure 5A bar graph showing the relative AsA content of Actinidia pubescens callus in CRISPR / Cas9-induced AceGGP3 mutants is provided. wt: wild type; ggp3#2, ggp3#11, ggp3#13, and ggp3#15 represent four gene-edited lines, respectively. Data are shown as mean + / - standard deviation (n=3). Significant differences were detected by t-test using GraphPad Prism 8 (*, p<0.05; **, p<0.01; ***, p<0.001).

[0105] Figure 6 Provided are bar graphs and line graphs showing the correlation between (A) AsA content (bars) and the expression of AceMYBS1 (lines), and (B) the correlation between the expression of AceGGP3 and AceMYBS1 in different developmental stages (every 20 days) of Actinidia pubescens fruit. Error bars represent + / - SD (n=3).

[0106] Figure 7 A provides a bar graph showing the results of dual luciferase assays in tobacco leaves, which shows that AceMYBS1 activates transcription of different length fragments of the AceGGP3 promoter (P2660, P2088, P1606, P1106). Empty vector (EV) is a control. Error bars: + / -SD. Significant differences were detected by t-test (**, p<0.01; ns: not significant). Figure 7 B shows the locations of two predicted binding targets of AceMYBS1 on the AceGGP3 promoter and an electrophoretic mobility shift assay (EMSA) showing binding of AceMYBS1 to the AceGGP3 promoter. Unlabeled probe was used as competitor. 100× and 300× represent the ratio of the competitor.

[0107] Figure 8 Provided are bar graphs showing the results of RT-qPCR analysis of (A) AceMYBS1 and (B) AceGGP3 transcripts, and (C) AsA content of Actinidia pubescens fruit (B) 7 days after infiltration with transiently expressed AceMYBS1 vector. EV: empty vector; OX-AceMYBS1: AceMYBS1 overexpression; TRV-AceMYBS1: AceMYBS1-antisense expression. The experiment was repeated three times, and each experiment contained three to six kiwifruits of each genotype. Error bars: + / - SD. Significant differences were detected by t-test (*, p<0.05; **, p<0.01; ***, p<0.001; ns: not significant).

[0108] Fig. 9Bar graphs are provided showing the results of RT-qPCR analysis of (A) AceMYBS1 and (B) AceGGP3; and (C) AsA content in callus of Actinidia pubescens transfected with the following vectors: EV: empty vector; OX-AceMYBS1: AceMYBS1 overexpression; TRV-AceMYBS1: AceMYBS1-antisense expression; TRV-AceGGP3: AceGGP3-antisense expression; TRV-AceGGP3+OX-AceMYBS1: AceMYBS1 overexpressed in the TRV-AceGGP3 background. All experiments were performed in triplicate. Error bars: + / - SD. Significant differences were detected by t-test (*, p<0.05; **, p<0.01; ***, p<0.001; ns: not significant).

[0109] Fig.10 Provided are bar graphs showing the results of RT-qPCR analysis of (A) AceMYBS1 and (B) AceGGP3 in wild type (WT) and six AceMYBS1 overexpressing transgenic kiwifruit (Actinidia quinata) lines: OE-AceMYBS1#1, OE-AceMYBS1#2, OE-AceMYBS1#3, OE-AceMYBS1#4, OE-AceMYBS1#5, OE-AceMYBS1#7.

[0110] Fig.11 Bar graphs are provided showing the results of (A) a bimolecular luminescent complementation (BiLC) assay showing the interaction of AceMYBS1 and AceGBF3 in vivo and (B) an in vitro pull-down assay showing the interaction between AceMYBS1 and AceGBF3. In (A), Agrobacterium clones containing the corresponding recombinant plasmids were combined 1:1 (v / v) and infiltrated into Nicotiana benthamiana leaves. Error bars show mean + / - SD; significant differences were detected by t-test: ***, p<0.001; ns: not significant. In (B), AceGBF3-GST protein was incubated with immobilized AceMYBS1-6xHis or 6xHis protein, and the immunoprecipitated fractions were detected by anti-GST antibody.

[0111] Fig.12Bar graphs showing the following results are provided: (A) Dual luciferase assay in tobacco leaves showing transcription of AceGGP3 activated by AceMYBS1 and AceGBF3 alone or together; (B) RT-qPCR analysis of AsA content and (C) expression level in transiently expressed Actinidia chinensis fruits 7 days after transformation; (D) Expression levels of AceGGP3 and (E) AsA content in transiently expressed Actinidia chinensis callus; and (F) Expression levels of NbGGP and (G) AsA content in transiently expressed tobacco leaves. EV: empty vector; OX-AceGBF3: AceGBF3 overexpression; TRV-AceGBF3: AceGBF3-antisense expression. The experiment was repeated three times, and each experiment contained three to six kiwifruit / genotype. All the above error bars represent standard deviations (+ / -SD), and three technical replicates were performed for each experimental group. Significant differences were detected by t-test (*, p<0.05; **, p<0.01; ***, p<0.001; ns: not significant). Different letters above the bars indicate significant differences obtained by one-way ANOVA test (p<0.05).

[0112] Fig.13 Provided are bar graphs showing (A) AceGGP3 expression levels and (B) AsA content determined by RT-qPCR analysis in Actinidia chinensis fruits transiently co-expressing AceMYBS1 and AceGBF3. EV: empty vector; TRV-AceMYBS1: AceMYBS1-antisense expression; OX-AceMYBS1: AceMYBS1 overexpression; TRV-AceMYBS1+TRV-AceGBF3: AceMYBS1 and AceGBF3 co-antisense expression; OX-AceMYBS1+OX-AceGBF3: co-overexpression of AceMYBS1 and AceGBF3; OX-AceMYBS1+TRV-AceGBF3: AceMYBS1-overexpression in an AceGBF3-antisense expression background; OX-AceGBF3+TRV-AceMYBS1: AceGBF3-overexpression in an AceMYBS1-antisense expression background. The experiment was repeated three times, and each experiment contained three to six kiwifruits / genotype. Significant differences were detected by t-test (*, p<0.05; **, p<0.01; ***, p<0.001; ns: not significant).

[0113] Fig.14Bar graphs showing (A) relative AsA content and (B) RT-qPCR analysis of AceGGP3 in wild-type (WT) and transgenic Actinidia chinensis overexpressing AceGBF3 are provided. All error bars above represent standard deviation (+ / -SD), and three technical replicates were performed for each experimental group. Significant differences were detected by t-test (*, p<0.05; **, p<0.01; ***, p<0.001; ns: not significant).

[0114] Fig.15 Bar graphs showing the average leaf AsA content (in mg / 100 g fresh weight) of (A) rice, (B) soybean and (C) Arabidopsis plants stably transformed to express AceMYBS1 are provided. Each transformed line is shown as a separate bar. Measurements were performed in triplicate and error bars represent standard deviations. In (A) and (B), asterisks represent significant differences from wild type according to Student's T test (p<0.05). In (C), different letters represent significantly different groupings (p<0.05) according to Student's T test. WT: wild type control plant. DETAILED DESCRIPTION

[0115] The present invention, in some embodiments thereof, relates to methods for producing host cells, including plant cells or plants, having increased L-ascorbic acid (AsA) and / or increased GGP translation, production and / or activity.

[0116] The present invention is based on the identification of two bZIP transcription factors, through genetic and molecular characterization as described herein, that are positive regulators of AsA biosynthesis by activating GGP transcription. The MYBS1-like transcription factor in kiwifruit was shown to bind to the promoter of GGP3 and, when overexpressed in kiwifruit, resulted in a significant increase in GGP3 expression and AsA accumulation. Overexpression of the GBF3 bZIP transcription factor also increased AsA content in an additive manner with MYBS1.

[0117] Identification of transcription factors that contribute to the regulation of AsA biosynthesis in plants allows the development of marker-assisted selection to breed plants with higher AsA content and allows the production of high levels of AsA by biotechnological means, such as biopharmaceuticals in crop plants and metabolic engineering of host cells such as yeast.

[0118] Therefore, according to one aspect of the present invention, a method for producing a plant cell or plant having increased L-ascorbic acid (AsA) content and / or increased GDP-L-galactose phosphorylase 3 (GGP) translation, production and / or activity is provided, the method comprising transforming a plant cell with a polynucleotide encoding a polypeptide having an amino acid sequence of any one of SEQ ID NOs: 1-102 and 348-356, or a variant of a polypeptide having at least about 70% sequence identity with a polypeptide having an amino acid sequence of any one of SEQ ID NOs: 1-102 and 348-356 (MYBS1-like protein).

[0119] In various embodiments, the method can further include transforming or co-transforming a second polynucleotide with the polynucleotide, the second polynucleotide encoding a polypeptide having an amino acid sequence of any one of SEQ ID NOs: 109-209 or a variant of a polypeptide having at least about 70% sequence identity with an amino acid sequence of any one of SEQ ID NOs: 109-209 (GBF3-like protein).

[0120] Applicants have identified a polynucleotide (SEQ ID No: 103) encoding a MYBS1-like transcription factor polypeptide (SEQ ID No: 1) and a polynucleotide (SEQ ID No: 210) encoding a GBF3-like transcription factor polypeptide from Actinidia pubescens (SEQ ID No: 109), as described in the Examples.

[0121] Applicants have shown that these polypeptides induce the expression of GGP3 and increase AsA levels in several species of Kiwifruit and Nicotiana.

[0122] The applicant also confirmed

[0123] a) MYBS1-like polypeptide sequences from a number of species that have significant sequence conservation with SEQ ID No: 1 and are variants of each other (SEQ ID Nos: 2-102 and 348-356), and

[0124] b) GBF3-like polypeptide sequences from many species share significant sequence conservation with SEQ ID No: 109 and are variants of each other (SEQ ID Nos: 110-209).

[0125] Disclosed herein are genetic constructs, vectors and plants containing polynucleotide sequences encoding MYBS1-like polypeptides (SEQ ID NOs: 1-102 and 348-356) or sequences encoding polypeptide sequences (SEQ ID NOs: 103-108) and / or polynucleotide sequences encoding GBF3-like polypeptides (SEQ ID NOs: 109-209) or sequences encoding polypeptide sequences (SEQ ID NO: 210).

[0126] In certain embodiments, plants and host cells comprising the gene constructs and vectors disclosed herein are provided.Preferably, the plants and host cells are stably transformed with the gene constructs and / or vectors.

[0127] In some embodiments, plants with altered GGP translation, production and / or activity relative to suitable control plants are provided, as well as plants with altered AsA content relative to suitable control plants. In some embodiments, plants with increased GGP translation, production and / or activity and increased AsA are provided. Preferably, the plant is stably transformed or modified.

[0128] In other embodiments, methods for producing such plants and methods for selecting such plants are provided.

[0129] Suitable control plants include untransformed plants of the same species or variety or plants transformed with a control construct.

[0130] 1. Polynucleotides and fragments

[0131] As used herein, the term "polynucleotide" means a single- or double-stranded deoxyribonucleotide or ribonucleotide polymer of any length but preferably of at least 15 nucleotides, and includes, by way of non-limiting example, coding and non-coding sequences, sense and antisense sequence complements, exons, introns, genomic DNA, cDNA, pre-mRNA, mRNA, rRNA, siRNA, miRNA, tRNA, ribozymes, recombinant polynucleotides, isolated and purified naturally occurring DNA or RNA sequences, synthetic RNA and DNA sequences, nucleic acid probes, primers and fragments of genes.

[0132] A "fragment" of a polynucleotide sequence provided herein is a subsequence of continuous nucleotides that can specifically hybridize to a target of interest, such as a sequence of at least 15 nucleotides in length. Fragments as disclosed herein include 15 nucleotides of continuous nucleotides of a polynucleotide as disclosed herein, preferably at least 20 nucleotides, more preferably at least 30 nucleotides, more preferably at least 50 nucleotides, more preferably at least 50 nucleotides, most preferably at least 60 nucleotides. Fragments of polynucleotide sequences can be used in antisense, gene silencing, triple helix or ribozyme technology, or as primers, probes, included in microarrays, or for polynucleotide-based selection methods disclosed herein.

[0133] The term "primer" refers to a short polynucleotide, usually with a free 3'OH group, which hybridizes to a template and is used to initiate polymerization of a polynucleotide complementary to the template. Such primers are preferably at least 5, more preferably at least 6, more preferably at least 7, more preferably at least 9, more preferably at least 10, more preferably at least 11, more preferably at least 12, more preferably at least 13, more preferably at least 14, more preferably at least 15, more preferably at least 16, more preferably at least 17, more preferably at least 18, more preferably at least 19, more preferably at least 20 nucleotides in length.

[0134] The term "probe" refers to a short polynucleotide used to detect a polynucleotide sequence complementary to the probe in a hybridization-based assay. The probe may consist of a "fragment" of a polynucleotide as defined herein. Preferably, such a probe is at least 5, more preferably at least 10, more preferably at least 20, more preferably at least 30, more preferably at least 40, more preferably at least 50, more preferably at least 100, more preferably at least 200, more preferably at least 300, more preferably at least 400, and most preferably at least 500 nucleotides in length.

[0135] 2. Peptides and fragments

[0136] As used herein, the term "polypeptide" encompasses amino acid chains of any length but preferably at least 5 amino acids, including full-length proteins, in which the amino acid residues are linked by covalent peptide bonds. The polypeptides disclosed herein may be purified natural products, or may be produced in part or in whole 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 a derivative thereof.

[0137] A "fragment" of a polypeptide is a subsequence of a polypeptide that performs the functions required for biological activity and / or provides a three-dimensional structure of the polypeptide. The term may refer to a polypeptide capable of performing the above-mentioned enzymatic activity, an aggregate of the polypeptide such as a dimer or other polymer, a fusion polypeptide, a polypeptide fragment, a polypeptide variant, or a derivative thereof.

[0138] The term "isolated" as applied to the polynucleotide or polypeptide sequences disclosed herein is used to refer to a sequence removed from its natural cellular environment. An isolated molecule can be obtained by any method or combination of methods, including biochemical, recombinant and synthetic techniques.

[0139] The term "recombinant" refers to a polynucleotide sequence that is removed from sequences surrounding it in its natural environment and / or recombined with sequences not found in its natural environment.

[0140] A "recombinant" polypeptide sequence is produced by translation from a "recombinant" polynucleotide sequence.

[0141] The term "derived from" with respect to a polynucleotide or polypeptide derived from a particular genus or species as disclosed herein means that the polynucleotide or polypeptide has the same sequence as a polynucleotide or polypeptide naturally occurring in that genus or species. Thus, a polynucleotide or polypeptide derived from a particular genus or species can be produced synthetically or recombinantly.

[0142] 3. Variants

[0143] As used herein, the term "variant" refers to a polynucleotide or polypeptide sequence different from a specifically identified sequence, wherein one or more nucleotides or amino acid residues are deleted, substituted or added. Variants can be naturally occurring allele variants or non-naturally occurring variants. Variants can be from the same species or from other species, and can encompass homologues, paralogues and orthologues. Variants as described herein can also be produced by site-directed mutagenesis of the coding sequence of a polypeptide, or by combining domains ("domain swaps") of the coding sequences of different naturally occurring polypeptides. The technology for modifying the gene encoding the functional polypeptides described herein is known, and in particular includes directed evolution techniques, site-directed mutagenesis techniques and random mutagenesis techniques, and can be used to increase the specific activity of a polypeptide, change substrate specificity, change expression levels, change subcellular locations or modify polypeptide-polypeptide interactions in a desired manner. Such modified polypeptides are considered to be variants.

[0144] In certain embodiments, variants of the polynucleotides and polypeptides disclosed herein have the same or similar biological activity as the polynucleotides or polypeptides disclosed herein.The term "variant" with respect to polynucleotides and polypeptides encompasses all forms of polynucleotides and polypeptides as defined herein.

[0145] 4. Polynucleotide variants

[0146] Variant polynucleotide sequences preferably exhibit at least 70%, more preferably at least 71%, more preferably at least 72%, more preferably at least 73%, more preferably at least 74%, more preferably at least 75%, more preferably at least 76%, more preferably at least 77%, more preferably at least 78%, more preferably at least 79%, more preferably at least 80%, more preferably at least 81%, more preferably at least 82%, more preferably at least 83%, more preferably at least 84%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, and most preferably at least 99% identity compared to the sequences disclosed herein. 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, more preferably at least 200 nucleotide positions, more preferably at least 300 nucleotide positions, more preferably at least 400 nucleotide positions, more preferably at least 500 nucleotide positions, and most preferably over the entire length of the polynucleotides disclosed herein.

[0147] Polynucleotide sequence identity can be determined in the following manner. 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.gov / blast / ), the subject polynucleotide sequence is compared to the candidate polynucleotide sequence using BLASTN (from the BLAST suite of programs, version 2.2.5 [November 2002]). The default parameters of bl2seq are used, except that filtering of low-complexity portions should be turned off.

[0148] The identity of a polynucleotide sequence can be checked using the following unix command line parameters:

[0149] bl2seq–i nucleotideseq1–j nucleotideseq2–FF–p blastn

[0150] The parameter -FF turns off filtering of low complexity parts. The parameter -p selects the appropriate algorithm for the sequence. The bl2seq program reports sequence identity as the number and percentage of identical nucleotides in the "identity=" line.

[0151] The polynucleotide sequence identity can also be calculated using a global sequence alignment program over the entire overlap length between the candidate and subject polynucleotide sequences (e.g., Needleman, SB and Wunsch, CD (1970) J. Mol. Biol. 48, 443-453). A complete implementation of the Needleman-Wunsch global alignment algorithm can be found in the needle program of the EMBOSS software package (Rice, P. Longden, I. and Bleasby, A. EMBOSS: The European Molecular Biology Open Software Suite, Trends in Genetics June 2000, vol 16, No 6. pp. 276-277), which can be obtained from http: / / www.hgmp.mrc.ac.uk / Software / EMBOSS / . The server of the European Bioinformatics Institute also provides the function of performing the EMBOSS-needle global alignment between two sequences online at http: / / www.ebi.ac.uk / emboss / align / .

[0152] Alternatively, the GAP program can be used, which calculates the 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.

[0153] Another method for calculating the % sequence identity of polynucleotides is based on the use of Clustal X to align the sequences being compared (Jeanmougin et al., 1998, Trends Biochem. Sci. 23, 403-5).

[0154] The polynucleotide variants disclosed herein also encompass those having similarity to one or more specifically identified sequences, i.e., those that are likely to retain functional equivalence to those sequences, and that could not reasonably be expected to occur by random chance. Such sequence similarity with respect to polypeptides can be determined using the publicly available bl2seq program from the BLAST suite of programs described above.

[0155] Polynucleotide sequences can be checked for similarity using the following unix command line arguments:

[0156] bl2seq–i nucleotideseq1–j nucleotideseq2–FF–p tblastx

[0157] The parameter -FF turns off filtering of low complexity parts. The parameter -p selects the appropriate algorithm for the sequence. The program finds regions of similarity between sequences and reports an "E value" for each such region, which is the expected number of times one can expect to see such a match by chance in a database of a fixed reference size containing random sequences. The size of the database is set by default in the bl2seq program. For small E values, much less than 1, the E value is approximately the probability of such a random match.

[0158] When compared to any of the specifically identified sequences, the variant polynucleotide sequence preferably exhibits less than 1×10 -10 More preferably, less than 1×10 -20 , preferably less than 1×10 -30 , preferably less than 1×10 -40 , preferably less than 1×10 -50 , more preferably less than 1×10 -60 , preferably less than 1×10 -70 , preferably less than 1×10 -80 , preferably less than 1×10 -90 and most preferably less than 1×10 -100 The E value of .

[0159] Alternatively, a variant polynucleotide as disclosed herein hybridizes under stringent conditions to a designated polynucleotide sequence or its complement.

[0160] The term "hybridize under stringent conditions" and its grammatical equivalents refer to the ability of a polynucleotide molecule to hybridize to a target polynucleotide molecule (e.g., a target polynucleotide molecule immobilized on a DNA or RNA blot (e.g., 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 and then increasing the stringency to the desired stringency.

[0161] For polynucleotide molecules greater than about 100 bases in length, typical stringent hybridization conditions are no more than 25 to 30°C (e.g., 10°C) lower than the melting point (Tm) of the natural 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). The Tm of a polynucleotide molecule 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 polynucleotides greater than 100 bases in length would be hybridization conditions such as pre-washing in a solution of 6X SSC, 0.2% SDS; hybridization overnight at 65°C, 6X SSC, 0.2% SDS; then washing twice at 65°C in 1X SSC, 0.1% SDS for 30 minutes each, and washing twice at 65°C in 0.2X SSC, 0.1% SDS for 30 minutes each.

[0162] For polynucleotide molecules less than 100 bases in length, exemplary stringent hybridization conditions are 5 to 10° C. below Tm. On average, polynucleotide molecules less than 100 bp in length have their Tm reduced by about (500 / oligonucleotide length)° C.

[0163] For DNA mimics called peptide nucleic acids (PNAs) (Nielsen et al., Science. 1991 Dec 6; 254(5037): 1497-500), Tm values ​​are higher than those of DNA-DNA or DNA-RNA hybrids and can be calculated using the formula described in Giesen et al., Nucleic Acids Res. 1998 Nov 1; 26(21): 5004-6. Exemplary stringent hybridization conditions for DNA-PNA hybrids less than 100 bases in length are 5 to 10°C below Tm.

[0164] Variant polynucleotides disclosed herein also include polynucleotides that differ from the sequences disclosed herein, but, due to the degeneracy of the genetic code, encode polypeptides having similar activities to the polypeptides encoded by the polynucleotides of the present invention. Sequence changes that do not change the amino acid sequence of the polypeptide are "silent variations." In addition to ATG (methionine) and TGG (tryptophan), other codons for the same amino acid can be changed by techniques recognized in the art, for example, to optimize codon usage in a particular host organism.

[0165] Polynucleotide sequence changes that result in conservative substitutions of one or several amino acids in the encoded polypeptide sequence without significantly changing its biological activity are also included in the present invention. The skilled person will be aware of methods for preparing phenotypically silent amino acid substitutions (see, e.g., Bowie et al., 1990, Science 247, 1306).

[0166] Variant polynucleotides due to silent variations and conservative substitutions in the encoded polypeptide sequence may be determined using the publicly available bl2seq program from the BLAST suite of programs (version 2.2.5 [November 2002]) from NCBI (ftp: / / ftp.ncbi.nih.gov / blast / ) via the tblastx algorithm as described previously.

[0167] The function of the polypeptides encoded by the variant polynucleotides disclosed herein as modifiers of GGP translation, production and / or activity can be evaluated, for example, by expressing such sequences in host cells and testing their activity, as described in the Examples herein. The function of the variants can also be tested for their ability to alter the AsA content in plants, also as described in the Examples section herein.

[0168] 5. Peptide variants

[0169] The term "variant" with respect to polypeptides encompasses naturally occurring, recombinant and synthetically produced polypeptides. Variant polypeptide sequences preferably exhibit at least 70%, more preferably at least 71%, more preferably at least 72%, more preferably at least 73%, more preferably at least 74%, more preferably at least 75%, more preferably at least 76%, more preferably at least 77%, more preferably at least 78%, more preferably at least 79%, more preferably at least 80%, more preferably at least 81%, more preferably at least 82%, more preferably at least 83%, more preferably at least 84%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, and most preferably at least 99% identity compared to the 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 as disclosed herein.

[0170] Peptide sequence identity can be determined in the following manner. The subject polypeptide sequence is compared to the candidate polypeptide sequence using BLASTP (from the BLAST suite of programs, Version 2.2.5 [November 2002]) in bl2seq, which is publicly available from NCBI (ftp: / / ftp.ncbi.nih.gov / blast / ). The default parameters of bl2seq are used, except that filtering of low complexity regions should be turned off.

[0171] The polypeptide sequence identity can also be calculated over the entire overlap length between the candidate and subject polypeptide sequences using a global sequence alignment program. EMBOSS-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 described above, are also suitable global sequence alignment programs for calculating polypeptide sequence identity.

[0172] Another method for calculating the % sequence identity of polypeptides is based on the use of Clustal X to align the sequences being compared (Jeanmougin et al., 1998, Trends Biochem. Sci. 23, 403-5.).

[0173] The polypeptide variants disclosed herein also encompass those that have similarity to one or more specifically identified sequences, i.e., those that are likely to retain functional equivalence to those sequences, and that cannot reasonably be expected to occur by random chance. Such sequence similarity for polypeptides can be determined using the publicly available bl2seq program from the NCBI BLAST suite of programs (version 2.2.5 [November 2002]) (ftp: / / ftp.ncbi.nih.gov / blast / ). The following unix command line parameters can be used to check the similarity of polypeptide sequences:

[0174] bl2seq–i peptideseq1–j peptideseq2-F F–p blastp

[0175] The parameter -FF turns off filtering of low-complexity parts. The parameter -p selects the appropriate algorithm for the sequence. The program finds regions of similarity between sequences and reports an "E value" for each such region, which is the expected number of times one can expect to see such a match by chance in a database of a fixed reference size including random sequences. For small E values, much less than one, this is approximately the probability of such a random match.

[0176] When compared to any of the specifically identified sequences, the variant polypeptide sequence preferably exhibits less than 1×10 -10 More preferably, less than 1×10 -20 , preferably less than 1×10 -30 , preferably less than 1×10 -40 , preferably less than 1×10 -50 , preferably less than 1×10 -60 , preferably less than 1×10 -70 , preferably less than 1×10 -80 , preferably less than 1×10 -90 and most preferably less than 1×10 -100 The E value of .

[0177] Conservative substitutions of one or several amino acids in a polypeptide sequence without significantly changing its biological activity are also included in the present invention. The skilled artisan will be aware of methods for making phenotypically silent amino acid substitutions (see, for example, Bowie et al., 1990, Science 247, 1306).

[0178] The function of the variant polypeptides disclosed herein as modifiers of GGP translation, production and / or activity can be evaluated, for example, by expressing such sequences in host cells and testing their activity, as described in the Examples herein. The function of the variants can also be tested for their ability to alter the AsA content in plants, also as described in the Examples section herein.

[0179] In various embodiments, the polypeptide or polypeptide variant may include one or more SANT / MYB domains. The SANT domain is a protein domain that allows many chromatin remodeling proteins to interact with histones (Boyer et al., 2002, Molecular Cell 10 (4): 935-942; Boyer et al., 2004, Nature Reviews Molecular Cell Biology 5 (2): 158-163). The SANT domain has an acidic predicted isoelectric point (pI), while the MYB domain has an alkaline pI (Ko et al., 2008, Molecular Cancer 7 (1): 77). The MYBS1 protein is predicted to have two SANT / MYB domains. For example, MYBS1 from Actinidia pubescens (SEQ ID No: 103) has an N-terminal SANT domain, which has a predicted acidic isoelectric point (pI), and a second SANT / MYB region, which has a predicted alkaline pI.

[0180] 6. Methods for identifying variants

[0181] Physical methods

[0182] Variant polypeptides can be identified using PCR-based methods (Mullis et al., ed., 1994 The Polymerase Chain Reaction, Birkhauser). Typically, the polynucleotide sequences of primers that can be used to amplify variants of polynucleotide molecules as disclosed herein by PCR can be based on sequences encoding conserved regions of corresponding amino acid sequences.

[0183] Alternatively, library screening methods well known to those skilled in the art can be used (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press, 1987). When variants of the probe sequence are identified, hybridization and / or washing stringency will generally be reduced relative to when an exact sequence match is sought.

[0184] Polypeptide variants may also be identified by physical methods, such as by screening expression libraries using antibodies raised against the polypeptides disclosed herein (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.

[0185] Computer-based methods

[0186] Variant sequences disclosed herein, including polynucleotide and polypeptide variants, can 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, etc.). 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 the sequence to be analyzed (i.e., the query sequence). The sequence comparison algorithm uses a scoring matrix to assign an overall score to each alignment.

[0187] An exemplary family of programs that can be used to identify variants in a sequence database is the BLAST suite of programs (version 2.2.5 [November 2002]), including BLASTN, BLASTP, BLASTX, tBLASTN and tBLASTX, which are publicly available from (ftp: / / ftp.ncbi.nih.gov / blast / ) or the National Center for Biotechnology Information (NCBI), the U.S. National Library of Medicine (Building 38A, Room 8N805, Bethesda, MD 20894 USA). The NCBI server also provides facilities for screening many publicly available sequence databases using programs. BLASTN compares a nucleotide query sequence to a nucleotide sequence database. BLASTP compares an amino acid query sequence to a protein sequence database. BLASTX compares a nucleotide query sequence translated in all reading frames to a protein sequence database. tBLASTN compares a protein query sequence to a nucleotide sequence database dynamically translated in all reading frames. tBLASTX compares a six-frame translation of a nucleotide query sequence to a six-frame translation of a nucleotide sequence database. The BLAST programs can be used with default parameters, or the parameters can be changed as desired to refine the screen.

[0188] The use of the BLAST family of algorithms (including BLASTN, BLASTP, and BLASTX) is described in Altschul et al., Nucleic Acids Res. 25:3389-3402, 1997.

[0189] "Hits" of a query sequence generated by BLASTN, BLASTP, BLASTX, tBLASTN, tBLASTX or a similar algorithm to one or more database sequences align and identify similar portions of the sequence. Hits are arranged in order of degree of similarity and length of sequence overlap. Hits to a database sequence typically represent an overlap over only a portion of the sequence length of the query sequence.

[0190] BLASTN, BLASTP, BLASTX, tBLASTN and tBLASTX algorithms also generate "expected" values ​​for alignments. The expected value (E) represents the number of hits that one can "expect" to see by chance when searching a database of the same size containing random contiguous sequences. The expected value is used as a significance threshold to determine whether a 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 on the alignment portion of the sequence, which has a similarity score that is simply accidental. For sequences with an E value of 0.01 or less on the alignment and matching portion, the probability of accidentally finding a match in the database is 1% or less using the BLASTN, BLASTP, BLASTX, tBLASTN or tBLASTX algorithms.

[0191] Multiple sequence alignment of a group of related sequences can be performed using CLUSTALW (Thompson, JD, Higgins, DG and Gibson, TJ (1994) CLUSTALW: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gappenalties and weight matrix choice. Nucleic Acids Research, 22: 4673-4680) CLUSTAL Omega (Sievers et al., (2011). Molecular Systems Biology 7: 539, https: / / www.ebi.ac.uk / Tools / msa / clustalo / ) or T-COFFEE (Cedric Notredame, Desmond G. Higgins, Jaap Heringa, T-Coffee: A novel method for fast and accurate multiple sequence alignment, J. Mol. Biol. (2000) 302: 205-217)) or PILEUP, using progressive pairwise alignments. (Feng and Doolittle, 1987, J. Mol. Evol. 25, 351).

[0192] Pattern recognition software applications can be used to find 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 identical motifs in the query sequence. MAST results are provided as a series of comparisons with appropriate statistics 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 for identifying the function of uncharacterized proteins translated from genomes or cDNA sequences.

[0193] The PROSITE database (www.expasy.org / prosite) contains biologically significant patterns and maps and is designed so that it can be used with appropriate computational tools to assign new sequences to known protein families or determine which known domains are present in a sequence (Falquet et al., 2002, Nucleic Acids Res. 30, 235). Prosearch is a tool that can search the SWISS-PROT and EMBL databases for given sequence patterns or signatures.

[0194] Another embodiment of the protein domain model database is Pfam (Sonnhammer et al., 1997, A comprehensive database of protein families based on seed alignments, Proteins, 28: 405-420; Finn et al., 2010, The Pfam protein families database', Nucl. Acids Res., 38: D211-D222). "Pfam" refers to a large collection of protein domains and protein families maintained by the Pfam consortium and available on several sponsored World Wide Web sites, including: pfam.xfam.org / (European Bioinformatics Institute) (EMBL-EBI). The latest version of Pfam is Pfam 35.0 (November 2021). Pfam domains and families are identified using multiple sequence alignments and hidden Markov models (HMMs). Pfam-A family or domain assignment is a high-quality assignment generated by using a curled seed alignment of a representative member of a protein family and a profile hidden Markov model based on the seed alignment. All identified sequences belonging to the family are then used to automatically generate a complete alignment of the family (Sonnhammer (1998) Nucleic Acids Research 26, 320-322; Bateman (2000) Nucleic Acids Research 26, 263-266; Bateman (2004) Nucleic Acids Research 32, Database Issue, D138-D141; Finn (2006) Nucleic Acids Research Database Issue 34, D247-251; Finn (2010) Nucleic Acids Research Database Issue 38, D21 1-222). Protein sequences can be queried against the HMM using the HMMER homology search software (e.g., HMMER2, HMMER3 or higher versions, hmmer.org) by accessing the Pfam database, for example, using the above-mentioned website. Significant matches that identify the queried protein as being in the pfam family (or as having a particular Pfam domain) are those matches whose median score is greater than or equal to the aggregation threshold for the Pfam domain. Expected values ​​(e-values) can also be used as criteria for including a query protein in Pfam or determining whether a query protein has a particular Pfam domain, where a low e-value (much less than 1.0, e.g., less than 0.1, or less than or equal to 0.01) indicates a low probability that the match is due to chance.

[0195] 7. Methods for isolating or producing polynucleotides

[0196] The polynucleotide molecules disclosed herein can also be separated by using a variety of techniques known to those of ordinary skill in the art. For example, such polynucleotides can be separated by using the polymerase chain reaction (PCR) described in Mullis et al., ed., 1994 The Polymerase Chain Reaction, Birkhauser, which is incorporated herein by reference. Polynucleotides as disclosed herein can be amplified using primers as defined herein, which are derived from polynucleotide sequences as disclosed herein.

[0197] Other methods for isolating polynucleotides as disclosed herein include using all or part of a polynucleotide having a sequence as described herein as a hybridization probe. The technique of hybridizing a labeled polynucleotide probe to a polynucleotide immobilized on a solid support such as a nitrocellulose filter or a nylon membrane can be used to screen a genomic or cDNA library. Exemplary hybridization and washing conditions are: hybridization at 65°C for 20 hours in 5.0×SSC, 0.5% sodium dodecyl sulfate, 1×Denhardt solution; washing in 1.0×SSC, 1% (w / v) sodium dodecyl sulfate (washing three times at 55°C, each for twenty minutes), and optionally washing once at 60°C in 0.5×SSC, 1% (w / v) sodium dodecyl sulfate (twenty minutes). Optional further washing (20 minutes) can be performed at 60°C under the conditions of 0.1×SSC, 1% (w / v) sodium dodecyl sulfate.

[0198] The polynucleotide fragments disclosed herein can be generated by techniques well known in the art, such as restriction endonuclease digestion, oligonucleotide synthesis, and PCR amplification.

[0199] In methods well known in the art, partial polynucleotide sequences can be used to identify corresponding full-length polynucleotide sequences. These methods include PCR-based methods, 5'RACE (Frohman MA, 1993, Methods Enzymol. 218: 340-56) and hybridization-based methods, computer / database-based methods. In addition, for example, inverse PCR allows unknown sequences (Triglia et al., 1998, Nucleic Acids Res 16, 8186, incorporated herein by reference) to be obtained from primers based on known regions that are located on the flanks of the polynucleotide sequences disclosed herein. The method uses several restriction enzymes to produce suitable fragments in the known regions of the gene. The fragments are then cyclized and used as PCR templates by intramolecular ligation. Divergent primers are designed from known regions. In order to physically assemble full-length clones, standard molecular biology methods (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press, 1987) can be used.

[0200] When generating transgenic plants from a particular species, it may be beneficial to transform such plants with one or more sequences derived from the species. The benefit may be to alleviate public concerns about cross-species transformation in generating transgenic organisms. In addition, when downregulation of a gene is a desired outcome, it may be desirable to utilize a sequence identical (or at least highly similar) to a sequence in a plant, for which reduced expression is desired. For these reasons, it is desirable to be able to identify and isolate orthologs of a particular gene in several different plant species.

[0201] Variants (including orthologs) can be identified by the methods described herein.

[0202] 8. Methods for isolating or producing polypeptides

[0203] The polypeptides disclosed herein, including variant polypeptides, can be prepared using peptide synthesis methods well known in the art, such as direct peptide synthesis using solid phase technology (e.g., Stewart et al., 1969, in Solid-Phase Peptide Synthesis, WH Freeman Co, San Francisco California), or automated synthesis, such as using Applied Biosystems 431A peptide synthesizer (Foster City, California). Mutant forms of polypeptides can also be generated during such synthesis.

[0204] The polypeptides and variant polypeptides disclosed herein may also be purified from natural sources using a variety of techniques well known in the art (eg, Deutscher, 1990, Ed, Methods in Enzymology, vol. 182, Guide to Protein Purification).

[0205] Alternatively, the polypeptides and variant polypeptides disclosed herein can be recombinantly expressed in a suitable host cell disclosed herein and isolated from the cell as described below.

[0206] 9. Constructs, vectors and their components

[0207] According to one embodiment, the polynucleotides useful in the methods according to some embodiments of the invention may be provided in a nucleic acid construct for transforming a plant or a host cell. Suitable plants and host cells are described herein.

[0208] The term "gene construct" refers to a polynucleotide molecule, typically a double-stranded DNA, into which another polynucleotide molecule (insertion polynucleotide molecule) may be inserted, such as but not limited to a cDNA molecule. The gene construct may contain the necessary elements that allow the insertion polynucleotide molecule to be transcribed and optionally the transcript to be translated into a polypeptide. The insertion polynucleotide molecule may be derived from a host cell, or may be derived from a different cell or organism and / or may be a synthetic or recombinant polynucleotide. Once in the host cell, the gene construct may be integrated into the host chromosome DNA. The gene construct may be connected to a vector.

[0209] The term "vector" refers to a polynucleotide molecule, usually double-stranded DNA, which is used to transport a gene construct into a host cell. The vector may be capable of replicating in at least one additional host system (eg, E. coli).

[0210] The term "expression construct" refers to a genetic construct that includes the necessary regulatory elements that allow transcription of an inserted polynucleotide molecule and optionally translation of the transcript into a polypeptide. An expression construct typically includes, in a 5' to 3' direction:

[0211] a) a promoter functional in the host cell into which the construct will be transformed,

[0212] b) a polynucleotide to be expressed, and

[0213] c) a terminator that is functional in the host cell into which the construct is transformed.

[0214] 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. A coding sequence is identified by the presence of a 5' translation initiation codon and a 3' translation termination codon. When inserted into a gene construct, a "coding sequence" is capable of being expressed when it is operably linked to a promoter and terminator sequence.

[0215] Because many microorganisms are capable of expressing multiple gene products from polycistronic mRNAs, multiple polypeptides can be expressed under the control of a single regulatory region in those microorganisms if desired.

[0216] "Operably linked" means that the sequence to be expressed is placed under the control of regulatory elements, including promoters, tissue-specific regulatory elements, temporal regulatory elements, enhancers, repressors and terminators. Typically, the translation start site of the translation reading frame of the coding sequence is located between 1 and about 50 nucleotides downstream of the regulatory region of the monocistronic gene.

[0217] "Regulatory region" refers to a nucleic acid having a nucleotide sequence that affects the initiation and rate of transcription or translation and the stability and / or mobility of the transcription or translation product. The regulatory region includes, but is not limited to, a promoter sequence, an enhancer sequence, a response element, a protein recognition site, an inducible element, a protein binding sequence, a 5' and 3' untranslated region (UTRs), a transcription start site, a termination sequence, a polyadenylation sequence, an intron and a combination thereof. The regulatory region generally includes at least one core (basal) promoter. The regulatory region may also include at least one control element, such as an enhancer sequence, an upstream element or an upstream activation region (UAR). By positioning the regulatory region and the coding sequence, the regulatory region is operably connected to the coding sequence so that the regulatory region effectively regulates the transcription or translation of the sequence. For example, in order to operably connect the coding sequence and the promoter sequence, the translation start site of the translation reading frame of the coding sequence is generally located between one and about fifty nucleotides downstream of the promoter. However, the regulatory region may be located up to about 5,000 nucleotides upstream of the translation start site, or about 2,000 nucleotides upstream of the transcription start site.

[0218] The choice of regulatory regions to be included depends on several factors, including but not limited to efficiency, selectivity, inducibility, desired expression levels, and preferential expression during certain culture stages. It is a routine matter for those skilled in the art to regulate the expression of coding sequences by appropriately selecting and positioning regulatory regions relative to the coding sequences. It should be understood that more than one regulatory region may be present, such as introns, enhancers, upstream activation regions, transcription terminators, and inducible elements.

[0219] The term "non-coding region" includes non-translated sequences upstream of the translation start site and downstream of the translation stop site. These sequences are also referred to as 5'UTR and 3'UTR, respectively. These sequences may include elements required for transcription initiation and termination and regulation of translation efficiency. The term "non-coding" also includes intron sequences within a genomic clone.

[0220] A terminator is a sequence that terminates transcription and is present at the 3' untranslated end of a gene downstream of the translated sequence.Terminators are important determinants of mRNA stability and in some cases have been found to have spatial regulatory functions.

[0221] The term "promoter" refers to a non-transcribed cis-regulatory element upstream of the coding region that regulates gene transcription. Promoters include cis-initiator elements and conserved boxes such as the TATA box that specify the transcription start site, as well as motifs bound by transcription factors.

[0222] A "transgene" is a polynucleotide taken from one organism and introduced into a different organism by transformation. The transgene may be derived from the same species or a different species than the species of the organism into which the transgene is introduced.

[0223] An "inverted repeat sequence" is a repeated sequence in which the second half of the repeat is in the complementary strand, e.g.

[0224] (5')GATCTA……TAGATC(3')

[0225] (3')CTAGAT……ATCTAG(5')

[0226] Read-through transcription will produce transcripts that undergo complementary base pairing to form a hairpin structure, provided that there are 3-5 bp spacers between the repeat regions.

[0227] 10. Methods for producing constructs and vectors

[0228] The genetic constructs disclosed herein include one or more polynucleotide sequences disclosed herein and / or polynucleotides encoding polypeptides disclosed herein, and can be used to transform, for example, bacteria, fungi, insects, mammals or plant organisms. The genetic constructs disclosed herein are intended to include expression constructs as defined herein.

[0229] Methods for making and using genetic constructs and vectors are well known in the art and are generally described in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press, 1987; Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing, 1987).

[0230] 11. Host Cells

[0231] In other embodiments, a host cell comprising a gene construct or vector disclosed herein is provided. In a preferred embodiment, the host cell is genetically modified to express one of the following:

[0232] a) a polynucleotide encoding a polypeptide or a variant of a polypeptide having an amino acid sequence of any one of SEQ ID NOs: 1-102 and 348-356, or

[0233] b) a polynucleotide comprising a nucleotide sequence selected from any one of SEQ ID NOs: 103-108 or a variant thereof,

[0234] and / or one of the following:

[0235] c) a polynucleotide encoding a polypeptide having an amino acid sequence of any one of SEQ ID NOs: 109 to 209 or a variant of the polypeptide, or

[0236] d) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 210, or a variant thereof.

[0237] Host cells comprising gene constructs as disclosed herein (such as expression constructs) can be used 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 disclosed herein. Such methods may involve culturing host cells in an appropriate culture medium under conditions suitable for or conducive to expression of the polynucleotides or polypeptides disclosed herein. The expressed recombinant polypeptide (which may optionally be secreted into the culture) can then be separated from the culture medium, host cells or culture medium by methods well known in the art (e.g., Deutscher, Ed, 1990, Methods in Enzymology, Vol 182, Guide to Protein Purification).

[0238] 12. Methods for producing plant cells and plants comprising constructs and vectors

[0239] In other embodiments, plant cells comprising a gene construct as disclosed herein, and plant cells modified to alter the expression of a polynucleotide or polypeptide as disclosed herein are provided. Plants comprising such cells are also provided.

[0240] Changes in GGP translation, production and / or activity can be altered in plants by methods according to some embodiments of the present invention. Such methods may involve transforming plant cells and plants with constructs designed to alter the expression of polynucleotides or polypeptides that alter GGP expression and / or AsA content in such plant cells and plants. Such methods also include transforming plant cells and plants with a combination of the constructs disclosed herein and one or more other constructs designed to alter the expression of one or more polynucleotides or polypeptides that regulate GGP activity and / or AsA content in such plant cells and plants.

[0241] Methods for transforming plant cells, plants and parts thereof with polypeptides are described in Draper et al., 1988, Plant Genetic Transformation and Gene Expression. A Laboratory Manual .Transgenic plants, including transformation techniques, are reviewed by 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. Galun and Breiman, 1997, Transgenic Plants. Imperial College Press, London.

[0242] Transformation can be transient or stable, as known in the art. Transient transformation results in the temporary introduction of nucleic acid into a cell, but the introduced nucleic acid is not integrated into the genome of the cell. Stable transformation results in modification of the cell genome that persists and can be passed on to subsequent generations of cells. Preferably, plant cells and plants are stably transformed.

[0243] In some embodiments, the plant cell to be transformed includes an endogenous GGP gene, preferably a functional GGP gene. For example, in some embodiments, the plant cell to be transformed has previously been transformed with a functional GGP gene. In other embodiments, the plant cell is co-transformed or subsequently transformed with a functional GGP gene. A functional GGP gene is a gene that encodes and expresses a functional GGP protein. A functional GGP protein is a protein that can perform one or more functions of a GGP protein, such as catalyzing the conversion of GDP-L-galactose to L-galactose 1-phosphate.

[0244] In some embodiments, the GGP gene is a GGP3 gene. In some embodiments, the GGP gene encodes a protein having at least about 70% sequence identity to SEQ ID No: 365, preferably at least about 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, at least about 99% or 100% sequence identity to SEQ ID No: 365.

[0245] In some embodiments, the GGP gene has a promoter including one or more MYBS1 binding sites, preferably two or more MYBS1 binding sites. In some embodiments, the methods described herein may further include gene editing the promoter of the endogenous GGP gene to provide a promoter including one or more MYBS1 binding sites. This can be achieved, for example, by modifying the endogenous GGP promoter. Alternatively, this can be achieved by replacing the endogenous promoter with a GGP promoter (e.g., SEQ ID No: 345) including one or more MYBS1 binding sites. In some embodiments, the methods described herein may further include transforming plant cells with the GGP gene. In some embodiments, the method may further include transforming plant cells with a GGP gene having a promoter, the promoter including one or more MYBS1 binding sites, preferably two or more MYBS1 binding sites.

[0246] MYBS1 binding site is any nucleotide sequence that MYBS1 protein can bind.MYBS1 binding site can be determined by many methods known in the art, such as using bioinformatics prediction (using, for example, JASPAR 2020 (Fornes et al., 2019, Nucleic Acids Research 48 (D1): D87-D92)) or using electrophoretic mobility shift assay (EMSA).Some exemplary methods for identifying MYBS1 binding sites are presented in Example 4.

[0247] Preferably, the one or more MYBS1 binding sites include the sequence TCTTATC or its reverse complement GATAAGA. In some embodiments, the GGP promoter has at least about 70% sequence identity to SEQ ID No: 345, preferably at least about 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, at least about 99% or 100% sequence identity to SEQ ID No: 345.

[0248] Plant genetic manipulation methods

[0249] Many plant transformation strategies are available (e.g., Birch, 1997, Ann Rev Plant Phys Plant Mol Biol, 48, 297, Hellens RP et al., (2000) Plant Mol Biol 42:819-32, Hellens R et al., Plant Meth 1:13). For example, strategies can be designed to increase the expression of polynucleotides / polypeptides in plant cells, organs, and / or at specific developmental stages at which they are normally expressed, or to ectopically express polynucleotides / polypeptides in cells, tissues, organs, and / or at specific developmental stages at which they are not normally expressed. The expressed polynucleotides / polypeptides can be derived from the plant species to be transformed or can be derived from a different plant species.

[0250] Genetic constructs used to express genes in transgenic plants generally include a promoter for driving expression of one or more cloned polynucleotides, a terminator, and a selectable marker sequence to detect the presence of the gene construct in the transformed plant.

[0251] The promoter suitable for constructs as described herein is functional in cells, tissues or organs of monocots or dicots, and includes cell, tissue and organ specific promoters, cell cycle specific promoters, time promoters, inducible promoters, constitutive promoters active in most plant tissues and recombinant promoters. The selection of promoters will depend on the temporal and spatial expression of the cloned polynucleotides, and is therefore desirable. The promoters can be those promoters usually associated with the target transgene, or promoters derived from genes of other plants, viruses and plant pathogenic bacteria and fungi. Those skilled in the art can select promoters suitable for modifying and regulating plant traits using gene constructs including polynucleotide sequences disclosed herein without too much experimentation. Examples of constitutive plant promoters include CaMV35S promoter, nopaline synthase promoter and octopine synthase promoter, and Ubi 1 promoter from corn. Plant promoters active in specific tissues, responding to internal developmental signals or external abiotic or biotic stresses are described in the scientific literature. Exemplary promoters are described, for example, in WO 02 / 00894, which is incorporated herein by reference.

[0252] In some embodiments, the promoter is active in leaves and / or roots. In some embodiments, the promoter is a tissue-specific promoter that is active in leaves and / or roots. In a preferred embodiment, the promoter is active in leaves. Promoters that are active in leaves and / or roots, including tissue-specific promoters, are known in the art.

[0253] Exemplary terminators commonly used in plant transformation gene constructs include, for example, the cauliflower mosaic virus (CaMV) 35S terminator, the Agrobacterium tumefaciens nopaline synthase or octopine synthase terminators, the zein gene terminator, the rice ADP-glucose pyrophosphorylase terminator, and the potato PI-II terminator.

[0254] Selectable markers commonly used in plant transformation include the neomycin phosphotransferase II gene (NPT II) that confers kanamycin resistance, the aadA gene that confers spectinomycin and streptomycin resistance, the phosphinothricin acetyltransferase (bar gene) that confers Ignite (AgrEvo) and Basta (Hoechst) resistance, and the hygromycin phosphotransferase gene (hpt) that confers hygromycin resistance.

[0255] Also contemplated is the use of gene constructs that include reporter genes (coding sequences that express host exogenous activity, typically enzymatic activity and / or a visible signal (e.g., luciferase, GUS, GFP), which can be used for promoter expression analysis in plants and plant tissues). Reporter gene literature is reviewed in Herrera-Estrella et al., 1993, Nature 303, 209, and Schrott, 1995, In: Gene Transfer to Plants (Potrykus, T., Spangenberg. Eds) Springer Verlag. Berlin, pp. 325-336.

[0256] The following are representative publications that disclose genetic transformation protocols that can be used to genetically transform the following plant species: rice (Alam et al., 1999, Plant Cell Rep. 18, 572); apple (Yao et al., 1995, Plant Cell Reports 14, 407-412); corn (U.S. Pat. Nos. 5,177,010 and 5,981,840); wheat (Ortiz et al., 1996, Plant Cell Rep. 15, 1996, 877); tomato (U.S. Pat. No. 5,159,135); potato (Kumar et al., 1996 Plant J. 9, :821); cassava (Li et al., 1996 Nat. Biotechnology 14, 736); lettuce (Michelmore et al., 1987, Plant Cell Rep. 6, 439); tobacco (Horsch et al., 1985, Science 227,1229); cotton (U.S. Pat. Nos. 5,846,797 and 5,004,863); grass (U.S. Pat. Nos. 5,187,073 and 6.020,539); mint (Niu et al., 1998, Plant Cell Rep. 17, 165); citrus subfamily (Pena et al., 1995, Plant Sci. 104, 183); caraway (Krens et al., 1997, Plant Cell Rep., 17,39); banana (U.S. Pat. No. 5,792,935); soybean (U.S. Pat. Nos. 5,416,011; 5,569,834; 5,824,877; 5,563,04455 and 5,968,830); pineapple (U.S. Pat. No. 5,952,543); poplar (U.S. Pat. No. 4,795,855); monocots (U.S. Pat. Nos. 5,591,616 and 6,037,522); Brassica (U.S. Pat. Nos. 5,188,958; 5,463,174 and 5,750,871); cereals (U.S. Pat. No. 6,074,877); pear (Matsuda et al., 2005, Plant Cell Rep. 24(1):45-51); plum (Ramesh et al., 2006 Plant Cell Rep. 24(1):45-51); Rep.25(8):821-8; Song and Sink 2005 Plant Cell Rep.2006;25(2):117-23; Gonzalez Padilla et al., 2003 Plant Cell Rep.22(1):38-45); strawberry (Oosumi et al., 2006 Planta.223(6):1219-30; Folta et al., 2006 Planta Apr 14; PMID:16614818), rose (Li et al., 2003), Rubus (Graham et al., 1995 Methods Mol Biol. 1995; 44:129-33), tomato (Dan et al., 2006, Plant Cell Reports V25:432-441), apple (Yao et al., 1995, Plant Cell Rep. 14, 407–412) and Actinidia eriantha (Wang et al., 2006, Plant Cell Rep. 25, 5:425-31). The present invention also contemplates transformation of other species. Suitable methods and protocols are available in the scientific literature. .

[0257] In one embodiment, a method of producing a plant cell or plant having increased L-ascorbic acid (AsA) content or increased GDP-L-galactose phosphorylase 3 (GGP) translation, production and / or activity is provided, the method comprising upregulating the expression of one of the following in the plant cell or plant:

[0258] a) a polynucleotide encoding a polypeptide or a variant of a polypeptide having an amino acid sequence of any one of SEQ ID NOs: 1-102 and 348-356, or

[0259] b) a polynucleotide comprising a nucleotide sequence selected from any one of SEQ ID NOs: 103-108 or a variant thereof,

[0260] and / or one of the following:

[0261] c) a polynucleotide encoding a polypeptide having an amino acid sequence of any one of SEQ ID NOs: 109 to 209 or a variant of the polypeptide, or

[0262] d) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 210, or a variant thereof.

[0263] Several methods known in the art can be used to change the expression of nucleotides and / or polypeptides as disclosed herein. Such methods include, but are not limited to, Tilling (Till et al., 2003, Methods Mol Biol, 2%, 205) and the so-called "Deletagene" technology (Li et al., 2001, Plant Journal 27 (3), 235).

[0264] Other methods may involve the use of sequence-specific nucleases that generate targeted double-stranded DNA breaks in the gene of interest. Examples of such methods include: zinc finger nucleases (Curtin et al., 2011, Sander et al., 2011), transcription activator-like effector nucleases or "TALENs" (Cermak et al., 2011, Mahfouz et al., 2011, Li et al., 2012), and LAGLIDADG homing endonucleases, also known as "meganucleases" (Tzfira et al., 2012).

[0265] Targeted genome editing using engineered nucleases such as clustered, regularly interspaced, short palindromic repeats (CRISPR) technology is an important new approach for producing RNA-guided nucleases such as Cas9 with customizable specificity. Genome editing mediated by these nucleases has been used to quickly, easily, and efficiently modify endogenous genes in a variety of biomedically important cell types and organisms that are traditionally challenging to genetically manipulate. Modified versions of the CRISPR-Cas9 system have been developed to recruit heterologous domains that can regulate endogenous gene expression or mark specific genomic loci in living cells (Sander and Joung, 2014). The technology is applicable to fungi (Nodvig et al., 2015).

[0266] Upregulating expression of a polypeptide in a plant, for example by genome editing, can be achieved by (i) replacing the endogenous sequence encoding the polypeptide of interest or a regulatory sequence under its control, and / or (ii) inserting a new gene encoding the polypeptide of interest in a targeted region of the genome, and / or (iii) introducing point mutations that result in upregulation of the endogenous gene encoding the polypeptide of interest (e.g., by altering regulatory sequences, such as promoters, enhancers, 5'-UTRs and / or 3'-UTRs, or mutations in the coding sequence).

[0267] In this way, an endogenous gene encoding a polypeptide or a variant of a polypeptide having an amino acid sequence of any one of SEQ ID NOs: 1-102 and 348-356 or 109-209, or comprising a nucleotide sequence selected from any one of SEQ ID NOs: 103-108 or 210 or a variant thereof, can be upregulated, resulting in increased AsA content and / or increased GGP translation, production and / or activity.

[0268] Antibodies or fragments thereof targeting specific polypeptides can also be expressed in plants to modulate the activity of the polypeptides (Jobling et al., 2003, Nat. Biotechnol., 21(1), 35). Transposon tagging methods can also be applied. In addition, peptides that interact with the polypeptides disclosed herein can be identified by techniques such as phage display (Dyax Corporation). Such interactive peptides can be expressed in plants or applied to plants to affect the activity of the polypeptides disclosed herein. The use of each of the above methods in altering the expression of nucleotides and / or polypeptides disclosed herein is particularly contemplated.

[0269] The terms "altered expression" and "altered expression" of a polynucleotide or polypeptide as disclosed herein are intended to encompass situations in which the genomic DNA corresponding to the polynucleotide as disclosed herein is modified, resulting in altered expression of the polynucleotide or polypeptide as disclosed herein. Modification of the genomic DNA may be by genetic transformation or other methods known in the art for inducing mutations. "Altered expression" may be associated with an increase or decrease in the amount of messenger RNA and / or polypeptide produced, and may also result in altered activity of the polypeptide due to changes in the sequence of the polynucleotides and polypeptides produced.

[0270] 13. Methods of selecting plants

[0271] Also provided are methods for selecting plants with altered GGP activity or AsA content. Such methods involve testing plants for altered expression of polynucleotides or polypeptides as disclosed herein. These methods can be applied to young or early developmental stages when altered GGP activity or AsA content may not necessarily be easy to measure.

[0272] The expression of polynucleotides (e.g., messenger RNA) is generally used as an indicator of corresponding polypeptide expression. Exemplary methods for measuring polynucleotide expression 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). Therefore, the polynucleotides disclosed herein or portions of the polynucleotides can be used as probes or primers as defined herein for identifying plants with altered GGP or AsA levels. The polynucleotides disclosed herein can be used as probes in hybridization experiments, or as primers in PCR-based experiments, designed to identify such plants.

[0273] Alternatively, antibodies can be generated for the polypeptides disclosed herein. Methods for generating 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 can be used in methods for detecting the expression of changes in the polypeptides disclosed herein. Such methods may include ELISA (Kemeny, 1991, A Practical Guide to ELISA, NY Pergamon Press) and Western analysis (Towbin and Gordon, 1994, J Immunol Methods, 72, 313).

[0274] These methods for analyzing polynucleotide or polypeptide expression and selecting plants with altered GGP activity or altered AsA content can be used in conventional breeding programs designed to produce varieties with altered GGP activity or AsA content.

[0275] 14. Plants

[0276] The term "plant" is intended to include the whole plant, any part of a plant, propagules and progeny of a plant.

[0277] The term "propagule" means any part of a plant that can be used for sexual or asexual reproduction or propagation, including seeds and cuttings.

[0278] A "transgenic" or "transformed" plant refers to a plant that contains new genetic material as a result of genetic manipulation or transformation. The new genetic material can be derived from a plant of the same species as the resulting transgenic or transformed plant or from a different species. Transformed plants include plants that are stably or transiently transformed with the new genetic material. Preferably, the transformed plant is stably transformed.

[0279] Plant and plant cell according to some embodiments of the present invention can grow and self or hybridize with different plant strains, and can identify the resulting hybrid with required phenotypic characteristics. Can grow two or more generations to ensure that the subject phenotypic characteristics are stably maintained and inherited. Plant and plant cell produced by these standard breeding methods also constitute an aspect of the present invention.

[0280] The function of the variant polynucleotides disclosed herein encoding MYBS1-like or GBF3-like transcription factors can be assessed, for example, by expressing such sequences in bacteria and testing the activity of the encoded protein, as described in the Examples section herein.

[0281] Changes in GGP activity and / or AsA content can also be changed in plants or plant cells by methods according to some embodiments of the present invention. Such methods may involve transforming plant cells and plants with constructs as disclosed herein, which are designed to change the expression of polynucleotides or polypeptides, which regulate the GGP activity and / or AsA content in such plant cells and plants. Such methods preferably also include transforming plant cells and plants with a combination of constructs disclosed herein and one or more other constructs, which are designed to change the expression of one or more other polynucleotides or polypeptides, which regulate the AsA content in such plant cells and plants.

[0282] Any plant is suitable for the present invention. The L-galactose biosynthetic pathway that produces AsA is present in all plants. The enzyme GDP-L-galactose phosphorylase (GGP) is essential to the pathway and is also present in all plants. Therefore, the methods of the present invention can be used to alter GGP expression and / or increase AsA production in any plant.

[0283] In various embodiments, the plant or plant cell is a gymnosperm species.

[0284] In another embodiment, the plant or plant cell is an angiosperm species.

[0285] In another embodiment, the plant or plant cell is a dicot species.

[0286] Plants and plant cells particularly useful in the methods of the invention disclosed herein include all plants and plant cells belonging to the green plant superfamily, in particular monocots and dicots, including forage or legume forage, ornamental plants, food crops, trees or shrubs selected from the list comprising Abrus precatorius, Acacia spp., Acer spp., Actinidia spp., Aesculus spp., Arachis duranensis, Arabidopsis spp., Arachis ipaensis, Betula spp., Brassica spp., Buddleja alternifolia, Cajanus cajan, Camellia sinensis, Capsicum spp., Carex littledalei), Carica papaya, Carya illinoinensis, Castanea mollissima, Catharanthus roseus, Cephalotus follicularis, Chenopodium quinoa, Cinnamomum cassia, Citrus clementina, Citrus sinensis, Citrus unshiu, Coffea arabica, Coronillia varia, Corchorus olitorius, Corylus heterophyll, Cotoneaster serotina, Crataegus spp., Cucumis spp., Cupressus spp., Cyathea spp. dealbata), quince (Cydonia oblonga), Cryptomeria japonica, Cymbopogon spp.), Dalbergia monetaria, Davallia divaricata, Desmodium spp., Dicksonia squarosa, Diheteropogon amplectens, Dioclea spp., Dolichos spp., Dorycnium rectum, Duriozibethinus, Echinochloa pyramidalis, Ehraffia spp., Eleusinecoracana, Eragrestis spp., Erythrina spp., Eucalypfus spp., Euclea schimperi, Eulalia villosa, Pagopyrum spp.), Feijoa sellowlana, Fragaria spp., Flemingia spp., Freycinetia banksli, Geranium thunbergii, Ginkgo biloba, Glycine javanica, Glycine max, Glycinesoja, Gliricidia spp., Gossypium anomalum, Gossypium barbadense, Gossypium darwinii, Gossypium hirsutum, Gossypium mustelinum, Gossypium raimondii, Gossypium stocksii, Greyillea spp., Guibourtia coleosperma), Hedysarum spp.), Helianthus annuus, Hemaffhiaaltissima, Herrania umbratical, Heteropogon contortus, Heveabrasiliensis, Hibiscus syriacus, Hordeum vulgare, Hyparrhenia rufa, Hypericum erectum, Hyperthelia dissoluta, Indigoincarnata, Ipomoea nil, Ipomoea triloba, Iris spp., Jatropha curcas, Juglans macrocarpa, Juglans regia, Juglans macrocarpa x Juglans regia, Lactuca saligna, Lactuca sativa, Leptarrhena pyrolifolia, Lespediza spp., Lettuca spp., Leucaena leucocephala, Loudetiab simplex, Lotonus bainesli, Lotus spp., Lupinus angustifolius, Macadamia integrifolia, Macrotyloma axillare, Malus spp., Manihot esculenta, Medicago saliva, Metasequoia glyptostroboides, Morella rubra, Morus notabilis, Mucunapruriens, Musa sapientum, Nicotiana spp., Nelumbo nucifera), Nyssa sinensis, Onobrychis spp., Ornithopus spp., Oryza spp.), Panicum hallii, Panicum virgatum, Papaver somniferum, Peltophorum africanum, Pennisetum spp., Persea gratissima, Petunia spp., Phalaenopsis equestris, Phaseolus spp., Phoenix canariensis, Phoenix dactylifera, Phormium cookianum, Photinia spp., Picea glauca, Pinus spp., Pisum sativam, Podocarpus totara, Pogonarthria fleckii, Pogonaffhriasquarrosa, Populus spp., Prosopis alba, Prosopis cineraria, Prunus armeniaca, Prunus avium, Prunus dulcis, Prunus mume, Prunus persica, Prunus yedoensis var. nudiflora, Douglas fir, Pterolobium stellatum, Punica granatum, Pyrus spp., Quercus spp., Rhamnella rubrinervis, Rhaphiolepsis umbellata, Rhodamnia argentea, Rhododendron griersonianum, Rhododendron simsii), Rhododendron williamsianum, Rhopalostylis sapida, Rhus natalensis, Ribes grossularia, Ribes spp.), Ricinus communis, Robinia pseudoacacia, Rosa spp., Rubus spp., Salix spp., Schyzachyrium sanguineum, Sciadopitys vefficillata, Sequoia sempervirens, Sequoiadendron giganteum, Solanum chilense, Solanum commersonii, Solanum lycopersicum, Solanum pennellii, Solanum tuberosum, Sorghum bicolor, Spatholobus suberectus, Spinacia spp., Sporobolus fimbriatus, Stiburus alopecuroides, Stylosanthos humilis), Tadehagi spp., Taxodium distichum, Telopea speciosissima, Trema orientale, Tetracentron sinense, Themeda triandra, Theobroma cacao, Trema orientale, Trifolium spp., Triticum spp., Tsuga heterophylla, Vaccinium spp., Vicia spp., Vigna angularis, Vigna radiata var.radiata), Vigna unguiculate, Vitis riparia, Vitis vinifera, Watsonia pyramidata, Zantedeschia aethiopica, Zea mays, Ziziphus jujuba, amaranth, artichoke, asparagus, broccoli, Brussels sprouts, cabbage, canola, carrot, cauliflower, celery, collard greens, flax, kale, lentil, oilseed rape, okra, onion, potato, rice, soybean, straw, sugar beet, sugar cane cane), sunflower, tomato, winter squash and tea, etc.

[0287] In some embodiments, the plant or plant cell can be a crop plant, such as a food crop or a biofuel crop. In one embodiment, the plant or plant cell can be a cereal, a bean or a fruit plant. In one embodiment, the plant or plant cell can be a cereal or a bean plant. In one embodiment, the plant or plant cell can be from Gramineae. In one embodiment, the cereal can be rice, wheat, oats, barley, triticale, rye, finger millet, Sonoran millet, sorghum or corn. In one embodiment, the cereal can be barley, wheat, rice or corn. In another embodiment, the leguminous plant can be alfalfa or soybean. In another embodiment, the fruit plant or plant cell can be apple, kiwi fruit or tomato. In one embodiment, the fruit plant or plant cell can be apple or kiwi fruit. In one embodiment, the plant or plant cell can be selected from kiwi fruit, corn, tomato, wheat, barley, tobacco, soybean, rice, apple, cotton, Brassica and alfalfa. In one embodiment, the plant or plant cell can be selected from Actinidia arguta, Actinidia chinensis, Actinidia pubescens, Arabidopsis thaliana, soybean, cotton, barley, apple, alfalfa, Nicotiana benthamiana, tobacco, rice, tomato, common wheat and corn. In one embodiment, the plant or plant cell can be rice, soybean or kiwi plant or plant cell. In one embodiment, the plant or plant cell can be selected from Actinidia arguta, Actinidia chinensis, Actinidia pubescens, soybean and rice.

[0288] In some embodiments, plants or plant cells grown specifically for "biomass" can be used. For example, suitable plants or plant cells include corn, switchgrass, sorghum, miscanthus, sugarcane, poplar, pine, wheat, rice, soybean, cotton, barley, turf grass, tobacco, potato, bamboo, rapeseed, sugar beet, sunflower, willow, and eucalyptus. In further embodiments, the plant or plant cell is Panicum virgatum, Arundo donax, Phalarisarundinacea, Miscanthus x giganteus, Miscanthus sp., Sicea Lespedeza cuneata, millet, Lolium multiflorum (Lolium sp.), Timothy, Kochia scoparia, forage soybean, alfalfa, clover, sun hemp, kenaf, bahia grass, Bermuda grass, Paspalum pubescens, Panicum serrata, Andropogon dasyphylla, Sorghum sp., Petunia (Dactylis sp.), Brachypodium distachyon, Bromus serrata, Orchard's grass, or Poa annua.

[0289] Alternatively, algae and other non-green plants may be used in the methods of some embodiments of the invention.

[0290] In some embodiments, the plant or plant cell is a fruit species. In one embodiment, the plant or plant cell is a Cucurbitaceae plant, such as Momordica grosvenori. In various embodiments, the fruit species is selected from the group comprising the following genera: Actinidia, Malus, Citrus, Fragaria and Vaccinium.

[0291] In another embodiment, the plant or plant cell is selected from the group consisting of Actinidia pubescens, cucumber, soybean, tomato, grape, Arabidopsis thaliana, apple, alfalfa, poplar, Actinidia arguta, Actinidia chinensis, strawberry, potato and corn. In another embodiment, the plant or plant cell is selected from the group consisting of Actinidia pubescens, cucumber, soybean, grape, Arabidopsis thaliana, apple, alfalfa, poplar, Actinidia arguta, Actinidia chinensis, strawberry and corn.

[0292] According to one embodiment, the plant or plant cell is a plant of the Rosaceae family, such as, but not limited to, apple trees, pear trees, oak trees, apricot trees, plum trees, cherry trees, peach trees, raspberry bushes, loquat trees, strawberry trees, almond trees, ornamental trees and shrubs (e.g., roses, spirea, heather, firethorn, rowan and hawthorn).

[0293] In one embodiment, the pear belongs to the genus Pyrus. Preferred pear species include: Pyrus calleryana, Pyrus caucasica, Pyrus communis, Pyrus elaeagrifolia, Pyrus hybrid cultivar, Pyrus pyrifolia, Pyrus salicifolia, Pyrus ussuriensis, and Pyrus x bretschneideri.

[0294] In one embodiment, the plant or plant cell is of the genus Malus. Preferred species of the genus Malus include: Malusaldenhamensis, Malus angustifolia, Malus asiatica, Malus baccata, Malus coronaria, Malus domestica, Malus doumeri, Malus florentina, Malus floribunda, Malus fusca, Malus halliana, Malus malushanensis, Malus hupehensis, Malus ioensis, Malus kansuensis, Malus mandshurica, Malus micromalus, Malus niedzwetzkyana, Malus ombrophilia, Malus orientalis, Malus prattii, Malus prunifolia, Malus truncatum ... pumila, Sargent's begonia (Malus sargentii), three-leaf begonia (Malus sieboldii), Xinjiang wild apple (Malus sieversii), European wild apple (Malus sylvestris), variable leaf begonia (Malus toringoides), mosaic begonia (Malus transitoria), Malus trilobata, Malus tschonoskii, Malus x domestica, Malus x domestica x Malussieversii, Malus x domestica x Pyrus communis Malus xiaojinensis, Dianchi begonia (Malus yunnanensis), ornamental begonia (Malus sp.), and European hawthorn (Mespilus germanica). In one embodiment, the plant species is apple (Malus domestica). In a specific embodiment, the plant is apple, three-leaf apple or Siberian begonia.

[0295] In another embodiment, the plant or plant cell is a plant of a Vitis species. Exemplary Vitis species include, but are not limited to, Vitis piasezkii maxim and Vitis saccharifera makino.

[0296] In one embodiment, the plant or plant cell is a plant from a species selected from the group including, but not limited to, Smilax (eg, Smilax glyciphylla) and Fragaria.

[0297] In another embodiment, the plant or plant cell is from a plant species selected from the group including, but not limited to, Brassica, Lycopersicon, and Solanum.

[0298] Particularly preferred vegetable plant species are: tomato (Solanum lycopersicum) (formerly Lycopersicon esculentum) and potato (Solanum tuberosum).

[0299] In another embodiment, the plant or plant cell is from a monocot species.

[0300] In another embodiment, the plant or plant cell is from a crop species selected from the group including, but not limited to, the genera Glycine max, Zea mays, Hordeum vulgare and Oryza sativa. Particularly preferred crop species are: rice, soybean and corn.

[0301] In various embodiments, the plant or plant cell is from a food crop or a biofuel crop. Species that can be used for food or biofuel production are known in the art. For example, species that can be used for biofuel production may include Miscanthus×giganteus, Cenchrus purpureus, coconut (Cocos nucifera L.), Jatropha (Jatropha L.) and castor (Ricinus communis L.).

[0302] In various embodiments, the plant or plant cell is selected from rice, wheat, oats, barley, triticale, rye, finger millet, Sonoran millet, sorghum, corn, banana, miscanthus, elephant grass or Uganda grass, coconut, sugarcane, cotton, sunflower, soybean, flax, sesame, jatropha, sugar beet, alfalfa, fodder brassica, rapeseed, mustard seed, almond, walnut, pecan, macadamia, peanut and castor bean.

[0303] Plants can be grouped by phylogenetic classification as described in APG IV (The Angiosperm Phylogeny Group et al. (2016), An update of the Angiosperm Phylogeny Group classification for the orders and families of flowering plants: APG IV, Botanical Journal of the Linnean Society 181(1), 1–20, https: / / doi.org / 10.1111 / boj.12385 ), which is incorporated herein by reference.

[0304] In another embodiment, the plant or plant cell is selected from the group consisting of Magnoliaceae, Monocots, Basal Eudicots, Euphorbiales, Pentacarales, Superrosaceae, Santalaceae, Berberis, Caryophyllales, Cornales, Heatherales, Campanulaceae, Cornales, Metteniusales, Metteniales, Boraginales, Gentianales, Vahliales, and Lamiales.

[0305] In another embodiment, the plant or plant cell is selected from the group consisting of Magnoliaceae, Monocots, Basal Eudicots, Euphorbiales, Pentacarales, Superrosaceae, Santalaceae, Berberis, Caryophyllales, Cornales, Heatherales, Campanulaceae, Cornales, Metteniusales, Gnaphalales, Boraginales, Gentianales, Vahliales, Lamiales, Twining Plants, Prunaceae, Sphenopalaceae, and Hydroleaceae.

[0306] In another embodiment, the plant or plant cell is selected from the family Magnoliaceae, Monocots, Basal Eudicots, Euphorbiales, Pentacarales, Leguminosae, Geraniales, Myrtales, Fusileales, Simsillales, Malvaceaes, Decapodales, Sapindales, Vitales, Saxifragales, Santalaceae, Berberis, Caryophyllaceae, Cornales, Heatherales, Campanulaceae, Cornales, Metteniusales, Gnaphalales, Boraginales, Gentianales, Vahliales, Lamiales, Twining Plants, Prunaceae, Sphenopalaceae, and Hydroleaceae.

[0307] In another embodiment, the plant or plant cell is selected from the group consisting of Magnoliaceae, Monocots, Basal Eudicots, Euphorbiales, Pentacarales, Leguminosae, Geraniales, Myrtales, Flintaceales, Simianales, Malvaceaes, Decapodales, Sapindales, Vitales, Saxifragales, Santalaceae, Berberis, Caryophyllales, Cornales, Heatherales, Campanulaceae, Cornales, Metteniusa les, Gnaphalales, Boraginales, Gentianales, Vahliales, Lamiales, Twining plants, Acerolaceae, Sphenantheraceae, Hydroleaceae, Hydroleaceae, Nasturtiumaceae, Moringaceae, Caricaceae, Ichthyaceae, Marigoldaceae, Echinopsaceae, Chenopodiaceae, Acanthaceae, Australopitheceae, Oleaceae, Cyclosporaceae, Acerolaceae, and Spiderwort family.

[0308] In some embodiments, the plant or plant cell comprises an endogenous GGP3 gene, preferably a functional GGP3 gene. In some embodiments, the plant or plant cell has previously been transformed with a functional GGP3 gene. In some embodiments, the plant or plant cell is co-transformed or subsequently transformed with a functional GGP3 gene. In some embodiments, the GGP3 gene has a promoter comprising one or more MYBS1 binding sites, more preferably two or more MYBS1 binding sites. Preferably, the MYBS1 binding site comprises the sequence TCTTATC or its reverse complementary sequence GATAAGA.

[0309] 15. Extraction and determination of AsA in plants

[0310] Also provided is a method for producing L-ascorbic acid (AsA) by extracting AsA from the plant of the present invention. AsA can be extracted from the plant as follows:

[0311] Frozen tissue samples were ground into fine powder in a cryogenic grinder at liquid nitrogen temperature. Approximately 200 mg of frozen powdered tissue was then suspended in 5 volumes of 7% metaphosphoric acid containing 2 mM TCEP (Pierce), vortexed for 20 seconds and incubated at 40°C in a heating block for 2 hours. TCEP was used in the extraction solution because it is a more effective reducing agent than DTT under acidic conditions, ensuring that all vitamin C is in the ascorbic acid reduced form. The extract was centrifuged at 4°C and 20 μL of the supernatant was injected into a rocket column and eluted with two solvents A (0.28% O-phosphoric acid, 0.1 mM EDTA and 0.25% methanol) and B (acetonitrile). L-ascorbic acid and other compounds were eluted using a 5-minute gradient to 90% B. Standards were run for each batch or 20 processed samples. AsA was calculated from the area under the absorbance at 240 nm curve at ~1 minute elution.

[0312] The method can be scaled up for AsA extraction on a larger scale using methods known to those skilled in the art.

[0313] The above methods should not be considered limiting, and suitable variations or substitutions will be apparent to those skilled in the art.

[0314] Example

[0315] 1. Materials and Methods

[0316] Plant material and growth conditions

[0317] Fruit samples were collected from field-grown plants in Hubei, China, during the growing seasons of 2016–2018. AsA content was determined in 48 Actinidia species and one hybrid population (Actinidia pubescens × Actinidia rutaecarpa) in 2017–2019. Six kiwifruit taxa (Actinidia pubescens, Actinidia chinensis, Actinidia deliciosa, Actinidia chinensis (original variant), Actinidia rutaecarpa, and Actinidia cylindrica) with >150-fold differences in AsA content were selected to investigate changes in AsA content during fruit development. Tissue culture materials of tobacco (Nicotiana benthamiana) and kiwifruit (Actinidia pubescens) were grown under long-day conditions (16 h light / 8 h dark) at 23–25 °C. Transgenic and gene-edited plants were potted and grown in a closed greenhouse-1 at the Wuhan Botanical Garden, Chinese Academy of Sciences, Hubei, China (14 h light / 10 h dark, 18 °C min / 30 °C max). Samples collected from a single plant were considered biological replicates.

[0318] Illumina RNA-seq and transcriptome analysis

[0319] Fruit RNA from 20 days after fruiting (DAF20) to DAF120 was extracted from Actinidia chinensis and Actinidia rutaecarpa, respectively. RNA was extracted using RNeasy Plant Mini Kits (Qiagen, Inc., USA). Library construction and sequencing were performed at BerryGenomics (www.berrygenomics.com) using the Illumina NovaSeq 2000 platform to obtain paired-end reads of 150 bp, and >20 million reads were generated for each sample. Sequencing adapters and low-quality raw reads were filtered out using Trimmomatic (DOI: 10.1093 / bioinformatics / btu170) with default parameters. The filtered clean reads were mapped to the Hongyang_v3 reference genome (http: / / kiwifruitgenome.org / ) by Hisat2 (DOI: 10.1038 / s41587-019-0201-4). The read counts and FPKM (fragments per kilobase of exon per million mapped fragments) of annotated genes were calculated by HTSeq (DOI: 10.1093 / bioinformatics / btu638.) and Stringtie2 (DOI: 10.1038 / nbt.3122), respectively. Differentially expressed genes between kiwifruit materials with AsA differentiation at the same developmental stage were identified by R package edgeR, with p value < 0.05, and Gene Ontology (GO) term enrichment was performed using Gene Ontology Resource (http: / / geneontology.org / ).

[0320] RNA extraction and quantitative RT-PCR analysis

[0321] Quantitative reverse transcription (qRT)-PCR was performed as previously described (Wang et al., Plant biotechnology journal 16(8):1424-1433.). Total RNA was isolated using an RNA extraction kit (Tiangen, Beijing, China), and single-stranded cDNA was obtained for all samples using a one-step gDNA removal and cDNA synthesis Supermix kit (TransGen, Beijing, China). Kiwifruit ACTIN (Achn107181) and protein phosphatase 2A (Achn381211) were used as reference genes for expression normalization. -ΔΔCt The method (Livak & Schmittgen, 2001) was used to calculate the relative expression of each gene. The primers used for qRT-PCR are provided in Table 1 below. All qPCR analyses were performed in three technical replicates.

[0322] AsA measurement

[0323] The total AsA concentration was measured using high performance liquid chromatography (HPLC) as previously described (Queval & Noctor, 2007 Anal Biochem 363(1):58-69; Li et al., 2016 Plant Molecular Biology 92(4):473-482).

[0324] Phylogenetic analysis and tree construction

[0325] Phylogenetic trees (Table 2) were constructed using MEGA7.0 (Kumar et al., 2016 Mol Biol Evol 33(7):1870-1874) software with sequences of kiwifruit and other species retrieved from the kiwifruit (http: / / kiwifruitgenome.org / ) and GenBank databases. Genetic distances were calculated using the Jukes-Cantor distance matrix, and evolutionary relationships were inferred using the neighbor-joining method with 1000 bootstrap resamplings.

[0326] Vector construction and genetic transformation of kiwifruit

[0327] The coding DNA sequences (CDS) of AceGGP3, AceMYBS1, AcrMYBS1 and AceGBF3 were amplified from cDNA from Actinidia pubescens or Actinidia rutaecarpa and cloned into overexpression vectors (POE-3Flag-DN, from Wuhan Botanical Garden Laboratory, 35S promoter-driven expression, and G418 (Geneticin, Invitrogen) or kanamycin selection marker) to produce 35S::AceGGP3, 35S::AceMYBS1, 35S::AcrMYBS1, 35S::AceGBF3, respectively.

[0328] AceGGP3 and AceMYBS1 were edited by CRISPR / Cas9 as described previously (Wang et al., 2018. Plant biotechnology journal 16(8):1424-1433). http: / / www.rgenome.net / ?tdsourcetag=s_pcqq_aiomsg ) Specific sgRNAs targeting AceGGP3 and AceMYBS1 were selected and cloned into CRISPR / Cas9 vectors to generate Cas9-AceGGP3 and Cas9-AceMYBS1 editing vectors. The recombinant plasmids were transformed into callus tissue of Actinidia pubescens using Agrobacterium-mediated transformation as previously described ( et al., 2009. Appl Biochem Biotechnol 158(2):470-475; Yuan, 2011 Proceedings of the 2011 International Conference on Future Computer Science and Application (FCSA 2011). 17-20; Wang et al., 2018. Plant biotechnology journal 16(4):844-855; Wang et al., 2018. Plant biotechnology journal 16(8):1424-1433). The primers used for vector construction and transgenic strain identification are listed in Table 1.

[0329] Transient expression assays in kiwifruit and tobacco

[0330] The antisense viral vectors AceGGP3-TRV2, AceMYBS1-TRV2, and AceGBF3-TRV2 were obtained by cloning the CDS of AceGGP3, AceMYBS1, and AceGBF3 into the TRV2 vector (Yu et al., 2019a), respectively. The TRV1 vector was used as an auxiliary plasmid. The TRV1 and TRV2 vectors were transformed into Agrobacterium tumefaciens GV3101A, respectively, and mixed with each other before injection into the callus and fruit of Actinidia chinensis. The same method was used for transient overexpression experiments of 35S-driven AceGGP3, AceMYBS1, and AceGBF3 vectors in Actinidia chinensis and Nicotiana benthamiana (tobacco) leaves.

[0331] Yeast one-hybrid (Y1H) assay

[0332] Yeast one-hybrid assay was performed as described previously (Lin et al., 2007. Science 318(5854):1302-1305; Cheng et al., 2021. The Plant cell 33(4):1229-1251.). The promoter sequences of AceGGP3 (2.6-kb, Table S2) and AcrGGP3 (2.5-kb, Table S3) were amplified and inserted into the corresponding sites of the reporter plasmid pLacZi (Clontech) to generate AceGGP3 pro::LacZ and AcrGGP3 pro::LacZ. The CDS of AceMYBS1 and AcrMYBS1 were cloned into the pJG4-5 vector (Clontech) to construct pJG-AceMYBS1 and pJG-AcrMYBS1, respectively. AceGGP3pro::LacZ or AcrGGP3pro::LacZ was co-transformed with pJG-AceMYBS1 or pJG-AcrMYBS1 into yeast strain EGY48 using an efficient yeast transformation method (Gietz & Schiestl, 2007). p53::LacZ+pJG-p53 was used as a positive control, and AceGGP3pro::LacZ or AcrGGP3pro::LacZ plus pJG4-5 empty vector was used as a negative control. Transformants were grown on SD / -Trp-Ura-deficient medium: 6.7 g L -1 Yeast nitrogen base, 20g.L -1 Galactose, 10g.L -1 Raffinose, 2g.L -1 Missing mixture - TRP-URA and 20 g.L -1 Agar. After sterilization at 121℃ for 15min, add 100mL 10×BU salt (containing 70g·L - 1 Na2HPO4·7H2O and 30g·L -1 NaH2PO4, pH=7.0) and 0.08 mg.mL-1X-gal for colorimetric screening. All yeast strains were incubated at 30°C for 3 days. The primers are listed in Table 1.

[0333] Yeast two-hybrid (Y2H) assay

[0334] The construction of kiwifruit cDNA library was performed by Wuhan Jinkairui Bioengineering Co., Ltd. (Wuhan, China) using mRNA from leaves and fruits for yeast two-hybrid experiments. The CDS of AceMYBS1 was cloned into the pGBKT7 vector (Clontech) as a bait plasmid. Y2H screening assay was performed as described in the BD Matchmaker Two-Hybrid Library Screening Kit user manual (Clontech). For the Y2H assay, AceGBF3 was cloned from the cDNA library and inserted into the pGADT7 vector (Clontech). The combination of recombinant plasmids was transferred into the yeast strain Y2H gold and grown on SD / -Trp-Leu and SD / -Trp-Leu-His-Ade deletion medium plates supplemented with 0.02 mg.L-1X-α-gal at 30 °C for 3 days. The primers are listed in Table 1.

[0335] Transcriptional activation assay

[0336] The full-length cDNAs of AceMYBS1 and AceGBF3 were amplified by PCR and fused into the pGBKT7 vector to obtain two constructs (BD-AceMYBS1 and BD-AceGBF3), which were then transformed into yeast strain AH109. Transcriptional activation analysis was performed as described (Geng & Liu, 2018. Journal of experimental botany 69(10):2677-2692).

[0337] Dual-luciferase (Dual-LUC) assay

[0338] For the Dual-LUC assay, the full-length promoters of AceGGP3 (2.6 kb; SEQ ID No: 345) and AcrGGP3 (2.5 kb; SEQ ID No: 346) or their truncated fragments were cloned into the pGreen0800-Luc vector (Hellens et al., 2005) to obtain the reporter gene: AceGGP3pro-2660::LUC (P 2660 ), AceGGP3pro-1106::LUC(P 1106 ), AceGGP3pro-1606::LUC(P 1606 ), AceGGP3pro-2088::LUC(P 2088) and AcrGGP3pro::LUC. 35S:AceMYBS1, 35S:AcrMYBS1 and 35S:AceGBF3 were transformed into Agrobacterium tumefaciens strain EHA105. The effector and reporter were mixed at 5:1 (v / v) and then infiltrated into 4-week-old tobacco leaves as described previously (Gao et al., 2020.Journal ofexperimental botany 71(12):3560-3574). After 2-3 days at 23 ° C, the promoter activity was determined by measuring the ratio of firefly luciferase to Renilla luciferase (LUC / REN) using a dual luciferase kit (TransGen, Beijing, China) with a chemiluminescent imaging system (Clinx, Shanghai, China).

[0339] Protein expression and electrophoretic mobility shift assay (EMSA)

[0340] The full-length AceMYBS1 CDS was inserted into a pET32a vector (Novagen) containing 6×His (fused at both the N-terminus and the C-terminus) and expressed using the E. coli strain BL21 (TransGen, Beijing, China) to produce recombinant AceMYBS1-His protein. The E. coli strain expressing 6×His was used as a negative control. E. coli was cultured at 16°C for 12 hours and then diluted into fresh medium at 1:100 (v / v) and grown for another 2-3 hours at 37°C. When cell growth reached the logarithmic growth phase, IPTG was added to a final concentration of 0.5 mM, and protein expression was induced at 16°C for 10-12 hours. The fusion protein was purified using Proteinlso Ni-NAT resin (TransGen, Beijing, China) according to the manufacturer's instructions. An oligonucleotide probe containing the AceMYBS1 binding sequence of AceGGP3 was synthesized (Table S1) and labeled with biotin, which was provided by JASPAR 2020 (http: / / jaspar.genereg.net / ) prediction. Double-stranded DNA probes were obtained by annealing two complementary oligonucleotides. The fusion protein AceMYBS1-His was mixed with the probe and incubated at room temperature for 20 minutes. DNA gel mobility shift assay was performed using an EMSA kit (Beyotime, China) according to the manufacturer's protocol.

[0341] Subcellular localization

[0342] AceMYBS1-YFP or AceGBF3-YFP mixed with NLS-mCherry-RFP was co-infiltrated into tobacco leaves with infiltration buffer as described previously (Gao et al., 2020. Journal of experimental botany 71(12):3560-3574). Fluorescence was observed by confocal microscopy (Leica TCS-SP8; excitation wavelengths were 510 nm for YFP and 552 nm for RFP) 48 h after infiltration.

[0343] Bimolecular fluorescence complementation assay (BiFC)

[0344] Full-length AceMYBS1 CDS with C-terminal YFP ( C YFP) fused, AceGBF3 and N-terminal YFP ( N YFP) fusion. The recombinant vector or control (empty vector) was transformed into Agrobacterium tumefaciens strain EHA105 and then co-transformed into onion epidermal cells with infiltration buffer (Zhu et al., 2020. The Plant Cell 32(10): 3155-3169). 48 hours after infiltration, YFP fluorescence was detected by confocal microscopy (Leica TCS-SP8; excitation wavelength of YFP: 510 nm and DAPI: 488 nm).

[0345] drop down

[0346] The full-length CDS of AceGBF3 was cloned into the pGEX-4T vector (GE Healthcare), where it was fused to the glutathione-S-transferase (GST) sequence. The recombinant vector was introduced into BL21 cells to produce AceGBF3-GST fusion protein, and the expressed AceGBF3-GST and AceMYBS1-6×His fusion proteins were purified as described previously (Xu, 2020). Aliquots (20 μL) of AceGBF3-GST and AceMYBS1-His proteins were mixed with 1 mL of binding buffer (50 mM Tris-HCl [pH = 7.5], 100 mM NaCl, 0.25% Triton-X100-[v / v], 35 mM β-mercaptoethanol), and then 50 μL Proteinlso GST resin or 50 μL Proteinlso Ni-NAT resin (TransGen, Beijing, China) were added, and the mixture was rotated at 4 ° C for 3-4 hours. The samples were washed four times with binding buffer, and expressed GST or 6×His was used as a negative control. 6× SDS protein loading buffer was added to the samples (to 1× final concentration), and the samples were denatured by boiling for 10 minutes before electrophoresis. After electrophoresis, the gel was analyzed by Western blot using anti-His (1:10,000 [v / v], Proteintech, 66005-1-Ig) and anti-GST (1:10,000 (v / v), Proteintech, 66002-2-Ig) antibodies.

[0347] Bimolecular luminescence complementation (BiLC) assay

[0348] BiLC was performed as described (Chen et al., 2008. Plant physiology 146(2):368-376). The full length of AceMYBS1 and AceGBF3 CDS was inserted into pCAMBIA1300-cLUC and pCAMBIA1300-nLUC vectors to generate AceMYBS1-cLUC and AceGBF3-nLUC constructs, respectively. Agrobacterium cultures carrying different constructs were mixed and co-transformed into tobacco leaves at 1:1 (v / v). Plants were incubated in the dark for 12 hours and then transferred to light conditions at 25°C for 48 hours. Before luciferase activity observation, transformed tobacco leaves were immersed in 0.15 mg.mL -1 The cells were incubated with potassium D-luciferin (Coolaber, China) for 2-3 min, and images were captured using a chemiluminescence imaging system (Clinx, Shanghai, China).

[0349] Statistical analysis

[0350] One-way analysis of variance (ANOVA) was performed using SPSS v20 (IBM Corp., Armonk, NY, USA), and Student's t-test was performed using GraphPad 8.0 software. Significant differences were detected by t-test. In the figures, the following symbols are used: *, P < 0.05; **, P < 0.01; and ***, P < 0.001. In the figures, different letters above the bars indicate significant groupings determined by ANOVA (P < 0.05), and data represent mean values, with error bars representing standard deviations.

[0351] Accession Number

[0352] The sequence information for this study was obtained from kiwifruit ( http: / / kiwifruitgenome.org / )、Nicotiana bunsenii( https: / / solgenomics.net / organism / Nicotiana_benthamiana / genome ) and the National Center for Biotechnology Information ( https: / / www.ncbi.nlm.nih.gov / ) database. In addition, the corresponding Acc gene model from Actinidia chinensis (genotype Red5) is also referred to and represents the PS1.1.69.0 version gene model (Pilkington et al., 2018, BMC Genomics 19). The GenBank accession numbers are listed in Table 2.

[0353] Table 1: Primers used for qRT-PCR, vector construction and transformation, and yeast-1-hybrid and yeast-2-hybrid.

[0354]

[0355]

[0356]

[0357]

[0358]

[0359]

[0360] Table 2: GenBank accession numbers.

[0361]

[0362]

[0363]

[0364] 2. Regulation of AsA biosynthesis in kiwifruit by GGP3

[0365] To understand the differences in AsA content among members of the genus Actinidia, HPLC was used to determine the AsA content in 48 species of Actinidia. The concentration varied greatly between species, ranging from 4.4 to 1185 mg.100 g -1 FW. Low vitamin C content (0-30mg100.g -1 FW), medium (30-200mg100.g -1 FW) and high (200-1200mg100 g -1 The AsA content of six Actinidia species representing low, medium, and high AsA concentrations during the growing season was determined ( Figure 1 ). The AsA content of Actinidia species with high and medium fruit AsA content accumulated rapidly after fertilization, reached a peak at the sampling point of 60 days after flowering (DAF60), and then decreased with fruit ripening, while the AsA content of Actinidia species with low AsA content remained at a low level throughout the growing season. Therefore, AsA biosynthesis during early fruit development is the main cause of AsA accumulation in Actinidia members, and differences in AsA synthesis during the stages may constitute the main determinant of AsA variation in Actinidia species.

[0366] To identify potential AsA-regulating genes, transcriptome sequencing was performed on fruits of Actinidia pubescens and Actinidia rutaecarpa at DAF20, DAF40, DAF60, and DAF120, which showed more than 30-fold differences in AsA content. A total of 24,415 differentially expressed genes (DEGs) were identified by pairwise comparison, and gene ontology (GO) enrichment analysis revealed that these DEGs were significantly enriched in biological processes related to responses to catalytic activity and biosynthetic processes (data not shown). Based on the increased transcript abundance in the L-galactose pathway, the transcript Actinidia suaveolens 32270 encoded by GGP was identified in Actinidia pubescens. RT-qPCR was further performed on the three GGP homologous genes, and correlation analysis was performed on the AsA content in fruits of Actinidia pubescens, Actinidia rutaecarpa, and their hybrids at different developmental stages ( Figure 2 , A and B). GGP3 (AceGGP3) was most highly expressed in Actinidia pubescens and was associated with high AsA content in the fruit.

[0367] GGP3 expression was also highly correlated with AsA concentration in A. pubescens × A. rutaecarpa hybrids (data not shown), and the expression of the AceGGP3 allele derived from A. pubescens was significantly higher than that of the A. rutaecarpa-derived allele AcrGGP3 (data not shown).

[0368] 3. Overexpression of AceGGP3 leads to a dramatic accumulation of AsA in kiwifruit

[0369] Transient expression assays were performed in kiwifruit and callus in tissue culture on grape using 35S-driven AceGGP3 overexpression and silencing constructs to confirm the function of AceGGP3 in AsA accumulation. At 7 days after infiltration, AceGGP3 expression and AsA content were significantly higher in infiltrated fruits than in control fruits ( Figure 3 A and B). In addition, silencing of AceGGP3 in the resulting plants resulted in a decrease in AsA by approximately 24.0% ( Figure 3 B). Similar results were obtained in transient assays in Actinidia pubescens calli, where overexpression of AceGGP3 significantly increased the AsA content (Figure 3C and D).

[0370] To further analyze the function of AceGGP3, transgenic kiwifruit lines were generated by Agrobacterium transformation of callus tissue. Five independent transgenic lines of Actinidia pubescens overexpressing AceGGP3 were obtained. Compared with wild-type (WT) plants, transgenic plants showed different levels of AceGGP3 gene expression - ranging from 3.1 to 8.1 times ( Figure 4 A), and the AsA content increased by 6.3 times, 20.0 times, 22.7 times, 16.7 times and 14.1 times, respectively ( Figure 4 B).

[0371] AceGGP3 was then mutated in kiwifruit by the CRISPR / Cas9 system, and the two targeting sites were located on the first and second exons of AceGGP3. A total of four G418-resistant lines were selected, which contained three types of homozygous mutant AceGGP3 genes (ggp3#2, 9bp deletion; ggp3#11 and #15, 1bp insertion; ggp3#13, 2bp insertion). The three mutations resulted in frameshift mutations or amino acid deletions, which induced premature termination or truncation of the predicted protein, resulting in the loss of function of the AceGGP3 gene. Compared with WT, the AsA content in ggp3 mutant fruits was reduced by 32.2%, 5.11% (not statistically significant), 18.2% and 15.9% ( Figure 5 ). The 1 bp insertion in gg3#11 was predicted to result in a nonsense frameshift, but the AsA level remained virtually unchanged. However, scanning of the ORFs in the mutant CDS showed that translation initiation from the fourth ATG codon downstream would produce a truncated form of GGP with 53 amino acids missing from the N-terminus, and the unchanged AsA content in gg3#11 indicated that the variant had functional GGP activity. Overall, the results indicate that up-regulation or down-regulation of AceGGP3 expression significantly affects AsA accumulation in Actinidia pubescens.

[0372] 4. AceMYBS1 acts as a transcriptional activator of AceGGP3

[0373] Through transcriptome analysis, the transcript Actinidia31027 (Actinidia31027) Actinidia chinensis (Hongyang) protein v3; Actinidia39811 (Actinidia39811): Actinidia chinensis (Hong yang) protein v3; Acc18653.1: LG16-18681898..18684251 [e=0]) was identified, and its expression was strongly correlated with that of Actinidia31027 (Actinidia32270) (GGP3; SEQ ID No: 347) (SEQ ID No: 204), and RT-qPCR analysis also showed that its expression was highly correlated with AceGGP expression and AsA content in Actinidia chinensis ( Figure 6A and B). A phylogenetic tree including the amino acid sequences of MYBs from kiwifruit 31027 and other plant species showed that kiwifruit 31027 was most closely related to TwMYBS1, as they shared 86% sequence identity, while it was most distant from CcMYB5 (not shown). Therefore, the protein has been named AceMYBS1. Multiple alignments performed on the SMART website (http: / / smart.embl-heidelberg.de / ) (Schultz et al., 1998, Proceedings of the National Academy of Sciences of the United States of America 95(11):5857-5864; Letunic et al., 2020, Nucleic Acids Research 49(D1):D458-D460) showed that kiwifruit 31027 contained two SANT / MYB regions. The SANT domain is a protein domain that allows many chromatin remodeling proteins to interact with histones (Boyer et al., 2002, Molecular Cell 10 (4): 935-942; Boyer et al., 2004, Nature Reviews Molecular Cell Biology 5 (2): 158-163), and because the SANT domain shares many similarities with the MYB DNA binding domain, and they are usually merged together. The SANT and MYB domains can be distinguished by the predicted isoelectric points (pi) of the domain peptides, where the histone interaction SANT domain has an acidic pI, while the MYB DNA binding domain has an alkaline pI (Ko et al., 2008, Molecular Cancer 7 (1): 77). The N-terminal SANT of kiwifruit 31027 has a predicted acidic isoelectric point (pi), and the second SANT / MYB region has a predicted alkaline pI (data not shown). Therefore, it is predicted that AceMYBS1 has a histone interaction domain on its N-terminal side and a MYB DNA binding domain in the middle of the protein.

[0374] To reveal the mechanism by which AceGGP3 is regulated by AceMYBS1, a Y1H assay was performed. All yeast cells grew well on SD / -Trp / -Ura medium, while only the positive control and the bait vector AceGGP3pro::LacZ co-transformed with the prey vector pJG-AceMYBS1 or pJG-AcrMYBS1 had blue cells on medium supplemented with X-gal (data not shown). Bioinformatics predictions were performed using JASPAR 2020 (Fornes et al., 2019, Nucleic Acids Research 48(D1):D87-D92). Among the many predicted MYBS1 cis-elements, two major cis-elements were identified within the AceGGP3 promoter (-2455 bp, TCTTATC; -1354 bp, TCTTATC). Four 5'-truncated AceGGP3 promoters (named P 1106 , P 1606 , P 2088 and P 2660 ), and the transcriptional activity was assessed using a dual LUC reporter system ( Figure 7 ). 1606 and P 2660 The activity is higher than that of P 1106 and P 2088 activity, indicating that the cis-element may be located between positions -1106-1606 bp and -2088-2660 bp, which is the same as the predicted position interval. However, regardless of which 5'-truncated AceGGP3 promoter was used to drive the reporter gene, AceMYBS1 co-transfected tobacco showed much higher relative luciferase LUC than AceGGP3 co-transfected tobacco, indicating that AceMYBS1 also binds to other regions of the AceGGP3 promoter ( Figure 7 A). To verify the binding specificity of AceMYBS1 to these motifs, we performed EMSAs and found that the AceMYBS1-HIS fusion protein could bind to DNA probes containing the motifs, whereas an unlabeled competitor probe effectively reduced the binding capacity of AceMYBS1 in a dose-dependent manner, and mutation of the core sequence abolished the binding ( Figure 7B). The above experiment was repeated in Actinidia rutaecarpa (with low AsA), and it was found that AcrMYBS1 was unable to bind to the AcrGGP3 promoter to regulate its expression (data not shown). The Actinidia rutaecarpa GGP3 promoter (SEQ ID No: 346) has many polymorphisms (deletions / insertions) compared to the promoter of Actinidia pubescens (SEQ ID No: 345), including putative MYBS1 binding sites. Without wishing to be bound by theory, the researchers hypothesized that since the AcrMYBS1 protein is almost identical to AceMYBS1 (97.7% identity at the amino acid level), the lack of binding may be due to a defect in the AcrGGP3 promoter rather than a non-functional MYBS1 protein. Therefore, the AsA content in Actinidia rutaecarpa can be increased by replacing or modifying the AcrGGP3 promoter alone or in combination with overexpression of MYBS1.

[0375] To explore the expression profile of AceMYBS1 in Actinidia pubescens, RT-qPCR was performed to assess transcript accumulation in the fruit. Consistent with AsA content, AceMYBS1 was highly expressed in the early fruit development stage and positively correlated with the expression of AceGGP3 ( Figure 6 A and B). Transient expression assays in Actinidia pubescens fruit confirmed that overexpression of AceMYBS1 increased the expression of AceGGP3 by 1.4-fold ( Figure 8 A and B), resulting in a 1.7-fold increase in AsA content in the fruit ( Figure 8 C). In contrast, the expression level of AceGGP3 in TRV-AceMYBS1 fruits was reduced by 30%. As expected, overexpression of AceMYBS1 in A. pubescens callus also resulted in an increase in AceGGP3 expression (1.8-fold) and AsA content (2.36-fold), and TRV-AceMYBS1-infected callus showed significantly reduced AceGGP3 expression and AsA content ( Fig. 9 AC). When AceGGP3 was inhibited by TRV-AceGGP3, AsA content did not increase, even when AceMYBS1 was overexpressed in A. pubescens callus and transgenic lines, indicating that AceMYBS1 is an upstream regulatory gene of AceGGP3.

[0376] To further understand the regulatory role of AceMYBS1, six stably overexpressing transgenic kiwifruit lines were generated. RT-qPCR confirmed that six independent transgenic lines accumulated high levels of AceMYBS1 transcripts (>32 to 55-fold; Fig.10 A), which successfully increased the expression level of AceGGP3 by 2.5 to 4 times ( Fig.10B). The AsA content increased by 1.4-fold, 1.6-fold, 2.0-fold, 1.5-fold, 1.7-fold and 1.8-fold of the control, respectively (data not shown). Using the CRISPR / Cas9 system, AceMYBS1 was further mutated in Actinidia chinensis. The two targeting sites were located within the first and third exons. A total of 5 independent AceMYBS1 mutants were identified (mybs1#12, 1bp deletion; mybs1#28, 26bp deletion; mybs1#30, 3bp deletion; mybs1#41, 1bp insertion; mybs1#53, 4bp deletion) (data not shown). The AsA content of the AceMYBS1-edited lines was reduced by 33.2%, 23.1%, 9.8%, 40.4% and 40.0%, respectively (data not shown). For mybs1#30, AsA content was only reduced by 9.8%; although the 3 bp deletion of the ATG was out of frame, the effect was only mild, as the results were virtually synonymous due to degeneracy, and the only difference from WT was the loss of aspartic acid at position 205 (just over 20 amino acids downstream of the MYB DNA binding domain). Taken together, these findings support that AceMYBS1 is a positive factor regulating AsA synthesis.

[0377] 5. AceGBF3 and AceMYBS1 synergistically upregulate the expression of AceGGP3 and increase the synthesis of AsA in kiwifruit

[0378] To identify AceMYBS1 interacting proteins, yeast two-hybrid (Y2H) screening was performed. Approximately 85 yeast transformants were screened, and 8 positive clones were identified as containing cDNA identical to their full-length sequences. The sequence encodes a bZIP protein (Actinidia 27344; SEQ ID No: 109), whose expression is highly correlated with AceGGP3 expression (SEQ ID No: 347); the protein was then named AceGBF3 based on bioinformatics analysis (data not shown). Further domain mapping analysis revealed that the N-terminal region of AceGBF3 (AceGBF3-N, amino acids 1-151) but not the C-terminal half (AceGBF3-C, amino acids 152-299) interacts with AceMYBS1 (data not shown). Additional transcriptional activity assays were then performed. Yeast cells transformed with BD-AceMYBS1 or BD-AceGBF3 but not the empty pGBKT7 vector grew normally on SD / -T / -H / -A / +X-α-Gal medium and turned blue (data not shown). Subcellular localization assays were performed in tobacco leaves transformed with nuclear markers to visualize the subcellular locations of AceMYBS1 and AceGBF3. Fluorescence of the YFP proteins fused to AceMYBS1 and AceGBF3 was detected only in the nucleus and merged perfectly with the nuclear markers (data not shown).

[0379] To further explore the potential interaction between AceGBF3 and AceMYBS1 proteins, three different approaches were used. First, the BiFC experiment was chosen as an in vivo assay using a cell biology approach. AceMYBS1 and AceGBF3 proteins were conjugated to the C-terminus of yellow fluorescent protein (YFP) (AceMYBS1- C YFP) and the N-terminus of YFP (AceGBF3- N YFP) and then transiently transformed onion epidermal cells. The YFP signal localized to the nucleus, indicating a tight interaction between AceMYBS1 and AceGBF3 as well as nuclear localization (data not shown). To confirm this interaction, AceMYBS1 was fused to the C-terminal half of LUC (AceMYBS1- C LUC), and fused AceGBF3 to the N-terminal half of LUC (AceGBF3- n LUC) and transiently expressed the construct in tobacco leaves. C LUC and AceGBF3- n LUC co-transformed leaves produced strong LUC signals ( Fig.11 A). Finally, the AceMYBS1-AceGBF3 physical interaction was confirmed by in vitro pull-down assay using recombinant purified proteins. When using GST resin, the AceGBF3-glutathione S-transferase (GST) fusion protein was precipitated with AceMYBS1-6×His, but not with 6×His alone ( Fig.11 B).

[0380] To test the hypothesis that AceGBF3 and AceMYBS1 co-regulate AsA synthesis, we performed dual LUC, overexpression, and virus-induced gene silencing experiments. These showed that AceMYBS1 and AceMYBS1 plus AceGBF3, but not AceGBF3 alone, were able to induce the expression of AceGGP3 (which increased by 3.74-fold and 6.67-fold, respectively) ( Fig.12 AceGBF3 in different plant materials (including kiwifruit ( Fig.12 B, C), callus ( Fig.12 D, E) and tobacco leaves ( Fig.12 F, H)) transient overexpression consistently promoted the accumulation of AsA and the expression of GGP3. In addition, co-overexpression of AceGBF3 together with AceMYBS1 cumulatively promoted the accumulation of AsA ( Fig.13 A, B).

[0381] When AceMYBS1 was silenced in Actinidia pubescens fruit, the expression level of AceGGP3 and the content of AsA were significantly reduced or unchanged, regardless of whether AceGBF3 was overexpressed or inhibited, confirming that AceGBF3 must interact with AceMYBS1 to regulate transcription. In addition, five transgenic Actinidia pubescens lines constitutively overexpressing AceGBF3 were generated, and their AceGGP3 transcript levels and AsA contents were increased by 2.11-3.38 times and 1.22-1.78 times, respectively, consistent with previous results ( Fig.14 A, B). The average AsA content in callus of AceGBF3 and AceMYBS1 co-expression lines (2-fold higher than that in WT callus) was significantly higher than that in callus of AceGBF3 (1.4-fold) and AceMYBS1 (1.6-fold) lines alone ( Fig.14 B). Taken together, these results suggest that AceGBF3 interacts with AceMYBS1 to co-regulate AceGGP3 expression, thereby forming an AceGBF3-AceMYBS1-AceGGP3 regulatory network that is involved in the synthesis and metabolism of AsA in kiwifruit.

[0382] 6. AceMYBS1 domain structure and genetics

[0383] AceMYBS1 identified herein is located on chr16, and the Chinese kiwifruit and hairy kiwifruit isoforms share 98% amino acid sequence identity and show no significant perturbations in the two predicted SANT domains (data not shown). In addition to AceMYBS1, there is another highly similar MYBS1-like gene that shares 87% amino acid identity on chr26, which is located within the recently identified hairy kiwifruit AsA supergene QTL interval (McCallum et al., 2019, Plants (Basel, Switzerland) 8 (7): 237). Alignment of the Chinese kiwifruit chromosome 26 and AceMYBS1 (chr16) protein sequences revealed that chromosomes 16 and 26 alleles share highly similar SANT / MYB domains, and the main difference is that 12 amino acids are missing in the chr26 allele in the unstructured region in the C-terminal third of the protein (data not shown). Unstructured regions are often associated with protein-protein binding interactions (Kragelund et al., 2012, Trends in Plant Science 17(11):625-632; Millard et al., 2019, Nucleic Acids Research 47(18):9592-9608). Protein disorder prediction using ODiNPred also supports SMART domain architecture prediction (data not shown) (Dass et al., 2020, Scientific Reports 10(1):14780). The high similarity, especially between the SANT and MYB domains, suggests that the chr26 AceMYBS1 gene should also regulate GGP3 expression, but it is expressed at very low levels in fruit and has little correlation with AsA levels (data not shown).

[0384] 7. Stable expression of AceMYBS1 in plants

[0385] Materials and methods

[0386] A plasmid T-DNA construct containing the AceMYBS1 gene (derived from Actinidia chinensis) as described in Li et al. (2022, New Phytologist 235(6): 2497-2497) was transformed into rice (Oryza sativa L.), soybean (Glycine max) and Arabidopsis thaliana using an Agrobacterium tumefaciens-mediated method. Stably transformed lines were selected by the PCR method. The mature leaf L-ascorbic acid content (AsA) of each line (triplicate plants) was then measured using HPLC as described previously (Li et al., 2022).

[0387] Rice (Oryza sativa L.)

[0388] AsA content was measured in 11 rice transformed lines and compared with wild-type non-transformed control (WT). Ten of the 11 lines had higher AsA content (p < 0.05), ranging from 1.5 to 89 times that of the non-transformed WT (Table 3; Fig.15 A).

[0389] Table 3: Leaf AsA content of stably transformed rice plants.

[0390] Strain No. Average AsA (mg / 100g fresh weight) AsA content relative to WT Standard Deviation T-test statistics #1 5.84 3.63 3.783 0.195 #2 2.50* 1.55 0.214 0.026 #8 144.26* 89.67 16.778 0.005 #9 5.54* 3.45 0.008 0.000 #16 21.12* 13.12 0.387 0.000 #17 3.45* 2.14 0.653 0.036 #18 3.35* 2.08 0.249 0.009 #19 4.62* 2.87 0.050 0.000 #20 2.42* 1.50 0.158 0.019 #21 2.93* 1.82 0.106 0.001 #26 2.48* 1.54 0.224 0.015 WT 1.61 1.00 0.040 -

[0391] *p<0.05.

[0392] Soybean (Glycine max)

[0393] AsA content was measured in five transformed lines of soybean and compared to the wild-type non-transformed control (WT). AsA content was higher in three of the five lines (p<0.05), with one line having reduced AsA. The variation ranged from 0.17 to 5.7 times that of the non-transformed WT (Table 4; Fig.15 B).

[0394] Table 4: Leaf AsA content of stably transformed soybean plants.

[0395] Strain No. Average AsA (mg / 100g fresh weight) AsA content relative to WT Standard Deviation T-test statistics #4 12.46* 1.99 1.287 0.005 #5 1.05* 0.17 0.087 0.035 #6 35.88* 5.74 3.464 0.002 #7 9.23* 1.48 0.618 0.040 #8 6.96 1.11 1.133 0.702 WT 6.25 1.00 1.682 -

[0396] *p<0.05.

[0397] Arabidopsis

[0398] AsA content was measured in 15 transformed Arabidopsis lines and compared to wild-type non-transformed controls (WT). Two lines showed a small increase (1.21-1.26 fold), while six lines had slightly decreased AsA (p<0.05). The variation was 0.75-1.26 fold of the non-transformed WT (Table 5; Fig.15 E). This shows that by generating many transformed lines and selecting plants showing the highest AsA content, it is possible to generate Arabidopsis plants with increased AsA content compared to the wild type.

[0399] Table 5: Leaf AsA content of stably transformed Arabidopsis plants.

[0400]

[0401]

[0402] *p<0.05.

[0403] discuss

[0404] The examples show that constitutive overexpression of AceMYBS1 can greatly increase AsA concentrations in plants. Very large increases were observed in some rice and soybean lines (~89 and ~5.7 times of wild-type controls). This constitutes the largest increase in AsA observed so far by transgenic methods (Bulley et al., 2012, Plant Biotechnology Journal 10(4): 390-397; Macknight et al., 2017, Current Opinion in Biotechnology 44: 153-160). In addition, since rice (monocotyledonous plants) and soybeans (dicotyledonous plants) are only distantly related, the examples show that overexpression of the MYBS1 gene can increase AsA concentrations in a variety of plant species.

[0405] Although the AsA content of each strain varies greatly, this is not unexpected. The expression of transgenes in plants is greatly affected by the genomic background into which they are inserted, and this will vary between individual transformed strains. It is common practice to produce many transformants and select the strain that shows the greatest effect. Therefore, it is not necessary for each transformed strain to show an equal increase in AsA content. On the contrary, the combination of transformation and selection can be used to provide plants with greatly increased AsA content, even if most transformed strains only show a moderate increase.

[0406] Overexpression of AceMYBS1 in Arabidopsis showed mixed results, but in two cases a modest increase in AsA was observed. As mentioned previously, the expression of the transgene can vary depending on the genomic context, and the experiments confirm that it is possible to increase AsA content in Arabidopsis by overexpressing the MYBS1 gene. By screening additional lines, greater increases in AsA content may be possible. This provides further evidence that the observed effects are broadly applicable to a variety of different plant species.

[0407] 8. Expression of MYBS1 from various plant species.

[0408] MYBS1 genes from various plant species will be stably expressed in Arabidopsis thaliana, and leaf AsA content will be measured.

[0409] The plant transformation vector pHex2s will be used, in which the MYBS1 transgene is driven by the 35s constitutive promoter. The MYBS1 transgenes to be used are listed in Table 6.

[0410] Table 6: MYBS1 gene.

[0411]

[0412]

[0413] Arabidopsis thaliana (Col0) will be transformed using Agrobacterium-mediated transformation using the floral dip method according to standard protocols.

[0414] Transformed lines will be identified and gene expression confirmed using qRT-PCR methods as described in Example 1. Transformed lines will also be assayed for ascorbic acid content as described in Example 1 and will also be tested for increased expression of the MYBS1 target gene GGP (GDP-galactose phosphorylase) using qRT-PCR.

[0415] It is not intended to limit the scope of the present invention to only the embodiments described above. As will be appreciated by those skilled in the art, many variations are possible without departing from the scope of the present invention as set forth in the appended claims.

[0416] Industrial Applications

[0417] The methods and tools described herein can be used to generate plants, including crops such as fruits and grains, with altered AsA content.

Claims

1. A method for producing a plant cell or plant with increased L-ascorbic acid (AsA) content, the method comprising transforming a plant cell with a polynucleotide encoding a polypeptide having an amino acid sequence of any one of SEQ ID NOs: 1-102 and 348-356, or a variant of the polypeptide having at least about 70% sequence identity with the polypeptide having the amino acid sequence of any one of SEQ ID NOs: 1-102 and 348-356.

2. A method for producing a plant cell or plant having increased translation, production and / or activity of GDP-L-galactose phosphorylase 3 (GGP), the method comprising transforming a plant cell with a polynucleotide encoding a polypeptide having an amino acid sequence of any one of SEQ ID NOs: 1-102 and 348-356, or a variant of the polypeptide having at least about 70% sequence identity to a polypeptide having the amino acid sequence of any one of SEQ ID NOs: 1-102 and 348-356.

3. A method for producing a plant cell or plant with increased L-ascorbic acid (AsA) content, the method comprising transforming a plant cell with a polynucleotide comprising a nucleotide sequence of any one of SEQ ID NOs: 103-108, or a variant thereof having at least about 70% sequence identity with a nucleotide sequence of any one of SEQ ID NOs: 103-108.

4. A method for producing a plant cell or plant having increased translation, production and / or activity of GDP-L-galactose phosphorylase 3 (GGP), the method comprising transforming a plant cell with a polynucleotide comprising a nucleotide sequence of any one of SEQ ID NOs: 103-108, or a variant thereof having at least about 70% sequence identity with a nucleotide sequence of any one of SEQ ID NOs: 103-108.

5. A method for producing a plant cell or plant having increased L-ascorbic acid (AsA) content or increased GDP-L-galactose phosphorylase 3 (GGP) translation, production and / or activity, the method comprising upregulating in a plant cell or expressing in a plant a polypeptide having an amino acid sequence of any one of SEQ ID NOs: 1-102 and 348-356, or a variant of the polypeptide having at least about 70% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 1-102 and 348-356.

6. A method for producing a plant cell or plant with increased L-ascorbic acid (AsA) content or increased GDP-L-galactose phosphorylase 3 (GGP) translation, production and / or activity, the method comprising upregulating in a plant cell or expressing in a plant a polynucleotide comprising a nucleotide sequence selected from any one of SEQ ID NOs: 103-108 or a variant having at least about 70% sequence identity with the nucleotide sequence of any one of SEQ ID NOs: 103-108.

7. The method according to any one of claims 1 to 6, wherein: The method also includes transforming or co-transforming a second polynucleotide with the polynucleotide, the second polynucleotide encoding a polypeptide having an amino acid sequence of any one of SEQ ID NOs: 109-209 or a variant of the polypeptide having at least about 70% sequence identity with an amino acid sequence of any one of SEQ ID NOs: 109-209.

8. The method according to any one of claims 1 to 4 or 7, wherein The method comprises stably transforming the plant cell with the polynucleotide or a variant thereof.

9. The method according to any one of claims 1 to 8, wherein: The method further comprises transforming or co-transforming with the polynucleotide a second polynucleotide having the nucleotide sequence of SEQ ID NO: 210, or a variant thereof having at least about 70% sequence identity with the nucleotide sequence of SEQ ID NO:

210.

10. The method according to any one of claims 1 to 9, wherein: The plant cell or plant is from a food or biofuel crop.

11. An isolated polynucleotide encoding a polypeptide having the amino acid sequence of any one of SEQ ID NOs: 1-4 and 102, or a variant of the polypeptide having at least about 75% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 1-4 and 102.

12. An isolated polynucleotide encoding a polypeptide having the amino acid sequence of any one of SEQ ID NOs: 109-153, 155-157, 159, 162-164, 166-174, 176, 178, 180-183, 185, 186, 188-190, 192-200, 202-204, and 206-209, or a variant of the polypeptide having at least about 75% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 109-153, 155-157, 159, 162-164, 166-174, 176, 178, 180-183, 185, 186, 188-190, 192-200, 202-204, and 206-209.

13. A gene construct comprising a polynucleotide encoding a polypeptide having an amino acid sequence of any one of SEQ ID NOs: 1-4 and 102 or a variant of the polypeptide having at least about 75% sequence identity with the amino acid sequence of any one of SEQ ID NOs: 1-4 and 102.

14. A genetic construct comprising a polynucleotide encoding a polypeptide having an amino acid sequence of any one of SEQ ID NOs: 109-153, 155-157, 159, 162-164, 166-174, 176, 178, 180-183, 185, 186, 188-190, 192-200, 202-204, and 206-209, or a variant of the polypeptide having at least about 75% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 109-153, 155-157, 159, 162-164, 166-174, 176, 178, 180-183, 185, 186, 188-190, 192-200, 202-204, and 206-209.

15. A gene construct comprising a polynucleotide comprising the nucleotide sequence of any one of SEQ ID NOs: 103-108, or a variant of the polypeptide having at least about 75% sequence identity with the nucleotide sequence of any one of SEQ ID NOs: 103-108.

16. A gene construct comprising a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 210, or a variant of the polypeptide having at least about 70% sequence identity with the nucleotide sequence of SEQ ID NO:

210.

17. The gene construct according to claim 13 or 15, wherein The construct further comprises a second polynucleotide a) the second polynucleotide encodes a polypeptide having an amino acid sequence of any one of SEQ ID NOs: 109-209, or a variant of the polypeptide having at least about 75% sequence identity with the amino acid sequence of any one of SEQ ID NOs: 109-209; and / or b) the second polynucleotide has the nucleotide sequence of SEQ ID NO: 210, or a variant of the polypeptide having at least about 70% sequence identity with the nucleotide sequence of SEQ ID NO:

210.

18. A host cell genetically modified to express a polynucleotide encoding a polypeptide having the amino acid sequence of any one of SEQ ID NOs: 1-102 and 348-356 or a variant of the polypeptide having at least about 70% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 1-102 and 348-356.

19. A host cell genetically modified to express a polynucleotide comprising the nucleotide sequence of any one of SEQ ID NOs: 103-108, or a variant thereof having at least about 70% sequence identity to the nucleotide sequence of any one of SEQ ID NOs: 103-108.

20. A plant cell genetically modified to express a polynucleotide encoding a polypeptide having an amino acid sequence of any one of SEQ ID NOs: 1-102 and 348-356 or a variant of said polypeptide having at least 70% sequence identity with said amino acid sequence of any one of SEQ ID NOs: 1-102 and 348-356.

21. A plant cell genetically modified to express a polynucleotide comprising a nucleotide sequence selected from any one of SEQ ID NOs: 103-108, or a variant thereof having at least 70% sequence identity with the nucleotide sequence of any one of SEQ ID NOs: 103-108.

22. The host cell or plant cell according to any one of claims 18 to 21, wherein The cell is genetically modified to express a second polynucleotide encoding a polypeptide having an amino acid sequence of any one of SEQ ID NOs: 109-209, or a variant of the polypeptide having at least about 70% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 109-209.

23. The host cell or plant cell according to any one of claims 18 to 21, wherein The cell is genetically modified to express a second polynucleotide having a nucleotide sequence of SEQ ID NO:210, or a variant thereof having at least about 70% sequence identity to the nucleotide sequence of SEQ ID NO:

210.

24. The method, isolated polynucleotide, genetic construct, host cell or plant cell according to any one of claims 1-11, 13, 15 and 17-23, wherein The polynucleotide encodes a polypeptide having the following amino acid sequence: a) any one of SEQ ID Nos: 1-102 and 348-356; b) any one of SEQ ID No: 1-4, 38, 41-45, 48, 49, 51, 52, 56, 60, 63, 66, 79, 80, 85, 89, 90, 92, 96, 97, 100-102 and 348-356; c) any one of SEQ ID Nos: 1-4, 49, 51, 66, 80, 85, 90, 92, 96, 102, and 348-356; d) any one of SEQ ID Nos: 1-4 and 102; e) any one of SEQ ID No: 1-4; or f) SEQ ID No: 1 or 102.

25. The method, isolated polynucleotide, genetic construct, host cell or plant cell according to any one of claims 1-11, 13, 15 and 17-23, wherein The polynucleotides encode variants of the polypeptide having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the following amino acid sequence: a) any one of SEQ ID Nos: 1-102 and 348-356; b) any one of SEQ ID No: 1-4, 38, 41-45, 48, 49, 51, 52, 56, 60, 63, 66, 79, 80, 85, 89, 90, 92, 96, 97, 100-102 and 348-356; c) any one of SEQ ID Nos: 1-4, 49, 51, 66, 80, 85, 90, 92, 96, 102, and 348-356; d) any one of SEQ ID Nos: 1-4 and 102; e) any one of SEQ ID No: 1-4; or f) SEQ ID No: 1 or 102.

26. The method, isolated polynucleotide, genetic construct, host cell or plant cell according to any one of claims 1-11, 13, 15 and 17-23, wherein The polynucleotide has the following nucleotide sequence: a) any one of SEQ ID NOs: 103-108, b) SEQ ID No: 103 or 105, or c) SEQ ID No:

103.

27. The method, isolated polynucleotide, genetic construct, host cell or plant cell according to any one of claims 1-11, 13, 15 and 17-23, wherein The polynucleotide encodes a variant having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the following nucleotide sequence: a) any one of SEQ ID NOs: 103-108, b) SEQ ID No: 103 or 105, or c) SEQ ID No:

103.

28. The method, isolated polynucleotide, genetic construct, host cell or plant cell according to any one of claims 8, 12, 17, 22 and 24-27, wherein The polynucleotide or the second polynucleotide encodes a polypeptide having the following amino acid sequence: a) any one of SEQ ID NOs: 109-209; b) any one of SEQ ID NOs: 109-114, 125, 126, 142, 177, 179, 187, 191 and 205; c) any one of SEQ ID NOs: 109-114 and 179; d) any one of SEQ ID NOs: 109-114; or e) SEQ ID NO:

109.

29. The method, isolated polynucleotide, genetic construct, host cell or plant cell according to any one of claims 8, 12, 17, 22 and 24-27, wherein The polynucleotide or the second polynucleotide encodes a variant having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the following amino acid sequence: a) any one of SEQ ID Nos: 109-209; b) any one of SEQ ID Nos: 109-114, 125, 126, 142, 177, 179, 187, 191 and 205; c) any one of SEQ ID Nos: 109-114 and 179; d) any one of SEQ ID Nos: 109-114; or e) SEQ ID NO:

109.

30. The method, isolated polynucleotide, genetic construct, host cell or plant cell according to any one of claims 8, 12, 17, 22 and 24-27, wherein The polynucleotide or the second polynucleotide has a nucleotide sequence of SEQ ID No:

210.

31. The method, isolated polynucleotide, genetic construct, host cell or plant cell according to any one of claims 8, 12, 17, 22 and 24-27, wherein The polynucleotide or the second polynucleotide encodes a variant having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence of SEQ ID No:

210.

32. A kit comprising a first gene construct according to any one of claims 13, 15, 24 and 25; and a second gene construct according to any one of claims 14, 16, 28 and 29.

33. A host cell comprising one or more genetic constructs according to any one of claims 13 to 17 and 24 to 31.

34. A plant comprising the plant cell of any one of claims 20, 21 and 24 to 31.

35. A method for selecting plants with altered GDP-L-galactose phosphorylase 3 (GGP) activity, the method comprising testing a plant for altered expression of a polynucleotide encoding a polypeptide having the amino acid sequence of any one of SEQ ID NOs: 1-102 and 348-356 or a variant of the polypeptide having at least 70% sequence identity with the polypeptide having the amino acid sequence of any one of SEQ ID NOs: 1-102 and 348-356.

36. A method for selecting a plant with altered GDP-L-galactose phosphorylase 3 (GGP) activity, the method comprising testing a plant for altered expression of a polynucleotide comprising a nucleotide sequence selected from any one of SEQ ID NOs: 103-108 or a variant thereof, wherein the variant comprises a sequence having at least 70% sequence identity to the nucleotide sequence of any one of SEQ ID NOs: 103-108.

37. A method for selecting plants with altered L-ascorbic acid (AsA) content, the method comprising testing a plant for altered expression of a polynucleotide encoding a polypeptide having the amino acid sequence of any one of SEQ ID NOs: 1-102 and 348-356 or a variant of the polypeptide having at least 70% sequence identity with the polypeptide having the amino acid sequence of any one of SEQ ID NOs: 1-102 and 348-356.

38. A method for selecting plants with altered L-ascorbic acid (AsA) content, the method comprising testing a plant for altered expression of a polynucleotide comprising a nucleotide sequence selected from any one of the sequences SEQ ID NOs: 103-108 or a variant thereof, the variant comprising a sequence having at least 70% sequence identity with the nucleotide sequence of any one of SEQ ID NOs: 103-108.

39. A plant cell or plant produced by the method of any one of claims 1 to 10 and 24-31.

40. A plant cell or plant selected by the method according to any one of claims 35-38.

41. A group or population of plants produced by the method of any one of claims 1 to 10 and 24-31.

42. A method of producing AsA, the method comprising extracting AsA from the host cell species according to any one of claims 18, 19 and 22-31.

43. A method for producing AsA, the method comprising extracting AsA from the plant cell or plant according to any one of claims 20 to 31.

Images