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WO2007006087A1 - Voies biosynthétiques de l’acide tartrique et l’acide ascorbique chez les plantes : rôle de la l-idonate déshydrogénase - Google Patents

Voies biosynthétiques de l’acide tartrique et l’acide ascorbique chez les plantes : rôle de la l-idonate déshydrogénase Download PDF

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Publication number
WO2007006087A1
WO2007006087A1 PCT/AU2006/000966 AU2006000966W WO2007006087A1 WO 2007006087 A1 WO2007006087 A1 WO 2007006087A1 AU 2006000966 W AU2006000966 W AU 2006000966W WO 2007006087 A1 WO2007006087 A1 WO 2007006087A1
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WIPO (PCT)
Prior art keywords
plant
polypeptide
polynucleotide
idonate
sequence
Prior art date
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Ceased
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PCT/AU2006/000966
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English (en)
Inventor
Seth Debolt
Douglas R. Cook
Christopher M. Ford
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
AUSTRALIAN DRIED FRUITS ASSOCIATION Inc
Grape And Wine Research & Development Corp
Minister For Primary Industries National Resources And Regional Development Represented By Department Of Primary Industries & Resources South Australia
State Of Victoria As Represented By Department Of Primary Industries
WINE GRAPE GROWERS' AUSTRALIA Inc
WINEMAKERS' FEDERATION OF AUSTRALIA Inc
Commonwealth Scientific and Industrial Research Organization CSIRO
State of Victoria
Australian Wine Research Institute Ltd
Horticulture Australia Ltd
Department of Primary Industries and Regional Development
Charles Sturt University
Adelaide University
University of California Berkeley
University of California San Diego UCSD
Original Assignee
AUSTRALIAN DRIED FRUITS ASSOCIATION Inc
Grape And Wine Research & Development Corp
Minister For Primary Industries National Resources And Regional Development Represented By Department Of Primary Industries & Resources South Australia
State Of Victoria As Represented By Department Of Primary Industries
WINE GRAPE GROWERS' AUSTRALIA Inc
WINEMAKERS' FEDERATION OF AUSTRALIA Inc
University of Adelaide
Commonwealth Scientific and Industrial Research Organization CSIRO
State of Victoria
Australian Wine Research Institute Ltd
Horticulture Australia Ltd
Department of Primary Industries and Regional Development
Charles Sturt University
University of California Berkeley
University of California San Diego UCSD
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Publication date
Priority claimed from AU2005903725A external-priority patent/AU2005903725A0/en
Application filed by AUSTRALIAN DRIED FRUITS ASSOCIATION Inc, Grape And Wine Research & Development Corp, Minister For Primary Industries National Resources And Regional Development Represented By Department Of Primary Industries & Resources South Australia, State Of Victoria As Represented By Department Of Primary Industries, WINE GRAPE GROWERS' AUSTRALIA Inc, WINEMAKERS' FEDERATION OF AUSTRALIA Inc, University of Adelaide, Commonwealth Scientific and Industrial Research Organization CSIRO, State of Victoria, Australian Wine Research Institute Ltd, Horticulture Australia Ltd, Department of Primary Industries and Regional Development, Charles Sturt University, University of California Berkeley, University of California San Diego UCSD filed Critical AUSTRALIAN DRIED FRUITS ASSOCIATION Inc
Publication of WO2007006087A1 publication Critical patent/WO2007006087A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine

Definitions

  • the present invention relates to the production of tartaric acid and ascorbic acid in plants.
  • the invention relates especially to an enzyme in the tartaric acid biosynthetic pathway in plants such as Vitis vinifera.
  • the present invention also provides methods for modulating the production of tartaric acid and ascorbic acid in plants for a variety of purposes.
  • Tartaric acid is a white crystalline organic acid, and is abundant in grapes. It is one of the main acids found in wine and is added as a flavouring in foods and beverages, and is used as an antioxidant. Tartaric acid is practically resistant to microbial attack in grape juice or wine and hence often chosen as the acid for addition to reduce grape juice and wine pH values. Furthermore, tartaric acid equilibria is important in wine production; for example, to control acidity, pH and tartrate stability.
  • TA biosynthesis begins with L-ascorbic acid (vitamin C, AA; Figure 1).
  • a key step in TA formation is cleavage of the six carbon intermediate either between position C2/C3 or C4/C5 (Saito et ah, 1997), depending on the plant species.
  • the former pathway yields oxalic acid (OxA) and TA (Geraniaceae), while the latter yields TA and 2C glycoaldehyde (Vitaceae) (Loewus, 1999; Banhegyi and Loewus, 2004).
  • TA biosynthesis represents an unusual fate for AA, and its accumulation to high levels in ripe berries (ca 7 mg g " fresh weight) suggests a highly active metabolic pathway that may compete for AA with those redox-associated functions more usually linked with in vivo AA pools.
  • Ascorbic acid is a dietary factor which must be present in the human diet to prevent scurvy, and which as an anti-oxidant has been identified as an agent that increases resistance to infection.
  • Ascorbic acid is used commercially, for example as a nutritional supplement, color fixing agent, flavouring and preservative in meats and other foods, anti-oxidant in bread dough, abscission of citrus fruit in harvesting and as a reducing agent in analytical chemistry.
  • Ascorbic acid also promotes human physiological functions such as the adsorption of iron, cold tolerance, the maintenance of the adrenal cortex, wound healing, the synthesis of polysaccharides and collagen, the formation of cartilage, dentine, bone and teeth, the maintenance of capillaries, and is useful as an antioxidant.
  • the inventors have identified a novel enzyme, having L-idonate dehydrogenase activity, in the tartaric acid biosynthetic pathway in Vitaceous plants.
  • the present invention provides a substantially purified polypeptide selected from: i) a polypeptide comprising an amino acid sequence as provided in SEQ ID NO:1, ii) a polypeptide comprising an amino acid sequence which is at least 35% identical to SEQ ID NO:1, and iii) a biologically active fragment of i) or ii), wherein the polypeptide has L-idonate dehydrogenase activity.
  • the polypeptide can be purified from a species of the Genus Vitis.
  • the polypeptide comprises an amino acid as provided in
  • polypeptide is a fusion protein further comprising at least one other polypeptide sequence.
  • the at least one other polypeptide may be a polypeptide that enhances the stability of a polypeptide of the present invention, or a polypeptide that assists in the purification of the fusion protein.
  • the invention provides an isolated polynucleotide comprising a sequence of nucleotides selected from: i) a sequence of nucleotides as provided in SEQ ID NO:2, ii) a sequence of nucleotides encoding a polypeptide of the invention, iii) a sequence of nucleotides which is at least 35% identical to SEQ ID NO:2, and iv) a sequence which hybridises to any one of i) to iii) under stringent conditions.
  • the polynucleotide encodes a polypeptide with L-idonate dehydrogenase activity.
  • the polynucleotide comprises a sequence of nucleotides as provided in SEQ ID NO: 14.
  • the present invention provides a method of reducing the level of the polypeptide of the first aspect endogenously produced by the plant.
  • This can be achieved by any means known in the art.
  • One example is by exposing the plant to an antisense polynucleotide or a catalytic polynucleotide which hybridizes to an rnRNA molecule encoding the polypeptide.
  • Another example is by exposing the plant to a dsRNA molecule that specifically down-regulates mRNA levels in a cell of an mRNA molecule encoding the polypeptide.
  • the invention provides an antisense polynucleotide which hybridises under physiological conditions to a polynucleotide comprising a sequence of nucleotides as provided in SEQ ID NO:2.
  • the invention provides a catalytic polynucleotide capable of cleaving a polynucleotide of the invention.
  • the catalytic polynucleotide is a ribozyme.
  • the invention provides an oligonucleotide which comprises at least 19 contiguous nucleotides of a polynucleotide of the invention.
  • the oligonucleotide comprises at least 19 contiguous nucleotides of SEQ ID NO:2.
  • the invention provides a double stranded RNA (dsRNA) molecule comprising an oligonucleotide of the invention, wherein the portion of the molecule that is double stranded is at least 19 base pairs in length and comprises said oligonucleotide.
  • dsRNA molecules include nucleic acids comprising the entire mRNA encoding a polypeptide of the invention, or fragments thereof as small as 19 base pairs in length.
  • the dsRNA molecule is expressed from a single promoter, wherein the strands of the double stranded portion are linked by a single stranded portion.
  • the invention provides a vector comprising the polynucleotide according to the invention.
  • the vectors may be, for example, a plasmid, virus, transposon or phage vector provided with an origin of replication, and preferably a promoter for the expression of the polynucleotide and optionally a regulator of the promoter.
  • the vector may contain one or more selectable markers, for example an ampicillin resistance gene in the case of a bacterial plasmid or a neomycin resistance gene for a mammalian expression vector.
  • the vector may be used in vitro, for example for the production of RNA or used to transfect or transform a host cell.
  • the vector is capable of replication in a plant cell, more preferably the cell of a grapevine.
  • the polynucleotide is operably linked to a promoter.
  • the invention provides a vector comprising or encoding the antisense polynucleotide of the invention, the catalytic polynucleotide of the invention, the oligonucleotide according to the invention, or the dsRNA molecule of the invention.
  • the invention provides a host cell comprising a vector according to the invention, or the isolated polynucleotide of the invention.
  • the invention provides a method of making the polypeptide of the invention, comprising the steps of:
  • the method further comprises the step of isolating said polypeptide.
  • Overexpression of a polypeptide of the invention can be used to produce plants with increased tartaric acid production when compared to a wild-type isogenic plant.
  • the invention provides a transgenic plant, the plant having been transformed with the polynucleotide according to the invention.
  • the polynucleotide is capable of expression to produce a polypeptide according to the invention.
  • the present invention provides a transgenic plant which has increased expression of a polypeptide having L-idonate dehydrogenase activity relative to a corresponding non-transgenic plant.
  • the transgenic plant comprises an extra copy of a polynucleotide encoding a polypeptide with L-idonate dehydrogenase activity.
  • the promoter region of an endogenous gene encoding a polypeptide with L-idonate dehydrogenase activity is manipulated such that expression levels of the gene are increased.
  • the polypeptide with L-idonate dehydrogenase activity comprises an amino acid sequence as provided in SEQ ID NO:1.
  • the polypeptide with L-idonate dehydrogenase activity comprises an amino acid sequence as provided in SEQ ID NO: 4.
  • the invention provides a transgenic plant, the plant having been transformed such that it produces the antisense polynucleotide of the invention, the catalytic polynucleotide of the invention, or a dsRNA molecule according to the invention.
  • the antisense polynucleotide, catalytic polynucleotide or dsRNA down-regulates the production of a polypeptide according to the invention which is endogenously produced by the plant.
  • a transgenic plant of the invention is a grapevine.
  • a method of altering tartaric acid production in a tartaric acid producing plant comprising manipulating said plant such that the production of a polypeptide is modified when compared to a wild-type plant, wherein the polypeptide has L-idonate dehydrogenase activity.
  • a method of altering vitamin C production in a tartaric acid producing plant comprising manipulating said plant such that the production of a polypeptide is modified when compared to a wild-type plant, wherein the polypeptide has L-idonate dehydrogenase activity.
  • the polypeptide comprises a sequence selected from: i) a polypeptide comprising an amino acid sequence as provided in SEQ ID NO:1, ii) a polypeptide comprising an amino acid sequence which is at least 35% identical to SEQ ID NO: 1 , and iii) a biologically active fragment of i) or ii), wherein the polypeptide has L-idonate dehydrogenase activity.
  • a substantially purified antibody, or fragment thereof, that specifically binds a polypeptide of the invention that specifically binds a polypeptide of the invention.
  • the antibody specifically binds a polypeptide as provided in SEQ ID NO: 1
  • a method of assessing the ability of a plant to produce tartaric acid comprising determining the level of expression, and/or sequence, of a polynucleotide of the plant, wherein the polynucleotide encodes a polypeptide having L-idonate dehydrogenase activity.
  • Any techniques known in the art can be used. Examples include, but are not limited to, RT-PCR 5 Northern blot analysis and sequencing.
  • the present invention provides a method of assessing the ability of a plant to produce tartaric acid, the method comprising analysing the production level, and/or activity, of a polypeptide having L-idonate dehydrogenase activity.
  • Examples include, but are not limited to, detection using an antibody of the invention, and performing an assay as described in Example 3.
  • the present invention provides a method of assessing the ability of a plant to produce vitamin C, the method comprising determining the level of expression, and/or sequence, of a polynucleotide of the plant, wherein the polynucleotide encodes a polypeptide having L-idonate dehydrogenase activity.
  • the present invention provides a method of assessing the ability of a plant to produce vitamin C, the method comprising analysing the production level, and/or activity, of a polypeptide having L-idonate dehydrogenase activity.
  • the present invention provides tartaric acid produced by a plant according to the invention.
  • the present invention provides a food or drink product produced from a plant according to the invention.
  • the product is selected from the group consisting of: wine, fruit juice and sultanas.
  • the invention provides vitamin C produced by a plant of the invention.
  • a method of producing tartaric acid comprising; i) growing a plant according to the invention, and ii) isolating tartaric acid from the plant.
  • the present invention provides a method of making wine, the method comprising; i) growing a plant according to the invention, ii) extracting juice from the fruit of the plant, and iii) making -wine from the juice.
  • the present invention provides a method of producing vitamin C, the method comprising; i) growing a plant according to the invention, and ii) isolating vitamin C from the plant.
  • the polypeptide of the invention can be used in screening procedures to identify agonists or antagonists thereof. Such agonists or antagonists could be applied to a plant that produces tartaric acid, such as a grapevine, to alter tartaric acid and/or vitamin C levels in said plant.
  • 3D modelling is performed when considering the crystal structure of the polypeptide.
  • a crystal of a polypeptide according to the invention is provided.
  • the present invention provides a method of screening for an agonist or antagonist of a polypeptide according to the invention, the method comprising using the structural coordinates of a crystal according to the invention to computationally evaluate a candidate compound for its ability to bind to the polypeptide.
  • a method of screening for an agonist or antagonist which modulates L-idonate dehydrogenase activity comprising contacting a polypeptide having L-idonate dehydrogenase activity with a candidate compound, and determining whether said compound increases or decreases the activity of the L-idonate dehydrogenase.
  • L-idonate dehydrogenase activity can be measured using techniques known in the art such as that provided in Example 3.
  • the present invention provides a method of modulating the tartaric acid content in a plant, or an extract thereof, the method comprising contacting the plant, or extract thereof, with an antagonist of L-idonate dehydrogenase activity, wherein the activity of L-idonate dehydrogenase is reduced and the tartaric acid content of the plant, or extract thereof, is reduced.
  • the present invention provides a method of modulating the tartaric acid content in a plant, or an extract thereof, the method comprising contacting the plant, or extract thereof, with an agonist of L-idonate dehydrogenase activity, wherein the activity of L-idonate dehydrogenase is increased and the tartaric acid content of the plant, or extract thereof, is increased.
  • a method of modulating the vitamin C content in a plant, or an extract thereof the method comprising contacting the plant, or extract thereof, with an antagonist of L-idonate dehydrogenase activity, wherein the activity of L-idonate dehydrogenase is reduced and the vitamin C content of the plant, or extract thereof, is increased.
  • the present invention provides a method of modulating the vitamin C content in a plant, or an extract thereof, the method comprising contacting the plant, or extract thereof, with an agonist of L-idonate dehydrogenase activity, wherein the activity of L-idonate dehydrogenase is increased and the vitamin C content of the plant, or extract thereof, is reduced.
  • the plant is Vitis sp.
  • the present invention provides an antagonist of L-idonate dehydrogenase activity identified by the method of the invention.
  • the present invention provides an agonist of L-idonate dehydrogenase activity identified by the method of the invention.
  • the present invention provides a method of producing tartaric acid, the method comprising exposing L-idonate to a substantially purified polypeptide according to the invention.
  • the method is performed using the host cell according to the invention or a cell-free expression system comprising the polypeptide according to the invention.
  • the present invention provides a method of providing vitamin C to a subject, the method comprising administering to the subject a plant, or portion thereof, of the invention, food or drink of the invention, and/or vitamin C of the invention.
  • any administration route can be used.
  • the plant, or portion thereof, food, drink, and/or vitamin C is administered orally.
  • the subject is a mammal. More preferably, the mammal is a human.
  • the subject is suffering from a condition associated with a deficiency in vitamin C such as, but not limited to, scurvy.
  • the present invention provides for the use of a plant, or portion thereof, of the invention, food or drink of the invention, and/or vitamin C of the invention for the manufacture of a medicament for treating or preventing a condition associated with a deficiency in vitamin C.
  • preferred features and characteristics of one aspect of the invention are applicable to many other aspects of the invention.
  • the word "comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.
  • the invention is hereinafter described by way of the following non-limiting
  • FIG. 1 The pathway for TA formation via AA in the grapevine. Ascorbate is cleaved to 2-keto L-gulonic acid which is oxidised to form L-idonate. L-idonate is converted to 5-keto D-gluconic acid by the activity of L-idonate dehydrogenase (IDN) with NAD + as a cofactor.
  • IDN L-idonate dehydrogenase
  • the C4/C5 cleavage of 5-keto D-gluconic acid most likely occurs via transketolase reaction to tartaric acid semi-aldehyde, which tartaric acid semi aldehyde dehydrogenase converts to L-tartaric acid.
  • Figure 2 A HPLC chromatogram showing the profile of V. vinifera at the retention time for TA, compared to A. acontifolia where no TA is detectable, b) Amplification of 8 candidate genes using cDNA and gDNA templates from TA accumulation and non-TA accumulating Vitaceous plants (1. V. vinifera and 2. A. acontifolia). Primers were designed to amplify products between 100-300 bp and amplicon sizes exactly matched the upper band; the lower band is non-specific primer dimers (below 50 bp). Lanes 2, 3, 4 and 8 were model validating control genes, these transcripts were identified as differentially expressed when and where TA biosynthesis was known to occur and these data show conserved expression in both templates.
  • Actin and ubiquitin were also used as control genes and were expressed in all templates (data not shown).
  • Lane 5 in both templates contains the amplicon for idonate dehydrogenase (IDN), present in the cDNA and gDNA of TA accumulating species and absent in non-TA accumulator.
  • Lane 4 was a unigene construct (containing oxidoreductase motif) that did not amplify under any conditions, most likely a chimera.
  • Lane 6 and 7 were other dehydrogenase domain containing pre veraison transcripts that amplified in both TA and non-TA accumulating templates, c) Quantitative RT-PCR of the gene encoding idonate dehydrogenase during berry development, showed a peak in expression 4 weeks post flowering and a lack of expression post veraison which correlates with exponential increase in tartaric acid accumulation Fig. 2d. d) HPLC measurement of TA over berry development measured in mg ml "1 berry, shows that TA biosynthesis increases exponentially 2-5 weeks post flowering. Figure 3.
  • Radiolabel tracer studies (Saito and Kasai, 1982; Saito and Kasai, 1984) (showed that a 14 C radiolabel placed on Cl of idonate and 5-keto gluconate both results in incorporation into the TA molecule only.
  • SEQ ID NO: 2 Coding region of IDN from Vitis vinifera.
  • SEQ ID NO: 4 Amino acid sequence of L-idonate dehydrogenase from E. coli.
  • SEQ ID NO's: 5-9 Polynucleotides for producing siRNA targeting IDN from V. vinifera as predicted by siRNA prediction software (siRNA Target Finder; http.7/www.ambion.com/techlib/misc/siRNA__fmder.html).
  • SEQ ID NO: 10 Forward primer to amplify V. vinifera IDN.
  • SEQ ID NO: 11 Reverse primer to amplify V. vinifera IDN.
  • SEQ ID NO: 12 Sorbitol dehydrogenase from Arabidopsis thaliana.
  • the term "grape” or “grapevine” refers to any species of the genus Vitis, including progenitors thereof, as well as progeny thereof produced by crosses with other species.
  • the term refers to the Vitis cultivars which are commonly referred to as Table or Raisin Grapes, such as Alden, Almeria, Anab-E- Shahi, Autumn Black, Beauty Seedless, Black Corinth, Black Damascus, Black Malvoisie, Black Prince, Blackrose, Bronx Seedless, Burgrave, Calmeria, Campbell Early, Canner, Cardinal, Catawba, Christmas, Concord, Dattier, Delight, Diamond, Dizmar, Duchess, Early Muscat, Emerald Seedless, Emperor, Exotic, Anthony de Lesseps, Fiesta, Flame seedless, Flame Tokay, Gasconade, Gold, Himrod, Hunisa, Hussiene, Isabella, Italia, July Muscat, Khandahar, Katta, Kourgane, Kish
  • a “tartaric acid producing plant” is a plant that belongs to a Family or Genus of plants, wherein members of the Family or Genus of plants include those that are known to produce tartaric acid. Examples of families with members that are known to produce tartaric acid are Vitaceae and Geraniaceae. Examples of genera with members known to produce tartaric acid include Vitis, Parthenocissus, Pelargonium, Ampelopsis, Bauhinia, Phaseolus, Coleus and Erodium.
  • a “tartaric acid producing plant” also includes plants that belong to these families and genera and that usually produce tartaric acid, but do not produce tartaric acid (for example the endogenous L-idonate dehydrogenase has been inactivated due to a mutation).
  • L-idonate dehydrogenase activity relates to a polypeptide in a tartaric acid biosynthetic pathway that is capable of converting L- idonate to 5-keto D-gluconate, and/or 5-keto D-gluconate, to L-idonate (see Figure 1). It is known to those skilled in the art that there are a number of synonyms of the term “ascorbic acid”. These include, but are not limited to, “ascorbate", “vitamin C”, “L-ascorbic acid” and "3-keto-L-gulofuranolactone".
  • Tartaric acid is known to those skilled in the art and include, by way of non-limiting examples, “L-tartaric acid”, “L-(+)-tartaric acid” and “natural tartaric acid”.
  • a wild-type plant is a plant that has not been altered by a method of the invention and/or does not comprise a transgene of the invention.
  • the manipulated plant is compared to a non-manipulated ("wild-type") member of the same species to determine the impact of the manipulation on tartaric acid production.
  • operably linked refers to a functional relationship between two or more nucleic acid (e.g., DNA) segments. Typically, it refers to the functional relationship of a transcriptional regulatory element to a transcribed sequence.
  • a promoter is operably linked to a coding sequence, such as a polynucleotide defined herein, if it stimulates or modulates the transcription of the coding sequence in an appropriate cell.
  • promoter transcriptional regulatory elements that are operably linked to a transcribed sequence are physically contiguous to the transcribed sequence, i.e., they are cis-acting.
  • some transcriptional regulatory elements, such as enhancers need not be physically contiguous or located in close proximity to the coding sequences whose transcription they enhance.
  • substantially purified polypeptide we mean a polypeptide that has generally been separated from the lipids, nucleic acids, other peptides, and other contaminating molecules with which it is associated in its native state.
  • the substantially purified polypeptide is at least 60% free, more preferably at least 75% free, and more preferably at least 90% free from other components with which it is naturally associated.
  • polypeptide and “protein” are generally used interchangeably.
  • the % identity of a polypeptide is determined by GAP (Needleman and
  • the query sequence is at least 15 amino acids in length, and the GAP analysis aligns the two sequences over a region of at least 15 amino acids. More preferably, the query sequence is at least 50 amino acids in length, and the GAP analysis aligns the two sequences over a region of at least 50 amino acids. More preferably, the query sequence is at least 100 amino acids in length and the GAP analysis aligns the two sequences over a region of at least 100 amino acids. Even more preferably, the query sequence is at least 250 amino acids in length and the GAP analysis aligns the two sequences over a region of at least 250 amino acids. More preferably, the two sequences are aligned over their entire length.
  • a "biologically active" fragment is a portion of a polypeptide of the invention which maintains a defined activity of the full-length polypeptide, namely possessing L-idonate dehydrogenase activity.
  • Biologically active fragments can be any size as long as they maintain the defined activity.
  • % identity figures higher than those provided above will encompass preferred embodiments.
  • the polypeptide/enzyme comprises an amino acid sequence which is at least 40%, more preferably at least 45%, more preferably at least 50%, more preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 76%, more preferably at least 80%, more preferably at least 85%, 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%, more preferably at least 99%, more preferably at least 99.1%, more preferably at least 99.2%, more preferably at least 99.3%, more preferably at least 99.4%, more preferably at least 99.5%, more preferably at least 99.6%, more preferably at least 99.7%, more preferably at least 99.
  • Amino acid sequence mutants of the polypeptides of the present invention can be prepared by introducing appropriate nucleotide changes into a nucleic acid of the present invention, or by in vitro synthesis of the desired polypeptide.
  • Such mutants include, for example, deletions, insertions or substitutions of residues within the amino acid sequence.
  • a combination of deletion, insertion and substitution can be made to arrive at the final construct, provided that the final peptide product possesses the desired characteristics .
  • Mutant (altered) peptides can be prepared using any technique known in the art.
  • a polynucleotide of the invention can be subjected to in vitro mutagenesis.
  • in vitro mutagenesis techniques include sub-cloning the polynucleotide into a suitable vector, transforming the vector into a "mutator" strain such as the E. coli XL-I red (Stratagene) and propagating the transformed bacteria for a suitable number of generations.
  • the polynucleotides of the invention are subjected to DNA shuffling techniques as broadly described by Harayama (1998). These DNA shuffling techniques may include genes related to those of the present invention, such as those from non-grape plants that produce tartaric acid. Products derived from mutated/altered DNA can readily be screened using techniques described herein to determine if they possess L-idonate dehydrogenase activity.
  • the location of the mutation site and the nature of the mutation will depend on characteristic(s) to be modified.
  • the sites for mutation can be modified individually or in series, e.g., by (1) substituting first with conservative amino acid choices and then with more radical selections depending upon the results achieved, (2) deleting the target residue, or (3) inserting other residues adjacent to the located site.
  • Amino acid sequence deletions generally range from about 1 to 15 residues, more preferably about 1 to 10 residues and typically about 1 to 5 contiguous residues.
  • Substitution mutants have at least one amino acid residue in the polypeptide molecule removed and a different residue inserted in its place.
  • the sites of greatest interest for substitutional mutagenesis include sites identified as the active site(s).
  • Other sites of interest are those in which particular residues obtained from various strains or species are identical. These positions may be important for biological activity. These sites, especially those falling within a sequence of at least three other identically conserved sites, are preferably substituted in a relatively conservative manner. Such conservative substitutions are shown in Table 1 under the heading of "exemplary substitutions".
  • unnatural amino acids or chemical amino acid analogues can be introduced as a substitution or addition into the polypeptides of the present invention.
  • Such amino acids include, but are not limited to, the D-isomers of the common amino acids, 2,4-diaminobutyric acid, ⁇ -amino isobutyric acid, 4- aminobutyric acid, 2-aminobutyric acid, 6-amino hexanoic acid, 2-amino isobutyric acid, 3 -amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, ⁇ -alanine, fluoro-amino acids, designer amino acids such as ⁇ -methyl amino acids, C ⁇ -methyl amino acids, N ⁇ -methyl
  • polypeptides of the present invention which are differentially modified during or after synthesis, e.g., by biotinylation, benzylation, glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc. These modifications may serve to increase the stability and/or bioactivity of the polypeptide of the invention.
  • Polypeptides of the present invention can be produced in a variety of ways, including production and recovery of natural polypeptides, production and recovery of recombinant polypeptides, and chemical synthesis of the polypeptides.
  • an isolated polypeptide of the present invention is produced by culturing a cell capable of expressing the polypeptide under conditions effective to produce the polypeptide, and recovering the polypeptide.
  • a preferred cell to culture is a recombinant cell of the present invention.
  • Effective culture conditions include, but are not limited to, effective media, bioreactor, temperature, pH and oxygen conditions that permit polypeptide production.
  • An effective medium refers to any medium in which a cell is cultured to produce a polypeptide of the present invention.
  • Such medium typically comprises an aqueous medium having assimilable carbon, nitrogen and phosphate sources, and appropriate salts, minerals, metals and other nutrients, such as vitamins.
  • Cells of the present invention can be cultured in conventional fermentation bioreactors, shake flasks, test tubes, microtiter dishes, and petri plates. Culturing can be carried out at a temperature, pH and oxygen content appropriate for a recombinant cell. Such culturing conditions are within the expertise of one of ordinary skill in the art.
  • isolated polynucleotide we mean a polynucleotide which has generally been separated from the polynucleotide sequences with which it is associated or linked in its native state. Preferably, the isolated polynucleotide is at least 60% free, more preferably at least 75% free, and more preferably at least 90% free from other components with which it is naturally associated.
  • polynucleotide is used interchangeably herein with the terms “nucleic acid molecule", “gene” and “mRNA”.
  • Polynucleotide refers to a oligonucleotide, polynucleotide or any fragment thereof. It may be DNA or RNA of genomic or synthetic origin, double-stranded or single-stranded, and combined with carbohydrate, lipids, protein, or other materials to perform a particular activity defined herein.
  • the query sequence is at least 45 nucleotides in length, and the GAP analysis aligns the two sequences over a region of at least 45 nucleotides.
  • the query sequence is at least 150 nucleotides in length, and the GAP analysis aligns the two sequences over a region of at least 150 nucleotides.
  • the query sequence is at least 300 nucleotides in length and the GAP analysis aligns the two sequences over a region of at least 300 nucleotides. More preferably, the two sequences are aligned over their entire length.
  • the polynucleotide comprises a polynucleotide sequence which is at least 40%, more preferably at least 45%, more preferably at least 50%, more preferably at least 55%, more preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 76%, more preferably at least 80%, more preferably at least 85%, 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%, more preferably at least 99%, more preferably at least 99.1%, more preferably at least 99.2%, more preferably at
  • a polynucleotide of the present invention may selectively hybridise to a polynucleotide that encodes a polypeptide of the present invention under stringent conditions. Furthermore, oligonucleotides of the present invention have a sequence that hybridizes selectively under stringent conditions to a polynucleotide of the present invention.
  • a denaturing agent such as formamide, for example, 50% (vol/vol) formamide with 0.1% bovine serum albumin, 0.1% Ficoll, 0.1% polyvinylpyrrolidone, 50 mM sodium phosphate buffer at pH 6.5 with 750 mM NaCl, 75 mM sodium citrate at 42 0 C; or (3) employ 50% formamide, 5 x SSC (0.75 MNaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 x Denhardt's solution, sonicated salmon sperm DNA (50 g/ml), 0.1% SDS and 10% dextran sulfate at 42 0 C in 0.2 x SSC and 0.1% S
  • formamide for example, 50% (vol/vol) formamide with 0.1% bovine serum albumin, 0.1% Ficoll, 0.1% polyvinylpyrrolidone, 50 mM sodium phosphate buffer at pH 6.5 with 750 m
  • Polynucleotides of the present invention may possess, when compared to naturally occurring molecules, one or more mutations which are deletions, insertions, or substitutions of nucleotide residues. Mutants can be either naturally occurring (that is to say, isolated from a natural source) or synthetic (for example, by performing site- directed mutagenesis or DNA shuffling on the nucleic acid as described above). It is thus apparent that polynucleotides of the invention can be either naturally occurring or recombinant.
  • Oligonucleotides of the present invention can be RNA, DNA, or derivatives of either.
  • the minimum size of such oligonucleotides is the size required for the formation of a stable hybrid between an oligonucleotide and a complementary sequence on a polynucleotide of the present invention.
  • the oligonucleotides are at least 15 nucleotides, more preferably at least 18 nucleotides, more preferably at least 19 nucleotides, more preferably at least 20 nucleotides, even more preferably at least 25 nucleotides in length.
  • the present invention includes oligonucleotides that can be used as, for example, probes to identify nucleic acid molecules, or primers to produce nucleic acid molecules. Oligonucleotides of the present invention used as a probe are typically conjugated with a label such as a radioisotope, an enzyme, biotin, a fluorescent molecule or a chemiluminescent molecule. Antisense Polynucleotides
  • antisense nucleic acid shall be taken to mean a DNA or RNA 5 or combination thereof, molecule that is complementary to at least a portion of a specific mRNA molecule of the invention and capable of interfering with a post-transcriptional event such as mRNA translation.
  • the use of antisense methods is well known in the art (see for example, G. Hartmann and S. Endres, Manual of Antisense Methodology, Kluwer (1999)).
  • the use of antisense techniques in plants has been reviewed by Bourque (1995) and Senior (1998). Bourque lists a large number of examples of how antisense sequences have been utilized in plant systems as a method of gene inactivation. She also states that attaining 100% inhibition of any enzyme activity may not be necessary as partial inhibition will more than likely result in measurable change in the system.
  • Senior (1998) states that antisense methods are now a very well established technique for manipulating gene expression.
  • an antisense polynucleotide which hybridises under physiological conditions means that the polynucleotide (which is fully or partially single stranded) is at least capable of forming a double stranded polynucleotide with mRNA encoding a protein as provided in SEQ ID NO:1 under normal conditions in a cell.
  • Antisense molecules may include sequences that correspond to the structural genes or for sequences that effect control over the gene expression or splicing event.
  • the antisense sequence may correspond to the targeted coding region of the genes of the invention, or the 5 '-untranslated region (UTR) or the 3'-UTR or combination of these. It may be complementary in part to intron sequences, which may be spliced out during or after transcription, preferably only to exon sequences of the target gene. In view of the generally greater divergence of the UTRs, targeting these regions provides greater specificity of gene inhibition.
  • the length of the antisense sequence should be at least 19 contiguous nucleotides, preferably at least 50 nucleotides, and more preferably at least 100, 200, 500 or 1000 nucleotides.
  • the full- length sequence complementary to the entire gene transcript may be used. The length is most preferably 100-2000 nucleotides.
  • the degree of identity of the antisense sequence to the targeted transcript should be at least 85%, preferably at least 90% and more preferably 95-100%.
  • the antisense RNA molecule may of course comprise unrelated sequences which may function to stabilize the molecule. Catalytic Polynucleotides
  • catalytic polynucleotide/nucleic acid refers to a DNA molecule or DNA-containing molecule (also known in the art as a "deoxyribozyme”) or an RNA or RNA-containing molecule (also known as a "ribozyme”) which specifically recognizes a distinct substrate and catalyzes the chemical modification of this substrate.
  • the nucleic acid bases in the catalytic nucleic acid can be bases A 3 C, G, T (and U for RNA).
  • the catalytic nucleic acid contains an antisense sequence for specific recognition of a target nucleic acid, and a nucleic acid cleaving enzymatic activity (also referred to herein as the "catalytic domain").
  • ribozymes that are particularly useful in this invention are the hammerhead ribozyme (Haseloff and Gerlach, 1988, Perriman et al, 1992) and the hairpin ribozyme (Shippy et al, 1999).
  • the ribozymes of this invention and DNA encoding the ribozymes can be chemically synthesized using methods well known in the art.
  • the ribozymes can also be prepared from a DNA molecule (that upon transcription, yields an RNA molecule) operably linked to an RNA polymerase promoter, e.g., the promoter for T7 RNA polymerase or SP6 RNA polymerase.
  • an RNA polymerase promoter e.g., the promoter for T7 RNA polymerase or SP6 RNA polymerase.
  • a nucleic acid molecule i.e., DNA or cDNA, coding for the ribozymes of this invention.
  • the ribozyme can be produced in vitro upon incubation with RNA polymerase and nucleotides.
  • the DNA can be inserted into an expression cassette or transcription cassette. After synthesis, the RNA molecule can be modified by ligation to a DNA molecule having the ability to stabilize the ribozyme and make it resistant to RNase.
  • RNA interference is particularly useful for specifically inhibiting the production of a particular protein.
  • RNAi RNA interference
  • Waterhouse et al. 1998 have provided a model for the mechanism by which dsRNA can be used to reduce protein production.
  • This technology relies on the presence of dsRNA molecules that contain a sequence that is essentially identical to the mRNA of the gene of interest or part thereof, in this case an mRNA encoding a polypeptide according to the invention
  • the dsRNA can be produced from a single promoter in a recombinant vector or host cell, where the sense and anti-sense sequences are flanked by an unrelated sequence which enables the sense and anti-sense sequences to hybridize to form the dsRNA molecule with the unrelated sequence forming a loop structure.
  • a DNA is introduced that directs the synthesis of an at least partly double stranded RNA product(s) with homology to the target gene to be inactivated.
  • the DNA therefore comprises both sense and antisense sequences that, when transcribed into RNA, can hybridize to form the double-stranded RNA region.
  • the sense and antisense sequences are separated by a spacer region that comprises an intron which, when transcribed into RNA, is spliced out.
  • the double-stranded region may comprise one or two RNA molecules, transcribed from either one DNA region or two. The presence of the double stranded molecule is thought to trigger a response from an endogenous plant system that destroys both the double stranded RNA and also the homologous RNA transcript from the target plant gene, efficiently reducing or eliminating the activity of the target gene.
  • the length of the sense and antisense sequences that hybridise should each be at least 19 contiguous nucleotides, preferably at least 30 or 50 nucleotides, and more preferably at least 100, 200, 500 or 1000 nucleotides.
  • the full-length sequence corresponding to the entire gene transcript may be used. The lengths are most preferably 100-2000 nucleotides.
  • the degree of identity of the sense and antisense sequences to the targeted transcript should be at least 85%, preferably at least 90% and more preferably 95-100%.
  • the RNA molecule may of course comprise unrelated sequences which may function to stabilize the molecule.
  • the RNA molecule may be expressed under the control of a RNA polymerase II or RNA polymerase III promoter. Examples of the latter include tRNA or snRNA promoters.
  • Preferred small interfering RNA ('siRNA”) molecules comprise a nucleotide sequence that is identical to about 19-21 contiguous nucleotides of the target mRNA.
  • the target mRNA sequence commences with the dinucleotide AA, comprises a GC-content of about 30-70% (preferably, 30-60%, more preferably 40- 60% and more preferably about 45%-55%), and does not have a high percentage identity to any nucleotide sequence other than the target in the genome of the plant (preferably grape) in which it is to be introduced, e.g., as determined by standard BLAST search.
  • siRNA molecules that target L-idonate dehydrogenase are selected from, but not limited to, the nucleotide sequences provided in SEQ ID NOs: 5 to 9.
  • Another molecular biological approach that may be used is co-suppression.
  • the mechanism of co-suppression is not well understood but is thought to involve post- transcriptional gene silencing (PTGS) and in that regard may be very similar to many examples of antisense suppression. It involves introducing an extra copy of a gene or a fragment thereof into a plant in the sense orientation with respect to a promoter for its expression.
  • the size of the sense fragment, its correspondence to target gene regions, and its degree of homology to the target gene are as for the antisense sequences described above.
  • the antisense, co-suppression or double stranded RNA molecules may also comprise a largely double-stranded RNA region, preferably comprising a nuclear localization signal, as described in WO 03/076619.
  • the largely double-stranded region is derived from a PSTVd type viroid or comprises at least 35 CUG trinucleotide repeats.
  • One embodiment of the present invention includes a recombinant vector, which comprises at least one isolated polynucleotide molecule of the present invention, inserted into any vector capable of delivering the polynucleotide molecule into a host cell.
  • a vector contains heterologous polynucleotide sequences, that is polynucleotide sequences that are not naturally found adjacent to polynucleotide molecules of the present invention and that preferably are derived from a species other than the species from which the polynucleotide molecule(s) are derived.
  • the vector can be either RNA or DNA 5 either prokaryotic or eukaryotic, and typically is a transposon (such as described in US 5,792,294), a virus or a plasmid.
  • One type of recombinant vector comprises a polynucleotide molecule of the present invention operatively linked to an expression vector.
  • the phrase operatively linked refers to insertion of a polynucleotide molecule into an expression vector in a manner such that the molecule is able to be expressed when transformed into a host cell.
  • an expression vector is a DNA or RNA vector that is capable of transforming a host cell and of effecting expression of a specified polynucleotide molecule.
  • the expression vector is also capable of replicating within the host cell.
  • Expression vectors can be either prokaryotic or eukaryotic, and are typically viruses or plasmids.
  • Expression vectors of the present invention include any vectors that function (i.e., direct gene expression) in recombinant cells of the present invention, including in bacterial, fungal, endoparasite, arthropod, animal, and plant cells. Particularly preferred expression vectors of the present invention can direct gene expression in plants cells, more preferably cells of a grapevine.
  • expression vectors of the present invention contain regulatory sequences such as transcription control sequences, translation control sequences, origins of replication, and other regulatory sequences that are compatible with the recombinant cell and that control the expression of polynucleotide molecules of the present invention.
  • recombinant molecules of the present invention include transcription control sequences. Transcription control sequences are sequences which control the initiation, elongation, and termination of transcription. Particularly important transcription control sequences are those which control transcription initiation, such as promoter, enhancer, operator and repressor sequences. Suitable transcription control sequences include any transcription control sequence that can function in at least one of the recombinant cells of the present invention. A variety of such transcription control sequences are known to those skilled in the art.
  • Particularly preferred transcription control sequences are promoters active in directing transcription in plants, either constitutively or stage and/or tissue specific, depending on the use of the plant or parts thereof.
  • These plant promoters include, but are not limited to, promoters showing constitutive expression, such as the 35S promoter of Cauliflower Mosaic Virus (CaMV), those for fruit-specific expression, such as the polygalacturonase (PG) promoter from tomato.
  • CaMV Cauliflower Mosaic Virus
  • PG polygalacturonase
  • Recombinant molecules of the present invention may also (a) contain secretory signals (i.e., signal segment nucleic acid sequences) to enable an expressed polypeptide of the present invention to be secreted from the cell that produces the polypeptide and/or (b) contain fusion sequences which lead to the expression of nucleic acid molecules of the present invention as fusion proteins.
  • suitable signal segments include any signal segment capable of directing the secretion of a polypeptide of the present invention.
  • Preferred signal segments include, but are not limited to, Nicotiana nectarin signal peptide (US 5,939,288), tobacco extensin signal, the soy oleosin oil body binding protein signal.
  • nucleic acid molecule of the present invention can be joined to a fusion segment that directs the encoded polypeptide to the proteosome, such as a ubiquitin fusion segment.
  • Recombinant molecules may also include intervening and/or untranslated sequences surrounding and/or within the nucleic acid sequences of nucleic acid molecules of the present invention.
  • Another embodiment of the present invention includes a recombinant cell comprising a host cell transformed with one or more recombinant molecules of the present invention. Transformation of a polynucleotide molecule into a cell can be accomplished by any method by which a polynucleotide molecule can be inserted into the cell. Transformation techniques include, but are not limited to, transfection, electroporation, microinjection, lipofection, adsorption, and protoplast fusion. A recombinant cell may remain unicellular or may grow into a tissue, organ or a multicellular organism.
  • Transformed polynucleotide molecules of the present invention can remain extrachromosomal or can integrate into one or more sites within a chromosome of the transformed (i.e., recombinant) cell in such a manner that their ability to be expressed is retained.
  • Suitable host cells to transform include any cell that can be transformed with a polynucleotide of the present invention.
  • Host cells of the present invention either can be endogenously (i.e., naturally) capable of producing polypeptides of the present invention or can be capable of producing such polypeptides after being transformed with at least one polynucleotide molecule of the present invention.
  • Host cells of the present invention can be any cell capable of producing at least one protein of the present invention, and include plant, bacterial, fungal (including yeast), parasite, arthropod, and cells.
  • the host cell is a plant cell, more preferably a cell of a grapevine.
  • Recombinant DNA technologies can be used to improve expression of a transformed polynucleotide molecule by manipulating, for example, the number of copies of the polynucleotide molecule within a host cell, the efficiency with which those polynucleotide molecules are transcribed, the efficiency with which the resultant transcripts are translated, and the efficiency of post-translational modifications.
  • Recombinant techniques useful for increasing the expression of polynucleotide molecules of the present invention include, but are not limited to, operatively linking polynucleotide molecules to high-copy number plasmids, integration of the polynucleotide molecule into one or more host cell chromosomes, addition of vector stability sequences to plasmids, substitutions or modifications of transcription control signals (e.g., promoters, operators, enhancers), substitutions or modifications of translational control signals (e.g., ribosome binding sites, Shine-Dalgarno sequences), modification of polynucleotide molecules of the present invention to correspond to the codon usage of the host cell, and the deletion of sequences that destabilize transcripts.
  • plant refers to whole plants, plant organs (e.g. leaves, stems roots, etc), seeds, plant cells and the like. Plants contemplated for use in the practice of the present invention include both monocotyledons and dicotyledons. Preferably, the transgenic plant is a grapevine.
  • Transgenic plants as defined in the context of the present invention include plants (as well as parts and cells of said plants) and their progeny which have been genetically modified using recombinant techniques to cause production of at least one polypeptide, antisense polynucleotide, catalytic polynucleotide or dsRNA, of the present invention in the desired plant or plant organ.
  • Transgenic plants can be produced using techniques known in the art, such as those generally described in A. Slater et ah, Plant Biotechnology - The Genetic Manipulation of Plants, Oxford University Press (2003), and P. Christou and H. Klee, Handbook of Plant Biotechnology, John Wiley and Sons (2004).
  • a polynucleotide of the present invention may be expressed constitutively in the transgenic plants during all stages of development.
  • the polypeptides, antisense polynucleotide, catalytic polynucleotide or dsRNA may be expressed in a stage-specific manner.
  • regulatory sequences which are known or are found to cause expression of a gene encoding a polypeptide, antisense polynucleotide, catalytic polynucleotide or dsRNA, of interest in plants may be used in the present invention.
  • the choice of the regulatory sequences used depends on the target plant and/or target organ of interest. Such regulatory sequences may be obtained from plants or plant viruses, or may be chemically synthesized. Such regulatory sequences are well known to those skilled in the art.
  • Terminor sequences and polyadenylation signals include any such sequence functioning as such in plants, the choice of which would be obvious to the skilled addressee.
  • An example of such sequences is the 3' flanking region of the nopaline synthase (nos) gene of ' Agrobacterium tumefaciens.
  • Several techniques are available for the introduction of an expression construct containing a nucleic acid sequence encoding a polypeptide, antisense polynucleotide, catalytic polynucleotide or dsRNA, of interest into the target plants. Such techniques include but are not limited to transformation of protoplasts using the calcium/polyethylene glycol method, electroporation and microinjection or (coated) particle bombardment.
  • transformation systems involving vectors are widely available, such as viral and bacterial vectors (e.g. from the genus Agrobacterium). After selection and/or screening, the protoplasts, cells or plant parts that have been transformed can be regenerated into whole plants, using methods known in the art. The choice of the transformation and/or regeneration techniques is not critical for this invention.
  • the plant is a grapevine. There are a number of teachings in the art on how to prepare transformed grapevines.
  • the invention also provides monoclonal or polyclonal antibodies to polypeptides of the invention or fragments thereof.
  • the present invention further provides a process for the production of monoclonal or polyclonal antibodies to polypeptides of the invention.
  • binds specifically refers to the ability of the antibody to bind to proteins of the present invention but not other known L-idonate dehydrogenase-like polypeptides such as provided in SEQ ID NO: 4.
  • epitope refers to a region of a polypeptide of the invention which is bound by the antibody.
  • An epitope can be administered to an animal to generate antibodies against the epitope, however, antibodies of the present invention preferably specifically bind the epitope region in the context of the entire polypeptide.
  • polyclonal antibodies are desired, a selected mammal (e.g., mouse, rabbit, goat, horse, etc.) is immunised with an immunogenic polypeptide such as that provided as SEQ ID NO:1. Serum from the immunised animal is collected and treated according to known procedures. If serum containing polyclonal antibodies contains antibodies to other antigens, the polyclonal antibodies can be purified by immunoaffmity chromatography. Techniques for producing and processing polyclonal antisera are known in the art. In order that such antibodies may be made, the invention also provides peptides of the invention or fragments thereof haptenised to another peptide for use as immunogens in animals.
  • an immunogenic polypeptide such as that provided as SEQ ID NO:1. Serum from the immunised animal is collected and treated according to known procedures. If serum containing polyclonal antibodies contains antibodies to other antigens, the polyclonal antibodies can be purified by immunoaffmity chromatography. Techniques for producing and processing polyclonal antisera are known
  • Monoclonal antibodies directed against polypeptides of the invention can also be readily produced by one skilled in the art.
  • the general methodology for making monoclonal antibodies by hybridomas is well known.
  • Immortal antibody-producing cell lines can be created by cell fusion, and also by other techniques such as direct transformation of B lymphocytes with oncogenic DNA, or transfection with Epstein- Barr virus.
  • Panels of monoclonal antibodies produced can be screened for various properties; i.e., for isotype and epitope affinity.
  • an alternative technique involves screening phage display libraries where, for example the phage express scFv fragments on the surface of their coat with a large variety of complementarity determining regions (CDRs).
  • CDRs complementarity determining regions
  • the term "antibody”, unless specified to the contrary, includes fragments of whole antibodies which retain their binding activity for a target antigen. Such fragments include Fv 5 F(ab') and F(ab')2 fragments, as well as single chain antibodies (scFv).
  • the antibodies and fragments thereof may be humanised antibodies, for example as described in EP-A-239400.
  • Antibodies of the invention may be bound to a solid support and/or packaged into kits in a suitable container along with suitable reagents, controls, instructions and the like.
  • antibodies of the present invention are detectably labeled.
  • Exemplary detectable labels that allow for direct measurement of antibody binding include radiolabels, fluorophores, dyes, magnetic beads, chemiluminescers, colloidal particles, and the like.
  • Examples of labels which permit indirect measurement of binding include enzymes where the substrate may provide for a coloured or fluorescent product.
  • Additional exemplary detectable labels include covalently bound enzymes capable of providing a detectable product signal after addition of suitable substrate.
  • suitable enzymes for use in conjugates include horseradish peroxidase, alkaline phosphatase, malate dehydrogenase and the like. Where not commercially available, such antibody-enzyme conjugates are readily produced by techniques known to those skilled in the art.
  • detectable labels include biotin, which binds with high affinity to avidin or streptavidin; fluorochromes (e.g., phycobiliproteins, phycoerythrin and allophycocyanins; fluorescein and Texas red), which can be used with a fluorescence activated cell sorter; haptens; and the like.
  • the detectable label allows for direct measurement in a plate luminometer, e.g., biotin.
  • Such labeled antibodies can be used in techniques known in the art to detect polypeptides of the invention.
  • L-idonate dehydrogenase may be employed in a screening process for compounds which activate (agonists) or inhibit (antagonists) the L-idonate dehydrogenase activity of the polypeptide.
  • potential antagonists include antibodies, oligosaccharides and derivatives thereof.
  • a potential antagonist includes a small molecule which binds to L- idonate dehydrogenase, making it inaccessible to a substrate of the polypeptide.
  • small molecules include, but are not limited to, small peptides or peptide- like molecules.
  • the small molecules may mimic the structure of a substrate of the L- idonate dehydrogenase.
  • agonists or antagonists which can be used to regulate L-idonate dehydrogenase activity are employed for purposes such as modulating the production of tartaric acid or vitamin C in Vitis vinifera, among others.
  • the invention also comprehends high-throughput screening (HTS) assays to identify compounds that interact with or inhibit the biological activity (i.e., affect enzymatic activity) of a polypeptide having L-idonate dehydrogenase activity.
  • HTS assays permit screening of large numbers of compounds in an efficient manner.
  • HTS assays are designed to identify "hits” or “lead compounds” having the desired property, from which modifications can be designed to improve the desired property. Chemical modification of the "hit” or “lead compound” is often based on an identifiable structure/activity relationship between the "hit” and the L-idonate dehydrogenase polypeptide.
  • the three-dimensional structure of a crystal comprising a polypeptide having L- idonate dehydrogenase activity can be used to identify antagonists or agonists through the use of computer modeling using a docking program such as GRAM, DOCK, or AUTODOCK (Dunbrack et ah, 1997).
  • Computer programs can also be employed to estimate the attraction, repulsion, and steric hindrance of a candidate compound to the polypeptide.
  • the tighter the fit e.g., the lower the steric hindrance, and/or the greater the attractive force
  • the more potent the potential agonist or antagonist will be since these properties are consistent with a tighter binding constant.
  • the more specificity in the design of a potential agonist or antagonist the more likely that it will not interfere with other proteins.
  • a potential compound could be obtained, for example, by screening a random peptide library produced by a recombinant bacteriophage, or a chemical library. A compound selected in this manner could be then be systematically modified by computer modeling programs until one or more promising potential compounds are identified.
  • the prospective agonist or antagonist can be placed into the activity assay described herein to test its effect on the activity of a polypeptide having L-idonate dehydrogenase activity.
  • any molecular biological technique known in the art which is capable of detecting a polymorphism/mutation/genetic variation or differential gene expression can be used in the methods of the present invention.
  • Such methods include, but are not limited to, the use of nucleic acid amplification, nucleic acid sequencing, nucleic acid hybridization with suitably labeled probes, single-strand conformational analysis (SSCA), denaturing gradient gel electrophoresis (DGGE), heteroduplex analysis (HET), chemical cleavage analysis (CCM), catalytic nucleic acid cleavage, or a combination thereof (see, for example, Lemieux, 2000).
  • the invention also includes the use of molecular marker techniques to detect polymorphisms closely linked to genes of the invention.
  • PCR polymerase chain reaction
  • PCR Methods for PCR are known in the art, and are taught, for example, in "PCR” (Ed. MJ. McPherson and S.G Moller (2000) BIOS Scientific Publishers Ltd, Oxford). PCR can be performed on cDNA obtained from reverse transcribing niRNA isolated from plant cells expressing, or that should be expressing, a gene of the invention. However, it will generally be easier if PCR is performed on genomic DNA isolated from a plant.
  • a primer is an oligonucleotide, usually of about 20 nucleotides long, with a minimum of about 15 and a maximum of about 50 nucleotides, that is capable of hybridising in a sequence specific fashion to the target sequence and being extended during the PCR.
  • Amplicons or PCR products or PCR fragments or amplification products are extension products that comprise the primer and the newly synthesized copies of the target sequences.
  • Multiplex PCR systems contain multiple sets of primers that result in simultaneous production of more than one amplicon.
  • Primers may be perfectly matched to the target sequence or they may contain internal mismatched bases that can result in the induction of restriction enzyme or catalytic nucleic acid recognition/cleavage sites in specific target sequences.
  • Primers may also contain additional sequences and/or modified or labelled nucleotides to facilitate capture or detection of amplicons. Repeated cycles of heat denaturation of the DNA, annealing of primers to their complementary sequences and extension of the annealed primers with polymerase result in exponential amplification of the target sequence.
  • target or target sequence or template refer to nucleic acid sequences which are amplified.
  • the TaqMan assay uses allele specific (ASO) probes with a donor dye on one end and an acceptor dye on the other end such that the dye pair interact via fluorescence resonance energy transfer (FRET).
  • a target sequence is amplified by PCR modified to include the addition of the labeled ASO probe.
  • the PCR conditions are adjusted so that a single nucleotide difference will effect binding of the probe. Due to the 5' nuclease activity of the Taq polymerase enzyme, a perfectly complementary probe is cleaved during PCR while a probe with a single mismatched base is not cleaved. Cleavage of the probe dissociates the donor dye from the quenching acceptor dye, greatly increasing the donor fluorescence.
  • the ASO probes contain complementary sequences flanking the target specific species so that a hairpin structure is formed.
  • the loop of the hairpin is complimentary to the target sequence while each arm of the hairpin contains either donor or acceptor dyes.
  • the hairpin structure brings the donor and acceptor dye close together thereby extinguishing the donor fluorescence.
  • the donor and acceptor dyes are separated with an increase in fluorescence of up to 900 fold.
  • Molecular beacons can be used in conjunction with amplification of the target sequence by PCR and provide a method for real time detection of the presence of target sequences or can be used after amplification.
  • Marker assisted selection is a well recognised method of selecting for heterozygous plants required when backcrossing with a recurrent parent in a classical breeding program.
  • the population of plants in each backcross generation will be heterozygous for the gene of interest, normally present in a 1:1 ratio in a backcross population, and the molecular marker can be used to distinguish the two alleles.
  • Grapes produced from transgenic grapevines of the invention are useful for the production of, for example, wine, raisins, sultanas, fresh table grapes, juice (and concentrate), and in canned products.
  • Tartaric acid produced by transgenic plants such as transgenic grapevines is, by means of non-limiting examples, important in wine production (for example, in controlling acidity, pH and tartrate stability) and also for use as a food acidulant
  • Food acidulants provide a wide range of functions. Not only do they provide acidity, but they contribute to flavour and assist in preservation by retarding microbial growth and enzymatic deterioration. Grapes are also a source of ascorbic acid.
  • Ascorbic acid (vitamin C) is a powerful water-soluble antioxidant that is vital for growth and maintenance of all tissue types in humans.
  • One important role of ascorbic acid is its involvement in the production of collagen, an essential cellular component for connective tissues, muscles, tendons, bones, teeth and skin. Collagen is also required for the repair of blood vessels, bruises, and broken bones.
  • Ascorbic acid helps regulate blood pressure, contributes to reduced cholesterol levels, and aids in the removal of cholesterol deposits from arterial walls.
  • Ascorbic acid also aids in the metabolisation of folic acid, regulates the uptake of iron, and is required for the conversion of the amino acids L-tyrosine and L- phenylalanine into noradrenaline.
  • tryptophan into serotonin, the neurohormone responsible for sleep, pain control, and well-being, also requires adequate supplies of ascorbic acid.
  • L-ascorbic acid can impair the production of collagen and lead to joint pain, anaemia, nervousness and retarded growth. Other effects are reduced immune response and increased susceptibility to infections.
  • the most extreme form of ascorbic acid deficiency is scurvy, a condition evidenced by swelling of the joints, bleeding gums, and the haemorrhaging of capillaries below the surface of the skin. If left untreated, scurvy is fatal.
  • Ascorbic acid is produced in all higher plants and in the liver or kidney of most higher animals, but not humans. Therefore, humans must have access to sufficient amounts of ascorbic acid from adequate dietary sources or supplements in order to maintain optimal health.
  • Example 1 Identification of a candidate gene in the tartaric acid biosynthetic pathway
  • Transcript data compiled from an extensive EST collection that relates to the expression of thousands of Vitis genes to many tissues and developmental stages of the grapevine were interrogated.
  • a structured query language was used to restrict the transcripts to candidates from cDNA libraries (56 cDNA libraries were used in the analysis) prepared from tissues in which tartaric acid biosynthesis maximally occurs (10 cDNA libraries met this criterion).
  • the method for identification of differentially expressed transcripts (Goes da Silva et ah, 2005) was modified to include transcripts with greater than 6 ESTs per transcript. Transcripts determined as differentially expressed in tissues in which tartaric acid biosynthesis is known to occur, including pre-veraison berries and young leaves, comprised a set of 87 genes.
  • Candidates identified by transcriptome profiling were analysed to identify putative protein functional domains and motifs via PFAM 5 NCBI, BRENDA and Interpro protein domain BLAST (Altschul et al. 1997) interfaces. Examination of proposed intermediates in the conversion of ascorbate to tartrate (Stafford, 1959) suggested the potential involvement of specific enzyme classes in these reactions; motifs representative of oxidoreductase family of proteins were sought as candidates. A set of 4 genes was selected based on the combined expression and protein homology criteria.
  • High performance liquid chromatography (HPLC) analysis was used to characterise the berry tartaric acid content of plants from 28 species of Vitaceae. Approximately 5 g fresh weight of grape berries was homogenised in a mortar and pestle with 5 mL of 0.5 M H 3 PO 4 , pH 1.5. The slurry was transferred to a 10 mL polypropylene centrifuge tube, and the volume adjusted to 10 mL with the same solvent and placed on a rotating mixer at room temperature for 2 h to ensure that all crystals were fully dissolved.
  • HPLC High performance liquid chromatography
  • a 2 mL aliquot was removed and placed into a 2 mL centrifuge tube, centrifuged at 14,000 rpm for 2 min at room temperature, passed through a 45 ⁇ m filter and the organic acids separated using HPLC.
  • the loading volume was 20 ⁇ L, loaded via an autosampler (Beckman System Gold, Model 507e) onto an HPLC column (Prevail OA organic acid 4.5 x 250 mm, Alltech Associates) fitted with a guard cartridge of the same material.
  • the mobile phase used was 25 mM KH 2 PO 4 adjusted to pH 2.0 with phosphoric acid, at a flow rate of 0.5 mL/min (Beckman System Gold, Model 126NM).
  • Detection of the organic acids was by UV absorbance at 210 nm using a diode array detector (Beckman System Gold, Model 168). Significant variation in berry tartaric acid levels was identified, including one species, Ampelopsis acontifo ⁇ ia, from which tartaric acid was absent ( Figure 2a).
  • rtPCR Reverse Transcriptase validation of gene expression Total RNA from Vitis vinifera, V. californica, Ampelopsis brevipedunculata and
  • A. acontifolia was extracted from washed grape berries and leaves as described (Iandolino, et a 2004). Genomic DNA was extracted (MoBio®) from V. vinifera, A. acontifolia and P. tricuspifolia. For candidate genes, internal 18-22mer primers were designed with annealing temperatures of approximately 55°C. Ubiquitin and actin sequences were used for amplification controls.
  • RT-PCR reactions comprised 1.5 pmol of each primer, 0.5 ⁇ l Taq DNA polymerase, 0.5 ⁇ l dNTPs, l ⁇ l MgCl (25 mM), l ⁇ l (500 ng ⁇ l-1) template mRNA, 2 ⁇ l 10x (Mg free) buffer, to 20 ⁇ l with dH2O.
  • Quantitative RT PCR used 10 ⁇ l BioRad® real time PCR reagent, l ⁇ l (500 ng ⁇ l '1 ) template cDNA, 8.4 ⁇ l dH2O and , 0.6 ⁇ l (1.5 pmol) F/R primer.
  • the open reading frame encoding IDN was PCR amplified from V. vinifera using the primers F'-GGGCATATGATGGGGAAAGGAGGCAACTCTG (SEQ ID NO: 10) and R'-CCGGATCCTTAGAGATTAAACATGACCTTG (SEQ ID NO: 11), cloned into ⁇ TOPO2.1 (Invitrogen) and sequenced (SEQ ID NO: 2).
  • the deduced protein of the gene (SEQ ID NO: 1) is 77% identical to plant NAD + -linked sorbitol dehydrogenases from a number of plant species ( Figure 3), but was found to be only 32% identical to the L-idonate dehydrogenase that occurs in the gluconate II pathway of E.
  • the same primers were used to amplify the IDN gene from Parthenocissus tricuspifolia (SEQ ID NO: 14).
  • the P. tricuspifolia gene contained 3 nucleotide base pair differences when compared to IDN from V. vinifera, with the nucleotide base pair changes resulting in a single amino acid change (SEQ ID NO: 13).
  • the IDN gene was amplified from V. vinifera RNA sampled 4 weeks post anthesis and cloned via pTOPO2.1 (Invitrogen) into pET14b
  • E. coli E. coli and used to transform BL21 (DE3) plysS (Novagen) for induction.
  • Cells 50 ml in 250 mL Erlenmeyer flask
  • ampicillin 100 ⁇ g mL " l
  • Cells were harvested by centrifugation 3 h post induction and resuspended in sonication buffer (4 ml/g wet cell paste; Clontech) containing 10 mM 2- mercaptoethanol followed by three freeze-thaw cycles in liquid nitrogen.
  • Protease cocktail was added (Roche) and cells disrupted by reciprocation through a 20 gauge needle followed by centrifugation at 13,500 g for 10 min at 4°C, and divided into soluble and insoluble fractions. Final purification of soluble material was achieved using Talon resin (Clontech). Overexpression and HIS-Tag purification of the candidate gene in E. coli yielded a 41 kDa recombinant protein.
  • Example 3 Enzyme Activity Assay Evidence from radioisotope studies (Saito and Kasai, 1982; 1984) in grape berries has shown that 14 C radiolabel on either idonate or 5-keto D-gluconate is incorporated into tartaric acid. Potential substrates were tested in an NAD + coupled activity assay.
  • the assays contained 15 ⁇ L purified protein extract pre-equilibrated to 30 0 C in 100 mM Tris HCl pH 8, 330 ⁇ mol NAD+ or NADH, in a glass cuvette zeroed at A340 nm before addition of 50 mmol substrate to a final volume of 150 ⁇ L.
  • the forward reaction (L-idonate to 5-keto gluconic acid) was followed by A340 nm increase; the reverse reaction, (5- keto-D-gluconic acid to L-idonate) by A340 nm decrease.
  • Substrates tested included L-idonate, 5-keto D-gluconic acid, D-sorbitol and D- gluconic acid.
  • the recombinant protein when tested in the NAD + coupled reaction, showed high substrate specificity and catalytic activity for L-idonate (Figure 4a) and, in the reverse reaction with NADH as the cofactor, for 5-keto D-gluconate as substrates (Figure 4b). No activity was observed if NADH was used as the cofactor with L- idonate, or if NAD + was used as the cofactor with 5-keto D-gluconate; furthermore, no activity was observed for either forward or reverse reactions with NADP + or NADPH, respectively, as the cofactor. D-gluconic acid and 2-keto L-gulonic acid (intermediates proposed to be involved in TA biosynthetic pathways) and AA were also tested as substrates with NAD + as cofactor; each displayed very low rates of NADH formation (Figure 4).
  • aconitifolia where TA accumulation does not occur and which lacks the gene encoding L-idonate dehydrogenase, AA accumulates to three times the concentration observed in the TA accumulating species V. vinifera ( Figure 7b). No corresponding accumulation of L-idonate was observed in berries of A. aconitifolia.
  • Table 2 Change in tartaric acid concentration (mg g "1 ) in V. vinifera berry slices incubated with 100 mM L-idonate, 5-keto D-gluconate, or water.
  • RNAi down-regulation of gene expression may be performed by first cloning sense and antisense of a 525 bp fragment of the L-idonate dehydrogenase gene (SEQ ID NO: 3), or another suitable fragment of the gene such as those described herein, into the pKANNIBAL vector (Wesley et ah, 2001) using standard gene cloning methods (Sambrook et al. supra). Where necessary, appropriate restriction sites are incorporated in to the oligonucleotide primers that are used to amplify each strand. The construct containing the sense and antisense strands and intron are then excised from pKANNIBAL by restriction digest and sub-cloned into pART27. pART27 is introduced in to an Agrobacterium strain by electroporation or tri- parental mating. The transformed Agrobacterium strain is then used to infect embryonic cell lines for transformation and regeneration in Vitis (Harmony (a rootstock) or Thompson seedless).
  • RNAi cassette in Vitis vinifera is confirmed using the methods described herein and known to those skilled in the art.
  • the level of production of tartaric acid and ascorbic acid in the grapevine transformed with the RNAi construct is determined using the methods of the invention as described herein.
  • the coding sequence of a gene of the invention is operably linked to a promoter, for example the CaMV 35S promoter, and a promoter
  • the promoter may be expressed constitutively throughout the plant, or in a tissue specific manner.
  • the promoter may be active in the berries of the plant.
  • Grapevines are transformed using the Agrobacterium-mediated transformation technique detailed in Example 6. Transgenic grapevines are identified and the level of production of tartaric acid and ascorbic acid are analyzed.
  • Tartaric acid and vitamin C levels in a plant can be modulated by altering the light conditions under which a tartaric acid producing plant is grown.
  • the effects of bunch shading on berry organic acid levels and transcript accumulation was tested by growing grapes under either ambient-light or box-shade conditions. The results reveal strong reductions in tartaric acid pools in shaded berry clusters when compared to unshaded controls (Figure 8A) and decrease in L-idonate dehydrogenase transcript (Figure 8B). By contrast, levels of malate and oxalate were unaffected by the shading treatment (Figure 8C).

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Abstract

La présente invention concerne une enzyme, la L-idonate déshydrogénase, dans la voie biosynthétique de l’acide tartrique chez les plantes. L’invention concerne en particulier de nouveaux polypeptides et des polynucléotides codant pour les polypeptides. La présente invention concerne en outre des procédés de modulation des niveaux d’acide tartrique et d’acide ascorbique chez les plantes, en particulier dans une vigne. La modulation de l’acide tartrique et de l’acide ascorbique chez les plantes peut être utilisée pour une variété de buts, par exemple pour augmenter ou diminuer les niveaux d’acide tartrique, ou pour augmenter les niveaux d’acide ascorbique chez une plante. L’invention concerne également des agonistes et des antagonistes de l’enzyme pour moduler la production d’acide tartrique et d’acide ascorbique dans les plantes, ou des extraits de ceux-ci.
PCT/AU2006/000966 2005-07-13 2006-07-07 Voies biosynthétiques de l’acide tartrique et l’acide ascorbique chez les plantes : rôle de la l-idonate déshydrogénase Ceased WO2007006087A1 (fr)

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WO2011074959A1 (fr) 2009-12-15 2011-06-23 Edwin Henricus Antonius Holman Végétaux transgéniques résistants à l'ozone

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011074959A1 (fr) 2009-12-15 2011-06-23 Edwin Henricus Antonius Holman Végétaux transgéniques résistants à l'ozone

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