WO2010028456A1 - Modification of fructan biosynthesis, increasing plant biomass, and enhancing productivity of biochemical pathways in a plant - Google Patents

Modification of fructan biosynthesis, increasing plant biomass, and enhancing productivity of biochemical pathways in a plant Download PDF

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Publication number
WO2010028456A1
WO2010028456A1 PCT/AU2009/001211 AU2009001211W WO2010028456A1 WO 2010028456 A1 WO2010028456 A1 WO 2010028456A1 AU 2009001211 W AU2009001211 W AU 2009001211W WO 2010028456 A1 WO2010028456 A1 WO 2010028456A1
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Prior art keywords
plant
fft
promoter
fusion
sst
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PCT/AU2009/001211
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French (fr)
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German Spangenberg
Aidyn Mouradov
Megan Elizabeth Griffith
Luciano Gaston Martelotto
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Agriculture Victoria Services Pty Ltd
Molecular Plant Breeding Nominees Ltd
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Priority claimed from AU2009902230A external-priority patent/AU2009902230A0/en
Application filed by Agriculture Victoria Services Pty Ltd, Molecular Plant Breeding Nominees Ltd filed Critical Agriculture Victoria Services Pty Ltd
Priority to AU2009291522A priority Critical patent/AU2009291522B2/en
Priority to MX2011002786A priority patent/MX2011002786A/en
Priority to JP2011526355A priority patent/JP5805534B2/en
Priority to EP09812549A priority patent/EP2337848A4/en
Priority to CA2737059A priority patent/CA2737059C/en
Priority to US13/063,992 priority patent/US9890387B2/en
Priority to NZ591777A priority patent/NZ591777A/en
Priority to BRPI0913562-6A priority patent/BRPI0913562B1/en
Publication of WO2010028456A1 publication Critical patent/WO2010028456A1/en
Priority to AU2015264827A priority patent/AU2015264827B2/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N15/8245—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 involving modified carbohydrate or sugar alcohol metabolism, e.g. starch biosynthesis
    • C12N15/8246—Non-starch polysaccharides, e.g. cellulose, fructans, levans
    • 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)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8222—Developmentally regulated expression systems, tissue, organ specific, temporal or spatial regulation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1051—Hexosyltransferases (2.4.1)
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

  • the present invention relates to the modification of fructan biosynthesis in plants and, more particularly, to methods of manipulating fructan biosynthesis in photosynthetic cells, and to related nucleic acids and constructs.
  • the present invention also relates to increasing plant biomass and, more particularly, to methods of enhancing biomass yield and/or yield stability, including shoot and/or root growth in a plant, and to related nucleic acids and constructs.
  • the present invention also relates to methods of enhancing the productivity of biochemical pathways and, more particularly, to fusion proteins in plants, and to related nucleic acids and constructs.
  • Fructans are a type of water-soluble carbohydrate whose primary function is to provide a readily accessible energy reserve for plant growth. Fructans are associated with various advantageous characters in grasses, such as cold and drought tolerance, increased tiller survival, enhanced persistence, good regrowth after cutting or grazing, improved recovery from stress, early spring growth and increased nutritional quality.
  • Fructan synthesis and metabolism in grasses and cereals is complex.
  • Fructans consist of linear or branched fructose chains attached to sucrose. The chain length of plant fructans ranges from three up to a few hundred fructose units. Different types of fructans can be distinguished based on the linkage types present. In perennial ryegrass three types of fructans have been identified: inulins, inulin neoseries and levan neoseries, with four fructosyltransferse (FT) enzymes involved in this fructan profile ( Figure 6).
  • FT fructosyltransferse
  • the enzyme 1-SST (sucrose: sucrose 1 -fructosyltransferase) catalyses the first step in fructan biosynthesis while the remaining enzymes elongate the growing fructose chain (1-FFT: fructan: fructan 1 -fructosyltransferase, 6G-FFT: 6-glucose fructosyltransferase, and 6-SFT: sucrose: fructose 6-fructosyltransferase).
  • the enzymes 1-FEH or 6-FEH reduce fructan chain length by releasing fructose molecules.
  • Fructans represent the major non-structural carbohydrate in 15% of plant species and play a key role in forage quality. Ruminant livestock grazing on high fructan diets show improved animal performance.
  • fructosyltransferase (FT) genes In grasses the level and composition of fructans has been increased in stems and leaf sheaths through the engineered expression of fructosyltransferase (FT) genes.
  • FT fructosyltransferase
  • Applicants have found that it is possible to nutritionally enhance plants eg. forage grasses and/or to increase plant biomass by spatial reprogramming of the fructan- biosynthesis pathway in photosynthetic cells. Using this process it is possible to drive fructan accumulation in leaf blades, the plant organs that are primarily grazed by livestock, and which may not normally accumulate fructans. Thus, accumulation of fructans, especially those exhibiting a high degree of polymerization ('high DP fructans'), provides more accessible nutrition for grazing animals. Fructans accumulate in the stems and leaf sheaths, with leaf fructans only forming during periods where CO 2 assimilation outperforms growth. Forage grasses may be nutritionally enhanced by expressing fructan genes in photosynthetic cells where sucrose is synthesised, thus driving fructan accumulation preferentially in leaf blades and providing more energy to grazing livestock.
  • Fructans in forage grasses contribute significantly to the readily available energy in the feed for grazing ruminant animals.
  • the fermentation processes in the rumen require considerable readily available energy.
  • the improvement of the readily available energy in the rumen can increase the efficiency of rumen digestion.
  • An increased efficiency in rumen digestion leads to an improved conversion of the forage protein fed to the ruminant animal into milk or meat, and to a reduction in nitrogenous waste.
  • Applicants have also found that reprogramming photosynthetic cells for extended life, for example by delaying leaf senescence, helps increase plant biomass.
  • Applicants have also found that it is possible to enhance the productivity of a biochemical pathway by co-ordinating enzymatic activity in the pathway by means of a genetic construct encoding a fusion, more preferably a translational fusion, of two or more enzymes from the pathway.
  • FT genes e.g. Lp1- SST and Lp6G-FFT
  • problems associated with differences in the expression patterns of these genes independently integrated into the plant genome may be alleviated, resulting in conversion of the sucrose molecules directly to fructans, those exhibiting a low degree of polymerisation ('low DP fructans') and a high degree of polymerization ('high DP fructans').
  • the FT proteins may physically associate with each other to form a metabolic channel for the efficient biosynthesis of fructans.
  • FT genes in photosynthetic cells leading to the accumulation of low and high DP fructans in photosynthetic cells may lead to a release from inhibition mechanisms of photosynthesis, facilitating solar energy capture and increased CO 2 fixation.
  • the present invention provides a method of manipulating fructan biosynthesis in photosynthetic cells of a plant, said method including introducing into said plant an effective amount of a genetic construct including a promoter, or a functionally active fragment or variant thereof, operatively linked to nucleic acids encoding one or more fructan biosynthetic enzymes, or functionally active fragments or variants thereof.
  • fructan biosynthesis is generally meant increasing fructan biosynthesis in a transformed plant relative to an untransformed control plant. However, for some applications it may be desirable to reduce or otherwise modify fructan biosynthesis in the transformed plant relative to the untransformed control plant. For example, it may be desirable to increase or decrease the activity of certain enzymes in the fructan biosynthetic pathway, in the transformed plant relative to the untransformed control plant.
  • photosynthetic cells are meant those cells of a plant in which photosynthesis takes place. Such cells generally contain the pigment chlorophyll and are otherwise known as green cells. Most photosynthetic cells are contained in the leaves of plants.
  • the genetic construct of the present invention is expressed in bundle sheath cells, more preferably in mesophyll and/or parenchymatous bundle sheath cells.
  • an effective amount is meant an amount sufficient to result in an identifiable phenotypic trait in said plant, or in a plant, plant seed or other plant part derived therefrom.
  • Such amounts can be readily determined by an appropriately skilled person, taking into account the type of plant, the route of administration and other relevant factors. Such a person will readily be able to determine a suitable amount and method of administration. See, for example, Maniatis et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, the entire disclosure of which is incorporated herein by reference.
  • 'genetic construct' is meant a recombinant nucleic acid molecule.
  • a 'promoter' is meant a nucleic acid sequence sufficient to direct transcription of an operatively linked nucleic acid sequence.
  • operatively linked is meant that the nucleic acid(s) and a regulatory sequence, such as a promoter, are linked in such a way as to permit expression of said nucleic acid under appropriate conditions, for example when appropriate molecules such as transcriptional activator proteins are bound to the regulatory sequence.
  • a regulatory sequence such as a promoter
  • an operatively linked promoter is upstream of the associated nucleic acid.
  • 'upstream' is meant in the 3' to 5' direction along the nucleic acid.
  • 'nucleic acid' is meant a chain of nucleotides capable of carrying genetic information.
  • the term generally refers to genes or functionally active fragments or variants thereof and/or other sequences in the genome of the organism that influence its phenotype.
  • the term 'nucleic acid' includes DNA (such as cDNA or genomic DNA) and RNA (such as mRNA or microRNA) that is single- or double-stranded, optionally containing synthetic, non-natural or altered nucleotide bases, synthetic nucleic acids and combinations thereof.
  • a 'nucleic acid encoding a fructan biosynthetic enzyme' is meant a nucleic acid encoding an enzyme of the fructan biosynthetic pathway in plants, for example fructosyltransferases such as sucrose:sucrose 1-fructosyltransferase (1-SST); fructan:fructan 1-fructosyltransferase (1-FFT); sucrose:fructan 6-fructosyltransferase (6- SFT); and fructan: fructan 6G-fructosyl transferase (6G-FFT); and fructoexohydrolases such as 1-fructoexohydrolase (1-FEH) and 6-fructoexohydrolase (6-FEH).
  • fructosyltransferases such as sucrose:sucrose 1-fructosyltransferase (1-SST); fructan:fructan 1-fructosyltransferas
  • the fragment or variant in relation to a promoter is meant that the fragment or variant (such as an analogue, derivative or mutant) is capable of directing transcription of an operatively linked nucleic acid.
  • Such variants include naturally occurring allelic variants and non-naturally occurring variants. Additions, deletions, substitutions and derivatizations of one or more of the nucleotides are contemplated so long as the modifications do not result in loss of functional activity of the fragment or. variant.
  • the functionally active fragment or variant has at least approximately 80% identity to the relevant part of the above mentioned sequence to which the fragment or variant corresponds, more preferably at least approximately 90% identity, even more preferably at least approximately 95% identity, most preferably at least approximately 98% identity.
  • the fragment has a size of at least 20 nucleotides, more preferably at least 50 nucleotides, more preferably at least 100 nucleotides, more preferably at least 200 nucleotides, more preferably at least 300 nucleotides.
  • a fructan biosynthetic enzyme By 'functionally active' in relation to the nucleic acid encoding a fructan biosynthetic enzyme is meant that the fragment or variant (such as an analogue, derivative or mutant) is capable of manipulating fructan biosynthesis in a plant by the method of the present invention, for example by being translated into an enzyme that is able to participate in the fructan biosynthetic pathway.
  • Such variants include naturally occurring allelic variants and non-naturally occurring variants. Additions, deletions, substitutions and derivatizations of one or more of the nucleotides are contemplated so long as the modifications do not result in loss of functional activity of the fragment or variant.
  • the functionally active fragment or variant has at least approximately 80% identity to the relevant part of the above mentioned sequence to which the fragment or variant corresponds, more preferably at least approximately 90% identity, even more preferably at least approximately 95% identity, most preferably at least approximately 98% identity.
  • Such functionally active variants and fragments include, for example, those having conservative nucleic acid changes.
  • nucleic acid changes is meant nucleic acid substitutions that result in conservation of the amino acid in the encoded protein, due to the degeneracy of the genetic code.
  • Such functionally active variants and fragments also include, for example, those having nucleic acid changes which result in conservative amino acid substitutions of one or more residues in the corresponding amino acid sequence.
  • Nonpolar Ala, VaI, Leu, lie, Pro, Met Phe, Trp
  • Uncharged polar GIy, Ser, Thr, Cys, Tyr, Asn, GIn
  • Proton Acceptor GIu, Asp, Thr, Ser, Tyr, Asn, GIn
  • Particularly preferred fragments and variants include one or more conserved sucrose binding/hydrolysis domains. Examples of such domains are shown in Figures 17, 18 and 36 hereto, for example (N/S)DP(N)G, FRDP and EC(I)D. Particularly preferred fragments and variants may also include one or more conserved amino acid domains found in Lolium FT, invertase and FEH sequences, for example as shown in Figures 17, 18 and 36 hereto.
  • the fragment has a size of at least 20 nucleotides, more preferably at least 50 nucleotides, more preferably at least 100 nucleotides, more preferably at least 200 nucleotides, more preferably at least 500 nucleotides.
  • the nucleic acid encoding one or more fructan biosynthetic enzymes is selected from the group consisting of genes encoding 1-SST, 1-FFT, 6-SFT and 6G- FFT, combinations thereof, and functionally active fragments and variants thereof.
  • the nucleic acid encodes a FT fusion protein of two or more of these fructan biosynthetic enzymes.
  • nucleic acid encoding one or more fructan biosynthetic enzymes encodes 1-SST and/or 6G-FFT, even more preferably a FT fusion protein of 1- SST and 6G-FFT, or functionally active fragments or variants thereof.
  • the nucleic acid encoding one or more fructan biosynthetic enzymes is isolated from or corresponds to a gene or genes from a species of interest. More preferably the gene or genes are from a ryegrass, fescue or wheat species, including Italian or annual ryegrass, perennial ryegrass, tall fescue, meadow fescue, red fescue, bread wheat and durum wheat. Even more preferably, the nucleic acid encoding one or more fructan biosynthetic enzymes is isolated from or corresponds to a gene from a Lolium species such as Lolium perenne or Lolium arundinaceum.
  • Lolium species such as Lolium perenne or Lolium arundinaceum.
  • Suitable nucleic acids encoding fructan biosynthetic enzymes are described in PCT/AU01/00705 and PCT/AU01/01275, the entire disclosures of which are incorporated herein by reference.
  • the nucleic acid encoding 1-SST includes a sequence selected from the group consisting of the sequence shown in SEQ ID NO: 11 of PCT/AU01/00705; and the nucleotide sequences encoding the polypeptide sequence shown in SEQ ID NO: 12 of PCT/AU01/00705; and functionally active fragments and variants thereof.
  • the nucleic acid encoding the 6G-FFT includes a sequence selected from the group consisting of the sequences shown in SEQ ID NO: 110 of PCT/AU01/01275, and Figure 7 hereto; and the nucleic acid sequences encoding the polypeptide sequences shown in SEQ ID NO: 111 of PCT/AU01/01275; and Figure 8 hereto; and functionally active fragments and variants thereof.
  • the nucleic acid encoding the 1-FFT includes a sequence selected from the group consisting of the sequences shown in SEQ ID NO: 3 of PCT/AU01 /00705, SEQ ID NOS: 103 and 105-109 of PCT/AU01/01275 and Figure 9 hereto; and the nucleotide sequences encoding the polypeptide sequences shown in SEQ ID No: 4 of PCT/AU01/00705, SEQ ID NO: 104 of PCT/AU01/01275 and Figure 10 hereto; and functionally active fragments and variants thereof.
  • Applicants have found that by generating a translational fusion of two FT genes as a single open reading frame, for example sucrose-sucrose 1-fructosyltransferase (Lp1- SST) and fructan-fructan 6G-fructosyltransferase (Lp6G-FFT) from Lolium perenne, a single mRNA transcript is produced which is translated as a single protein, with combined enzyme activities.
  • a translational fusion of two FT genes e.g. LpI-SST and Lp6G-FFT
  • problems associated with differences in the expression patterns of these two genes independently integrated in to the plant genome may be alleviated, resulting in the conversion of sucrose to low and high DP fructans.
  • the nucleic acid encoding the FT fusion protein of 1-SST and 6G-FFT includes a sequence selected from the group consisting of the sequences shown in Figures 12 and 14 hereto and the nucleic acid sequences encoding the polypeptide sequences shown in Figures 13 and 15 hereto; and functionally active fragments and variants thereof.
  • the genetic construct includes a sequence selected from the group consisting of the sequences shown in Figures 24 to 27, 31 , 32, 35, 36, 38 and 41 to 47 hereto; and functionally active fragments and variants thereof.
  • the present invention provides a method of enhancing productivity of a biochemical pathway in a plant, said method including introducing into said plant an effective amount of a genetic construct including nucleic acids encoding two or more enzymes from said pathway, or functionally active fragments or variants thereof.
  • said nucleic acids are linked to form a fusion gene encoding a fusion protein of said two or more enzymes.
  • a 'biochemical pathway' is meant a plurality of chemical reactions occurring within a cell which are catalysed by more than one enzyme or enzyme subunit and result in the conversion of a substrate into a product. This includes, for example, a situation in which two or more enzyme subunits (each being a discrete protein coded by a separate gene) combine to form a processing unit that converts a substrate into a product.
  • a 'biochemical pathway' is not constrained by temporal or spatial sequentiality.
  • 'enhancing productivity' is generally meant that the amount of product of the biochemical pathway, or the rate of production of the product, is increased in a transformed plant relative to an untransformed control plant.
  • a 'fusion protein' is meant a hybrid or chimeric protein produced recombinantly by expressing a fusion gene including two or more linked nucleic acids which originally encoded separate proteins, or functionally active fragments or variants thereof.
  • a 'fusion', 'translational fusion' or 'fusion gene' is meant that two or more nucleic acids are linked in such a way as to permit expression of the fusion protein, preferably as a translational fusion. This typically involves removing the stop codon from a nucleic acid sequence coding for a first protein, then appending the nucleic acid sequence of a second protein in a frame. The FT fusion gene is then expressed by a cell as a single protein.
  • the protein may be engineered to include the full sequence of both original proteins, or a functionally active fragment or variant of either or both.
  • the genetic construct may also include a nucleic acid sequence encoding a linker between the two linked nucleic acids.
  • a 'linker' is any chemical, synthetic, carbohydrate, lipid, polypeptide molecule (or combination thereof) positioned between and joined to two adjacent active fragments in a fusion protein.
  • a preferred linker of the invention is a flexible linker, such as a polypeptide chain consisting of one or more amino acid residues joined by amino acid bonds to the two active fragments.
  • a (GIy 4 Ser) 3 linker may be positioned between the two active fragments in the fusion protein.
  • the fragment or variant in relation to the nucleic acids encoding two or more enzymes from a biochemical pathway is meant that the fragment or variant (such as an analogue, derivative or mutant) is capable of enhancing productivity of the biochemical pathway in a plant by the method of the present invention.
  • Such variants include naturally occurring allelic variants and non-naturally occurring variants. Additions, deletions, substitutions and derivatizations of one or more of the nucleotides are contemplated so long as the modifications do not result in loss of functional activity of the fragment or variant.
  • the functionally active fragment or variant has at least approximately 80% identity to the relevant part of the above mentioned sequence to which the fragment or.
  • Such functionally active variants and fragments include, for example, those having conservative nucleic acid changes.
  • nucleic acid changes By 'conservative nucleic acid changes' is meant nucleic acid substitutions that result in conservation of the same amino acid in the encoded protein, due to the degeneracy of the genetic code.
  • Such functionally active variants and fragments also include, for example, those having nucleic acid changes which result in conservative amino acid substitutions of one or more residues in the corresponding amino acid sequence.
  • By 'conservative amino acid substitutions' is meant the substitution of an amino acid by another one of the same class, the classes being as follows:
  • Nonpolar Ala, VaI, Leu, He, Pro, Met Phe, Trp
  • Uncharged polar GIy, Ser, Thr, Cys, Tyr, Asn, GIn
  • Proton Acceptor GIu, Asp, Thr, Ser, Tyr, Asn, GIn
  • Particularly preferred fragments and variants include one or more conserved sucrose binding/hydrolysis domains. Examples of such domains are shown in Figures 17, 18 and 36 hereto, for example (N/S)DP(N)G, FRDP and EC(I)D.
  • Particularly preferred fragments and variants may also include one or more conserved amino acid domains found in Lolium FT, invertase and FEH sequences, for example as shown in Figures 17, 18 and 36 hereto.
  • the fragment has a size of at least 20 nucleotides, more preferably at least 50 nucleotides, more preferably at least 100 nucleotides, more preferably at least 200 nucleotides, more preferably at least 500 nucleotides.
  • the biochemical pathway is a fructan biosynthetic pathway.
  • the two or more enzymes from said pathway are selected from the group consisting of enzymes of the fructan biosynthetic pathway in plants, for example fructosyltransferases such as sucrose:sucrose 1 -fructosyltransferase (1-SST); fructan:fructan 1 -fructosyltransferase (1-FFT); sucrose:fructan 6-fructosyltransferase (6- SFT); and fructan: fructan 6G-fructosyl transferase (6G-FFT); and fructoexohydrolases such as 1-fructoexohydrolase (1-FEH) and 6-fructoexohydrolase (6-FEH).
  • fructosyltransferases such as sucrose:sucrose 1 -fructosyltransferase (1-SST); fructan:fructan 1 -fructosyltransferase (1-FFT); sucrose:
  • the nucleic acids encoding a FT fusion protein include two or more nucleic acids selected from the group consisting of genes encoding 1-SST, 1-FFT, 6-SFT and 6G-FFT, and functionally active fragments and variants thereof, linked to form a FT fusion gene.
  • the nucleic acids are optionally connected by a linker such as a flexible linker.
  • the nucleic acids encoding a FT fusion protein include two or more nucleic acids linked to form a FT fusion protein of 1-SST and 6G-FFT, or functionally active fragments or variants thereof, optionally connected by a linker such as a flexible linker.
  • the genes encoding enzymes of the fructan biosynthetic pathway are isolated from or correspond to genes from a ryegrass or fescue species, including Italian or annual ryegrass, perennial ryegrass, tall fescue, meadow fescue and red fescue. Even more preferably, the genes encoding enzymes of the fructan biosynthetic pathway are isolated from or correspond to genes from a Lolium species such as Lolium perenne or Lolium arundinaceum.
  • Suitable nucleic acids encoding fructan biosynthetic enzymes are described in PCT/AU01/00705 and PCT/AU01/01275, the entire disclosures of which are incorporated herein by reference.
  • the nucleic acid encoding 1-SST includes a sequence selected from the group consisting of the sequence shown in SEQ ID NO: 11 of PCT/AU01/00705; and the nucleotide sequences encoding the polypeptide sequence shown in SEQ ID NO: 12 of PCT/AU01 /00705; and functionally active fragments and variants thereof.
  • the nucleic acid encoding the 6G-FFT includes a sequence selected from the group consisting of the sequences shown in SEQ ID NO: 110 of PCT/AU01/01275, and Figure 7 hereto; and the nucleic acid sequences encoding the polypeptide sequences shown in SEQ ID NO: 111 of PCT/AU01/01275; and Figure 8 hereto; and functionally active fragments and variants thereof.
  • the nucleic acid encoding the 1-FFT includes a sequence selected from the group consisting of the sequences shown in SEQ ID NO: 3 of PCT/AU01 /00705, SEQ ID NOS: 103 and 105-109 of PCT/AU01/01275 and Figure 9 hereto; and the nucleotide sequences encoding the polypeptide sequences shown in SEQ ID No: 4 of PCT/AU01/00705, SEQ ID NO: 104 of PCT/AU01/01275 and Figure 10 hereto; and functionally active fragments and variants thereof.
  • the nucleic acid encoding the of 1-SST and 6G- FFT includes a sequence selected from the group consisting of the sequences shown in Figures 12 and 14 hereto and the nucleic acid sequences encoding the polypeptide sequences shown in Figures 13 and 15 hereto; and functionally active fragments and variants thereof.
  • the genetic construct includes a sequence selected from the group consisting of the sequences shown in Figures 24 to 27, 31 , 32, 35, 36, 38 and 41 to 475 hereto, and functionally active fragments and variants thereof.
  • the promoter used in the constructs and methods of the present invention may be a constitutive, tissue specific or inducible promoter.
  • the promoter is a light-regulated promoter, more preferably a photosynthetic promoter.
  • a 'light regulated promoter' is meant a promoter capable of mediating gene expression in response to light stimulus.
  • a 'photosynthetic promoter' is meant a promoter capable of mediating expression of a gene encoding a protein involved in a photosynthetic pathway in plants.
  • Photosynthetic promoters are active in a large group of cells including leaf blades, the upper and outer stem (>55% cells);
  • sucrose producing cells meophyll and parenchymatous bundle sheath cells
  • Frutosyltransferase activity will remove sucrose from the source thereby preventing feedback suppression on photosynthesis, and may facilitate increases in CO2 fixation;
  • Particularly preferred light-regulated promoters include a ribulose-1 , 5-bisphosphate carboxylase/oxygtenase small subunit (RbcS) promoter and a chlorophyll a/b binding protein (CAB) promoter, and functionally active fragments and variants thereof.
  • the light-regulated promoter may be from any suitable plant species including monocotyledonous plants [such as maize, rice, wheat, barley, sorghum, sugarcane, forage grasses, bioenergy grasses], dicotyledonous plants (such as Arabidopsis, soybean, canola, cotton, alfalfa and tobacco) and gymnosperms.
  • monocotyledonous plants such as maize, rice, wheat, barley, sorghum, sugarcane, forage grasses, bioenergy grasses
  • dicotyledonous plants such as Arabidopsis, soybean, canola, cotton, alfalfa and tobacco
  • gymnosperms such as Arabidopsis, soybean, canola, cotton, alfalfa and tobacco
  • the light-regulated promoter is isolated from or corresponds to a promoter from a ryegrass or fescue species, including Italian or annual ryegrass, perennial ryegrass, tall fescue, meadow fescue and red fescue. Even more preferably, the light regulated promoter is isolated from or corresponds to a promoter from a Lolium species such as Lolium perenne or Lolium arundinaceum.
  • the light-regulated promoter is isolated from or corresponds to a promoter from Arabidopsis, even more preferably Arabidopsis tha liana.
  • the RbcS promoter includes a sequence selected from the group consisting of the sequence shown in Figure 5 hereto, and functionally active fragments and variants thereof.
  • the RbcS promoter includes a sequence selected from the group consisting of the sequence shown in Figure 38 hereto, and functionally active fragments and variants thereof.
  • the CAB promoter includes a sequence selected from the group consisting of the sequence shown in Figure 4 hereto, and functionally active fragments and variants thereof.
  • the promoter may be a constitutive promoter, such as a ubiquitin (Ubi) promoter.
  • Ubi promoter includes a sequence selected from the group consisting of the sequence shown in Figure 41 hereto, and functionally active fragments and variants thereof.
  • the genetic constructs of the present invention may be introduced into the plants by any suitable technique.
  • Techniques for incorporating the genetic constructs of the present invention into plant cells are well known to those skilled in the art. Such techniques include v4grobactem//77-mediated introduction, Rhizobium-mediated introduction, electroporation to tissues, cells and protoplasts, protoplast fusion, injection into reproductive organs, injection into immature embryos and high velocity projectile introduction to cells, tissues, calli, immature and mature embryos, biolistic transformation, Whiskers transformation, and combinations thereof.
  • the choice of technique will depend largely on the type of plant to be transformed, and may be readily determined by an appropriately skilled person.
  • Cells incorporating the genetic constructs of the present invention may be selected, as . described below, and then cultured in an appropriate medium to regenerate transformed plants, using techniques well known in the art.
  • the culture conditions such as temperature, pH and the like, will be apparent to the person skilled in the art.
  • the resulting plants may be reproduced, either sexually or asexually, using methods well known in the art, to produce successive generations of transformed plants.
  • the methods of the present invention may be applied to a variety of plants, including monocotyledons [such as grasses (e.g. forage, turf and bioenergy grasses including perennial ryegrass, tall fescue, Italian ryegrass, red fescue, reed canarygrass, big bluestem, cordgrass, napiergrass, wildrye, wild sugarcane, Miscanthus, switchgrass), corn or maize, rice, wheat, barley, sorghum, sugarcane, rye, oat)], dicotyledons [such as Arabidopsis, tobacco, soybean, canola, alfalfa, potato, cassava, clovers (e.g. white clover, red clover, subterranean clover), vegetable brassicas, lettuce, spinach] and gymnosperms.
  • monocotyledons such as grasses (e.g. forage, turf and bioenergy grasses including perennial ryegrass, tall fescue, Italian
  • a genetic construct capable of manipulating fructan biosynthesis in photosynthetic cells of a plant, said genetic construct including a light-regulated promoter, or functionally active fragment or variant thereof, operatively linked to nucleic acids encoding one or more fructan biosynthetic enzymes, or functionally active fragments or variants thereof.
  • a genetic construct capable of enhancing productivity of a biochemical pathway in a plant said genetic construct including nucleic acids encoding two or more enzymes from said pathway, or functionally active fragments or variants thereof.
  • said nucleic acids are linked to form a fusion gene encoding a fusion protein of said two or more enzymes.
  • the genetic constructs according to the various aspects of the present invention may be vectors.
  • a 'vector' is meant a genetic construct used to transfer genetic material to a target cell.
  • the vector may be of any suitable type and may be viral or non-viral.
  • the vector may be an expression vector.
  • Such vectors include chromosomal, non-chromosomal and synthetic nucleic acid sequences, eg. derivatives of plant viruses; bacterial plasmids; derivatives of the Ti plasmid from Agrobacterium tumefaciens; derivatives of the Ri plasmid from Agrobacterium rhizogenes; phage DNA; yeast artificial chromosomes; bacterial artificial chromosomes; binary bacterial artificial chromosomes; vectors derived from combinations of plasmids and phage DNA.
  • any other vector may be used as long as it is replicable or integrative or viable in the plant cell.
  • the genetic construct may further include a terminator; said promoter, gene and terminator being operably linked.
  • the promoter, gene and terminator may be of any suitable type and may be endogenous to the target plant cell or may be exogenous, provided that they are functional in the target plant cell.
  • terminators which may be employed in the genetic constructs of the present invention are also well known to those skilled in the art.
  • the terminator may be from the same gene as the promoter sequence or a different gene.
  • Particularly suitable terminators are polyadenylation signals, such as the (CaMV)35S polyA and other terminators from the nopaline synthase (nos) and the octopine synthase (ocs) genes.
  • the genetic construct in addition to the promoter, the gene and the terminator, may include further elements necessary for expression of the nucleic acid, in different combinations, for example vector backbone, origin of replication (ori), multiple cloning sites, spacer sequences, enhancers, introns (such as the maize Ubiquitin Ubi intron), antibiotic resistance genes and other selectable marker genes [such as the neomycin phosphotransferase (nptll) gene, the hygromycin phosphotransferase (hph) gene, the phosphinothricin acetyltransferase (bar or pat) gene], and reporter genes (such as beta- glucuronidase (GUS) gene (gusA)].
  • the genetic construct may also contain a ribosome binding site for translation initiation.
  • the genetic construct may also include appropriate sequences for amplifying expression.
  • the genetic construct may further include a nucleic acid sequence encoding a linker between the two linked nucleic acids, as hereinbefore described.
  • the genetic construct is substantially purified or isolated.
  • substantially purified is meant that the genetic construct is free of the genes, which, in the naturally- occurring genome of the organism from which the nucleic acid or promoter of the invention is derived, flank the nucleic acid or promoter.
  • the term therefore includes, for example, a genetic construct which is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote; or which exists as a separate molecule (eg. a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences.
  • the substantially purified genetic construct is at least approximately 90% pure, more preferably at least approximately 95% pure, even more preferably at least approximately 98% pure.
  • isolated means that the material is removed from its original environment (eg. the natural environment if it is naturally occurring).
  • a naturally occurring nucleic acid present in a living plant is not isolated, but the same nucleic acid separated from some or all of the coexisting materials in the natural system, is isolated.
  • nucleic acids could be part of a vector and/or such nucleic acids could be part of a composition, and still be isolated in that such a vector or composition is not part of its natural environment.
  • the presence of the genetic construct in transformed cells may be determined by other techniques well known in the art, such as PCR (polymerase chain reaction), Southern blot hybridisation analysis, histochemical assays (e.g. GUS assays), thin layer chromatography (TLC), northern and western blot hybridisation analyses.
  • PCR polymerase chain reaction
  • Southern blot hybridisation analysis histochemical assays (e.g. GUS assays)
  • TLC thin layer chromatography
  • Applicant has also found that the methods of the present invention may result in enhanced biomass in the transformed plant relative to an untransformed control plant.
  • This enhanced biomass may in turn be used as a selection tool for identifying transformed plants. This has the advantage that in some circumstances there may be no need to include an antibiotic resistance or other marker to select for transformants, where subsequent removal of such markers (and for the creation of marker-free plants) may present difficulties.
  • 'enhancing biomass' or 'enhanced biomass' is meant enhancement of, increase in, or increased stability of biomass yield, including shoot and/or root growth, in a transformed plant relative to an untransformed control plant.
  • one or more growth characteristics selected from the group consisting of plant height, herbage dry weight, total leaf area, cumulative leaf area, leaf growth dynamics (ie.
  • number of leaves over time may be enhanced, increased or more stable in a transformed plant relative to an untransformed control plant.
  • This technique is particularly applicable to plants that are substantially genetically uniform or genetically identical or exhibit small phenotype differences in biomass prior to transformation.
  • a method of enhancing biomass in a plant including introducing into said plant an effective amount of a genetic construct including a promoter, or a functionally active fragment or variant thereof, operatively liked to nucleic acids encoding one or more fructan biosynthetic enzymes, or functionally active fragments or variants thereof.
  • the promoter is a light regulated promoter.
  • a method of enhancing biomass in a plant including introducing into said plant an effective, amount of a genetic construct including nucleic acids encoding two or more enzymes from a biochemical pathway in said plant, or functionally active fragments or variants thereof.
  • a method of enhancing biomass in a plant including introducing into said plant effective amounts of genetic construct capable of manipulating fructan biosynthesis in photosynthetic cells of the plant and a genetic construct capable of manipulating senescence in the plant.
  • the genetic constructs may be introduced into the plant by any suitable technique, as hereinbefore described, and may be introduced concurrently, sequentially or separately.
  • the genetic construct capable of manipulating fructan biosynthesis includes a promoter, or a functionally active fragment or variant thereof, operatively linked to
  • the promoter is a light regulated promoter.
  • the genetic construct capable of manipulating senescence in the plant is capable of manipulating senescence in photosynthetic cells of the plant.
  • the genetic construct capable of manipulating senescence includes a MYB gene promoter or modified MYB gene promoter, or a functionally active fragment or variant thereof, operatively linked to a gene encoding an enzyme involved in biosynthesis of a cytokinin, or a functionally active fragment or variant thereof.
  • Manipulating senescence generally relates to delaying senescence in the transformed plant or cells or organs of the transformed plant, eg photosynthetic cells, relative to an untransformed control plant. However, for some applications it may be desirable to promote or otherwise modify senescence in the plant. Senescence may be promoted or otherwise modified for example, by utilizing an antisense gene.
  • the MYB gene promoter may be of any suitable type.
  • the MYB gene promoter is a MYB32 gene promoter.
  • the MYB gene promoter is from Arabidopsis, more preferably Arabidopsis thaliana.
  • the MYB gene promoter includes a nucleotide sequence selected from the group consisting of the sequence shown in Sequence ID No: 1 of PCT/AU01/01092 and functionally active fragments and variants thereof.
  • a suitable promoter is described in Li et al., Cloning of three MYB-like genes from Arabidopsis (PGR 99-138) Plant Physiology 121 :313 (1999), the entire disclosure of which is incorporated herein by reference.
  • modified MYB gene promoter a promoter normally associated with a MYB gene, which promoter is modified to delete or inactivate one or more root specific motifs and/or pollen specific motifs in said promoter.
  • the modified MYB gene promoter is a modified MYB32 gene promoter.
  • the modified MYB gene promoter is modified from the MYB gene promoter from Arabidopsis, or more preferably Arabidopsis thaliana.
  • a suitable promoter which may be modified according to the present invention is described in Li et al., Cloning of three MYB-like genes from Arabidposis (PGR 99-138) Plant Physiology 121 :313 (1999), the entire disclosure of which is incorporated herein by reference.
  • root specific motif a sequence of 3-7 nucleotides, preferably 4-6 nucleotides, more preferably 5 nucleotides, which directs expression of any associated gene in the roots of a plant.
  • the root specific motif includes a consensus sequence ATATT or AATAT.
  • polystyrene-specific motif a sequence of 3-7 nucleotides, preferably 4-6 nucleotides, more preferably 4 or 5 nucleotides, which directs expression of an associated gene in the pollen of a plant.
  • the pollen specific motif includes a consensus sequence selected from the group consisting of TTTCT, AGAAA, TTCT and AGAA.
  • a root or pollen specific motif may be inactivated by adding, deleting, substituting or derivatizing one or more nucleotides within the motif, so that it no longer has the preferred consensus sequence.
  • the modified MYB gene promoter includes a nucleotide sequence selected from the group consisting of the sequences show in SEQ ID NOS: 2, 3 and 4 of US 11/789,526 and functionally active fragments and variants thereof.
  • a “gene encoding an enzyme involved in biosynthesis of a cytokinin” is meant a gene encoding an enzyme involved in the synthesis of cytokinins such kinetin, zeatin and benzyl adenine, for example a gene encoding isopentyl transferase (IPT), or IPT-like gene such as the sho gene (eg. from petunia).
  • IPT isopentyl transferase
  • sho gene eg. from petunia
  • the gene is an isopentenyl transferase (IPT) gene or sho gene.
  • the gene is from a species selected from the group consisting of Agrobacte ⁇ um, more preferably Agrobactehum tumefaciens; Lotus, more preferably Lotus japonicus; and Petunia, more preferably Petunia hybrida.
  • the gene includes a nucleotide sequence selected from the group consisting of the sequences shown in SEQ ID NOS: 5, 7 and 9 of US 11/789,526, sequences encoding the polypeptides shown in SEQ ID NOS: 6, 8 and 10 of US 11/789,526, and functionally active fragments and variants thereof.
  • the present invention also provides a method of selecting for transformed plants, said method including introducing into said plants an effective amount of a genetic construct including a promoter, or a functionally active fragment or variant thereof, operatively liked to nucleic acids encoding one or more fructan biosynthetic enzymes, or functionally active fragments or variants thereof and selecting plants with enhanced biomass.
  • a genetic construct including a promoter, or a functionally active fragment or variant thereof, operatively liked to nucleic acids encoding one or more fructan biosynthetic enzymes, or functionally active fragments or variants thereof and selecting plants with enhanced biomass.
  • the promoter is a light regulated promoter.
  • transgenic plant cell, plant, plant seed or other plant part with modified fructan biosynthetic characteristics or enhanced biomass relative to an untransformed control plant in a further aspect of the present invention there is provided a transgenic plant cell, plant, plant seed or other plant part with modified fructan biosynthetic characteristics or enhanced biomass relative to an untransformed control plant.
  • modified fructan biosynthetic characteristics is meant that the transformed plant exhibits increased fructan biosynthesis and/or contains increased levels of soluble carbohydrate relative to an untransformed control plant.
  • the a transgenic plant cell, plant, plant seed or other plant part with enhanced biomass has an increase in biomass of at least approximately 10%, more preferably at least approximately 20%, more preferably at least approximately 30%, more preferably at least approximately 40% relative to an untransformed control plant.
  • biomass may be increased by between approximately 10% and 300%, more preferably between approximately 20% and 200%, more preferably between approximately 30% and 100%, more preferably between approximately 40% and 80% relative to an untransformed control plant.
  • plant height may be increased by between approximately 10% and 300%, more preferably between approximately 20% and 200%, more preferably between approximately 30% and 100%, more preferably between approximately 40% and 80% relative to an untransformed control plant.
  • herbage dry weight may be increased by between approximately 10% and 600%, more preferably between approximately 20% and 400%, more preferably between approximately 30% and 300%, more preferably between approximately 40% and 200% relative to an untransformed control plant.
  • the transgenic plant cell, plant, plant seed or other plant part with modified fructan biosynthetic characteristics has an increase in soluble carbohydrate of least approximately 10%, more preferably at least approximately 20%, more preferably at least approximately 30%, more preferably at least approximately 40% relative to an untransformed control plant.
  • soluble carbohydrates may be increased by between approximately 10% and 300%, more preferably between approximately 20% and 200%, more preferably between approximately 30% and 100%, more preferably between approximately 40% and 80% relative to an untransformed control plant.
  • fructan concentration may be increased between approximately 10% and 600%, more preferably between approximately 20% and 400%, more preferably between approximately 30% and 200%, more preferably between approximately 40% and 150% relative to an untransformed control plant.
  • said plant cell, plant, plant seed or other plant part includes a genetic construct or vector according to the present invention.
  • the transgenic plant cell, plant, plant seed or other plant part is produced by a method according to the present invention.
  • the present invention also provides a transgenic plant, plant seed or other plant part derived from a plant cell of the present invention and including a genetic construct or vector of the present invention.
  • the present invention also provides a transgenic plant, plant seed or other plant part derived from a plant of the present invention and including a genetic construct or vector of the present invention.
  • the transgenic plant cell, plant, plant seed or other plant part is a Lolium species, more preferably Lolium perenne or Lolium arundinaceum.
  • the transgenic plant cell, plant, plant seed or other plant part is a cereal grain, more preferably a Triticum species, more preferably wheat (Triticum aestivum).
  • the present invention enables the production of transgenic perennial ryegrass plants with increased fructans in leaf blades, vigorous growth and/or greater tolerance to abiotic stress, for improved nutrition for grazing animals.
  • the present invention also enables the production of transgenic wheat plants with increased fructans, vigorous growth, and/or tolerance to abiotic stress, for increased mass of usable carbohydrates, eg. for bio-fuel production or animal feed.
  • Plant cell is meant any self-propagating cell bounded by a semi-permeable membrane and containing a plastid. Such a cell also requires a cell wall if further propagation is desired.
  • Plant cell includes, without limitation, algae, cyanobacteria, seeds suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen and microspores.
  • transgenic is meant any cell which includes a DNA sequence which is inserted by artifice into a cell and becomes part of the genome of the organism which develops from that cell.
  • the transgenic organisms are generally transgenic plants and the DNA (transgene) is inserted by artifice into either the nuclear or plastidic genome.
  • a fusion protein comprising two or more enzymes of a biochemical pathway in a plant, or functionally active fragments or variants thereof.
  • the fragment or variant has one or more of the biological properties of the corresponding protein from which the fragment or variant is derived. Additions, deletions, substitutions and derivatizations of one or more of the amino acids are contemplated so long as the modifications do not result in loss of functional activity of the fragment or variant.
  • the fragment or variant has at least approximately 80% identity to the relevant part of the above mentioned sequence to which the fragment or variant corresponds, more preferably at least approximately 90% identity, more preferably at least approximately 95% identity, most preferably at least approximately 98% identity.
  • Such functionally active variants and fragments include, for example, those having conservative amino acid substitutions of one or more residues in the corresponding amino acid sequence.
  • the fragment has a size of at least 10 amino acids, more preferably at least 20 amino acids, more preferably at least 50 amino acids, more preferably at least 100 amino acids, more preferably at least 200 amino acids.
  • the biochemical pathway is in the fructan biosynthetic pathway.
  • the two or more enzymes from said pathway are selected from the group consisting of enzymes of the fructan biosynthetic pathway in plants, for example fructosyltransferases such as sucrose:sucrose 1-fructosyltransferase (1-SST); fructan :fructan 1-fructosyltransferase (1-FFT); sucrose:fructan 6-fructosyltransferase (6- SFT); and fructan: fructan 6G-fructosyl transferase (6G-FFT); and fructoexohydrolases such as 1-fructoexohydrolase (1-FEH) and 6-fructoexohydrolase (6-FEH).
  • fructosyltransferases such as sucrose:sucrose 1-fructosyltransferase (1-SST); fructan :fructan 1-fructosyltransferase (1-FFT); sucrose:fructan 6-f
  • the fusion protein is a FT fusion protein of 1-SST and 6G-FFT, or functionally active fragments or variants thereof.
  • the two or more enzymes from said pathway correspond to enzymes from a ryegrass or fescue species, including Italian or annual ryegrass, perennial ryegrass, tall fescue, meadow fescue and red fescue. Even more preferably, the two or more enzymes from said pathway correspond to enzymes from a Lolium species such as Lolium perenne or Lolium arundinaceum.
  • Suitable fructan biosynthetic enzymes are described in PCT/AU01 /00705 and PCT/AU01/01275, the entire disclosures of which are incorporated herein by reference.
  • the 1-SST includes an amino acid sequence shown in SEQ ID NO: 12 of PCT/AU01 /00705, or a functionally active fragment or variant thereof.
  • the 6G-FFT includes an amino acid sequence shown in SEQ ID NO: 111 of PCT/AU01/01275 or Figure 8 hereto, or functionally active fragments or variants thereof.
  • the 1-SST 6G-FFT FT fusion protein includes an amino acid sequence shown in Figure 13 or 15 hereto, or functionally active fragments or variants thereof.
  • FIG. 1 Model for targeted expression of fructan biosynthesis genes in photosynthetic cells in leaf blades. Expression of fructosyl transferase (FT) genes is driven by photosynthetic promoters. Fructan biosynthesis then occurs in sucrose producing, photosynthetic cells. Pyramiding with modification of cytokinin biosynthesis to delay leaf senescence, thus extending life of photosynthetic cells that are engineered to synthesise fructans and leading to increased biomass production.
  • FT fructosyl transferase
  • FIG. 1 The expression of the RuBisCO Small subunit gene in perennial ryegrass is light regulated as shown by quantitative real-time RT-PCR. Tissue sampling occurred every four hours. Boxes represent periods of daylight.
  • FIG. 3 In silico expression patterns of the Ribulose-1 ,5-bisphosphate carboxylase/oxygenase Small subunit (LpRbcS) and Chlorophyll a/b Binding Protein (LpCAB) in perennial ryegrass shows that it is most abundant in vegetative tissues.
  • LpRbcS contig LPCL9_C359
  • LpRbcS contig LPCL1112_C12
  • 19 ESTs In silico expression patterns of the Ribulose-1 ,5-bisphosphate carboxylase/oxygenase Small subunit (LpRbcS) and Chlorophyll a/b Binding Protein (LpCAB) in perennial ryegrass shows that it is most abundant in vegetative tissues.
  • LpRbcS contig LPCL9_C359
  • LpRbcS contig LPCL1112_C12
  • Figure 5 Nucleotide sequences of LpRbcS promoter (SEQ ID No: 2).
  • Figure 6 Schematic representation of the fructan biosynthetic pathway in some grasses.
  • FIG. 1 Nucleotide sequence of Lp6G-FFT open reading frame (SEQ ID No: 3).
  • FIG. 1 Deduced amino acid sequence of Lp6G-FFT (SEQ ID No: 4).
  • Figure 11 Diagrammatic representation of the strategy used to generate the translational FT fusion of the LpI-SST and Lp6G-FFT fructosyl transferase genes (Lp1- SST_Lp6G-FFT).
  • Figure 15 Deduced amino acid sequence of Lp1-SST_Lp6G-FFT FT fusion 3 (SEQ ID No: 10).
  • Figure 16 Diagrammatic representation of the strategy to be used to generate the different translational FT fusions of the LpI-SST, Lp6G-FFT and LpI-FFT fructosyl transferase genes.
  • FIG. 1 (A) and (B) Hypothetical model of the interaction of FT proteins to form a transmebrane protein.
  • C Representation of the key protein domains in Lp1-SST-6G- FFT proteins. Box1 : (N/S)DPNG; Box2: RDP and Box3: EC represent the highly conserved domains involved in substrate (sucrose) binding and hydrolysis.
  • Crosses (X) represent the highly conserved amino acid sequences (domains) found among the FT, invertase and FEH sequences from Lolium species.
  • FIG. 19 Functional analysis of fructan:fructan 6G-fructosyltransferase (Lp6G-FFT).
  • HPAEC Water soluble carbohydrate
  • FIG. 20 Base destination vector, pPZP200-ubi:bar-nos R4 R3, used in Multisite Gateway recombinational cloning.
  • FIG. 21 Outline of the procedure for the in planta transient expression system. Agrobacterium cultures are prepared that harbour the expression constructs. These are injected into tobacco leaves. After three days post filtration expression of the proteins are tested. Upper right panel shows GUS activity, lower right panel shows example of water soluble carbohydrate separation by HPAEC.
  • HPAEC High performance anion exchange chromatography
  • Destination vectors of wheat RuBisCO promoter driving expression of (A) LpI-SST, (B) Lp6G-FFT, (C) Lp1SST_Lp6GFFT FT fusion 1 , (D) Lp1SST_Lp6GFFT FT fusion 3, and (E) the GUS marker gene.
  • FIG. 24 Sequence of TaRbcS::l_p1-SST::TaRbcS expression cassette (SEQ ID No: 34).
  • the regulatory sequences, TaRbcS promoter and terminator are indicated in italics and underlined italics, respectively.
  • the ORF sequence is indicated in regular font and the start (ATG) and stop (TAG) codons are shaded.
  • FIG. 25 Sequence of TaRbcS::Lp6GFFT::TaRbcS expression cassette (SEQ ID No: 35).
  • the regulatory sequences, TaRbcS promoter and terminator are indicated in italics and underlined italics, respectively.
  • the ORF sequence is indicated in regular font and the start (ATG) and stop (TAG) codons are shaded.
  • FIG. 26 Sequence of TaRbcS::Lp1-SST_Lp6G-FFT::TaRbcS FT fusion 1 expression cassette (SEQ ID No: 36).
  • the regulatory sequences, TaRbcS promoter and terminator are indicated in italics and underlined italics, respectively.
  • the ORF sequence is indicated in regular font and the start (ATG) and stop (TAG) codons are shaded.
  • FIG. 27 Sequence of TaRbcS::Lp1-SST_Lp6G-FFT::TaRbcS FT fusion 3 expression cassette (SEQ ID No: 37).
  • the regulatory sequences, TaRbcS promoter and terminator are indicated in italics and underlined italics, respectively.
  • the ORF sequence is indicated in regular font and the start (ATG) and stop (TAG) codons are shaded.
  • Figure 28 Vector pBlueScript SK harbouring the LpFT4 3' terminator sequence, pBS- LpFT4.
  • FIG. 29 (A) The plasmid pBS-Lp1-SST::FT4 and (B) the plasmid pBS-LpRbcS::Lp1- SST::LpFT4.
  • Figure 30 (A) The plasmid pBS-LpCAB::LpFT4 and (B) the plasmid pBS-LpCAB::Lp6G- FFT::LpFT4.
  • FIG. 31 Sequence of LpRbcS::Lp1-SST::LpFT4 expression cassette (SEQ ID No: 38).
  • the regulatory sequences, LpRbcS promoter and LpFT4 terminator are indicated in italics and underlined italics, respectively.
  • the ORF sequence is indicated in regular font and the start (ATG) and stop (TAG) codons are shaded.
  • FIG 32 Sequence of LpCAB::Lp6G-FFT::LpFT4 expression cassette (SEQ ID No: 39).
  • the regulatory sequences, LpRbcS promoter and LpFT4 terminator are indicated in italics and underlined italics, respectively.
  • the ORF sequence is indicated in regular font and the start (ATG) and stop (TAG) codons are shaded.
  • Figure 33 The plasmid PCR Blunt -Lp1-SST_Lp6G-FFT FT fusion.
  • Figure 34 Destination vectors containing the ryegrass RuBisCO (LpRbcs) promoter driving FT fusions 1 and 3.
  • LpRbcs ryegrass RuBisCO
  • FIG. 35 Sequence of LpRbcS::Lp1-SST_Lp6G-FFT::LpFT4 FT fusion 1 expression cassette (SEQ ID No: 40).
  • the regulatory sequences, LpRbcS promoter and LpFT4 terminator are indicated in italics and underlined italics, respectively.
  • the ORF sequence is indicated in regular font and the start (ATG) and stop (TAG) codons are shaded.
  • FIG 36 Sequence of LpRbcS::Lp1-SST_Lp6G-FFT::LpFT4 FT fusion 3 expression cassette (SEQ ID No: 41 ).
  • the regulatory sequences, LpRbcS promoter and LpFT4 terminator are indicated in italics and underlined italics, respectively.
  • the ORF sequence is indicated in regular font and the start (ATG) and stop (TAG) codons are shaded.
  • the amino acid sequence is indicated in bold (SEQ ID No: 42).
  • FIG. 37 Destination vector containing the Arabidopsis RuBisCO (AtRbcS) promoter driving FT fusion 3, pPZP200_AtRbcS::Lp1-SST_6G-FFT::nos FT fusion 3.
  • Figure 38 Sequence of the AtRbcS::Lp1-SST-6G-FFT::nos FT fusion 3 expression construct (SEQ ID No: 43).
  • Figure 39 Details of the base vector pBlueScript SK(-) from Promega, with the positions of the restriction endonuclease sites for cloning indicated.
  • Figure 40 Vector backbone used for construction of p-Ubi::Lp1-SST::35S and p- Ubi::Lp6G-FFT::35S (Ye et a/., 2001 ).
  • FIG 41 Representative sequence of a constitutive (Ubi) promoter combined with a FT fusion protein and a terminator sequence (SEQ ID No: 44).
  • the regulatory sequences, Ubi promoter and LpFT4 terminator are indicated in italics and underlined italics, respectively.
  • the ORF sequence is indicated in regular font and the start (ATG) and stop (TAG) codons are shaded.
  • Figure 42 Representative sequence of a constitutive ((CAMV)35S 2 ) promoter combined with a FT fusion protein and a terminator sequence (SEQ ID No: 45).
  • the regulatory sequences, (CAMV)35S 2 promoter and LpFT4 terminator are indicated in italics and underlined italics, respectively.
  • the ORF sequence is indicated in regular font and the start (ATG) and stop (TAG) codons are shaded.
  • FIG 43 Representative sequence of a constitutive (RUBQ2) promoter combined with a FT fusion protein and a terminator sequence (SEQ ID No: 46).
  • the regulatory sequences, RUBQ2i promoter and LpFT4 terminator are indicated in italics and underlined italics, respectively.
  • the ORF sequence is indicated in regular font and the start (ATG) and stop (TAG) codons are shaded.
  • Figure 44 Representative sequence of a constitutive (OsActi ) promoter combined with a FT fusion protein and a terminator sequence (SEQ ID No: 47).
  • the regulatory sequences, OsActi promoter and LpFT4 terminator are indicated in italics and underlined italics, respectively.
  • the ORF sequence is indicated in regular font and the start (ATG) and stop (TAG) codons are shaded.
  • FIG 45 Representative sequence of a tissue specific (tuber) promoter (Cathlnh) combined with a FT fusion protein and a terminator sequence (SEQ ID No: 48).
  • the regulatory sequences, Cathlnh promoter and LpFT4 terminator are indicated in italics and underlined italics, respectively.
  • the ORF sequence is indicated in regular font and the start (ATG) and stop (TAG) codons are shaded.
  • Figure 46 Representative sequence of a stress regulated (Atrd29a) promoter combined with a FT fusion protein and a terminator sequence (SEQ ID No: 49).
  • the regulatory sequences, Atrd29a promoter and LpFT4 terminator are indicated in italics and underlined italics, respectively.
  • the ORF sequence is indicated in regular font and the start (ATG) and stop (TAG) codons are shaded.
  • Figure 47 Representative sequence of a sucrose regulated (16R) promoter combined with a FT fusion protein and a terminator sequence (SEQ ID No: 50).
  • the regulatory sequences, 16R promoter and LpFT4 terminator are indicated in italics and underlined italics, respectively.
  • the ORF sequence is indicated in regular font and the start (ATG) and stop (TAG) codons are shaded.
  • FIG 48 Plant regeneration phenotypes of transgenic perennial ryegrass after co- transformation with the TaRbcS promoter light-regulated gene constructs (Table 1) and the pAcH1 vector, with selection on hygromycin.
  • the plants that contain either of the TaRbcS::Lp1-SST_Lp6G-FFT::TaRbcS FT fusion constructs show growth advantage under in vitro culture conditions thus allowing for their early identification and screening (far right column).
  • FIG. 49 Plant regeneration phenotypes of transgenic perennial ryegrass after co- transformation with the LpRbcS promoter light-regulated gene constructs with selection on hygromycin.
  • the plants contain either the LpRbcS::Lp1-SST::LpFT4 or the LpRbcS::Lp1-SST_Lp6G-FFT::LPFT4 FT fusion 1 / 3 constructs.
  • the plants that contain the FT fusion constructs show growth advantage under in vitro culture conditions
  • Figure 50 Mature plant phenotypes under glasshouse conditions. Representative samples of transgenic perennial ryegrass plants at the vegetative stage. The TaRbcS::Lp1-SST_Lp6G-FFT::LpFT4 FT fusion transgenic perennial ryegrass plants show enhanced growth performance with larger leaves, enhanced tillers, increased root growth compared to control, non-transgenic perennial ryegrass plants. The plants were trimmed equally three weeks earlier. Close up micrographs of the leaf blades indicate and increase leaf diameter in FT fusion transgenics. Figure 51. Representative samples of transgenic perennial ryegrass mature plant phenotypes (4 weeks) under field conditions. The FT fusion transgenic perennial ryegrass plants show enhanced growth performance with larger leaves, enhanced tillers, increased root growth compared to control LpI-SST transgenic perennial ryegrass plants.
  • Figure 52 Representative examples of phenotypic biomass scores (1- least biomass to 5- most biomass) of transgenic perennial ryegrass plants expressing FT fusion transgenes under field conditions.
  • Figure 53 Leaf phenotypes of transgenic perennial ryegrass. Representative samples of hand sections of leaf blades at vegetative stage. Left shows comparison of whole leaf sections, right magnified areas of leaf sections. Ad-Adaxial, Ab-abaxial.
  • HPAEC Biochemical analysis of fructan level and composition present in stable transgenic TaRbcS::Lp1-SST_Lp6G-FFT::TaRbcS FT fusion 3, TaRbcS::Lp1- SST::TaRbcS, TaRbcS::Lp6G-FFT::TaRbcS perennial ryegrass plants and control perennial ryegrass plants harbouring only the selectable marker (hph gene).
  • HPAEC Biochemical analysis of total fructans present in whole tillers of (A) TaRbcS::Lp1-SST_Lp6G-FFT::TaRbcS FT fusion 1 , (B) TaRbcS::Lp1-SST_Lp6G- FFT::TaRbcS FT fusion 3, (C) TaRbcS::Lp1-SST::TaRbcS, and (D) TaRbcS::6G- FFT::TaRbcS transgenic perennial ryegrass plants compared to control perennial ryegrass plants (lanes 6' and 1'), harbouring only the selectable marker (hph gene).
  • HPAEC Biochemical analysis
  • HPAEC Biochemical analysis of 1-kestose present in whole tillers of (A) TaRbcS::Lp1-SST_Lp6G-FFT::TaRbcS FT fusion 1 , (B) TaRbcS::Lp1-SST_Lp6G- FFT::TaRbcS FT fusion 3, (C) TaRbcS::Lp1-SST::TaRbcS, and (D) TaRbcS::6G- FFT::TaRbcS transgenic perennial ryegrass plants compared to control perennial ryegrass plants (lanes 6' and 1'), harbouring only the selectable marker (hph gene).
  • HPAEC Biochemical analysis
  • FIG. 57 Biochemical analysis (HPAEC) of sucrose present in whole tillers of (A) TaRbcS::Lp1-SST_Lp6G-FFT::TaRbcS FT fusion 1 , (B) TaRbcS::Lp1-SST_Lp6G- FFT::TaRbcS FT fusion 3, (C) TaRbcS::Lp1-SST::TaRbcS, and (D) TaRbcS::6G- FFT::TaRbcS transgenic perennial ryegrass plants compared to a control perennial ryegrass plants (lanes 6' and 1'), harbouring only the selectable marker (hph gene).
  • HPAEC Biochemical analysis
  • Figure 58 Fructan levels in whole tillers and leaf blades in wild-type (control) and FT fusion and LpRbcS ::Lp1-SST transgenic perennial ryegrass plants grown under field conditions and harvested in December 2009.
  • Figure 59 Fructan composition in leaf blades of wild-type and LpRbcS::Lp1-SST transgenic perennial ryegrass plants grown under field conditions. Box 1 represents low DP fructan (DP up to 10-15). Box 2 represents high DP fructan (DP higher than 10-15).
  • FIG. 60 Transgene expression in whole tillers of LpRbcS FT fusion and LpRbcS::Lp1-SST transgenic perennial ryegrass plants grown under field conditions. Samples were collected in November (white bars) and December (black bars) 2009. Samples were normalised against endogenous histone expression and are presented as number of transcript copies per 35ng of RNA.
  • FIG 61 Phenotypic analysis of the transgenic perennial ryegrass after 7 weeks (A-C) and 12 weeks (D-E) propagation in potting mix from a single tiller.
  • Figure 62 Quantitative phenotypic analysis of the transgenic TarbcS::Lp1-SST_Lp6G- FFT::Tarbcs FT fusion 1 and TarbcS::Lp1-SST_Lp6G-FFT::Tarbcs FT fusion 3 plants after 7 weeks (white bars) and 12 weeks (black bars) growth. Measurements were conducted for plant height (A), leaf width (B) and tiller number (C) compared to the average of 8 control plants expressing only the hph gene.
  • FIG 63 Transgenic perennial ryegrass plants expressing LXR ® technology alone (AtMYB32::IPT), LpRbcS::Lp1-SST_Lp6G-FFT::LpFT4 FT fusion 3 alone, as well as LXR ® and LpRbcS::Lp1-SST_Lp6G-FFT::LpFT4 FT fusion 3 together under glasshouse conditions.
  • Figure 64 Herbage dry weight analysis of GOI-ve control (average of five lines) and independent FT fusion alone or the FT fusion plus LXR ® transgenic perennial ryegrass plants, grown under glasshouse conditions and collected 6 weeks post-trim,.
  • Figure 65 Fructan levels in leaf blades of GOI-ve control (average of five lines) and independent FT fusion alone or FT fusion plus LXR ® transgenic perennial ryegrass plants, grown under glasshouse conditions.
  • FIG. 66 Transgenic tall fescue plants expressing LpRbcS::l_p1-SST_Lp6G- FFT::LpFT4 FT fusion 3 under glasshouse conditions.
  • FIG. 67 Herbage dry weight analysis of glass house grown GOI-ve control (average of five lines) and independent FT fusion alone or FT fusion plus LXR ® transgenic tall fescue plants.
  • Figure 68 Tiller number of glass house grown GOI-ve control (average of five lines) and independent FT fusion alone or FT fusion plus LXR ® transgenic tall fescue plants.
  • Figure 69 Fructan accumulation in leaf blades of glass house grown GOI-ve control (average of five lines) and independent transgenic tall fescue lines expressing the FT fusion.
  • FIG. 70 Plant regeneration phenotypes of transgenic wheat plants after transformation with the light-regulated gene constructs.
  • the transgenic wheat plants growing in vitro that contain the Lp1-SST_Lp6G-FFT FT fusion construct show growth advantage under in vitro culture conditions thus allowing for their early identification and screening.
  • the superior growth phenotype of the transgenic wheat FT fusion lines was observed during the early stages of in vitro plant regeneration conducted on tissue culture plates.
  • Figure 74 Phenotypic analysis of transgenic wheat plants expressing LXR ® technology alone (AtMYB3::IPT::35S), TaRbcS::Lp1-SST_l_p6G-FFT::TaRbcS FT fusion 3 alone, as well as LXR ® and TaRbcS::Lp1-SST_Lp6G-FFT::TaRbcS FT fusion 3 together under glasshouse conditions.
  • Figure 75 Fructan accumulation and tiller number in transgenic wheat plants containing either FT fusion constructs alone or LXR ® plus FT fusion constructs, as compared to transformed gene of interest minus (GOI-) controls.
  • FIG 76 Fructan accumulation in T 1 GOI-ve control, FT fusion alone and LXR ® plus FT fusion transgenic wheat plants nine weeks after sowing.
  • the fructan level in the control represents the data average obtained from six GOI-ve plants.
  • FIG. 77 Phenotype of transgenic white clover plants expressing LXR ® , AtRbcS::Lp1- SST-6G_FF::nos FT fusion or LXR ® plus AtRbcS::Lp1-SST-6G_FF::nos FT fusion constructs as compared to transformed GOI minus controls.
  • FIG. 78 Transgene expression levels of the FT fusion transgene driven by the AtRbcS promoter in white clover plants. Controls were wild type plants. Samples were normalised against endogenous histone expression and are presented as number of transcript copies per 35ng of RNA.
  • Figure 79 Fructan accumulation in wild-type control, AtRbcS FT fusion and AtRbcS FT fusion plus LXR ® transgenic white clover lines.
  • Figure 80 Phenotype of transgenic Arabidopsis plants expressing LXR ® , AtRbcS::Lp1- SST-6G_FF::nos FT fusion or LXR ® plus AtRbcS::Lp1-SST-6G_FF::nos FT fusion constructs as compared to transformed GOI minus controls.
  • FIG. 81 Transgene expression levels of the FT fusion transgene driven by the AtRbcS promoter in Arabidopsis plants. Controls were wild type plants. Samples were normalised against endogenous histone expression and are presented as number of transcript copies per 35ng of RNA.
  • FIG. 82 Transgenic T 2 FT fusion Arabidopsis plants grown in soil.
  • Figure 83 Leaves from A. white clover, B. canola and C. wheat plants displaying delayed leaf senescence (leaves from LXR ® transgenic plants, lower images) as compared to negative control plants (leaves from control plants, upper images) 7 - 20 days following detachment of leaves from plants.
  • Figure 84 Positive selection of perennial ryegrass transgenic plants by screening of in vitro growth phenotype on plates without antibiotic selection.
  • A-C CaIIi in dark for 8 weeks after transformation;
  • D-F 1 week after transfer to light.
  • Figure 85 Embryogenic perennial ryegrass calli bombarded with gold particles alone (control) and gold particles covered with TaRbcS FT fusion vector prior to, and four weeks post, transfer to light.
  • Figure 86 Embryogenic perennial ryegrass calli bombarded with gold particles alone (control) and gold particles covered with TaRbcS FT fusion 1 alone, TaRbcS FT fusion
  • Ribulose-1 ,5-bisphosphate carboxylase/oxygenase Small subunit (RbcS) is a well- characterised light-regulated gene in higher plants.
  • the bread wheat Triticum aestivum
  • TaRbcS regulatory sequences promoter and terminator
  • a 695 bp promoter fragment from sequence previously published containing the TATA signal from the TaRbcS gene was PCR-amplified.
  • a 1700 bp fragment of the Arabidopsis thaliana Ribulose-1 ,5-bisphosphate carboxylase/oxygenase Small subunit (AtRbcS) promoter sequence has previously been cloned. Primers will be designed to amplify a smaller fragment containing the TATA signal from the AtRbcS promoter for use in expression vectors.
  • RbcS and Chlorophyll a/b Binding Protein are well characterised light-regulated genes in higher plants.
  • the abundance of LpRbcS mRNA transcripts in perennial ryegrass by quantitative real time PCR is illustrated in Figure 2.
  • LpRbcS and LpCAB genes were chosen for promoter discovery and isolation in perennial ryegrass.
  • Publicly available cDNA sequences (LpRbcS, EC778430 and LpCAB, EC778438) were used as query sequences in a BLAST search of the perennial ryegrass EST database in our in-house database.
  • both genes are members of multigene families, several contigs (each contig represents an individual gene) were identified in our perennial ryegrass EST collection.
  • Nine contigs were identified to be homologous to the published LpRbcS cDNA sequence and thirteen contigs were found to be homologous to the LpCAB cDNA sequence.
  • LpRbcS LpCL9_C359
  • LpCAB LpCL1112_C12
  • the I-Core box and SORLIPs cis- regulatory sequences were present in the CAB promoter. SORLIPs were found to be over-represented in light-induced promoters in Arabidopsis (Hudson, et al., 2003).
  • Table 1 The position of the c/s-regulatory sequences identified by the PLACE database. Common cis-acting regulatory sequences are listed (Schaffner, et al., 1991 ; Terzaghi, et al., 1995; Martinez-Hernandez, et al., 2002; Hudson, et al., 2003). Positions noted are the first nucleotide in the sequence relative to the ATG. (n.p. - not present).
  • Lolium perenne cDNA clones encoding sequences for LpI-SST and Lp6G-FFT have previously been isolated from a perennial ryegrass cDNA library (Chalmers, et al., 2003; Chalmers, et al., 2005).
  • the complete gene sequences of the isolated perennial ryegrass fructosyltransferase homologues are available, and nucleotide and protein sequences for LpI-SST are disclosed in International patent application PCT AU01/00705 (SEQ ID NOS 11 and 12).
  • Partial sequence for Lp6G-FFT is disclosed in International patent PCT/AU/01/01275 SEQ IDs 109 and 110, for nucleotide and amino acid sequences respectively.
  • LpI-FFT Lp6-SFT
  • Lp6-SST Lp6-SST
  • the cDNA sequence for LpI-FFT has been isolated from perennial ryegrass ( Figure 9) and the amino acid sequence is represented in Figure 10.
  • primers based on this sequence could be used to amplify the full-length cDNA by PCR for cloning and use in the present invention as described below.
  • a genetic FT fusion was created between the open reading frames for LpI-SST and Lp6G-FFT, following the procedure depicted in Figure 11.
  • the LpI-SST gene was PCR-amplified with a GATEWAY recombination site incorporated in the forward primer.
  • a sequence that codes for three glycine residues followed by a Hind III site was incorporated into the reverse primer, with the stop codon removed.
  • the Lp6G-FFT gene was PCR-amplified with a Hind III site followed by sequence that codes for three glycine residues and the gene specific sequence without the ATG.
  • the reverse primer for the Lp6G-FFT gene was flanked by a second GATEWAY recombination site.
  • the primer sequences are provided in Table 2.
  • the purified fragments were digested with Hind III and the ligated product was cloned into the Invitrogen GATEWAY pDONR221 Entry vector.
  • FT fusion 1 one sequence was confirmed to be the predicted product, with eight amino acids in the linker joining the two genes ( Figures 12 and 13).
  • another sequence FT fusion 3 contained two consecutive Hind III sites, which would result in the addition of another two amino acids, giving a total of ten amino acids between the two FT genes upon translation ( Figures 14 and 15).
  • Table 2 Primer sequences used to amplify the PCR fragments used to generate the translational FT fusion of the LpI-SST and Lp6G-FFT fructosyltransferase genes (FT
  • SJlajjej sequences are gene-specific, bold and underlined (Hind III RE site) sequences are nucleotides introduced to generate the linker region, and italic nucleotides represent the recombination-specific sequences.
  • Lp1-SST_Lp6G-FFT the FT proteins physically associate with each other to form a FT fusion protein which contains three transmembrane domains as designated by SOSUI, a classification and secondary structure prediction of membrane proteins database (Table 4, Figures 17 and 18).
  • Plant FTs have a high degree of amino acid homology to the vacuolar, acid invertases (b- fructosidases) which are the members of the glycoside hydrolase family 32. (GH32) and share three highly conserved regions characterised by the motifs (N/S)DPNG (also called b-fructosidase motif), RDP, and EC (Altenbach et al., 2005) ( Figures 17, 18 and 36).
  • N/S)DPNG also called b-fructosidase motif
  • RDP b-fructosidase motif
  • EC Altenbach et al., 2005
  • Another common feature of plant FTs and vacuolar invertases is that they usually are composed of a large and a small subunit due to posttranslational processing. The large subunit, which harbours all three conserved motifs mentioned above, determines the catalytic specificity (Altenbach et al., 2004).
  • LpI-FFT LpI-FFT
  • Lp6-SFT Lp6-SST
  • Lp6-SST Lp6G-FFT
  • Lp1-SST (Lp1-FFT/ Lp6-SFT/Lp-SST)
  • FT fusion could also be created using a similar methodology ( Figure 16B). It is proposed that the triplicate fusion would be constructed to incorporate the genes Lp1- SST, Lp6G-FFT and LpI-FFT, Lp6-SFT or Lp6-SST. By altering the primer sequences used to join the two FT genes together it is possible to change the linker size and potentially add up to approximately 30 amino acids. FT proteins could physically associate with each other to form a metabolic channel, therefore the distance separating the FT genes within the translational fusion may affect protein function. FT fusion proteins preferably contain the sequences which represent the domains which are highly conserved among the FT, INV and FEH proteins from Lolium perenne plants indicated in Figures 17, 18 and 36.
  • the cDNA sequence encoding the LpI-SST mature protein has been previously expressed in Pichia pastoris for functional characterisation (Chalmers, et al., 2003) and the conversion of sucrose to 1-kestose by expression of this protein was demonstrated.
  • the recombinant Lp6G-FFT enzyme was produced from single colonies of transformed P. pastoris inoculated into a pre-culture medium and induced by the addition of methanol for a 45 hr duration. The supernatant was concentrated and samples were incubated with 100 mM sucrose overnight. The carbohydrates produced were analysed by HPAEC according to Chalmers et al., 2003, using fructan extracts from onion as a control ( Figure 19).
  • This methodology relies on the generation of individual Entry plasmids containing, either the promoter, gene of interest (GOI), or terminator sequences flanked by recombination sites.
  • the recombination sites facilitate the directional triple insertion of each of the Entry plasmids into a Gateway-enabled destination vector, by recombination.
  • the final vector is then sequenced and used directly for plant co-transformation with a plasmid, or expression cassette, for expression of a plant selectable marker.
  • the FT fusion 1 and 3 ORFs were cloned under the control of the enhanced cauliflower mosaic virus (CAMV)35S 2 promoter (Kay, et al., 1987), using the Multisite GatewayTM Technology recombination system (see www.lnvitroqen.com for product manual) into Agrobacterium binary vector (Figure 21) (Hajdukiewicz, et al., 1994).
  • Agrobacterium binary vector Figure 21
  • Gateway Entry vectors were constructed for the (CAMV)35S 2 promoter, the TaRbcS terminator sequence, as well as FT fusion 1 and 3 ORFs.
  • the cloned fragments were sequence-verified and used for three-way recombination cloning with the cloned GOI cDNA sequences into the pPZP200-ubi:bar-nos R4 R3 destination vector.
  • constructs also included the Lp6G-FFT and LpI-SST single ORF driven by the (CAMV)35S 2 promoter as controls.
  • the LpI-FFT (or Lp6-SFT, Lp6- SSJ) single ORF is also cloned in the same manner.
  • the GUS ORF was used for confirmation of expression. The following constructs were made.
  • pPZP200-AtrbcS Lp6G-FFT::35S
  • pPZP200-AtrbcS :(Lp1-FFT/ Lp6-SFT/Lp-SST)::35S
  • transient expression of the constructs containing chimeric Lp1- SST, Lp6G-FFT and FT fusion protein genes driven by the (CaMV)35S promoter was conducted in tobacco plants, as they do not naturally produce fructans.
  • the method involved Agro-infiltration of the individual constructs into N. benthamiana leaves (Kapila, et al., 1997; Wydro, et al., 2006) followed by biochemical analysis by anion exchange chromatography.
  • a diagram of the transient expression procedure is illustrated in Figure 21.
  • LXR ® technology is based on vectors containing one cytokinin biosynthesis gene encoding isopentenyl transferase (IPT) for delayed leaf senescence under the control of the AtMYB32 gene promoter.
  • IPT isopentenyl transferase
  • the LXR ® vector for biolistic transformation was constructed utilising GatewayTM Multisite technology. Details of the binary vector pBS- ubi::bar::nos_AtMYB32_IPT_35S are described in International patent application PCT/AU01/01092.
  • a 695 kb promoter fragment from sequence previously published containing the TATA signal from the TaRbcS gene was PCR-amplified with GatewayTM (Invitrogen) recombination sites at the primer flanks. The fragment was cloned into the Invitrogen pDONRP4-P1 R Entry vector using GatewayTM recombination technology.
  • the 696 bp TaRbcS gene termination signal sequence (Sasanuma, 2001 ) was also PCR-amplified using primers with recombination sites and cloned into the Invitrogen pDONRP2-P3R Entry vector.
  • the cloned fragments were sequence-verified and used for three-way recombination cloning with the cloned GOI cDNA sequences into the pDEST-R4R3 destination vector: pDESTR1-R2R-Lp1-SST, pDESTR1-R2- Lp6G-FFT, and pDESTP1-P2R-Lp1-SST_Lp6G-FFT gene FT fusion expression vectors.
  • the following constructs for photosynthetic-regulation of expression of fructosyltransferases by the TaRbcS promoter to be used are outlined below and graphically depicted in Figure 23.
  • Constructs containing a ryegrass photosynthetic promoter were produced by conventional cloning methods.
  • the 693 base pair (bp) fructosyltransferase 4 gene (LpFT4) termination sequence (Lidgett, et al., 2002) was amplified by PCR using gene specific primers containing the restriction endonuclease (RE) site EcoR I at the 5' end of the forward PCR primer.
  • EcoR V and Xma I endonuclease restriction sites were incorporated at the 3' end of the reverse PCR primer.
  • the PCR product was cloned into the EcoR I and Xma I restriction endonuclease sites of the pBlueScript SK(-) vector DNA (Short, et al., 1988), resulting in the plasmid pBS-LpFT4 ( Figure 28).
  • the LpRbcS promoter was amplified using gene specific primers containing the endonuclease restriction sites Xho I and EcoR V at the 5' end of the forward primer and an EcoR I restriction site was incorporated in the 3' end of the reverse primer.
  • the 610 bp PCR product was cloned into the pBS-LpFT4 plasmid digested with EcoR I and Xho I, resulting in the plasmid pBS-LpRbcS::LpFT4 ( Figure 29A).
  • the LpI-SST coding region was amplified from a cDNA template (Chalmers et al., 2003) with EcoR I sites flanking both forward and reverse PCR primers, and cloned into the EcoR I site of pBS- LpRbcS::LpFT4 vector, generating the final construct pBS-LpRbcS::Lp1-SST::LpFT4 ( Figure 29B). Sequence of the expression cassette, indicating promoter and terminator, as well as ORF is provided in Figure 31.
  • the expression cassette containing the L. perenne sequences may be excised from the plasmid vector DNA using the EcoR V restriction endonuclease. Following agarose gel electrophoresis, the resulting DNA fragment is purified from the agarose matrix prior to being used for plant transformation to produce DNA with out vector backbone sequences.
  • the plasmid pBS-LpFT4 ( Figure 28) containing the 693 base pair LpFT4 terminator sequence was prepared as outlined above.
  • the LpCAB promoter fragment of 870 base pairs was amplified with a forward PCR primer containing the Xho I and EcoR V sites and a reverse PCR primer containing the EcoR I restriction site. This fragment was cloned in the Xho I and EcoR I sites of pBS-LpFT4, generating the pBS-LpCAB::LpFT4 plasmid ( Figure 30A).
  • the Lp6G-FFT coding region was amplified from a cDNA template (Chalmers, et al., 2005) with EcoR I sites flanking both forward and reverse PCR primers, and cloned into the EcoR I site of pBS-LpCAB::LpFT4 vector, generating the final construct pBS-LpCAB::Lp6G-FFT::LpFT4 ( Figure 30B). Sequence of the expression cassette, indicating promoter and terminator, as well as ORF is provided in Figure 32.
  • the DNA expression cassette may be excised from the plasmid vector DNA using the EcoR V restriction endonuclease.
  • the resulting DNA fragment is purified from the agarose matrix prior to being used for plant transformation to produce DNA without vector backbone sequences.
  • the translational FT fusion between the genes LpI-SST and Lp6G-FFT was amplified from pDEST-TaRbcS::Lp1-SST_l_p6G- FFT::TaRbcS FT fusion 1 and 3 plasmids ( Figure 23C-D) using primers specific for a region just outside the ORF, with EcoR I restriction sites engineered in the 3' region on both the forward and reverse primers.
  • the 3920 bp ORF was PCR amplified and cloned into pCR®-Blunt vector (Invitrogen) to produce PCR Blunt-Lp1-SST-Lp6G-FFT FT fusion ( Figure 33). It was then excised using EcoR I restriction enzymes to remove the vector-specific sequences, and cloned into the pBS-LpRbcS::LpFT4 plasmid ( Figure 29A) at the EcoR I restriction site, generating the pBS-LpRbcS::Lp1-SST_Lp6G- FFT::LpFT4 ( Figure 34).
  • Sequence of the expression cassette of FT fusion 1 and 3, indicating relevant domains (FT fusion 3), is provided in Figure 35 and 36, respectively.
  • the DNA expression cassette may be excised from the plasmid vector DNA using the EcoR V restriction endonuclease. Following agarose gel electrophoresis, the resulting DNA fragment is purified from the agarose matrix prior to being used for plant transformation to produce DNA without vector backbone sequences.
  • a construct containing an Arabidopsis photosynthetic promoter driving expression of FT fusion 3 was produced using Multisite Gateway cloning methods -pPZP200_AtRbcS:: Lp1-SST_6G-FFT::35S FT fusion 3 ( Figure 37).
  • the sequence of the AtRbcS::Lp1- SST_6G-FFT::nos FT fusion 3 expression cassette is provided in Figure 38.
  • Ubi ubiquitin
  • a cDNA copy of the candidate genes LpI-SST and Lp6G-FFT was amplified by PCR as described by Chalmers et. al. (2003) and cloned into the pBlueScript SK(-) vector ( Figure 39).
  • the cDNA fragments were excised using the restriction endonucleases Xho I and Xba I, and then blunt-end cloned into the BamH I site of p-Ubi-35S vector ( Figure 40).
  • the p-Ubi-35S binary plant expression vector has been previously described in other transformation experiments of L. multiflorum (Ye et al., 2001 ).
  • Constructs containing the cauliflower mosaic virus 35S promoter The constructs for regulation of expression of fructosyltransferases under the control of the enhanced cauliflower mosaic virus (CAMV)35S 2 promoter (Kay, et al., 1987), are described in a previous section and are outlined below.
  • Promoters with tissue-specificity are desirable to drive expression of transgenes in crops to target accumulation in particular tissues/ organs and to avoid unwanted expression elsewhere.
  • Examples of different promoters to drive transgene expression for different objectives are presented in Table 5.
  • Representative examples of promoters for constitutive (Ubi, (CAMV)35S 2 , RUBQ2, OsActi ), tuber and stolon specific (Cathlnh), stress regulated (Atrd29a) and sucrose responsive (14-3-3 protein family 16R) linked to FT fusions are presented in Figures 42 - 48, respectively.
  • photosynthetic promoters have been shown to be strong regulators of expression of transgenes in light-responsive tissues.
  • Advantages of photosynthetic promoters for expressing fructan biosynthesis genes include that they are active in the large group of cells of the leaves and upper part of the stems which accounts the majority of the plants biomass. They are not constitutively expressed, however their expression pattern temporally and spatially overlaps with sucrose accumulation.
  • the following vectors are transformed singly or in groups (double and triple) to assess synergistic responses of co-expression required for the generation of low and high DP fructans.
  • the genetic constructs of the present invention may be introduced into plant cells by transduction, transfection, transformation or gene targeting.
  • Such techniques include ⁇ g/Obacfer/um-mediated introduction, electroporation of tissues, cells and protoplasts,, protoplast fusion, injection into reproductive organs, injection into immature embryos and high velocity projectile introduction to cells, tissues, calli, immature and mature embryos, microinjection into cells and protoplasts, polyethylene glycol mediated direct gene transfer into protoplasts, biolistic transformation, Whiskers transformation and combinations thereof.
  • the choice of technique depends largely on the type of plant to be transformed and the appropriate vector for the method chosen are used.
  • Cells incorporating the genetic constructs of the present invention may be selected, as directed by the vectors used, and then cultured in an appropriate medium to regenerate transformed plants, using techniques well established. The resulting plants may be reproduced, either sexually or asexually, to produce successive generations of transformed plants.
  • the present invention may be applied to a variety of plants, including monocotyledons [such as wheat, corn or maize, rice, barley, sorghum, sugarcane, oats, rye, grasses
  • monocotyledons such as wheat, corn or maize, rice, barley, sorghum, sugarcane, oats, rye, grasses
  • Triticum aestivum (wheat), C3 grasses containing native fructans such as Lolium perenne (ryegrass) and Lolium arundinaceum (tall fescue), as well as Paspalum dilatatum (paspalum) a C4 perennial apomitic grass with no native fructans.
  • the invention may also be applied to dicots such as Arabidopsis thaliana, Brassica napus
  • the candidate genes are inserted into the plant genome by particle bombardment using whole plasmids so vector backbone sequences may also be incorporated into the genome.
  • Transgenic plant tissues are recovered by survival on tissue culture media containing a selective agent. Agrobacterium-med ' iated transformation of dicots eg Arabidopsis, tobacco, canola and white clover
  • Agrobacte ⁇ um-mediated transformation takes advantage of the natural pathogenic activity of the soil bacterium Agrobacterium tumefaciens.
  • A. tumefaciens infects the roots & stems of dicotyledonous plants resulting in infection directed by the tumor inducing (Ti) plasmid by the insertion of specific genes (T-DNA) into the genome of infected plant cells.
  • Ti tumor inducing
  • T-DNA specific genes
  • Biolistic co-transformation of perennial ryegrass with the vectors containing the TaRbcS and LpRbcS regulatory sequences, driving the expression of individual fructan genes or as a FT translational fusion, and the pAcH1 vector for hygromycin resistance was conducted on embryogenic calli for perennial ryegrass.
  • the pAcH1 vector was previously constructed and has been used successfully in plant transformation experiments (Bilang, et al., 1991 ; Spangenberg, et al., 1995a; Spangenberg, et al., 1995b; Ye, et al., 1997; Bai, et al., 2001).
  • the GUS marker gene was also cloned as a positive control. Table 6 summarises the transformation and molecular analysis for the generation of these lines.
  • Table 6 Summary of production of transgenic perennial ryegrass plants for expression of LpI-SST and Lp6G-FFT and FT fusion ORFs under control of photosynthetic promoter from wheat.
  • Codonuclease "Cassette DNA" containing L. perenne sequences was excised from the plasmid vectors pBS-LpRbcS::Lp1-SST::LpFT4, pBS-LpCAB::Lp6G-FFT::LpFT4 and pBS-l_pRbcS::Lp1- SST_Lp6G-FFT::LpFT4 ( Figures 29, 30 and 34 respectively) using the EcoR V restriction endonuclease. Following agarose gel electrophoresis, the resulting DNA fragment was purified from the agarose gel prior to being used for plant transformation to produce DNA without vector backbone sequences. The pAcH1 vector previously constructed and used successfully in plant transformation experiments was also digested with restriction enzymes to produce a DNA fragment for the expression of the selectable marker only.
  • Table 7 Summary of transformation progress for production of transgenic perennial ryegrass plants for expression of LpI-SST and Lp6G-FFT and FT fusion ORFs under control of ryegrass photosynthetic promoters.
  • transgenic perennial ryegrass plants differences in growth phenotypes were noticed between the lines. Both the tissue culture regenerants and corresponding soil grown plants from both of the FT fusion 1 and FT fusion 3 transgenic plants showed a superior growth performance phenotype compared to the transgenic plants containing either a single fructan biosynthesis gene or control plants containing only the selectable marker, hph. Phenotypic examples of transgenic perennial ryegrass plants in tissue culture are displayed for the TaRbcS promoter and
  • the plants showing the superior growth performance phenotype were confirmed to contain the FT gene of interest.
  • the superior growth performance phenotype of the transgenic FT fusion 1 and FT fusion 3 plants was first observed during the early stages of plant regeneration conducted on plates. Specifically just 12 days after incubation under lights the transgenic calli showed further developed green shoots. The fast growth rate of the FT fusion transgenic plants became more evident 22 days after transferring to rooting media. Transgenic plants containing either FT fusion 1 or FT fusion 3 constructs showed clearly greater numbers of tillers. In addition, the FT fusion transgenic plants consistently showed a higher tiller density per plant compared to control plants in vitro ( Figures 48 - 49).
  • FT fusion 1 and FT fusion 3 lines Following transfer to soil and propagation under glasshouse conditions more specific differences were observed between the FT fusion 1 and FT fusion 3 lines. Even though both FT fusion plants displayed enhanced growth performance, FT fusion 1 plants had longer, thicker and a slightly darker green leaf blades. Also the plants were physically more robust with thicker leaf sheaths and leaf blades. FT fusion 3 lines continued to grow faster than the other control plants with longer leaf blades and more vigorous tiller growth, but the leaf morphology was more similar to wild-type. An increase in root biomass was also observed in both FT fusion 1 and FT fusion 3 soil grown transgenic perennial ryegrass plants (Figure 50).
  • the control transgenic plants harbouring either the LpI-SST or Lp6G-FFT as single genes did not show the level of increased growth rate that was observed in the FT fusion 1 and 3 transgenic plants. Their appearance is similar to each other, although some developed more vigorously than the transgenic plants containing either GUS or hph ( Figure 50).
  • the levels of 1-kestose were up to 4 times higher in FT fusion 1 lines (up to 3.7 ⁇ g/mg of DW, total fructans: 20.5 ⁇ g/mg of DW and sucrose 51.2 ⁇ g/mg of DW.), and 3 times higher in FT fusion 3 lines (2.4 ⁇ g/mg of DW, total fructans: 26.0 ⁇ g/mg of DW and sucrose 49.8 ⁇ g/mg of DW) compared to the hph controls (Figure 55A-B).
  • transgenic perennial ryegrass was also evaluated under field conditions for total fructan level and composition ( Figures 58 & 59) and transgene expression (Figure 60C).
  • the control and transgenic perennial ryegrass plants were sampled repeatedly throughout the field trial growing season. Biochemical analysis of wild-type controls and independent transformants was conducted to show the level of total fructan per plant.
  • Figure 58 illustrates fructan levels in milligrams (mg) per gram (g) of dry weight (DW) transgenic and wild-type field grown whole tillers and leaf blades.
  • the transgenic FT fusion 1 and fusion 3 lines showed up to an 80% increase in plant height (43 cm), up to 60% increase in leaf width (4 mm), and up to 3 fold increase in tiller number (6 tillers). After 12 weeks the control plants were, on average, 43 cm tall, leaf blades width was 3.5 mm, with 5 tillers per plant produced. Over the same period of time the transgenic FT fusion 1 and fusion 3 plants had grown up to 62 cm tall (43% increase compared to controls). The leaf width was up to 6 mm (70% increase) and the maximum number of tillers observed was 16 per plant (220% increase) ( Figure 62).
  • the control was calculated as the average of five independent 'gene of interest' negative (GOI-ve) plants. Both FT fusion and FT fusion plus LXR ® transgenic plants produced plants with a dry weight higher (up to two fold) than the average level for the control (Figure 64).
  • Table 9 Summary of transformation progress for tall fescue with photosynthetic- regulated expression of FT fusion 3 and/ or LXR ® .
  • the control was calculated as the average of five independent GOI-ve plants.
  • Tiller number experiments were also conducted to establish the growth vigour of individual transgenic plants. Both tall fescue transgenic and GOI-ve control plants, at the 5 tiller stage, were trimmed as mentioned above and left growing under glasshouse conditions for 6 weeks before tiller numbers were counted. The tiller number in the control represents the average tiller number obtained from five independent GOI-ve plants. Transgenic lines of FT fusion alone and FT fusion plus LXR ® tall fescue plants showed up to a two fold increase in tiller number compared to the average value of the control plants ( Figure 68). Transgenic tall fescue plants (5 tillers) were trimmed (as indicated above) and grown under glasshouse conditions for 6 weeks when leaf blades were collected and freeze- dried for fructan analysis.
  • the average fructan level in controls represents data obtained from five independent GOI-ve plants.
  • Transgenic lines of FT fusion tall fescue plants show a dramatic increase (between three to five fold) in fructan accumulation in leaf blades compared to the average fructan level in GOI-ve control plants ( Figure 69).
  • Biolistic co-transformation of wheat with the vectors containing the photosynthetic promoter regulatory sequences, driving the expression of individual fructan genes or as a translational FT fusion, and a vector containing a chimeric Ubi::bar::nos selectable marker gene for glufosinate resistance (pAcH25) was conducted on wheat embryogenic calli.
  • a transformation vector has been constructed for biolistic transformation of wheat containing the chimeric AtMYB32::IPT::35S with a chimeric Ubi::bar::nos selectable marker gene for glufosinate resistance.
  • Genetic transformation of wheat with LXR ® vector was based on biolistic transformation of embryogenic calli from Triticum aestivum L Bobwhite 26 wheat line as described in International patent application PCT/AU01/01092.
  • the candidate gene was inserted into the wheat genome by particle bombardment using whole plasmids so vector backbone sequences may also be incorporated into the genome.
  • Transgenic plant tissues were recovered by survival on tissue culture media containing a selective agent.
  • transgenic plants were generated that contain both fructan biosynthetic genes driven by light-regulated promoters and the LXR ® technology for re-programmed fructan biosynthesis in photosynthetic cells and extended life of photosynthetic cells.
  • Table 8 summarises the transformation and molecular analysis for the generation of these transgenic plants.
  • Table 10 Summary of transformation progress for production of transgenic wheat plants for expression of LpI-SST and Lp6G-FFT and FT fusion ORFs under control of photosynthetic promoters of wheat and in combination with LXR ® technology for re- programmed fructan biosynthesis in photosynthetic cells and extended life of photosynthetic cells.
  • tissue culture regenerants from both of the FT fusion 1 and FT fusion 3 transgenic plants showed a superior vigour phenotype compared to control plants.
  • the superior growth phenotype of the transgenic FT fusion 1 and FT fusion 3 plants was first observed during the early stages of in vitro plant regeneration conducted on tissue culture plates. Following biolistic transformation calli were kept for two weeks on tissue culture plates in the dark and then transferred to light conditions. Approximately 6 weeks after incubation under light conditions the transformed calli showed more fully developed green shoots and the roots of the FT fusion transgenic regenerants grew at an extremely advanced rate (Figure 70).
  • FT fusion transgenic plants showed an obvious early increase in tiller number at around 2 months as compared to null controls (up to 5 tillers compare to one tiller observed in control plants).
  • the width of the leaves of the some of the plants was 4-5 mm compare to control plants 2-3 mm.
  • the FT fusion transgenics consistently showed a higher tiller density per plant compared to control lines ( Figure 71 ).
  • transgenic wheat plants that contain the FT fusion constructs continued to show an increase in tiller number as compared to control plants ( Figure 72).
  • transgenic wheat plants that contain the LXR ® technology construct showed an increase in tiller number as compared to control plants under glasshouse conditions ( Figure 73A). They also showed and increase of photosynthetic tissue after 35 days under glasshouse conditions ( Figure 73B).
  • the candidate gene expression construct was inserted into the Paspalum dilatatum genome by particle bombardment using whole plasmids so vector backbone sequences may also be incorporated into the genome.
  • Transgenic plant tissues were recovered by survival on tissue culture media containing a selective agent.
  • LXR ® transgenic plants display a superior growth phenotype.
  • Transgenic Paspalum dilatatum plants expressing the IPT gene under control of the AtMYB32 promoter revealed an enhanced biomass accumulation.
  • the distinctive growth phenotype may be used as a selection tool for identifying transformed plants in combination with co-transformed vectors.
  • Transformation vectors also contained a chimeric 35S::nptll::35S or 35S::hph::35S as selectable marker genes.
  • Transgenic white clover Trifolium repens
  • Arabidopsis thaliana plants expressing LXR ® technology alone AtMYB3::IPT
  • AtRbcS::Lp1-SST_Lp6G-FFT::35S FT fusion alone AtMYB3::IPT
  • AtRbcS::Lp1-SST_Lp6G-FFT::35S FT fusion alone as well as LXR ® technology and the AtRbcS::Lp1-SST_Lp6G-FFT::35S FT fusion together have been produced ( Figures 77 and 80).
  • Tables 11 and 12 summarise the transformation and molecular analysis for the generation of these lines, respectively.
  • transgenic Arabidopsis plants Quantitative RT-PCR was used to confirm transformants and detect expression levels of the AtRbcS FT fusion in selected lines (Figure 81 ).
  • Transgenic T 2 FT fusion Arabidopsis plants grown in soil are shown in Figure 82.
  • Gene of interest negative plants (GOI-ve) are also presented and show no phenotypic difference to FT fusion transgenic plants shown to express the transgene.
  • Binary vectors were also used for yAgfrofeacte ⁇ ' u/77-mediated transformation of Brassica napus (canola) hypocotyl segments (Patent PCT/AU01/01092).
  • transgenic LXR ® dicot plants A functionally active fragment of the AtMYB32 promoter was used to drive IPT expression in transgenic white clover and canola plants as described in International patent application PCT/AU01/01092.
  • Outcomes observed from the LXR ® technology in dicot plants have been delayed leaf senescence; enhanced leaf growth dynamics; reduced stolon death; enhanced biomass production; increased cumulative green leaf area; increased seed yield; enhanced drought tolerance; increased shading tolerance; enhanced herbage quality reflected by enhanced ruminal fermentation kinetics and higher dry matter digestibility.
  • Transgenic plants display a delayed leaf senescence phenotype.
  • the regulation of developmental senescence may be assessed by simulating and initiating artificial aging of detached leaves in vitro on moist filter paper. Incubation of detached leaves in darkness is highly effective in inducing Senescence Associated Genes (SAGS), leaf yellowing and chlorophyll loss (Weaver and Amasino, 2001).
  • SAGS Senescence Associated Genes
  • the transgenic plants displayed a significant delay of leaf senescence as compared to leaves from control plants 7 - 20 days following detachment.
  • transgenic plants have been generated that contain both, fructan biosynthetic genes (FT including FT fusion genes) under control of light- ' regulated, photosynthetic promoters for re-programming fructan biosynthesis in photosynthetic cells and LXR ® technology through co-expression of IPT gene driven by the AtMYB32 promoter for extending life of the photosynthetic cells.
  • FT fructan biosynthetic genes
  • photosynthetic promoters for re-programming fructan biosynthesis in photosynthetic cells
  • LXR ® technology through co-expression of IPT gene driven by the AtMYB32 promoter for extending life of the photosynthetic cells.
  • Example 16 Use of the distinctive growth phenotype as a selection tool to identify transgenic plants in vitro
  • the superior growth phenotype of the transgenic FT fusion 1 or FT fusion 3 plants was observed in all plant types to which it was transformed (eg perennial ryegrass and wheat). In both ryegrass and wheat it was first observed during the early stages of plant regeneration conducted in plates. In the experiments conducted without antibiotic selection, strong shoot induction has been observed at the stage when after bombardment the calli have been kept in dark conditions for 8 weeks. ( Figure 84 A-C). After transferring the plates to light conditions (7 days after transfer) strong shoot induction was observed in the transgenic plants and much lower level of shoot regeneration was detected in control plants ( Figure 84 D-F).
  • sucrose- regulated or constitutive promoters could be used as a selection tool for the identification of transformed plants at the tissue culture stage.
  • Expression of the FT fusion protein may be also driven by a set of promoters, which are active due to the high concentration of sucrose that exists in tissue culture medium, and much less active at the low sucrose levels present in soil-grown plants. This transgene may subsequently be segregated away from the transgenic plants in successive generations.
  • the increased biomass of the transformed plants to be used as the selective agent should not require an antibiotic resistance marker for the selection process, enabling the production of a market ready product.
  • Plants were regenerated without antibiotic selection and kept 2 weeks under dark conditions and then transferred to light conditions (16/8hr light/dark photo-period). The plant's growth was examined prior to transfer to light and weekly for five weeks under light conditions. Calli were kept under progressively starving conditions on the same plate for five weeks (Callus induction medium: MS full strength + 250mg/l_ L-asparagine +2.5mg/L 2.4-D+ 6% sucrose +0.7% agar).
  • co-transformation efficiency is determined for a variety of transformation events which include a vector containing an antibiotic selectable marker.
  • These co- transformation events include:
  • a second round of co-transformation events also takes place without an antibiotic selectable marker on selection free media.
  • These co-transformation events include:
  • FT fusion regulated by a photosynthetic promoter plus LXR ® plus dsRED marker Selection for increased growth rate of shoots and/or roots takes place and the presence of the transgene for each double or triple co-transformation event is determined.
  • the presence of the dsRED marker gene is easily determined by visualisation of fluorescence and helps determine the co-tranformation efficiency for each of the transformation events. Comparison of the co-transformation efficiencies determined with and without selectable marker aids in establishing the efficacy of using a superior phenotype as a selection tool.
  • Bai, Y., et al. (2001 ). "Genetic transformation of elite turf-type cultivars of Tall Fescue.” International Turfgrass Society Research Journal 9: 129-136.

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Abstract

The present invention relates to the modification of fructan biosynthesis in plants and, more particularly, to methods of manipulating fructan biosynthesis in photosynthetic cells, and to related nucleic acids and constructs. The present invention also relates to increasing plant biomass and, more particularly, to methods of enhancing biomass yield and/or yield stability, including shoot and/or root growth in a plant, and to related nucleic acids and constructs. The present invention also relates to methods of enhancing the productivity of biochemical pathways and, more particularly, to fusion proteins in plants, and to related nucleic acids and constructs.

Description

MODIFICATION OF FRUCTAN BIOSYNTHESIS, INCREASING PLANT BIOMASS, AND ENHANCING PRODUCTIVITY OF BIOCHEMICAL PATHWAYS IN A PLANT
Field of the invention
The present invention relates to the modification of fructan biosynthesis in plants and, more particularly, to methods of manipulating fructan biosynthesis in photosynthetic cells, and to related nucleic acids and constructs.
The present invention also relates to increasing plant biomass and, more particularly, to methods of enhancing biomass yield and/or yield stability, including shoot and/or root growth in a plant, and to related nucleic acids and constructs.
The present invention also relates to methods of enhancing the productivity of biochemical pathways and, more particularly, to fusion proteins in plants, and to related nucleic acids and constructs.
Background of the invention
Fructans are a type of water-soluble carbohydrate whose primary function is to provide a readily accessible energy reserve for plant growth. Fructans are associated with various advantageous characters in grasses, such as cold and drought tolerance, increased tiller survival, enhanced persistence, good regrowth after cutting or grazing, improved recovery from stress, early spring growth and increased nutritional quality.
Fructan synthesis and metabolism in grasses and cereals is complex. Fructans consist of linear or branched fructose chains attached to sucrose. The chain length of plant fructans ranges from three up to a few hundred fructose units. Different types of fructans can be distinguished based on the linkage types present. In perennial ryegrass three types of fructans have been identified: inulins, inulin neoseries and levan neoseries, with four fructosyltransferse (FT) enzymes involved in this fructan profile (Figure 6). The enzyme 1-SST (sucrose: sucrose 1 -fructosyltransferase) catalyses the first step in fructan biosynthesis while the remaining enzymes elongate the growing fructose chain (1-FFT: fructan: fructan 1 -fructosyltransferase, 6G-FFT: 6-glucose fructosyltransferase, and 6-SFT: sucrose: fructose 6-fructosyltransferase). The enzymes 1-FEH or 6-FEH (fructoexohydrolase) reduce fructan chain length by releasing fructose molecules.
Fructans represent the major non-structural carbohydrate in 15% of plant species and play a key role in forage quality. Ruminant livestock grazing on high fructan diets show improved animal performance.
In grasses the level and composition of fructans has been increased in stems and leaf sheaths through the engineered expression of fructosyltransferase (FT) genes.
However, manipulating biochemical pathways by manipulating the activity of enzymes in the pathways may be difficult because of the ways in which the various enzymes and their substrates may interact.
Thus, it would be desirable to have improved methods of manipulating biochemical pathways, particularly in plants. For example, it would be desirable to have methods of manipulating fructan biosynthesis in plants, including grass species such as Lolium and Festuca and cereals such as wheat and maize, thereby facilitating the production of eg. forage grasses with improved herbage quality, leading to improved pasture production, improved animal production and reduced environmental pollution, bioenergy grasses with enhanced biomass yield e.g. for bioethanol production, and eg. cereals with increased grain and biomass yield.
Nucleic acid sequences encoding some of the enzymes involved in the fructan biosynthetic pathway have been isolated for certain species of plants. For example, PCT/AU01/00705 to the present applicants, describes fructosyltransferase homologues from Lolium and Festuca. However, there remains a need for materials useful in the modification of fructan biosynthesis in plants, and also to engineer fructan accumulation in different parts of the plant. It is an object of the present invention to overcome, or at least alleviate, one or more of the difficulties or deficiencies associated with the prior art. Summary of the invention
Applicants have found that it is possible to nutritionally enhance plants eg. forage grasses and/or to increase plant biomass by spatial reprogramming of the fructan- biosynthesis pathway in photosynthetic cells. Using this process it is possible to drive fructan accumulation in leaf blades, the plant organs that are primarily grazed by livestock, and which may not normally accumulate fructans. Thus, accumulation of fructans, especially those exhibiting a high degree of polymerization ('high DP fructans'), provides more accessible nutrition for grazing animals. Fructans accumulate in the stems and leaf sheaths, with leaf fructans only forming during periods where CO2 assimilation outperforms growth. Forage grasses may be nutritionally enhanced by expressing fructan genes in photosynthetic cells where sucrose is synthesised, thus driving fructan accumulation preferentially in leaf blades and providing more energy to grazing livestock.
Fructans in forage grasses contribute significantly to the readily available energy in the feed for grazing ruminant animals. The fermentation processes in the rumen require considerable readily available energy. The improvement of the readily available energy in the rumen can increase the efficiency of rumen digestion. An increased efficiency in rumen digestion leads to an improved conversion of the forage protein fed to the ruminant animal into milk or meat, and to a reduction in nitrogenous waste.
Applicants have also found that reprogramming photosynthetic cells for extended life, for example by delaying leaf senescence, helps increase plant biomass.
Applicants have also found that it is possible to enhance the productivity of a biochemical pathway by co-ordinating enzymatic activity in the pathway by means of a genetic construct encoding a fusion, more preferably a translational fusion, of two or more enzymes from the pathway.
While applicant does not wish to be restricted by theory, it is thought that by bringing two enzymes in a pathway into close proximity, for example by expressing a translational fusion, expression of the individual enzymes may be co-ordinated thereby improving the efficiency of the pathway. For example, by expressing a translational fusion of two or more FT genes (e.g. Lp1- SST and Lp6G-FFT), problems associated with differences in the expression patterns of these genes independently integrated into the plant genome may be alleviated, resulting in conversion of the sucrose molecules directly to fructans, those exhibiting a low degree of polymerisation ('low DP fructans') and a high degree of polymerization ('high DP fructans'). Furthermore, the FT proteins may physically associate with each other to form a metabolic channel for the efficient biosynthesis of fructans.
Furthermore, expression of FT genes in photosynthetic cells leading to the accumulation of low and high DP fructans in photosynthetic cells may lead to a release from inhibition mechanisms of photosynthesis, facilitating solar energy capture and increased CO2 fixation.
Thus, applicants have found that reprogramming photosynthetic cells for extended life and enhanced fructan biosynthesis facilitates solar energy capture and increases plant biomass production including shoot and/or root growth.
Furthermore, since accumulation of low and high DP fructans has been associated with plants' tolerance to abiotic stress such as cold and drought; and since enhanced root growth and/or delayed leaf senescence has also been implicated in plants' tolerance of drought stress, reprogramming photosynthetic cells for extended life and/or enhanced fructan biosynthesis may facilitate yield stability and/or plants' tolerance of abiotic stresses.
Accordingly, in one aspect, the present invention provides a method of manipulating fructan biosynthesis in photosynthetic cells of a plant, said method including introducing into said plant an effective amount of a genetic construct including a promoter, or a functionally active fragment or variant thereof, operatively linked to nucleic acids encoding one or more fructan biosynthetic enzymes, or functionally active fragments or variants thereof.
By 'manipulating fructan biosynthesis' is generally meant increasing fructan biosynthesis in a transformed plant relative to an untransformed control plant. However, for some applications it may be desirable to reduce or otherwise modify fructan biosynthesis in the transformed plant relative to the untransformed control plant. For example, it may be desirable to increase or decrease the activity of certain enzymes in the fructan biosynthetic pathway, in the transformed plant relative to the untransformed control plant.
By 'photosynthetic cells' is meant those cells of a plant in which photosynthesis takes place. Such cells generally contain the pigment chlorophyll and are otherwise known as green cells. Most photosynthetic cells are contained in the leaves of plants. Preferably, the genetic construct of the present invention is expressed in bundle sheath cells, more preferably in mesophyll and/or parenchymatous bundle sheath cells.
By 'an effective amount' is meant an amount sufficient to result in an identifiable phenotypic trait in said plant, or in a plant, plant seed or other plant part derived therefrom. Such amounts can be readily determined by an appropriately skilled person, taking into account the type of plant, the route of administration and other relevant factors. Such a person will readily be able to determine a suitable amount and method of administration. See, for example, Maniatis et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, the entire disclosure of which is incorporated herein by reference.
By 'genetic construct' is meant a recombinant nucleic acid molecule.
By a 'promoter' is meant a nucleic acid sequence sufficient to direct transcription of an operatively linked nucleic acid sequence.
By 'operatively linked' is meant that the nucleic acid(s) and a regulatory sequence, such as a promoter, are linked in such a way as to permit expression of said nucleic acid under appropriate conditions, for example when appropriate molecules such as transcriptional activator proteins are bound to the regulatory sequence. Preferably an operatively linked promoter is upstream of the associated nucleic acid.
By 'upstream' is meant in the 3' to 5' direction along the nucleic acid.
By 'nucleic acid' is meant a chain of nucleotides capable of carrying genetic information. The term generally refers to genes or functionally active fragments or variants thereof and/or other sequences in the genome of the organism that influence its phenotype. The term 'nucleic acid' includes DNA (such as cDNA or genomic DNA) and RNA (such as mRNA or microRNA) that is single- or double-stranded, optionally containing synthetic, non-natural or altered nucleotide bases, synthetic nucleic acids and combinations thereof.
By a 'nucleic acid encoding a fructan biosynthetic enzyme' is meant a nucleic acid encoding an enzyme of the fructan biosynthetic pathway in plants, for example fructosyltransferases such as sucrose:sucrose 1-fructosyltransferase (1-SST); fructan:fructan 1-fructosyltransferase (1-FFT); sucrose:fructan 6-fructosyltransferase (6- SFT); and fructan: fructan 6G-fructosyl transferase (6G-FFT); and fructoexohydrolases such as 1-fructoexohydrolase (1-FEH) and 6-fructoexohydrolase (6-FEH).
By 'functionally active fragment or variant' in relation to a promoter is meant that the fragment or variant (such as an analogue, derivative or mutant) is capable of directing transcription of an operatively linked nucleic acid. Such variants include naturally occurring allelic variants and non-naturally occurring variants. Additions, deletions, substitutions and derivatizations of one or more of the nucleotides are contemplated so long as the modifications do not result in loss of functional activity of the fragment or. variant. Preferably the functionally active fragment or variant has at least approximately 80% identity to the relevant part of the above mentioned sequence to which the fragment or variant corresponds, more preferably at least approximately 90% identity, even more preferably at least approximately 95% identity, most preferably at least approximately 98% identity. Preferably the fragment has a size of at least 20 nucleotides, more preferably at least 50 nucleotides, more preferably at least 100 nucleotides, more preferably at least 200 nucleotides, more preferably at least 300 nucleotides.
By 'functionally active' in relation to the nucleic acid encoding a fructan biosynthetic enzyme is meant that the fragment or variant (such as an analogue, derivative or mutant) is capable of manipulating fructan biosynthesis in a plant by the method of the present invention, for example by being translated into an enzyme that is able to participate in the fructan biosynthetic pathway. Such variants include naturally occurring allelic variants and non-naturally occurring variants. Additions, deletions, substitutions and derivatizations of one or more of the nucleotides are contemplated so long as the modifications do not result in loss of functional activity of the fragment or variant. Preferably the functionally active fragment or variant has at least approximately 80% identity to the relevant part of the above mentioned sequence to which the fragment or variant corresponds, more preferably at least approximately 90% identity, even more preferably at least approximately 95% identity, most preferably at least approximately 98% identity. Such functionally active variants and fragments include, for example, those having conservative nucleic acid changes.
By 'conservative nucleic acid changes' is meant nucleic acid substitutions that result in conservation of the amino acid in the encoded protein, due to the degeneracy of the genetic code. Such functionally active variants and fragments also include, for example, those having nucleic acid changes which result in conservative amino acid substitutions of one or more residues in the corresponding amino acid sequence.
By 'conservative amino acid substitutions' is meant the substitution of an amino acid by another one of the same class, the classes being as follows:
Nonpolar: Ala, VaI, Leu, lie, Pro, Met Phe, Trp
Uncharged polar: GIy, Ser, Thr, Cys, Tyr, Asn, GIn
Acidic: Asp, GIu
Basic: Lys, Arg, His
Other conservative amino acid substitutions may also be made as follows:
Aromatic: Phe, Tyr, His
Proton Donor: Asn, GIn, Lys, Arg, His, Trp
Proton Acceptor: GIu, Asp, Thr, Ser, Tyr, Asn, GIn
Particularly preferred fragments and variants include one or more conserved sucrose binding/hydrolysis domains. Examples of such domains are shown in Figures 17, 18 and 36 hereto, for example (N/S)DP(N)G, FRDP and EC(I)D. Particularly preferred fragments and variants may also include one or more conserved amino acid domains found in Lolium FT, invertase and FEH sequences, for example as shown in Figures 17, 18 and 36 hereto.
Preferably the fragment has a size of at least 20 nucleotides, more preferably at least 50 nucleotides, more preferably at least 100 nucleotides, more preferably at least 200 nucleotides, more preferably at least 500 nucleotides.
Preferably, the nucleic acid encoding one or more fructan biosynthetic enzymes is selected from the group consisting of genes encoding 1-SST, 1-FFT, 6-SFT and 6G- FFT, combinations thereof, and functionally active fragments and variants thereof. Preferably, the nucleic acid encodes a FT fusion protein of two or more of these fructan biosynthetic enzymes.
Even more preferably the nucleic acid encoding one or more fructan biosynthetic enzymes encodes 1-SST and/or 6G-FFT, even more preferably a FT fusion protein of 1- SST and 6G-FFT, or functionally active fragments or variants thereof.
Preferably, the nucleic acid encoding one or more fructan biosynthetic enzymes is isolated from or corresponds to a gene or genes from a species of interest. More preferably the gene or genes are from a ryegrass, fescue or wheat species, including Italian or annual ryegrass, perennial ryegrass, tall fescue, meadow fescue, red fescue, bread wheat and durum wheat. Even more preferably, the nucleic acid encoding one or more fructan biosynthetic enzymes is isolated from or corresponds to a gene from a Lolium species such as Lolium perenne or Lolium arundinaceum.
Suitable nucleic acids encoding fructan biosynthetic enzymes are described in PCT/AU01/00705 and PCT/AU01/01275, the entire disclosures of which are incorporated herein by reference.
In a particularly preferred embodiment the nucleic acid encoding 1-SST includes a sequence selected from the group consisting of the sequence shown in SEQ ID NO: 11 of PCT/AU01/00705; and the nucleotide sequences encoding the polypeptide sequence shown in SEQ ID NO: 12 of PCT/AU01/00705; and functionally active fragments and variants thereof. In a particularly preferred embodiment the nucleic acid encoding the 6G-FFT includes a sequence selected from the group consisting of the sequences shown in SEQ ID NO: 110 of PCT/AU01/01275, and Figure 7 hereto; and the nucleic acid sequences encoding the polypeptide sequences shown in SEQ ID NO: 111 of PCT/AU01/01275; and Figure 8 hereto; and functionally active fragments and variants thereof.
In a particularly preferred embodiment, the nucleic acid encoding the 1-FFT includes a sequence selected from the group consisting of the sequences shown in SEQ ID NO: 3 of PCT/AU01 /00705, SEQ ID NOS: 103 and 105-109 of PCT/AU01/01275 and Figure 9 hereto; and the nucleotide sequences encoding the polypeptide sequences shown in SEQ ID No: 4 of PCT/AU01/00705, SEQ ID NO: 104 of PCT/AU01/01275 and Figure 10 hereto; and functionally active fragments and variants thereof.
Applicants have found that by generating a translational fusion of two FT genes as a single open reading frame, for example sucrose-sucrose 1-fructosyltransferase (Lp1- SST) and fructan-fructan 6G-fructosyltransferase (Lp6G-FFT) from Lolium perenne, a single mRNA transcript is produced which is translated as a single protein, with combined enzyme activities. By expressing a translational fusion of two FT genes (e.g. LpI-SST and Lp6G-FFT), problems associated with differences in the expression patterns of these two genes independently integrated in to the plant genome may be alleviated, resulting in the conversion of sucrose to low and high DP fructans.
In a particularly preferred embodiment the nucleic acid encoding the FT fusion protein of 1-SST and 6G-FFT includes a sequence selected from the group consisting of the sequences shown in Figures 12 and 14 hereto and the nucleic acid sequences encoding the polypeptide sequences shown in Figures 13 and 15 hereto; and functionally active fragments and variants thereof.
In a particularly preferred embodiment, the genetic construct includes a sequence selected from the group consisting of the sequences shown in Figures 24 to 27, 31 , 32, 35, 36, 38 and 41 to 47 hereto; and functionally active fragments and variants thereof.
In a further aspect, the present invention provides a method of enhancing productivity of a biochemical pathway in a plant, said method including introducing into said plant an effective amount of a genetic construct including nucleic acids encoding two or more enzymes from said pathway, or functionally active fragments or variants thereof.
Preferably, said nucleic acids are linked to form a fusion gene encoding a fusion protein of said two or more enzymes.
By a 'biochemical pathway' is meant a plurality of chemical reactions occurring within a cell which are catalysed by more than one enzyme or enzyme subunit and result in the conversion of a substrate into a product. This includes, for example, a situation in which two or more enzyme subunits (each being a discrete protein coded by a separate gene) combine to form a processing unit that converts a substrate into a product. A 'biochemical pathway' is not constrained by temporal or spatial sequentiality.
By 'enhancing productivity' is generally meant that the amount of product of the biochemical pathway, or the rate of production of the product, is increased in a transformed plant relative to an untransformed control plant. However, for some applications it may be desirable to reduce or otherwise modify the amount of product of the biochemical pathway or the rate of production of the product in the transformed plant relative to the untransformed control plant. For example, it may be desirable to increase or decrease the amount of an intermediate of the pathway, or its rate of production, in a transformed plant relative to an untransformed control plant.
By a 'fusion protein' is meant a hybrid or chimeric protein produced recombinantly by expressing a fusion gene including two or more linked nucleic acids which originally encoded separate proteins, or functionally active fragments or variants thereof.
By a 'fusion', 'translational fusion' or 'fusion gene' is meant that two or more nucleic acids are linked in such a way as to permit expression of the fusion protein, preferably as a translational fusion. This typically involves removing the stop codon from a nucleic acid sequence coding for a first protein, then appending the nucleic acid sequence of a second protein in a frame. The FT fusion gene is then expressed by a cell as a single protein. The protein may be engineered to include the full sequence of both original proteins, or a functionally active fragment or variant of either or both. The genetic construct may also include a nucleic acid sequence encoding a linker between the two linked nucleic acids. A 'linker' is any chemical, synthetic, carbohydrate, lipid, polypeptide molecule (or combination thereof) positioned between and joined to two adjacent active fragments in a fusion protein. A preferred linker of the invention is a flexible linker, such as a polypeptide chain consisting of one or more amino acid residues joined by amino acid bonds to the two active fragments. For example, a (GIy4 Ser)3 linker may be positioned between the two active fragments in the fusion protein.
By 'functionally active' in relation to the nucleic acids encoding two or more enzymes from a biochemical pathway is meant that the fragment or variant (such as an analogue, derivative or mutant) is capable of enhancing productivity of the biochemical pathway in a plant by the method of the present invention. Such variants include naturally occurring allelic variants and non-naturally occurring variants. Additions, deletions, substitutions and derivatizations of one or more of the nucleotides are contemplated so long as the modifications do not result in loss of functional activity of the fragment or variant. Preferably the functionally active fragment or variant has at least approximately 80% identity to the relevant part of the above mentioned sequence to which the fragment or. variant corresponds more preferably at least approximately 90% identity, even more preferably at least approximately 95% identity, most preferably at least approximately 98% identity. Such functionally active variants and fragments include, for example, those having conservative nucleic acid changes. By 'conservative nucleic acid changes' is meant nucleic acid substitutions that result in conservation of the same amino acid in the encoded protein, due to the degeneracy of the genetic code. Such functionally active variants and fragments also include, for example, those having nucleic acid changes which result in conservative amino acid substitutions of one or more residues in the corresponding amino acid sequence. By 'conservative amino acid substitutions' is meant the substitution of an amino acid by another one of the same class, the classes being as follows:
Nonpolar: Ala, VaI, Leu, He, Pro, Met Phe, Trp
Uncharged polar: GIy, Ser, Thr, Cys, Tyr, Asn, GIn
Acidic: Asp, GIu Basic: Lys, Arg, His
Other conservative amino acid substitutions may also be made as follows:
Aromatic: Phe, Tyr, His
Proton Donor: Asn, GIn1 Lys, Arg, His, Trp
Proton Acceptor: GIu, Asp, Thr, Ser, Tyr, Asn, GIn
Particularly preferred fragments and variants include one or more conserved sucrose binding/hydrolysis domains. Examples of such domains are shown in Figures 17, 18 and 36 hereto, for example (N/S)DP(N)G, FRDP and EC(I)D.
Particularly preferred fragments and variants may also include one or more conserved amino acid domains found in Lolium FT, invertase and FEH sequences, for example as shown in Figures 17, 18 and 36 hereto.
Preferably the fragment has a size of at least 20 nucleotides, more preferably at least 50 nucleotides, more preferably at least 100 nucleotides, more preferably at least 200 nucleotides, more preferably at least 500 nucleotides.
Preferably, the biochemical pathway is a fructan biosynthetic pathway.
Preferably, the two or more enzymes from said pathway are selected from the group consisting of enzymes of the fructan biosynthetic pathway in plants, for example fructosyltransferases such as sucrose:sucrose 1 -fructosyltransferase (1-SST); fructan:fructan 1 -fructosyltransferase (1-FFT); sucrose:fructan 6-fructosyltransferase (6- SFT); and fructan: fructan 6G-fructosyl transferase (6G-FFT); and fructoexohydrolases such as 1-fructoexohydrolase (1-FEH) and 6-fructoexohydrolase (6-FEH).
Even more preferably, the nucleic acids encoding a FT fusion protein include two or more nucleic acids selected from the group consisting of genes encoding 1-SST, 1-FFT, 6-SFT and 6G-FFT, and functionally active fragments and variants thereof, linked to form a FT fusion gene. The nucleic acids are optionally connected by a linker such as a flexible linker. Even more preferably, the nucleic acids encoding a FT fusion protein include two or more nucleic acids linked to form a FT fusion protein of 1-SST and 6G-FFT, or functionally active fragments or variants thereof, optionally connected by a linker such as a flexible linker.
Preferably, the genes encoding enzymes of the fructan biosynthetic pathway are isolated from or correspond to genes from a ryegrass or fescue species, including Italian or annual ryegrass, perennial ryegrass, tall fescue, meadow fescue and red fescue. Even more preferably, the genes encoding enzymes of the fructan biosynthetic pathway are isolated from or correspond to genes from a Lolium species such as Lolium perenne or Lolium arundinaceum.
Suitable nucleic acids encoding fructan biosynthetic enzymes are described in PCT/AU01/00705 and PCT/AU01/01275, the entire disclosures of which are incorporated herein by reference.
In a particularly preferred embodiment the nucleic acid encoding 1-SST includes a sequence selected from the group consisting of the sequence shown in SEQ ID NO: 11 of PCT/AU01/00705; and the nucleotide sequences encoding the polypeptide sequence shown in SEQ ID NO: 12 of PCT/AU01 /00705; and functionally active fragments and variants thereof.
In a particularly preferred embodiment the nucleic acid encoding the 6G-FFT includes a sequence selected from the group consisting of the sequences shown in SEQ ID NO: 110 of PCT/AU01/01275, and Figure 7 hereto; and the nucleic acid sequences encoding the polypeptide sequences shown in SEQ ID NO: 111 of PCT/AU01/01275; and Figure 8 hereto; and functionally active fragments and variants thereof.
In a particularly preferred embodiment, the nucleic acid encoding the 1-FFT includes a sequence selected from the group consisting of the sequences shown in SEQ ID NO: 3 of PCT/AU01 /00705, SEQ ID NOS: 103 and 105-109 of PCT/AU01/01275 and Figure 9 hereto; and the nucleotide sequences encoding the polypeptide sequences shown in SEQ ID No: 4 of PCT/AU01/00705, SEQ ID NO: 104 of PCT/AU01/01275 and Figure 10 hereto; and functionally active fragments and variants thereof. In a particularly preferred embodiment the nucleic acid encoding the of 1-SST and 6G- FFT includes a sequence selected from the group consisting of the sequences shown in Figures 12 and 14 hereto and the nucleic acid sequences encoding the polypeptide sequences shown in Figures 13 and 15 hereto; and functionally active fragments and variants thereof.
In a particularly preferred embodiment, the genetic construct includes a sequence selected from the group consisting of the sequences shown in Figures 24 to 27, 31 , 32, 35, 36, 38 and 41 to 475 hereto, and functionally active fragments and variants thereof.
The promoter used in the constructs and methods of the present invention may be a constitutive, tissue specific or inducible promoter. In a preferred embodiment, the promoter is a light-regulated promoter, more preferably a photosynthetic promoter. By a
'light regulated promoter' is meant a promoter capable of mediating gene expression in response to light stimulus. By a 'photosynthetic promoter' is meant a promoter capable of mediating expression of a gene encoding a protein involved in a photosynthetic pathway in plants.
Less fructans accumulate in mature leaf blades than in leaf sheaths and stems. In order to specifically increase the level of fructans in leaf blades, a strategic approach has been devised that co-ordinately expresses fructan biosynthesis genes in photosynthetic cells (Figure 1 ). The use of light-regulated or photosynthetic promoters may provide the following advantages:
• Photosynthetic promoters are active in a large group of cells including leaf blades, the upper and outer stem (>55% cells);
• They are active in sucrose producing cells (mesophyll and parenchymatous bundle sheath cells);
• Their expression pattern temporally and spatially overlaps with sucrose accumulation;
• Frutosyltransferase activity will remove sucrose from the source thereby preventing feedback suppression on photosynthesis, and may facilitate increases in CO2 fixation; Particularly preferred light-regulated promoters include a ribulose-1 , 5-bisphosphate carboxylase/oxygtenase small subunit (RbcS) promoter and a chlorophyll a/b binding protein (CAB) promoter, and functionally active fragments and variants thereof.
The light-regulated promoter may be from any suitable plant species including monocotyledonous plants [such as maize, rice, wheat, barley, sorghum, sugarcane, forage grasses, bioenergy grasses], dicotyledonous plants (such as Arabidopsis, soybean, canola, cotton, alfalfa and tobacco) and gymnosperms.
Preferably, the light-regulated promoter is isolated from or corresponds to a promoter from a ryegrass or fescue species, including Italian or annual ryegrass, perennial ryegrass, tall fescue, meadow fescue and red fescue. Even more preferably, the light regulated promoter is isolated from or corresponds to a promoter from a Lolium species such as Lolium perenne or Lolium arundinaceum.
In another embodiment, preferably the light-regulated promoter is isolated from or corresponds to a promoter from Arabidopsis, even more preferably Arabidopsis tha liana.
In a particularly preferred embodiment, the RbcS promoter includes a sequence selected from the group consisting of the sequence shown in Figure 5 hereto, and functionally active fragments and variants thereof.
In a particularly preferred embodiment, the RbcS promoter includes a sequence selected from the group consisting of the sequence shown in Figure 38 hereto, and functionally active fragments and variants thereof.
In another particularly preferred embodiment, the CAB promoter includes a sequence selected from the group consisting of the sequence shown in Figure 4 hereto, and functionally active fragments and variants thereof. In another preferred embodiment, the promoter may be a constitutive promoter, such as a ubiquitin (Ubi) promoter. In a particularly preferred embodiment, the Ubi promoter includes a sequence selected from the group consisting of the sequence shown in Figure 41 hereto, and functionally active fragments and variants thereof.
The genetic constructs of the present invention may be introduced into the plants by any suitable technique. Techniques for incorporating the genetic constructs of the present invention into plant cells (for example by transduction, transfection, transformation or gene targeting) are well known to those skilled in the art. Such techniques include v4grobactem//77-mediated introduction, Rhizobium-mediated introduction, electroporation to tissues, cells and protoplasts, protoplast fusion, injection into reproductive organs, injection into immature embryos and high velocity projectile introduction to cells, tissues, calli, immature and mature embryos, biolistic transformation, Whiskers transformation, and combinations thereof. The choice of technique will depend largely on the type of plant to be transformed, and may be readily determined by an appropriately skilled person.
Cells incorporating the genetic constructs of the present invention may be selected, as . described below, and then cultured in an appropriate medium to regenerate transformed plants, using techniques well known in the art. The culture conditions, such as temperature, pH and the like, will be apparent to the person skilled in the art. The resulting plants may be reproduced, either sexually or asexually, using methods well known in the art, to produce successive generations of transformed plants.
The methods of the present invention may be applied to a variety of plants, including monocotyledons [such as grasses (e.g. forage, turf and bioenergy grasses including perennial ryegrass, tall fescue, Italian ryegrass, red fescue, reed canarygrass, big bluestem, cordgrass, napiergrass, wildrye, wild sugarcane, Miscanthus, switchgrass), corn or maize, rice, wheat, barley, sorghum, sugarcane, rye, oat)], dicotyledons [such as Arabidopsis, tobacco, soybean, canola, alfalfa, potato, cassava, clovers (e.g. white clover, red clover, subterranean clover), vegetable brassicas, lettuce, spinach] and gymnosperms.
In a further aspect of the present invention, there is provided a genetic construct capable of manipulating fructan biosynthesis in photosynthetic cells of a plant, said genetic construct including a light-regulated promoter, or functionally active fragment or variant thereof, operatively linked to nucleic acids encoding one or more fructan biosynthetic enzymes, or functionally active fragments or variants thereof.
In a still further aspect of the present invention there is provided a genetic construct capable of enhancing productivity of a biochemical pathway in a plant said genetic construct including nucleic acids encoding two or more enzymes from said pathway, or functionally active fragments or variants thereof.
Preferably, said nucleic acids are linked to form a fusion gene encoding a fusion protein of said two or more enzymes.
In preferred embodiments, the genetic constructs according to the various aspects of the present invention may be vectors.
By a 'vector' is meant a genetic construct used to transfer genetic material to a target cell.
The vector may be of any suitable type and may be viral or non-viral. The vector may be an expression vector. Such vectors include chromosomal, non-chromosomal and synthetic nucleic acid sequences, eg. derivatives of plant viruses; bacterial plasmids; derivatives of the Ti plasmid from Agrobacterium tumefaciens; derivatives of the Ri plasmid from Agrobacterium rhizogenes; phage DNA; yeast artificial chromosomes; bacterial artificial chromosomes; binary bacterial artificial chromosomes; vectors derived from combinations of plasmids and phage DNA. However, any other vector may be used as long as it is replicable or integrative or viable in the plant cell.
In a preferred embodiment of this aspect of the invention, the genetic construct may further include a terminator; said promoter, gene and terminator being operably linked.
The promoter, gene and terminator may be of any suitable type and may be endogenous to the target plant cell or may be exogenous, provided that they are functional in the target plant cell.
A variety of terminators which may be employed in the genetic constructs of the present invention are also well known to those skilled in the art. The terminator may be from the same gene as the promoter sequence or a different gene. Particularly suitable terminators are polyadenylation signals, such as the (CaMV)35S polyA and other terminators from the nopaline synthase (nos) and the octopine synthase (ocs) genes.
The genetic construct, in addition to the promoter, the gene and the terminator, may include further elements necessary for expression of the nucleic acid, in different combinations, for example vector backbone, origin of replication (ori), multiple cloning sites, spacer sequences, enhancers, introns (such as the maize Ubiquitin Ubi intron), antibiotic resistance genes and other selectable marker genes [such as the neomycin phosphotransferase (nptll) gene, the hygromycin phosphotransferase (hph) gene, the phosphinothricin acetyltransferase (bar or pat) gene], and reporter genes (such as beta- glucuronidase (GUS) gene (gusA)]. The genetic construct may also contain a ribosome binding site for translation initiation. The genetic construct may also include appropriate sequences for amplifying expression.
In particular, the genetic construct may further include a nucleic acid sequence encoding a linker between the two linked nucleic acids, as hereinbefore described.
Those skilled in the art will appreciate that the various components of the genetic construct are operably linked, so as to result in expression of said nucleic acid. Techniques for operably linking the components of the genetic construct of the present invention are well known to those skilled in the art. Such techniques include the use of linkers, such as synthetic linkers, for example including one or more restriction enzyme sites.
Preferably, the genetic construct is substantially purified or isolated. By 'substantially purified' is meant that the genetic construct is free of the genes, which, in the naturally- occurring genome of the organism from which the nucleic acid or promoter of the invention is derived, flank the nucleic acid or promoter. The term therefore includes, for example, a genetic construct which is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote; or which exists as a separate molecule (eg. a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. It also includes a genetic construct which is part of a hybrid gene encoding additional polypeptide sequence. Preferably, the substantially purified genetic construct is at least approximately 90% pure, more preferably at least approximately 95% pure, even more preferably at least approximately 98% pure.
The term "isolated" means that the material is removed from its original environment (eg. the natural environment if it is naturally occurring). For example, a naturally occurring nucleic acid present in a living plant is not isolated, but the same nucleic acid separated from some or all of the coexisting materials in the natural system, is isolated.
Such nucleic acids could be part of a vector and/or such nucleic acids could be part of a composition, and still be isolated in that such a vector or composition is not part of its natural environment.
As an alternative to use of a selectable marker gene to provide a phenotypic trait for selection of transformed host cells, the presence of the genetic construct in transformed cells may be determined by other techniques well known in the art, such as PCR (polymerase chain reaction), Southern blot hybridisation analysis, histochemical assays (e.g. GUS assays), thin layer chromatography (TLC), northern and western blot hybridisation analyses.
Applicant has also found that the methods of the present invention may result in enhanced biomass in the transformed plant relative to an untransformed control plant. This enhanced biomass may in turn be used as a selection tool for identifying transformed plants. This has the advantage that in some circumstances there may be no need to include an antibiotic resistance or other marker to select for transformants, where subsequent removal of such markers (and for the creation of marker-free plants) may present difficulties.
By 'enhancing biomass' or 'enhanced biomass' is meant enhancement of, increase in, or increased stability of biomass yield, including shoot and/or root growth, in a transformed plant relative to an untransformed control plant. For example, one or more growth characteristics selected from the group consisting of plant height, herbage dry weight, total leaf area, cumulative leaf area, leaf growth dynamics (ie. number of leaves over time), number of shoots, number of tillers, number of roots, root mass or weight, shoot mass or weight, root length, shoot length, stolon length, number of tubers, tuber weight, number of flowers, number of fruits, number of seeds, seed weight, fruit weight, percentage of flowering plants and seed yield per flower or per area sown; may be enhanced, increased or more stable in a transformed plant relative to an untransformed control plant.
This technique is particularly applicable to plants that are substantially genetically uniform or genetically identical or exhibit small phenotype differences in biomass prior to transformation.
Accordingly, in a further aspect of the present invention, there is provided a method of enhancing biomass in a plant, said method including introducing into said plant an effective amount of a genetic construct including a promoter, or a functionally active fragment or variant thereof, operatively liked to nucleic acids encoding one or more fructan biosynthetic enzymes, or functionally active fragments or variants thereof. Preferably, the promoter is a light regulated promoter.
In a still further aspect of the present invention there is provided a method of enhancing biomass in a plant, said method including introducing into said plant an effective, amount of a genetic construct including nucleic acids encoding two or more enzymes from a biochemical pathway in said plant, or functionally active fragments or variants thereof.
In a still further aspect of the present invention, there is provided a method of enhancing biomass in a plant, said method including introducing into said plant effective amounts of genetic construct capable of manipulating fructan biosynthesis in photosynthetic cells of the plant and a genetic construct capable of manipulating senescence in the plant.
The genetic constructs may be introduced into the plant by any suitable technique, as hereinbefore described, and may be introduced concurrently, sequentially or separately.
Preferably the genetic construct capable of manipulating fructan biosynthesis includes a promoter, or a functionally active fragment or variant thereof, operatively linked to
> nucleic acids encoding one or more fructan biosynthetic enzymes, or functionally active fragments or variants thereof. Preferably the promoter is a light regulated promoter.
Preferably the genetic construct capable of manipulating senescence in the plant is capable of manipulating senescence in photosynthetic cells of the plant. Preferably the genetic construct capable of manipulating senescence includes a MYB gene promoter or modified MYB gene promoter, or a functionally active fragment or variant thereof, operatively linked to a gene encoding an enzyme involved in biosynthesis of a cytokinin, or a functionally active fragment or variant thereof.
Suitable genetic constructs or vectors are described in International patent application PCT/AU01/01092 and United States patent application 11/789,526, the entire disclosures of which are incorporated herein by reference.
"Manipulating senescence" generally relates to delaying senescence in the transformed plant or cells or organs of the transformed plant, eg photosynthetic cells, relative to an untransformed control plant. However, for some applications it may be desirable to promote or otherwise modify senescence in the plant. Senescence may be promoted or otherwise modified for example, by utilizing an antisense gene.
The MYB gene promoter may be of any suitable type. Preferably the MYB gene promoter is a MYB32 gene promoter. Preferably the MYB gene promoter is from Arabidopsis, more preferably Arabidopsis thaliana. Most preferably the MYB gene promoter includes a nucleotide sequence selected from the group consisting of the sequence shown in Sequence ID No: 1 of PCT/AU01/01092 and functionally active fragments and variants thereof.
A suitable promoter is described in Li et al., Cloning of three MYB-like genes from Arabidopsis (PGR 99-138) Plant Physiology 121 :313 (1999), the entire disclosure of which is incorporated herein by reference.
By a "modified MYB gene promoter" is meant a promoter normally associated with a MYB gene, which promoter is modified to delete or inactivate one or more root specific motifs and/or pollen specific motifs in said promoter.
Preferably the modified MYB gene promoter is a modified MYB32 gene promoter. Preferably the modified MYB gene promoter is modified from the MYB gene promoter from Arabidopsis, or more preferably Arabidopsis thaliana.
A suitable promoter which may be modified according to the present invention is described in Li et al., Cloning of three MYB-like genes from Arabidposis (PGR 99-138) Plant Physiology 121 :313 (1999), the entire disclosure of which is incorporated herein by reference.
By a "root specific motif is meant a sequence of 3-7 nucleotides, preferably 4-6 nucleotides, more preferably 5 nucleotides, which directs expression of any associated gene in the roots of a plant.
Preferably the root specific motif includes a consensus sequence ATATT or AATAT.
By a "pollen specific motif is meant a sequence of 3-7 nucleotides, preferably 4-6 nucleotides, more preferably 4 or 5 nucleotides, which directs expression of an associated gene in the pollen of a plant.
Preferably the pollen specific motif includes a consensus sequence selected from the group consisting of TTTCT, AGAAA, TTCT and AGAA.
A root or pollen specific motif may be inactivated by adding, deleting, substituting or derivatizing one or more nucleotides within the motif, so that it no longer has the preferred consensus sequence.
Preferably the modified MYB gene promoter includes a nucleotide sequence selected from the group consisting of the sequences show in SEQ ID NOS: 2, 3 and 4 of US 11/789,526 and functionally active fragments and variants thereof.
By a "gene encoding an enzyme involved in biosynthesis of a cytokinin" is meant a gene encoding an enzyme involved in the synthesis of cytokinins such kinetin, zeatin and benzyl adenine, for example a gene encoding isopentyl transferase (IPT), or IPT-like gene such as the sho gene (eg. from petunia). Preferably the gene is an isopentenyl transferase (IPT) gene or sho gene. In a preferred embodiment, the gene is from a species selected from the group consisting of Agrobacteήum, more preferably Agrobactehum tumefaciens; Lotus, more preferably Lotus japonicus; and Petunia, more preferably Petunia hybrida.
Most preferably the gene includes a nucleotide sequence selected from the group consisting of the sequences shown in SEQ ID NOS: 5, 7 and 9 of US 11/789,526, sequences encoding the polypeptides shown in SEQ ID NOS: 6, 8 and 10 of US 11/789,526, and functionally active fragments and variants thereof.
The present invention also provides a method of selecting for transformed plants, said method including introducing into said plants an effective amount of a genetic construct including a promoter, or a functionally active fragment or variant thereof, operatively liked to nucleic acids encoding one or more fructan biosynthetic enzymes, or functionally active fragments or variants thereof and selecting plants with enhanced biomass. Preferably the promoter is a light regulated promoter.
In a further aspect of the present invention there is provided a transgenic plant cell, plant, plant seed or other plant part with modified fructan biosynthetic characteristics or enhanced biomass relative to an untransformed control plant.
By "modified fructan biosynthetic characteristics" is meant that the transformed plant exhibits increased fructan biosynthesis and/or contains increased levels of soluble carbohydrate relative to an untransformed control plant.
In a preferred embodiment the a transgenic plant cell, plant, plant seed or other plant part with enhanced biomass has an increase in biomass of at least approximately 10%, more preferably at least approximately 20%, more preferably at least approximately 30%, more preferably at least approximately 40% relative to an untransformed control plant.
For example, biomass may be increased by between approximately 10% and 300%, more preferably between approximately 20% and 200%, more preferably between approximately 30% and 100%, more preferably between approximately 40% and 80% relative to an untransformed control plant.
For example, plant height may be increased by between approximately 10% and 300%, more preferably between approximately 20% and 200%, more preferably between approximately 30% and 100%, more preferably between approximately 40% and 80% relative to an untransformed control plant.
For example, herbage dry weight may be increased by between approximately 10% and 600%, more preferably between approximately 20% and 400%, more preferably between approximately 30% and 300%, more preferably between approximately 40% and 200% relative to an untransformed control plant.
In a preferred embodiment, the transgenic plant cell, plant, plant seed or other plant part with modified fructan biosynthetic characteristics has an increase in soluble carbohydrate of least approximately 10%, more preferably at least approximately 20%, more preferably at least approximately 30%, more preferably at least approximately 40% relative to an untransformed control plant.
For example, soluble carbohydrates may be increased by between approximately 10% and 300%, more preferably between approximately 20% and 200%, more preferably between approximately 30% and 100%, more preferably between approximately 40% and 80% relative to an untransformed control plant.
For example, fructan concentration may be increased between approximately 10% and 600%, more preferably between approximately 20% and 400%, more preferably between approximately 30% and 200%, more preferably between approximately 40% and 150% relative to an untransformed control plant.
Preferably said plant cell, plant, plant seed or other plant part includes a genetic construct or vector according to the present invention. Preferably the transgenic plant cell, plant, plant seed or other plant part is produced by a method according to the present invention.
The present invention also provides a transgenic plant, plant seed or other plant part derived from a plant cell of the present invention and including a genetic construct or vector of the present invention.
The present invention also provides a transgenic plant, plant seed or other plant part derived from a plant of the present invention and including a genetic construct or vector of the present invention.
Preferably, the transgenic plant cell, plant, plant seed or other plant part is a Lolium species, more preferably Lolium perenne or Lolium arundinaceum. Preferably, the transgenic plant cell, plant, plant seed or other plant part is a cereal grain, more preferably a Triticum species, more preferably wheat (Triticum aestivum).
For example, the present invention enables the production of transgenic perennial ryegrass plants with increased fructans in leaf blades, vigorous growth and/or greater tolerance to abiotic stress, for improved nutrition for grazing animals.
The present invention also enables the production of transgenic wheat plants with increased fructans, vigorous growth, and/or tolerance to abiotic stress, for increased mass of usable carbohydrates, eg. for bio-fuel production or animal feed.
By 'plant cell' is meant any self-propagating cell bounded by a semi-permeable membrane and containing a plastid. Such a cell also requires a cell wall if further propagation is desired. Plant cell, as used herein includes, without limitation, algae, cyanobacteria, seeds suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen and microspores.
By 'transgenic' is meant any cell which includes a DNA sequence which is inserted by artifice into a cell and becomes part of the genome of the organism which develops from that cell. As used herein, the transgenic organisms are generally transgenic plants and the DNA (transgene) is inserted by artifice into either the nuclear or plastidic genome.
In a further aspect of the present invention there is provided a fusion protein comprising two or more enzymes of a biochemical pathway in a plant, or functionally active fragments or variants thereof.
By 'functionally active' in this context is meant that the fragment or variant has one or more of the biological properties of the corresponding protein from which the fragment or variant is derived. Additions, deletions, substitutions and derivatizations of one or more of the amino acids are contemplated so long as the modifications do not result in loss of functional activity of the fragment or variant. Preferably the fragment or variant has at least approximately 80% identity to the relevant part of the above mentioned sequence to which the fragment or variant corresponds, more preferably at least approximately 90% identity, more preferably at least approximately 95% identity, most preferably at least approximately 98% identity. Such functionally active variants and fragments include, for example, those having conservative amino acid substitutions of one or more residues in the corresponding amino acid sequence.
Preferably the fragment has a size of at least 10 amino acids, more preferably at least 20 amino acids, more preferably at least 50 amino acids, more preferably at least 100 amino acids, more preferably at least 200 amino acids.
Preferably, the biochemical pathway is in the fructan biosynthetic pathway.
Preferably, the two or more enzymes from said pathway are selected from the group consisting of enzymes of the fructan biosynthetic pathway in plants, for example fructosyltransferases such as sucrose:sucrose 1-fructosyltransferase (1-SST); fructan :fructan 1-fructosyltransferase (1-FFT); sucrose:fructan 6-fructosyltransferase (6- SFT); and fructan: fructan 6G-fructosyl transferase (6G-FFT); and fructoexohydrolases such as 1-fructoexohydrolase (1-FEH) and 6-fructoexohydrolase (6-FEH).
Even more preferably, the fusion protein is a FT fusion protein of 1-SST and 6G-FFT, or functionally active fragments or variants thereof.
Preferably, the two or more enzymes from said pathway correspond to enzymes from a ryegrass or fescue species, including Italian or annual ryegrass, perennial ryegrass, tall fescue, meadow fescue and red fescue. Even more preferably, the two or more enzymes from said pathway correspond to enzymes from a Lolium species such as Lolium perenne or Lolium arundinaceum.
Suitable fructan biosynthetic enzymes are described in PCT/AU01 /00705 and PCT/AU01/01275, the entire disclosures of which are incorporated herein by reference.
In a particularly preferred embodiment the 1-SST includes an amino acid sequence shown in SEQ ID NO: 12 of PCT/AU01 /00705, or a functionally active fragment or variant thereof.
In a particularly preferred embodiment the 6G-FFT includes an amino acid sequence shown in SEQ ID NO: 111 of PCT/AU01/01275 or Figure 8 hereto, or functionally active fragments or variants thereof. In a particularly preferred embodiment the 1-SST 6G-FFT FT fusion protein includes an amino acid sequence shown in Figure 13 or 15 hereto, or functionally active fragments or variants thereof.
Detailed description of the embodiments
The present invention will now be more fully described with reference to the accompanying examples and drawings. It should be understood, however, that the description following is illustrative only and should not be taken in any way as a restriction on the generality of the invention described above.
In the Figures:
Figure 1. Model for targeted expression of fructan biosynthesis genes in photosynthetic cells in leaf blades. Expression of fructosyl transferase (FT) genes is driven by photosynthetic promoters. Fructan biosynthesis then occurs in sucrose producing, photosynthetic cells. Pyramiding with modification of cytokinin biosynthesis to delay leaf senescence, thus extending life of photosynthetic cells that are engineered to synthesise fructans and leading to increased biomass production.
Figure 2. The expression of the RuBisCO Small subunit gene in perennial ryegrass is light regulated as shown by quantitative real-time RT-PCR. Tissue sampling occurred every four hours. Boxes represent periods of daylight.
Figure 3. In silico expression patterns of the Ribulose-1 ,5-bisphosphate carboxylase/oxygenase Small subunit (LpRbcS) and Chlorophyll a/b Binding Protein (LpCAB) in perennial ryegrass shows that it is most abundant in vegetative tissues. LpRbcS (contig LPCL9_C359) is represented by the 47 ESTs and LpRbcS (contig LPCL1112_C12) is represented by 19 ESTs.
Figure 4. Nucleotide sequences of LpCAB promoter (SEQ ID No: 1 ).
Figure 5. Nucleotide sequences of LpRbcS promoter (SEQ ID No: 2). Figure 6. Schematic representation of the fructan biosynthetic pathway in some grasses.
Figure 7. Nucleotide sequence of Lp6G-FFT open reading frame (SEQ ID No: 3).
Figure 8. Deduced amino acid sequence of Lp6G-FFT (SEQ ID No: 4).
Figure 9. Nucleotide sequence of LpI-FFT open reading frame (SEQ ID No: 5).
Figure 10. Deduced amino acid sequence of LpI-FFT (SEQ ID No: 6).
Figure 11. Diagrammatic representation of the strategy used to generate the translational FT fusion of the LpI-SST and Lp6G-FFT fructosyl transferase genes (Lp1- SST_Lp6G-FFT).
Figure 12. Nucleotide sequence of Lp1-SST_Lp6G-FFT FT fusion 1 open reading frame (SEQ ID No: 7).
Figure 13. Deduced amino acid sequence of Lp1-SSTJ_p6G-FFT FT fusion 1 (SEQ ID No: 8).
Figure 14. Nucleotide sequence of Lp1-SST_Lp6G-FFT FT fusion 3 open reading frame (SEQ ID No: 9).
Figure 15. Deduced amino acid sequence of Lp1-SST_Lp6G-FFT FT fusion 3 (SEQ ID No: 10).
Figure 16. Diagrammatic representation of the strategy to be used to generate the different translational FT fusions of the LpI-SST, Lp6G-FFT and LpI-FFT fructosyl transferase genes.
Figure 17. (A) and (B) Hypothetical model of the interaction of FT proteins to form a transmebrane protein. (C) Representation of the key protein domains in Lp1-SST-6G- FFT proteins. Box1 : (N/S)DPNG; Box2: RDP and Box3: EC represent the highly conserved domains involved in substrate (sucrose) binding and hydrolysis. Crosses (X) represent the highly conserved amino acid sequences (domains) found among the FT, invertase and FEH sequences from Lolium species. LS-large subunit, SU-Small subunit. Representation of the active domains within the amino acid sequence of the Lp1- SST_Lp6G-FFT FT fusion 3 protein can be found in Figure 36.
Figure 18. Amino acid alignment of FT, INV and FEH from Lolium perenne (SEQ ID
Nos: 11-33). The key protein domains found among the FT, invertase and FEH sequences, such as (N/S)DPNG, RDP and EC, which represent the highly conserved domains involved in substrate (sucrose) binding and hydrolysis, are bold underlined and labelled. Highly conserved amino acid domains found among the FT, invertase and FEH sequences from Lolium species are underlined. Representation of the active domains within the amino acid sequence of the Lp1-SST_Lp6G-FFT FT fusion 3 protein can be found in Figure 36.
Figure 19. Functional analysis of fructan:fructan 6G-fructosyltransferase (Lp6G-FFT). A. Plasmid map of Lp6G-FFT in the yeast expression vector. B. Excreted protein from yeast containing either pPICZαA::Lp6G-FFT or pPICZαA vector only separated by polyacrylamide gel electrophoresis. C. Water soluble carbohydrate (WSC) traces after high pressure anion exchange chromatography (HPAEC). WSC were isolated from onion, or solution of 1-kestose incubated with either Lp6G-FFT purified protein (pPICZαA::Lp6G-FFT) or vector only control (pPICZαA).
Figure 20. Base destination vector, pPZP200-ubi:bar-nos R4 R3, used in Multisite Gateway recombinational cloning.
Figure 21. Outline of the procedure for the in planta transient expression system. Agrobacterium cultures are prepared that harbour the expression constructs. These are injected into tobacco leaves. After three days post filtration expression of the proteins are tested. Upper right panel shows GUS activity, lower right panel shows example of water soluble carbohydrate separation by HPAEC.
Figure 22. High performance anion exchange chromatography (HPAEC) is used to separate and quantify carbohydrates using standards (1-kestose), and to quantify the amount of total fructans extracted from a control plant (35S::GUS) and transgenic plants transiently over-expressing LpI-SST (35S::1-SST), Lp6G-FFT (35S::6G-FFT) and the FT fusion (35S::Lp1-SST_Lp6G-FFT). Figure 23. Destination vectors of wheat RuBisCO promoter driving expression of (A) LpI-SST, (B) Lp6G-FFT, (C) Lp1SST_Lp6GFFT FT fusion 1 , (D) Lp1SST_Lp6GFFT FT fusion 3, and (E) the GUS marker gene.
Figure 24. Sequence of TaRbcS::l_p1-SST::TaRbcS expression cassette (SEQ ID No: 34). The regulatory sequences, TaRbcS promoter and terminator are indicated in italics and underlined italics, respectively. The ORF sequence is indicated in regular font and the start (ATG) and stop (TAG) codons are shaded.
Figure 25. Sequence of TaRbcS::Lp6GFFT::TaRbcS expression cassette (SEQ ID No: 35). The regulatory sequences, TaRbcS promoter and terminator are indicated in italics and underlined italics, respectively. The ORF sequence is indicated in regular font and the start (ATG) and stop (TAG) codons are shaded.
Figure 26. Sequence of TaRbcS::Lp1-SST_Lp6G-FFT::TaRbcS FT fusion 1 expression cassette (SEQ ID No: 36). The regulatory sequences, TaRbcS promoter and terminator are indicated in italics and underlined italics, respectively. The ORF sequence is indicated in regular font and the start (ATG) and stop (TAG) codons are shaded.
Figure 27. Sequence of TaRbcS::Lp1-SST_Lp6G-FFT::TaRbcS FT fusion 3 expression cassette (SEQ ID No: 37). The regulatory sequences, TaRbcS promoter and terminator are indicated in italics and underlined italics, respectively. The ORF sequence is indicated in regular font and the start (ATG) and stop (TAG) codons are shaded.
Figure 28. Vector pBlueScript SK harbouring the LpFT4 3' terminator sequence, pBS- LpFT4.
Figure 29. (A) The plasmid pBS-Lp1-SST::FT4 and (B) the plasmid pBS-LpRbcS::Lp1- SST::LpFT4.
Figure 30. (A) The plasmid pBS-LpCAB::LpFT4 and (B) the plasmid pBS-LpCAB::Lp6G- FFT::LpFT4.
Figure 31. Sequence of LpRbcS::Lp1-SST::LpFT4 expression cassette (SEQ ID No: 38). The regulatory sequences, LpRbcS promoter and LpFT4 terminator are indicated in italics and underlined italics, respectively. The ORF sequence is indicated in regular font and the start (ATG) and stop (TAG) codons are shaded.
Figure 32. Sequence of LpCAB::Lp6G-FFT::LpFT4 expression cassette (SEQ ID No: 39). The regulatory sequences, LpRbcS promoter and LpFT4 terminator are indicated in italics and underlined italics, respectively. The ORF sequence is indicated in regular font and the start (ATG) and stop (TAG) codons are shaded.
Figure 33. The plasmid PCR Blunt -Lp1-SST_Lp6G-FFT FT fusion.
Figure 34. Destination vectors containing the ryegrass RuBisCO (LpRbcs) promoter driving FT fusions 1 and 3. (A) pBS-LpRbcS::Lp1-SST_Lp6G-FFT::LpFT4 FT fusioni and (B) pBS-LpRbcS::Lp1-SST-Lp6G-FFT::LpFT4 FT fusion 3.
Figure 35. Sequence of LpRbcS::Lp1-SST_Lp6G-FFT::LpFT4 FT fusion 1 expression cassette (SEQ ID No: 40). The regulatory sequences, LpRbcS promoter and LpFT4 terminator are indicated in italics and underlined italics, respectively. The ORF sequence is indicated in regular font and the start (ATG) and stop (TAG) codons are shaded.
Figure 36. Sequence of LpRbcS::Lp1-SST_Lp6G-FFT::LpFT4 FT fusion 3 expression cassette (SEQ ID No: 41 ). The regulatory sequences, LpRbcS promoter and LpFT4 terminator are indicated in italics and underlined italics, respectively. The ORF sequence is indicated in regular font and the start (ATG) and stop (TAG) codons are shaded. The amino acid sequence is indicated in bold (SEQ ID No: 42). Domains are highlighted as follows: the boxes indicate the highly conserved motifs in the family of the 32 glycoside hydrolases including invertases, fructosyltransferases and fructan exohydrolases which are involved in substrate binding and hydrolysis: double underlines show trans-membrane domains; and shaded boxes represent conservative domains among 32 glycoside hydrolases.
Figure 37. Destination vector containing the Arabidopsis RuBisCO (AtRbcS) promoter driving FT fusion 3, pPZP200_AtRbcS::Lp1-SST_6G-FFT::nos FT fusion 3.
Figure 38. Sequence of the AtRbcS::Lp1-SST-6G-FFT::nos FT fusion 3 expression construct (SEQ ID No: 43). Figure 39. Details of the base vector pBlueScript SK(-) from Promega, with the positions of the restriction endonuclease sites for cloning indicated.
Figure 40. Vector backbone used for construction of p-Ubi::Lp1-SST::35S and p- Ubi::Lp6G-FFT::35S (Ye et a/., 2001 ).
Figure 41. Representative sequence of a constitutive (Ubi) promoter combined with a FT fusion protein and a terminator sequence (SEQ ID No: 44). The regulatory sequences, Ubi promoter and LpFT4 terminator are indicated in italics and underlined italics, respectively. The ORF sequence is indicated in regular font and the start (ATG) and stop (TAG) codons are shaded.
Figure 42. Representative sequence of a constitutive ((CAMV)35S2) promoter combined with a FT fusion protein and a terminator sequence (SEQ ID No: 45). The regulatory sequences, (CAMV)35S2 promoter and LpFT4 terminator are indicated in italics and underlined italics, respectively. The ORF sequence is indicated in regular font and the start (ATG) and stop (TAG) codons are shaded.
Figure 43. Representative sequence of a constitutive (RUBQ2) promoter combined with a FT fusion protein and a terminator sequence (SEQ ID No: 46). The regulatory sequences, RUBQ2i promoter and LpFT4 terminator are indicated in italics and underlined italics, respectively. The ORF sequence is indicated in regular font and the start (ATG) and stop (TAG) codons are shaded.
Figure 44. Representative sequence of a constitutive (OsActi ) promoter combined with a FT fusion protein and a terminator sequence (SEQ ID No: 47). The regulatory sequences, OsActi promoter and LpFT4 terminator are indicated in italics and underlined italics, respectively. The ORF sequence is indicated in regular font and the start (ATG) and stop (TAG) codons are shaded.
Figure 45. Representative sequence of a tissue specific (tuber) promoter (Cathlnh) combined with a FT fusion protein and a terminator sequence (SEQ ID No: 48). The regulatory sequences, Cathlnh promoter and LpFT4 terminator are indicated in italics and underlined italics, respectively. The ORF sequence is indicated in regular font and the start (ATG) and stop (TAG) codons are shaded. Figure 46. Representative sequence of a stress regulated (Atrd29a) promoter combined with a FT fusion protein and a terminator sequence (SEQ ID No: 49). The regulatory sequences, Atrd29a promoter and LpFT4 terminator are indicated in italics and underlined italics, respectively. The ORF sequence is indicated in regular font and the start (ATG) and stop (TAG) codons are shaded.
Figure 47. Representative sequence of a sucrose regulated (16R) promoter combined with a FT fusion protein and a terminator sequence (SEQ ID No: 50). The regulatory sequences, 16R promoter and LpFT4 terminator are indicated in italics and underlined italics, respectively. The ORF sequence is indicated in regular font and the start (ATG) and stop (TAG) codons are shaded.
Figure 48. Plant regeneration phenotypes of transgenic perennial ryegrass after co- transformation with the TaRbcS promoter light-regulated gene constructs (Table 1) and the pAcH1 vector, with selection on hygromycin. The plants that contain either of the TaRbcS::Lp1-SST_Lp6G-FFT::TaRbcS FT fusion constructs show growth advantage under in vitro culture conditions thus allowing for their early identification and screening (far right column).
Figure 49. Plant regeneration phenotypes of transgenic perennial ryegrass after co- transformation with the LpRbcS promoter light-regulated gene constructs with selection on hygromycin. The plants contain either the LpRbcS::Lp1-SST::LpFT4 or the LpRbcS::Lp1-SST_Lp6G-FFT::LPFT4 FT fusion 1 / 3 constructs. The plants that contain the FT fusion constructs show growth advantage under in vitro culture conditions
Figure 50. Mature plant phenotypes under glasshouse conditions. Representative samples of transgenic perennial ryegrass plants at the vegetative stage. The TaRbcS::Lp1-SST_Lp6G-FFT::LpFT4 FT fusion transgenic perennial ryegrass plants show enhanced growth performance with larger leaves, enhanced tillers, increased root growth compared to control, non-transgenic perennial ryegrass plants. The plants were trimmed equally three weeks earlier. Close up micrographs of the leaf blades indicate and increase leaf diameter in FT fusion transgenics. Figure 51. Representative samples of transgenic perennial ryegrass mature plant phenotypes (4 weeks) under field conditions. The FT fusion transgenic perennial ryegrass plants show enhanced growth performance with larger leaves, enhanced tillers, increased root growth compared to control LpI-SST transgenic perennial ryegrass plants.
Figure 52. Representative examples of phenotypic biomass scores (1- least biomass to 5- most biomass) of transgenic perennial ryegrass plants expressing FT fusion transgenes under field conditions.
Figure 53. Leaf phenotypes of transgenic perennial ryegrass. Representative samples of hand sections of leaf blades at vegetative stage. Left shows comparison of whole leaf sections, right magnified areas of leaf sections. Ad-Adaxial, Ab-abaxial.
Figure 54. Biochemical analysis (HPAEC) of fructan level and composition present in stable transgenic TaRbcS::Lp1-SST_Lp6G-FFT::TaRbcS FT fusion 3, TaRbcS::Lp1- SST::TaRbcS, TaRbcS::Lp6G-FFT::TaRbcS perennial ryegrass plants and control perennial ryegrass plants harbouring only the selectable marker (hph gene).
Figure 55. Biochemical analysis (HPAEC) of total fructans present in whole tillers of (A) TaRbcS::Lp1-SST_Lp6G-FFT::TaRbcS FT fusion 1 , (B) TaRbcS::Lp1-SST_Lp6G- FFT::TaRbcS FT fusion 3, (C) TaRbcS::Lp1-SST::TaRbcS, and (D) TaRbcS::6G- FFT::TaRbcS transgenic perennial ryegrass plants compared to control perennial ryegrass plants (lanes 6' and 1'), harbouring only the selectable marker (hph gene).
Figure 56. Biochemical analysis (HPAEC) of 1-kestose present in whole tillers of (A) TaRbcS::Lp1-SST_Lp6G-FFT::TaRbcS FT fusion 1 , (B) TaRbcS::Lp1-SST_Lp6G- FFT::TaRbcS FT fusion 3, (C) TaRbcS::Lp1-SST::TaRbcS, and (D) TaRbcS::6G- FFT::TaRbcS transgenic perennial ryegrass plants compared to control perennial ryegrass plants (lanes 6' and 1'), harbouring only the selectable marker (hph gene).
Figure 57. Biochemical analysis (HPAEC) of sucrose present in whole tillers of (A) TaRbcS::Lp1-SST_Lp6G-FFT::TaRbcS FT fusion 1 , (B) TaRbcS::Lp1-SST_Lp6G- FFT::TaRbcS FT fusion 3, (C) TaRbcS::Lp1-SST::TaRbcS, and (D) TaRbcS::6G- FFT::TaRbcS transgenic perennial ryegrass plants compared to a control perennial ryegrass plants (lanes 6' and 1'), harbouring only the selectable marker (hph gene).
Figure 58. Fructan levels in whole tillers and leaf blades in wild-type (control) and FT fusion and LpRbcS ::Lp1-SST transgenic perennial ryegrass plants grown under field conditions and harvested in December 2009.
Figure 59. Fructan composition in leaf blades of wild-type and LpRbcS::Lp1-SST transgenic perennial ryegrass plants grown under field conditions. Box 1 represents low DP fructan (DP up to 10-15). Box 2 represents high DP fructan (DP higher than 10-15).
Figure 60. Transgene expression in whole tillers of LpRbcS FT fusion and LpRbcS::Lp1-SST transgenic perennial ryegrass plants grown under field conditions. Samples were collected in November (white bars) and December (black bars) 2009. Samples were normalised against endogenous histone expression and are presented as number of transcript copies per 35ng of RNA.
Figure 61. Phenotypic analysis of the transgenic perennial ryegrass after 7 weeks (A-C) and 12 weeks (D-E) propagation in potting mix from a single tiller. TarbcS::Lp1- SST_Lp6G-FFT::Tarbcs FT fusion 1 (A, D) and TarbcS::Lp1-SST_Lp6G-FFT::Tarbcs FT fusion 3 (B) plants, show greater leaf length and number of tillers in fusion plants compared to the control plants expressing only the hph gene (C, E)
Figure 62. Quantitative phenotypic analysis of the transgenic TarbcS::Lp1-SST_Lp6G- FFT::Tarbcs FT fusion 1 and TarbcS::Lp1-SST_Lp6G-FFT::Tarbcs FT fusion 3 plants after 7 weeks (white bars) and 12 weeks (black bars) growth. Measurements were conducted for plant height (A), leaf width (B) and tiller number (C) compared to the average of 8 control plants expressing only the hph gene.
Figure 63. Transgenic perennial ryegrass plants expressing LXR® technology alone (AtMYB32::IPT), LpRbcS::Lp1-SST_Lp6G-FFT::LpFT4 FT fusion 3 alone, as well as LXR® and LpRbcS::Lp1-SST_Lp6G-FFT::LpFT4 FT fusion 3 together under glasshouse conditions. Figure 64. Herbage dry weight analysis of GOI-ve control (average of five lines) and independent FT fusion alone or the FT fusion plus LXR® transgenic perennial ryegrass plants, grown under glasshouse conditions and collected 6 weeks post-trim,.
Figure 65. Fructan levels in leaf blades of GOI-ve control (average of five lines) and independent FT fusion alone or FT fusion plus LXR® transgenic perennial ryegrass plants, grown under glasshouse conditions.
Figure 66. Transgenic tall fescue plants expressing LpRbcS::l_p1-SST_Lp6G- FFT::LpFT4 FT fusion 3 under glasshouse conditions.
Figure 67. Herbage dry weight analysis of glass house grown GOI-ve control (average of five lines) and independent FT fusion alone or FT fusion plus LXR® transgenic tall fescue plants.
Figure 68. Tiller number of glass house grown GOI-ve control (average of five lines) and independent FT fusion alone or FT fusion plus LXR® transgenic tall fescue plants.
Figure 69. Fructan accumulation in leaf blades of glass house grown GOI-ve control (average of five lines) and independent transgenic tall fescue lines expressing the FT fusion.
Figure 70. Plant regeneration phenotypes of transgenic wheat plants after transformation with the light-regulated gene constructs. The transgenic wheat plants growing in vitro that contain the Lp1-SST_Lp6G-FFT FT fusion construct show growth advantage under in vitro culture conditions thus allowing for their early identification and screening. The superior growth phenotype of the transgenic wheat FT fusion lines was observed during the early stages of in vitro plant regeneration conducted on tissue culture plates. Six weeks after incubation under light conditions the calli showed further developed in vitro growing tillers/shoots (panel A) and more specifically further developed in vitro growing roots (panel B) in the transgenic wheat plants growing in vitro that contain the Lp1-SST_Lp6G-FFT FT fusion construct compared to the control plants.
Figure 71. The transgenic wheat plants that contain the TaRbcS::Lp1-SST_Lp6G- FFT::TaRbcS FT fusion construct showed an obvious early increase in tiller number as compared to control plants growing under (A) 2 months in in vitro conditions and (B) under glasshouse conditions.
Figure 72. Transgenic wheat plants that contain FT fusion constructs showed an obvious early increase in tiller number as compared to control plants growing under glasshouse conditions.
Figure 73. The transgenic wheat plants that contain LXR® technology showed an obvious early increase in tiller number as compared to control plants under glasshouse conditions (A). They also showed and increase of photosynthetic tissue after 35 days under glasshouse conditions (B).
Figure 74. Phenotypic analysis of transgenic wheat plants expressing LXR® technology alone (AtMYB3::IPT::35S), TaRbcS::Lp1-SST_l_p6G-FFT::TaRbcS FT fusion 3 alone, as well as LXR® and TaRbcS::Lp1-SST_Lp6G-FFT::TaRbcS FT fusion 3 together under glasshouse conditions.
Figure 75. Fructan accumulation and tiller number in transgenic wheat plants containing either FT fusion constructs alone or LXR® plus FT fusion constructs, as compared to transformed gene of interest minus (GOI-) controls.
Figure 76. Fructan accumulation in T1 GOI-ve control, FT fusion alone and LXR® plus FT fusion transgenic wheat plants nine weeks after sowing. The fructan level in the control represents the data average obtained from six GOI-ve plants.
Figure 77. Phenotype of transgenic white clover plants expressing LXR®, AtRbcS::Lp1- SST-6G_FF::nos FT fusion or LXR® plus AtRbcS::Lp1-SST-6G_FF::nos FT fusion constructs as compared to transformed GOI minus controls.
Figure 78. Transgene expression levels of the FT fusion transgene driven by the AtRbcS promoter in white clover plants. Controls were wild type plants. Samples were normalised against endogenous histone expression and are presented as number of transcript copies per 35ng of RNA.
Figure 79. Fructan accumulation in wild-type control, AtRbcS FT fusion and AtRbcS FT fusion plus LXR® transgenic white clover lines. Figure 80. Phenotype of transgenic Arabidopsis plants expressing LXR®, AtRbcS::Lp1- SST-6G_FF::nos FT fusion or LXR® plus AtRbcS::Lp1-SST-6G_FF::nos FT fusion constructs as compared to transformed GOI minus controls.
Figure 81. Transgene expression levels of the FT fusion transgene driven by the AtRbcS promoter in Arabidopsis plants. Controls were wild type plants. Samples were normalised against endogenous histone expression and are presented as number of transcript copies per 35ng of RNA.
Figure 82. Transgenic T2 FT fusion Arabidopsis plants grown in soil.
Figure 83. Leaves from A. white clover, B. canola and C. wheat plants displaying delayed leaf senescence (leaves from LXR® transgenic plants, lower images) as compared to negative control plants (leaves from control plants, upper images) 7 - 20 days following detachment of leaves from plants.
Figure 84. Positive selection of perennial ryegrass transgenic plants by screening of in vitro growth phenotype on plates without antibiotic selection. A-C. CaIIi in dark for 8 weeks after transformation; D-F. 1 week after transfer to light.
Figure 85. Embryogenic perennial ryegrass calli bombarded with gold particles alone (control) and gold particles covered with TaRbcS FT fusion vector prior to, and four weeks post, transfer to light.
Figure 86. Embryogenic perennial ryegrass calli bombarded with gold particles alone (control) and gold particles covered with TaRbcS FT fusion 1 alone, TaRbcS FT fusion
3 alone, LXR alone, as well as TaRbcS FT fusion plus LXR vectors five weeks after transfer to light. Molecular analysis positive lines: TaRbcS FT fusion 1 # 1 , 2, 3, 4, 7, 6,
12, 13, 14, 16, 17; TaRbcS FT fusion 3 # 1 , 2, 3, 5, 8, 10, 11 , 12, 13; TaRbcS FT fusion
1 plus LXR # 1 , 2, 7, 12 (TaRbcS FT fusion 1 alone); # 8, 14 (TaRbcS FT fusion 1 plus LXR). Example 1
Isolation of photosynthetic promoters
Cloning of a photosynthetic promoter from bread wheat
The Ribulose-1 ,5-bisphosphate carboxylase/oxygenase Small subunit (RbcS) is a well- characterised light-regulated gene in higher plants. The bread wheat (Triticum aestivum), TaRbcS regulatory sequences (promoter and terminator) have previously been cloned (Zeng, et al., 1995; Sasanuma, 2001 ). A 695 bp promoter fragment from sequence previously published containing the TATA signal from the TaRbcS gene (NCBI accession number AB042069) was PCR-amplified.
Cloning of a photosynthetic promoter from Arabidopsis
A 1700 bp fragment of the Arabidopsis thaliana Ribulose-1 ,5-bisphosphate carboxylase/oxygenase Small subunit (AtRbcS) promoter sequence has previously been cloned. Primers will be designed to amplify a smaller fragment containing the TATA signal from the AtRbcS promoter for use in expression vectors.
Discovery and cloning of photosynthetic promoters from perennial ryegrass
The expression of RbcS and Chlorophyll a/b Binding Protein (CAB) are well characterised light-regulated genes in higher plants. The abundance of LpRbcS mRNA transcripts in perennial ryegrass by quantitative real time PCR is illustrated in Figure 2.
Both LpRbcS and LpCAB genes were chosen for promoter discovery and isolation in perennial ryegrass. Publicly available cDNA sequences (LpRbcS, EC778430 and LpCAB, EC778438) were used as query sequences in a BLAST search of the perennial ryegrass EST database in our in-house database. As both genes are members of multigene families, several contigs (each contig represents an individual gene) were identified in our perennial ryegrass EST collection. Nine contigs were identified to be homologous to the published LpRbcS cDNA sequence and thirteen contigs were found to be homologous to the LpCAB cDNA sequence. Two contigs, LPCL9_C359 (LpRbcS) and LpCL1112_C12 (LpCAB), representing the genes of the promoters to be isolated, contained (47) and (19) EST sequences, respectively. These sequences came from a variety of libraries representing a range of different tissues. This data was used for in silico expression analysis and indicated that both genes are only expressed in photosynthetic tissues (Figure 3).
DNA sequence alignments for each of the gene family members were performed, and gene-specific primers were designed for contigs LpRbcS_C359 and LpCAB_C12 and used to screen perennial ryegrass BAC DNA pools by PCR. The BAC clones were identified and sequenced. Primers were designed and the Lolium perenne specific promoter regulatory sequences were cloned, sequenced (Figures 4 and 5) and the cis- regulatory sequences specific for photosynthetic promoters were identified by PLACE (http://www.dna.affrc.go.jp/PLACE/) (Table 1). The sequences included the I-Box motif and the GT1 box for RbcS (Terzaghi, et al., 1995; Martinez-Hernandez, et al., 2002). In addition 16/ 19 nucleotides of the LpRbcS sequence shared homology with the monocot RbcS Consensus sequence (Schaffner, et al., 1991 ). The I-Core box and SORLIPs cis- regulatory sequences were present in the CAB promoter. SORLIPs were found to be over-represented in light-induced promoters in Arabidopsis (Hudson, et al., 2003).
Table 1. The position of the c/s-regulatory sequences identified by the PLACE database. Common cis-acting regulatory sequences are listed (Schaffner, et al., 1991 ; Terzaghi, et al., 1995; Martinez-Hernandez, et al., 2002; Hudson, et al., 2003). Positions noted are the first nucleotide in the sequence relative to the ATG. (n.p. - not present).
Figure imgf000041_0001
These L. perenne specific promoter regulatory sequences were subsequently used in the construction of backbone-free expression cassettes with fructan biosynthesis genes. Example 2
Isolation of fructan biosynthesis genes
Isolation of fructan biosynthesis genes from Lolium perenne
The Lolium perenne cDNA clones encoding sequences for LpI-SST and Lp6G-FFT have previously been isolated from a perennial ryegrass cDNA library (Chalmers, et al., 2003; Chalmers, et al., 2005). The complete gene sequences of the isolated perennial ryegrass fructosyltransferase homologues are available, and nucleotide and protein sequences for LpI-SST are disclosed in International patent application PCT AU01/00705 (SEQ ID NOS 11 and 12). Partial sequence for Lp6G-FFT is disclosed in International patent PCT/AU/01/01275 SEQ IDs 109 and 110, for nucleotide and amino acid sequences respectively. The full- length clone was PCR amplified from a cDNA, cloned and sequenced (Figure 7). When the Lp6G-FFT ORF was compared with the published Lp6G-FFT from L. perenne 23 nucleotide changes were noted. Comparison of the predicted protein sequences revealed only two changes between the two amino acid sequences (Figure 8).
Other FT genes that may be used and, either transformed singly or co-transformed with LpI-SST and Lp6G-FFT include LpI-FFT, Lp6-SFT and Lp6-SST. The cDNA sequence for LpI-FFT has been isolated from perennial ryegrass (Figure 9) and the amino acid sequence is represented in Figure 10. As an example, primers based on this sequence could be used to amplify the full-length cDNA by PCR for cloning and use in the present invention as described below.
Other homologous proteins can be found by screening databases such as EMBL (http://www.ebi.ac.uk/Tools/index.htm) or the National Center for Biotechnology Information (NCBI, http://www.ncbi.nlm.nih.gov/blast/Blast.cgitf). In such a database search, for example the sequences described in Figures 7 - 10 are set as a query, using default parameter settings set for the database. For example, for protein sequence alignments (Blastp) with NCBI these settings are as follows: limit entrez=not activated; filter=low complexity activated; expect value=10; word size=3; matrix=BLOSUM; gapcostsexistence-11 ,extension=1. Such database searches can be used for finding proteins with domains contained in FTs (using default parameters). Example 3
Creation of translational FT fusion proteins
Cloning of FT translational FT fusion
A genetic FT fusion was created between the open reading frames for LpI-SST and Lp6G-FFT, following the procedure depicted in Figure 11. The LpI-SST gene was PCR-amplified with a GATEWAY recombination site incorporated in the forward primer. A sequence that codes for three glycine residues followed by a Hind III site was incorporated into the reverse primer, with the stop codon removed. The Lp6G-FFT gene was PCR-amplified with a Hind III site followed by sequence that codes for three glycine residues and the gene specific sequence without the ATG. The reverse primer for the Lp6G-FFT gene was flanked by a second GATEWAY recombination site. The primer sequences are provided in Table 2. The purified fragments were digested with Hind III and the ligated product was cloned into the Invitrogen GATEWAY pDONR221 Entry vector. When the resultant pENTRY1-Lp1-SST-Lp6G-FFT-2 entry clones were sequenced, one sequence (FT fusion 1 ) was confirmed to be the predicted product, with eight amino acids in the linker joining the two genes (Figures 12 and 13). Whereas, another sequence (FT fusion 3) contained two consecutive Hind III sites, which would result in the addition of another two amino acids, giving a total of ten amino acids between the two FT genes upon translation (Figures 14 and 15). Table 2. Primer sequences used to amplify the PCR fragments used to generate the translational FT fusion of the LpI-SST and Lp6G-FFT fructosyltransferase genes (FT
Lp1-SST_Lp6G-FFT). SJlajjej sequences are gene-specific, bold and underlined (Hind III RE site) sequences are nucleotides introduced to generate the linker region, and italic nucleotides represent the recombination-specific sequences.
Figure imgf000043_0001
By using the primer sequences outlined in Table 3 it is possible to create a new FT fusion reversing the order to Lp6G-FFT-Lp1SST using the same method as illustrated above.
Table 3. Primer sequences used to amplify the PCR fragments used to generate the translational FT fusion of the LpI-SST and Lp6G-FFT fructosyl transferase genes (Lp6G-FFT-Lp1-SST). j|JSj|| sequences are gene-specific, bold and underlined (Hind III RE site) sequences are nucleotides introduced to generate the linker region, and italic nucleotides represent the recombination-specific sequences.
Figure imgf000044_0001
In Lp1-SST_Lp6G-FFT the FT proteins physically associate with each other to form a FT fusion protein which contains three transmembrane domains as designated by SOSUI, a classification and secondary structure prediction of membrane proteins database (Table 4, Figures 17 and 18).
Table 4. FT fusion 1 / 3 transmembrane domains as indicated by SOSUI, a Classification and Secondary Structure Prediction of Membrane Proteins database (http://bp.nuap.nagoya-u.ac.ip/sosui/sosui submit.html)
Figure imgf000045_0001
Structural features of plant fructosyltransferases
Plant FTs have a high degree of amino acid homology to the vacuolar, acid invertases (b- fructosidases) which are the members of the glycoside hydrolase family 32. (GH32) and share three highly conserved regions characterised by the motifs (N/S)DPNG (also called b-fructosidase motif), RDP, and EC (Altenbach et al., 2005) (Figures 17, 18 and 36). Another common feature of plant FTs and vacuolar invertases is that they usually are composed of a large and a small subunit due to posttranslational processing. The large subunit, which harbours all three conserved motifs mentioned above, determines the catalytic specificity (Altenbach et al., 2004).
The other FT genes LpI-FFT, Lp6-SFT and Lp6-SST may also be used in combination with LpI-SST or Lp6G-FFT to produce a selection of translational FT fusions, by the scheme outlined in Figure 16A, as indicated below.
Lp6G-FFT::Lp1-SST
Lp1-SST::(Lp1-FFT/ Lp6-SFT/Lp-SST)
(LpI-FFT/ Lp6-SFT/Lp-SST)::Lp1-SST A triplicate FT fusion could also be created using a similar methodology (Figure 16B). It is proposed that the triplicate fusion would be constructed to incorporate the genes Lp1- SST, Lp6G-FFT and LpI-FFT, Lp6-SFT or Lp6-SST. By altering the primer sequences used to join the two FT genes together it is possible to change the linker size and potentially add up to approximately 30 amino acids. FT proteins could physically associate with each other to form a metabolic channel, therefore the distance separating the FT genes within the translational fusion may affect protein function. FT fusion proteins preferably contain the sequences which represent the domains which are highly conserved among the FT, INV and FEH proteins from Lolium perenne plants indicated in Figures 17, 18 and 36.
Example 4
Transient assays of fructan biosynthesis gene function
Function of LpI-SST, Lp6G-FFT and FT fusion protein
The cDNA sequence encoding the LpI-SST mature protein has been previously expressed in Pichia pastoris for functional characterisation (Chalmers, et al., 2003) and the conversion of sucrose to 1-kestose by expression of this protein was demonstrated.
Similarly, the Lp6G-FFT cDNA was cloned into the expression vector pPICZαA
(Invitrogen) that contains a methanol-inducible promoter and the Saccharomyces cerevisiae α-factor sequence to enable secretion of the recombinant protein for isolation for functional characterisation. The recombinant Lp6G-FFT enzyme was produced from single colonies of transformed P. pastoris inoculated into a pre-culture medium and induced by the addition of methanol for a 45 hr duration. The supernatant was concentrated and samples were incubated with 100 mM sucrose overnight. The carbohydrates produced were analysed by HPAEC according to Chalmers et al., 2003, using fructan extracts from onion as a control (Figure 19).
Generation of vectors for transient gene expression assays
A number of vectors were constructed using Invitrogen Multisite Gateway™ technology
(see www.lnvitoqen.com for product manual) based on recombinational cloning. This methodology relies on the generation of individual Entry plasmids containing, either the promoter, gene of interest (GOI), or terminator sequences flanked by recombination sites. The recombination sites facilitate the directional triple insertion of each of the Entry plasmids into a Gateway-enabled destination vector, by recombination. The final vector is then sequenced and used directly for plant co-transformation with a plasmid, or expression cassette, for expression of a plant selectable marker.
In order to test the function of the FT fusion protein, the FT fusion 1 and 3 ORFs were cloned under the control of the enhanced cauliflower mosaic virus (CAMV)35S2 promoter (Kay, et al., 1987), using the Multisite Gateway™ Technology recombination system (see www.lnvitroqen.com for product manual) into Agrobacterium binary vector (Figure 21) (Hajdukiewicz, et al., 1994).
Gateway Entry vectors were constructed for the (CAMV)35S2 promoter, the TaRbcS terminator sequence, as well as FT fusion 1 and 3 ORFs. The cloned fragments were sequence-verified and used for three-way recombination cloning with the cloned GOI cDNA sequences into the pPZP200-ubi:bar-nos R4 R3 destination vector. In addition, constructs also included the Lp6G-FFT and LpI-SST single ORF driven by the (CAMV)35S2 promoter as controls. As an example, the LpI-FFT (or Lp6-SFT, Lp6- SSJ) single ORF is also cloned in the same manner. As a control the GUS ORF was used for confirmation of expression. The following constructs were made.
pPZP200-35S2::Lp6G-FFT::TaRbcS
pPZP200-35S2::Lp1 -SST::TaRbcS
• pPZP200-35S2::(Lp1-FFT/ Lp6-SFT/Lp-SST)::TaRbcS
pPZP200-35S2::Lp1-SST::6G-FFT::TaRbcS (FT fusion 1 and 3)
pPZP200-35S2::GUS::TaRbcS
Utilising Invitrogen Multisite Gateway™ Technology the following vectors are created containing the Atrbcs photosynthetic promoter and the (CAMV)35S terminator for use in transient assays.
pPZP200-AtrbcS::Lp1-SST::35S
pPZP200-AtrbcS:: Lp6G-FFT::35S pPZP200-AtrbcS::(Lp1-FFT/ Lp6-SFT/Lp-SST)::35S
pPZP200-AtrbcS::Lp1-SST::6G-FFT::35S (FT fusion 1 and 3)
Function of LpI-SST, Lp6G-FFT and FT fusion protein in transient transgene expression assays For proof-of-f u notion transient expression of the constructs containing chimeric Lp1- SST, Lp6G-FFT and FT fusion protein genes driven by the (CaMV)35S promoter was conducted in tobacco plants, as they do not naturally produce fructans. The method involved Agro-infiltration of the individual constructs into N. benthamiana leaves (Kapila, et al., 1997; Wydro, et al., 2006) followed by biochemical analysis by anion exchange chromatography. A diagram of the transient expression procedure is illustrated in Figure 21. Three days after the injection the plant material was harvested and the water- soluble carbohydrates were extracted using a hot water extraction method. The extracts were separated using high performance anion exchange chromatography (HPAEC). The results show production of fructans, with the increased production of both 1-kestose and 6G-kestose by the FT fusion protein (Figure 22). An equivalent experiment is used to assess the function constructs containing chimeric LpI-SST, Lp6G-FFT and FT fusion protein genes driven by the AtRbcS promoter.
Agro-infiltration using a combination of vectors for transcriptional co- transformation To assess the function of the fructan biosynthesis when transcriptionally co-ordinated together in a cell, triple agro-infiltration experiments are conducted using the groups of vectors outlined below. The transient expression procedure as illustrated in Figure 21 is used to insert three vectors together in the same plant tissue. Three days after the injection, the plant material is harvested and the water-soluble carbohydrates extracted using a hot water extraction method. The extracts are separated using high performance anion exchange chromatography (HPAEC). The results indicate the differences resulting from the independent expression of three fructan biosynthesis genes in the plant genome. pPZP200-35S2::Lp6G-FFT::TaRbcS +
pPZP200-35S2::l_p1-SST::TaRbcS +
pPZP200-35S2::(Lp1-FFT/ Lp6-SFT/Lp-SST)::TaRbcS
pPZP200-AtRbcS::Lp1-SST::35S +
• pPZP200-AtRbcS:: Lp6G-FFT::35S +
pPZP200-AtRbcS::(Lp1-FFT/ Lp6-SFT/Lp-SST)::35S
Agro-infiltration using a FT fusion vectors for translational co-transformation
By comparison to the transcriptional co-transformation experiments are conducted to compare translational co-transformation by conducting transient assays with the vectors that have previously been discussed and are indicated below.
pPZP200-35S2::l_p1-SST_6G-FFT::TaRbcS (FT fusion 1 and 3)
pPZP200-AtRbcS::l_p1-SST_6G-FFT::35S (FT fusion 1 and 3)
Example 5
Generation of vectors for stable transformation and production of transgenic plants
Production of LXR® vector for biolistic and Agrobacterium-med'tated transformation
LXR® technology is based on vectors containing one cytokinin biosynthesis gene encoding isopentenyl transferase (IPT) for delayed leaf senescence under the control of the AtMYB32 gene promoter. The LXR® vector for biolistic transformation was constructed utilising Gateway™ Multisite technology. Details of the binary vector pBS- ubi::bar::nos_AtMYB32_IPT_35S are described in International patent application PCT/AU01/01092.
The production of the LXR® vectors for Agrobacterium-med iated transformation is disclosed in International patent application PCT/AU01/01092. The candidate gene constructs were fully sequenced and the vectors were generated for Agrobacteήum mediated transformation following strict quality assurance protocols.
Constructs containing a wheat photosynthetic promoter
A 695 kb promoter fragment from sequence previously published containing the TATA signal from the TaRbcS gene (NCBI accession number AB042069) was PCR-amplified with Gateway™ (Invitrogen) recombination sites at the primer flanks. The fragment was cloned into the Invitrogen pDONRP4-P1 R Entry vector using Gateway™ recombination technology. The 696 bp TaRbcS gene termination signal sequence (Sasanuma, 2001 ) was also PCR-amplified using primers with recombination sites and cloned into the Invitrogen pDONRP2-P3R Entry vector. The cloned fragments were sequence-verified and used for three-way recombination cloning with the cloned GOI cDNA sequences into the pDEST-R4R3 destination vector: pDESTR1-R2R-Lp1-SST, pDESTR1-R2- Lp6G-FFT, and pDESTP1-P2R-Lp1-SST_Lp6G-FFT gene FT fusion expression vectors. The following constructs for photosynthetic-regulation of expression of fructosyltransferases by the TaRbcS promoter to be used are outlined below and graphically depicted in Figure 23. Expression cassette sequences for pDEST- TaRbcS::Lp1-SST::TaRbcS, pDEST-TaRbcS::Lp6G-FFT::TaRbcS and pDEST- TaRbcS::Lp1-SST_Lp6G-FFT::TaRbcS FT fusioni and 3 are provided in Figures 24 - 27.
pDEST-TaRbcS-Lpi-SST-TaRbcS
• pDEST-TaRbcS-LpβG-FFT-TaRbcS
pDEST-TaRbcS::Lp1-SST_Lp6G-FFT::TaRbcS FT fusion 1 and 3
pDEST-TaRbcSiiGUSiiTaRbcS
Constructs containing a ryegrass photosynthetic promoter
Constructs containing a ryegrass photosynthetic promoter were produced by conventional cloning methods. The 693 base pair (bp) fructosyltransferase 4 gene (LpFT4) termination sequence (Lidgett, et al., 2002) was amplified by PCR using gene specific primers containing the restriction endonuclease (RE) site EcoR I at the 5' end of the forward PCR primer. EcoR V and Xma I endonuclease restriction sites were incorporated at the 3' end of the reverse PCR primer. The PCR product was cloned into the EcoR I and Xma I restriction endonuclease sites of the pBlueScript SK(-) vector DNA (Short, et al., 1988), resulting in the plasmid pBS-LpFT4 (Figure 28).
The LpRbcS promoter was amplified using gene specific primers containing the endonuclease restriction sites Xho I and EcoR V at the 5' end of the forward primer and an EcoR I restriction site was incorporated in the 3' end of the reverse primer. The 610 bp PCR product was cloned into the pBS-LpFT4 plasmid digested with EcoR I and Xho I, resulting in the plasmid pBS-LpRbcS::LpFT4 (Figure 29A). The LpI-SST coding region was amplified from a cDNA template (Chalmers et al., 2003) with EcoR I sites flanking both forward and reverse PCR primers, and cloned into the EcoR I site of pBS- LpRbcS::LpFT4 vector, generating the final construct pBS-LpRbcS::Lp1-SST::LpFT4 (Figure 29B). Sequence of the expression cassette, indicating promoter and terminator, as well as ORF is provided in Figure 31. The expression cassette containing the L. perenne sequences may be excised from the plasmid vector DNA using the EcoR V restriction endonuclease. Following agarose gel electrophoresis, the resulting DNA fragment is purified from the agarose matrix prior to being used for plant transformation to produce DNA with out vector backbone sequences.
The plasmid pBS-LpFT4 (Figure 28) containing the 693 base pair LpFT4 terminator sequence was prepared as outlined above. The LpCAB promoter fragment of 870 base pairs was amplified with a forward PCR primer containing the Xho I and EcoR V sites and a reverse PCR primer containing the EcoR I restriction site. This fragment was cloned in the Xho I and EcoR I sites of pBS-LpFT4, generating the pBS-LpCAB::LpFT4 plasmid (Figure 30A). The Lp6G-FFT coding region was amplified from a cDNA template (Chalmers, et al., 2005) with EcoR I sites flanking both forward and reverse PCR primers, and cloned into the EcoR I site of pBS-LpCAB::LpFT4 vector, generating the final construct pBS-LpCAB::Lp6G-FFT::LpFT4 (Figure 30B). Sequence of the expression cassette, indicating promoter and terminator, as well as ORF is provided in Figure 32. The DNA expression cassette may be excised from the plasmid vector DNA using the EcoR V restriction endonuclease. Following agarose gel electrophoresis, the resulting DNA fragment is purified from the agarose matrix prior to being used for plant transformation to produce DNA without vector backbone sequences. To generate an expression construct, the translational FT fusion between the genes LpI-SST and Lp6G-FFT was amplified from pDEST-TaRbcS::Lp1-SST_l_p6G- FFT::TaRbcS FT fusion 1 and 3 plasmids (Figure 23C-D) using primers specific for a region just outside the ORF, with EcoR I restriction sites engineered in the 3' region on both the forward and reverse primers. The 3920 bp ORF was PCR amplified and cloned into pCR®-Blunt vector (Invitrogen) to produce PCR Blunt-Lp1-SST-Lp6G-FFT FT fusion (Figure 33). It was then excised using EcoR I restriction enzymes to remove the vector-specific sequences, and cloned into the pBS-LpRbcS::LpFT4 plasmid (Figure 29A) at the EcoR I restriction site, generating the pBS-LpRbcS::Lp1-SST_Lp6G- FFT::LpFT4 (Figure 34). Sequence of the expression cassette of FT fusion 1 and 3, indicating relevant domains (FT fusion 3), is provided in Figure 35 and 36, respectively. The DNA expression cassette may be excised from the plasmid vector DNA using the EcoR V restriction endonuclease. Following agarose gel electrophoresis, the resulting DNA fragment is purified from the agarose matrix prior to being used for plant transformation to produce DNA without vector backbone sequences.
The constructs for photosynthetic-regulation of expression of fructosyltransferases by L perenne promoter sequences are outlined below.
pBS-LpRbcS::Lp1-SST::LpFT4
pBS-LpCAB::Lp6G-FFT::LpFT4
• pBS-LpRbcS::Lp1-SST_Lp6G-FFT::LpFT4 FT fusion 1 and 3
Constructs containing an Arabϊdopsis photosynthetic promoter
A construct containing an Arabidopsis photosynthetic promoter driving expression of FT fusion 3 was produced using Multisite Gateway cloning methods -pPZP200_AtRbcS:: Lp1-SST_6G-FFT::35S FT fusion 3 (Figure 37). The sequence of the AtRbcS::Lp1- SST_6G-FFT::nos FT fusion 3 expression cassette is provided in Figure 38.
Constructs containing a constitutive ubiquitin promoter
Constructs containing the promoter and first intron of the maize (Zea mays) ubiquitin (Ubi) gene (Christensen et al., 1992) were produced by conventional cloning methods. The Ubi promoter is considered a constitutive promoter, but expression is highest in young actively growing grass tissues (Rooke et al., 2000).
A cDNA copy of the candidate genes LpI-SST and Lp6G-FFT was amplified by PCR as described by Chalmers et. al. (2003) and cloned into the pBlueScript SK(-) vector (Figure 39). The cDNA fragments were excised using the restriction endonucleases Xho I and Xba I, and then blunt-end cloned into the BamH I site of p-Ubi-35S vector (Figure 40). The p-Ubi-35S binary plant expression vector has been previously described in other transformation experiments of L. multiflorum (Ye et al., 2001 ). The p-Ubi::Lp1- SST::35S and p-Ubi::Lp6G-FFT::35S clones containing the DNA insert in the required 5' to 3' orientation were confirmed by DNA sequencing. A representative sequence of the constitutive (Ubi) promoter combined with a FT fusion protein and a terminator sequence is provided in Figure 41. A similar method is used to construct p-Ubi::Lp1- FFT::35S clones.
The constructs for photosynthetic-regulation of expression of fructosyltransferases by the Ubi promoter sequences are outlined below.
p-Ubi::Lp1-SST::35S
p-Ubi::Lp6G-FFT::35S
p-Ubi::(Lp1-FFT/ Lp6-SFT/Lp-SST)::35S
Constructs containing the cauliflower mosaic virus 35S promoter The constructs for regulation of expression of fructosyltransferases under the control of the enhanced cauliflower mosaic virus (CAMV)35S2 promoter (Kay, et al., 1987), are described in a previous section and are outlined below.
pPZP200-35S2: :Lp6G-FFT: :Ta RbcS
pPZP200-35S2::Lp1-SST::TaRbcS
• pPZP200-35S2::(Lp1-FFT/ Lp6-SFT/Lp-SST)::TaRbcS
pPZP200-35S2::Lp1-SST_6G-FFT::TaRbcS FT fusioni and 3 Constructs containing tissue specific or regulated promoters
Promoters with tissue-specificity are desirable to drive expression of transgenes in crops to target accumulation in particular tissues/ organs and to avoid unwanted expression elsewhere. Examples of different promoters to drive transgene expression for different objectives are presented in Table 5. Representative examples of promoters for constitutive (Ubi, (CAMV)35S2, RUBQ2, OsActi ), tuber and stolon specific (Cathlnh), stress regulated (Atrd29a) and sucrose responsive (14-3-3 protein family 16R) linked to FT fusions are presented in Figures 42 - 48, respectively.
Table 5. Examples of different promoters to drive transgene expression.
Figure imgf000054_0001
Xylem phenylalanine ammonialyase Nicotiana . Keller and gene 2, PAL2 benthamiana Baumgartner
(tobacco) (1991 )
4-coumarate:coenzyme A Nicotiana . Hauffe et al. ligase. 4CL benthamiana (1993)
(tobacco)
Figure imgf000055_0001
Figure imgf000056_0001
Several photosynthetic promoters have been shown to be strong regulators of expression of transgenes in light-responsive tissues. Advantages of photosynthetic promoters for expressing fructan biosynthesis genes include that they are active in the large group of cells of the leaves and upper part of the stems which accounts the majority of the plants biomass. They are not constitutively expressed, however their expression pattern temporally and spatially overlaps with sucrose accumulation.
Using a combination of vectors for transcriptional co-transformation
The following vectors are transformed singly or in groups (double and triple) to assess synergistic responses of co-expression required for the generation of low and high DP fructans.
pDEST-TaRbcS::Lp1 -SST::TaRbcS
pBS-LpRbcS::Lp1-SST::LpFT4
p-Ubi::Lp1-SST::35S pPZP200-35S2::Lp1-SST::TaRbcS
pDEST-TaRbcS::Lp6G-FFT::TaRbcS
pBS-LpCAB::Lp6G-FFT::LpFT4
p-Ubi::Lp6G-FFT::35S
• pPZP200-35S2::Lp6G-FFT::TaRbcS
pDEST-TaRbcS::(Lp1-FFT/ Lp6-SFT/Lp-SST)::TaRbcS
p-Ubi::(Lp1-FFT/ Lp6-SFT/Lp-SST)::35S
pPZP200-35S2::(Lp1-FFT/ Lp6-SFT/Lp-SST)::TaRbcS
Using FT fusion vectors for translational co-transformation To make comparisons with the transcriptional co-transformations as indicated above, translational co-transformation experiments are also conducted with the FT fusion vectors that have previously been discussed and are indicated below.
pDEST-TaRbcSi±pi-SSTJ-pθG-FFTiiTaRbcS FT fusioni and 3
pBS-LpRbcS::Lp1-SST_Lp6G-FFT::LpFT4 FT fusioni and 3
• pPZP200-35S2::Lp1-SST_6G-FFT::TaRbcS FT fusion1 and 3
Example 6
Production of stable transgenic plants by transformation Transformation of plants
The genetic constructs of the present invention may be introduced into plant cells by transduction, transfection, transformation or gene targeting. Such techniques include ^g/Obacfer/um-mediated introduction, electroporation of tissues, cells and protoplasts,, protoplast fusion, injection into reproductive organs, injection into immature embryos and high velocity projectile introduction to cells, tissues, calli, immature and mature embryos, microinjection into cells and protoplasts, polyethylene glycol mediated direct gene transfer into protoplasts, biolistic transformation, Whiskers transformation and combinations thereof. The choice of technique depends largely on the type of plant to be transformed and the appropriate vector for the method chosen are used.
Cells incorporating the genetic constructs of the present invention may be selected, as directed by the vectors used, and then cultured in an appropriate medium to regenerate transformed plants, using techniques well established. The resulting plants may be reproduced, either sexually or asexually, to produce successive generations of transformed plants.
The present invention may be applied to a variety of plants, including monocotyledons [such as wheat, corn or maize, rice, barley, sorghum, sugarcane, oats, rye, grasses
(e.g. forage, turf and bioenergy grasses including perennial ryegrass, tall fescue, Italian ryegrass, red fescue, reed canary-grass, big bluestem, cordgrass, napiergrass, switchgrass, wildrye, wild sugarcane, Miscanthus, Paspalum)], dicotyledons [such as
Arabidopsis, tobacco, soybean, canola, alfalfa, cotton, potato, tomato, tobacco, clovers (e.g. white clover, red clover, subterranean clover), vegetable brassicas, lettuce, spinach] and gymnosperms. In particular, invention may be applied to cereals such as
Triticum aestivum (wheat), C3 grasses containing native fructans such as Lolium perenne (ryegrass) and Lolium arundinaceum (tall fescue), as well as Paspalum dilatatum (paspalum) a C4 perennial apomitic grass with no native fructans. The invention may also be applied to dicots such as Arabidopsis thaliana, Brassica napus
(canola), Nicotiana benthamiana (tobacco) and Trifolium repens (white clover).
Biolistic transformation of monocots eg wheat, perennial ryegrass, tall fescue and paspalum
The candidate genes are inserted into the plant genome by particle bombardment using whole plasmids so vector backbone sequences may also be incorporated into the genome. Transgenic plant tissues are recovered by survival on tissue culture media containing a selective agent. Agrobacterium-med'iated transformation of dicots eg Arabidopsis, tobacco, canola and white clover
Agrobacteήum-mediated transformation takes advantage of the natural pathogenic activity of the soil bacterium Agrobacterium tumefaciens. A. tumefaciens infects the roots & stems of dicotyledonous plants resulting in infection directed by the tumor inducing (Ti) plasmid by the insertion of specific genes (T-DNA) into the genome of infected plant cells. The candidate genes were inserted into the plant genome by Agrobacterium-med'iated transformation using binary vectors based on the Ti plasmids.
Example 7
Production of transgenic perennial grasses
Use of constructs containing photosynthetic promoters
Biolistic co-transformation of perennial ryegrass with the vectors containing the TaRbcS and LpRbcS regulatory sequences, driving the expression of individual fructan genes or as a FT translational fusion, and the pAcH1 vector for hygromycin resistance was conducted on embryogenic calli for perennial ryegrass. The pAcH1 vector was previously constructed and has been used successfully in plant transformation experiments (Bilang, et al., 1991 ; Spangenberg, et al., 1995a; Spangenberg, et al., 1995b; Ye, et al., 1997; Bai, et al., 2001). The GUS marker gene was also cloned as a positive control. Table 6 summarises the transformation and molecular analysis for the generation of these lines.
Table 6. Summary of production of transgenic perennial ryegrass plants for expression of LpI-SST and Lp6G-FFT and FT fusion ORFs under control of photosynthetic promoter from wheat.
Figure imgf000059_0001
Figure imgf000060_0001
"Cassette DNA" containing L. perenne sequences was excised from the plasmid vectors pBS-LpRbcS::Lp1-SST::LpFT4, pBS-LpCAB::Lp6G-FFT::LpFT4 and pBS-l_pRbcS::Lp1- SST_Lp6G-FFT::LpFT4 (Figures 29, 30 and 34 respectively) using the EcoR V restriction endonuclease. Following agarose gel electrophoresis, the resulting DNA fragment was purified from the agarose gel prior to being used for plant transformation to produce DNA without vector backbone sequences. The pAcH1 vector previously constructed and used successfully in plant transformation experiments was also digested with restriction enzymes to produce a DNA fragment for the expression of the selectable marker only.
Biolistic co-transformation of perennial ryegrass with the vectors containing the L. perenne regulatory sequences, driving the expression of individual fructan genes or as a translational FT fusion, and the pAcH1 expression cassette for hygromycin resistance was conducted on embryogenic calli for perennial ryegrass. Table 7 summarises the transformation and molecular analysis for the generation of these lines.
Table 7. Summary of transformation progress for production of transgenic perennial ryegrass plants for expression of LpI-SST and Lp6G-FFT and FT fusion ORFs under control of ryegrass photosynthetic promoters.
Figure imgf000060_0002
Example 8
Characterisation of transgenic perennial grasses
Characterisation of transgenic FT and FT fusion perennial ryegrass plants
During the regeneration of the transgenic perennial ryegrass plants differences in growth phenotypes were noticed between the lines. Both the tissue culture regenerants and corresponding soil grown plants from both of the FT fusion 1 and FT fusion 3 transgenic plants showed a superior growth performance phenotype compared to the transgenic plants containing either a single fructan biosynthesis gene or control plants containing only the selectable marker, hph. Phenotypic examples of transgenic perennial ryegrass plants in tissue culture are displayed for the TaRbcS promoter and
LpRbcS FT fusion transgenics in Figures 48 -51.
The plants showing the superior growth performance phenotype were confirmed to contain the FT gene of interest. The superior growth performance phenotype of the transgenic FT fusion 1 and FT fusion 3 plants was first observed during the early stages of plant regeneration conducted on plates. Specifically just 12 days after incubation under lights the transgenic calli showed further developed green shoots. The fast growth rate of the FT fusion transgenic plants became more evident 22 days after transferring to rooting media. Transgenic plants containing either FT fusion 1 or FT fusion 3 constructs showed clearly greater numbers of tillers. In addition, the FT fusion transgenic plants consistently showed a higher tiller density per plant compared to control plants in vitro (Figures 48 - 49).
Following transfer to soil and propagation under glasshouse conditions more specific differences were observed between the FT fusion 1 and FT fusion 3 lines. Even though both FT fusion plants displayed enhanced growth performance, FT fusion 1 plants had longer, thicker and a slightly darker green leaf blades. Also the plants were physically more robust with thicker leaf sheaths and leaf blades. FT fusion 3 lines continued to grow faster than the other control plants with longer leaf blades and more vigorous tiller growth, but the leaf morphology was more similar to wild-type. An increase in root biomass was also observed in both FT fusion 1 and FT fusion 3 soil grown transgenic perennial ryegrass plants (Figure 50). The control transgenic plants harbouring either the LpI-SST or Lp6G-FFT as single genes did not show the level of increased growth rate that was observed in the FT fusion 1 and 3 transgenic plants. Their appearance is similar to each other, although some developed more vigorously than the transgenic plants containing either GUS or hph (Figure 50).
A similar phenotype to that observed in the glasshouse was also observed in the field. The FT fusion transgenic plants showed a more vigorous growth phenotype with increased number of tillers and longer leaf blades (Figure 51 ). The field trial transgenic plants were analysed for biomass production (Table 8). Biomass was assessed, as outlined in Figure 52, ranging from a score of 1 having the least biomass to 5 having the most.
Table 8. Percent of plants indicating the range of biomass scores per genotype observed under field trial growth conditions.
Figure imgf000062_0001
Leaf blades from individual plants were cut and hand sectioned (Figure 53). Obvious differences seen were in the amount of chloroplasts in each cell, and the number of cells with chloroplasts: being more in both of the transgenic FT fusion plants than in the control plants. In addition, chloroplasts were present in cells located on the abaxial side
(lower part of the leaf) of transgenic plants, despite that both plants were grown under the same light conditions in the growth room. Sometimes it was observed that control plants produced more chloroplasts in mesophyll cells located on the adaxial side (upper side which face the light source) than on the abaxial side, whereas the transgenic plants most often produced near-equal number of chloroplasts on both sides.
Biochemical analysis by HPAEC of water soluble carbohydrates extracted from independent transformants harbouring the TaRbcS::Lp1-SST_Lp6G-FFT::TaRbcS FT fusion 1 , TaRbcS::Lp1-SST_Lp6G-FFT::TaRbcS FT fusion 3, TaRbcS::Lp1-
SST::TaRbcS, TaRbcS:: Lp6G-FFT::TaRbcS, and two control lines (hph onlyj showed that the FT fusion 1 and FT fusion 3 transgenic plants contained significantly higher levels of total fructans (Figure 54), showing up to 2.5 fold increase over the control lines (Figure 54). In addition, the levels of 1-kestose were up to 4 times higher in FT fusion 1 lines (up to 3.7 μg/mg of DW, total fructans: 20.5 μg/mg of DW and sucrose 51.2 μg/mg of DW.), and 3 times higher in FT fusion 3 lines (2.4 μg/mg of DW, total fructans: 26.0 μg/mg of DW and sucrose 49.8 μg/mg of DW) compared to the hph controls (Figure 55A-B). In the TaRbcS::Lp1-SST::TaRbcS plants 1-Kestose has increased up to 2.9 μg/mg of DW (a 3-fold increase) whereas total fructan content only increased 0.5 fold to 14 μg/mg of DW. In contrast 1-kestose levels in the TaRbcS::Lp6G-FFT::TaRbcS transgenic plant lines showed marginal increases up to 1.6 μg/mg of DW for 1-kestose (up to 0.5 fold) and only one line showed a small increase in total fructans to 10 μg/mg of DW (Figures 55C-D and 56C-D). Analysis of sucrose contents of all the lines revealed that some of the high fructan lines also showed an increase in total sucrose content (Figure 57).
The transgenic perennial ryegrass was also evaluated under field conditions for total fructan level and composition (Figures 58 & 59) and transgene expression (Figure 60C). The control and transgenic perennial ryegrass plants were sampled repeatedly throughout the field trial growing season. Biochemical analysis of wild-type controls and independent transformants was conducted to show the level of total fructan per plant. Figure 58 illustrates fructan levels in milligrams (mg) per gram (g) of dry weight (DW) transgenic and wild-type field grown whole tillers and leaf blades. Multiple individual FT fusion and LpRbcS::Lp1-SST transgenic plants were identified with fructan concentrations between 80 to 120 percent higher than the corresponding the wild-type (WT) control plants in both whole tiller and leaf blade samples (Figure 58). Representative results on the composition of fructans in leaf blades of three LpRbcS::Lp1-SST transgenic perennial ryegrass plants as compared to wild-type controls are shown in Figure 59. The results indicate an increased level of low DP fructans in transgenic plants expressing LpRbcS::Lp1-SST (Box 1 , Figure 59).
Transgene expression was detected in representative LpRbcS FT fusion and LpRbcS::Lp1SST transgenic perennial ryegrass plants analysed by quantitative reverse transcription PCR (qRT-PCR) (Figure 60). In order to quantify the increase in biomass single tillers were separated from each of the To transgenic lines and control lines, and propagated in potting mix under glasshouse conditions. After 7 weeks and 12 weeks each plant was analysed for plant height, leaf blade width and total tiller number (Figures 61 and 62). After 7 weeks the control plants showed an average height of 24 cm, the average leaf width was 2.5 mm, and each plant had an average of two tillers. The transgenic FT fusion 1 and fusion 3 lines, however, showed up to an 80% increase in plant height (43 cm), up to 60% increase in leaf width (4 mm), and up to 3 fold increase in tiller number (6 tillers). After 12 weeks the control plants were, on average, 43 cm tall, leaf blades width was 3.5 mm, with 5 tillers per plant produced. Over the same period of time the transgenic FT fusion 1 and fusion 3 plants had grown up to 62 cm tall (43% increase compared to controls). The leaf width was up to 6 mm (70% increase) and the maximum number of tillers observed was 16 per plant (220% increase) (Figure 62).
Characterisation of transgenic LXR® and transgenic FT fusion plus LXR® perennial ryegrass plants
Co-transformation of the FT fusion and LXR® technology produced an enhanced growth phenotype. Plants grown under glasshouse conditions showed an increased number of tillers and an enhanced root biomass compared to control and LXR® alone transgenic plants (Figure 65).
Dry weight experiments of plant tissue were conducted to establish the biomass of individual FT fusion and LXR® transgenic plants. Transgenic perennial ryegrass plants grown under glasshouse conditions were trimmed 5 mm below the lowest leaf sheath at the 10 tiller stage. After 6 weeks all plant biomass from a height of 5 cm above the soil level was harvested into paper bags, oven-dried and weighed on a precision balance.
The control was calculated as the average of five independent 'gene of interest' negative (GOI-ve) plants. Both FT fusion and FT fusion plus LXR® transgenic plants produced plants with a dry weight higher (up to two fold) than the average level for the control (Figure 64).
Biochemical analysis of GOI-ve controls and independent transformants was also conducted to show levels of total fructan per plant. Fructan levels in the leaf blades of FT fusion alone, as well as FT fusion plus LXR® transgenic plants showed up to a six fold increase compared to the average value of the control plants (Figure 65).
Characterisation of transgenic FT fusion tall fescue plants
Transformation of tall fescue grass with the vectors containing the L. perenne regulatory sequences, driving the FT translational fusion, and the pAcH1 expression cassette for hygromycin resistance was conducted. Transgenic tall fescue plants grown under glasshouse conditions showed an increased number of tillers and an enhanced root biomass compared to control transgenic plants (Figure 66).
Characterisation of transgenic LXR® and transgenic FT fusion plus LXR® tall fescue plants
Transgenic tall fescue (Lolium arundinaceum cv Jesup S3) plants expressing LpRbcS FT fusion 3 alone, TaRbcS FT fusion 3 alone, as well as TaRbcS FT fusion 3 plus LXR® technology (AtMYB32::IPT) together have been produced. Table 9 summarises the transformation and molecular analysis for the generation of these lines.
Table 9. Summary of transformation progress for tall fescue with photosynthetic- regulated expression of FT fusion 3 and/ or LXR®.
Figure imgf000066_0001
Dry weight experiments of plant tissue were conducted to establish the biomass of individual transgenic plants. Transgenic tall fescue plants grown under glasshouse conditions were trimmed 5 mm below the lowest leaf sheath at the 5 tiller stage. After 6 weeks all plant biomass from a height of 5 cm above the soil level was harvested into paper bags, oven-dried and weighed on a precision balance.
The control was calculated as the average of five independent GOI-ve plants. Transgenic FT fusion alone and FT fusion plus LXR® tall fescue plants both showed a two fold increase in herbage dry weight as compared to the average value of the control plants (Figure 67).
Tiller number experiments were also conducted to establish the growth vigour of individual transgenic plants. Both tall fescue transgenic and GOI-ve control plants, at the 5 tiller stage, were trimmed as mentioned above and left growing under glasshouse conditions for 6 weeks before tiller numbers were counted. The tiller number in the control represents the average tiller number obtained from five independent GOI-ve plants. Transgenic lines of FT fusion alone and FT fusion plus LXR® tall fescue plants showed up to a two fold increase in tiller number compared to the average value of the control plants (Figure 68). Transgenic tall fescue plants (5 tillers) were trimmed (as indicated above) and grown under glasshouse conditions for 6 weeks when leaf blades were collected and freeze- dried for fructan analysis. The average fructan level in controls represents data obtained from five independent GOI-ve plants. Transgenic lines of FT fusion tall fescue plants show a dramatic increase (between three to five fold) in fructan accumulation in leaf blades compared to the average fructan level in GOI-ve control plants (Figure 69).
Example 9
Production of transgenic wheat plants
Transformation of light-regulated promoter expressing single fructan genes or the FT translational fusion
Biolistic co-transformation of wheat with the vectors containing the photosynthetic promoter regulatory sequences, driving the expression of individual fructan genes or as a translational FT fusion, and a vector containing a chimeric Ubi::bar::nos selectable marker gene for glufosinate resistance (pAcH25) was conducted on wheat embryogenic calli.
Transformation of AtMYB32 promoter and IPT gene for delayed senescence
A transformation vector has been constructed for biolistic transformation of wheat containing the chimeric AtMYB32::IPT::35S with a chimeric Ubi::bar::nos selectable marker gene for glufosinate resistance. Genetic transformation of wheat with LXR® vector was based on biolistic transformation of embryogenic calli from Triticum aestivum L Bobwhite 26 wheat line as described in International patent application PCT/AU01/01092. The candidate gene was inserted into the wheat genome by particle bombardment using whole plasmids so vector backbone sequences may also be incorporated into the genome. Transgenic plant tissues were recovered by survival on tissue culture media containing a selective agent.
Production of transgenic plants for re-programmed fructan biosynthesis in photosynthetic cells and extended life of photosynthetic cells
Using the methods outlined above transgenic plants were generated that contain both fructan biosynthetic genes driven by light-regulated promoters and the LXR® technology for re-programmed fructan biosynthesis in photosynthetic cells and extended life of photosynthetic cells. Table 8 summarises the transformation and molecular analysis for the generation of these transgenic plants.
Table 10. Summary of transformation progress for production of transgenic wheat plants for expression of LpI-SST and Lp6G-FFT and FT fusion ORFs under control of photosynthetic promoters of wheat and in combination with LXR® technology for re- programmed fructan biosynthesis in photosynthetic cells and extended life of photosynthetic cells.
Figure imgf000068_0001
Example 10
Characterisation of transgenic wheat plants Characterisation of transgenic FT fusion wheat plants
During the regeneration of the transgenic wheat plants differences in in vitro growth phenotypes were noticed. The tissue culture regenerants from both of the FT fusion 1 and FT fusion 3 transgenic plants showed a superior vigour phenotype compared to control plants.
The superior growth phenotype of the transgenic FT fusion 1 and FT fusion 3 plants was first observed during the early stages of in vitro plant regeneration conducted on tissue culture plates. Following biolistic transformation calli were kept for two weeks on tissue culture plates in the dark and then transferred to light conditions. Approximately 6 weeks after incubation under light conditions the transformed calli showed more fully developed green shoots and the roots of the FT fusion transgenic regenerants grew at an extremely advanced rate (Figure 70).
The fast growth rate of the FT fusion transgenic plants became more evident after transferring to rooting media. FT fusion transgenic plants showed an obvious early increase in tiller number at around 2 months as compared to null controls (up to 5 tillers compare to one tiller observed in control plants). The width of the leaves of the some of the plants was 4-5 mm compare to control plants 2-3 mm. In addition, the FT fusion transgenics consistently showed a higher tiller density per plant compared to control lines (Figure 71 ).
Following transfer to soil and propagation under glasshouse conditions the transgenic wheat plants that contain the FT fusion constructs continued to show an increase in tiller number as compared to control plants (Figure 72).
Characterisation of transgenic LXR® and FT fusion plus LXR® wheat plants
The transgenic wheat plants that contain the LXR® technology construct showed an increase in tiller number as compared to control plants under glasshouse conditions (Figure 73A). They also showed and increase of photosynthetic tissue after 35 days under glasshouse conditions (Figure 73B).
Co-transformation of the FT fusion construct and LXR® technology produced an enhanced growth phenotype of glasshouse grown plants. Some of the plants also showed an obvious late senescence (at 40 days) under glasshouse conditions (Figure 74). Transgenic wheat plants expressing the FT fusion construct and the FT fusion construct plus LXR® also showed an enhanced level of fructans in leaves and an increased number of tillers as compared to control plants under glasshouse conditions (Figure 75).
Biochemical analysis of GOI-ve controls, FT fusion, as well as FT fusion plus LXR® independent Ti wheat transformants, grown under glass house conditions, was conducted to show levels of total fructan per plant. A dramatic increase in fructan level (up to five fold) was detected for both transgenic lines (Figure 76). Example 11
Production of transgenic Paspalum dilatatum plants
Transformation of IPT gene under control of AtMYB32 promoter for delayed leaf senescence Genetic transformation of Paspalum dilatatum (apomictic dallisgrass) was based on biolistic transformation as described in International patent application PCT/AU01/01092.
The candidate gene expression construct was inserted into the Paspalum dilatatum genome by particle bombardment using whole plasmids so vector backbone sequences may also be incorporated into the genome. Transgenic plant tissues were recovered by survival on tissue culture media containing a selective agent.
Transformation of FT translational fusion under control of light-regulated promoter for engineering fructan biosynthesis in photosynthetic cells
Genetic transformation of Paspalum dilatatum with photosynthetic regulated fructan biosynthesis genes is conducted using the same method as was used to produce the LXR® transgenic Paspalum dilatatum plants.
Example 12
Characterisation of transgenic Paspalum dilatatum plants
LXR® transgenic plants display a superior growth phenotype. Transgenic Paspalum dilatatum plants expressing the IPT gene under control of the AtMYB32 promoter revealed an enhanced biomass accumulation. During the regeneration of the putative transgenic P. dilatatum plants differences in growth phenotypes were noticed showing a superior growth phenotype compared to control plants. The distinctive growth phenotype may be used as a selection tool for identifying transformed plants in combination with co-transformed vectors. Example 13
Production of transgenic dicotyledonous plants
Transformation of LXR® and FT fusion plus LXR® dicot plants
Binary vectors containing the FT fusion and LXR® technology have been generated for Agrobacterium-med\ated transformation of dicot plants. Transformation vectors also contained a chimeric 35S::nptll::35S or 35S::hph::35S as selectable marker genes.
Production of transgenic dicot plants
Transgenic white clover (Trifolium repens) and Arabidopsis thaliana plants expressing LXR® technology alone (AtMYB3::IPT), AtRbcS::Lp1-SST_Lp6G-FFT::35S FT fusion alone, as well as LXR® technology and the AtRbcS::Lp1-SST_Lp6G-FFT::35S FT fusion together have been produced (Figures 77 and 80). Tables 11 and 12 summarise the transformation and molecular analysis for the generation of these lines, respectively.
Table 11 Summary of transformation progress for white clover with Arabidopsis photosynthetic-regulated expression of FT fusion and/ or LXR®
Figure imgf000071_0001
Table 12 Summary of transformation progress for Arabidopsis with Arabidopsis photosynthetic-regulated expression of FT fusion and/or LXR®
Figure imgf000071_0002
Characterisation of transgenic white clover plants
Quantitative RT-PCR was used to confirm transformants and detect expression levels of the AtRbcS FT fusion in selected lines (Figure 78). These lines, showing expression of the transgene also demonstrated an increased level of fructans (Figure 68B). No expression was detected in control lines (Figure 78).
Biochemical analysis by HPAEC of water soluble carbohydrates extracted from independent transformants expressing AtRbcS FT fusion alone, AtRbcS FT fusion plus LXR® and GOI-ve control lines was conducted to show levels of total fructans per plant. AtRbcS FT fusion and AtRbcS FT fusion plus LXR® transgenic lines showed a two fold increase of fructan accumulation in leaves higher than that observed in the controls (Figure 79).
Characterisation of transgenic Arabidopsis plants Quantitative RT-PCR was used to confirm transformants and detect expression levels of the AtRbcS FT fusion in selected lines (Figure 81 ). Transgenic T2 FT fusion Arabidopsis plants grown in soil are shown in Figure 82. Gene of interest negative plants (GOI-ve) are also presented and show no phenotypic difference to FT fusion transgenic plants shown to express the transgene.
Binary vectors were also used for yAgfrofeacteπ'u/77-mediated transformation of Brassica napus (canola) hypocotyl segments (Patent PCT/AU01/01092).
Example 14
Characterisation of transgenic dicotyledonous plants
Characterisation of transgenic LXR® dicot plants A functionally active fragment of the AtMYB32 promoter was used to drive IPT expression in transgenic white clover and canola plants as described in International patent application PCT/AU01/01092. Outcomes observed from the LXR® technology in dicot plants have been delayed leaf senescence; enhanced leaf growth dynamics; reduced stolon death; enhanced biomass production; increased cumulative green leaf area; increased seed yield; enhanced drought tolerance; increased shading tolerance; enhanced herbage quality reflected by enhanced ruminal fermentation kinetics and higher dry matter digestibility. Transgenic plants display a delayed leaf senescence phenotype.
The regulation of developmental senescence may be assessed by simulating and initiating artificial aging of detached leaves in vitro on moist filter paper. Incubation of detached leaves in darkness is highly effective in inducing Senescence Associated Genes (SAGS), leaf yellowing and chlorophyll loss (Weaver and Amasino, 2001). Figure
83 demonstrates detached senescence assay data associated with expression of the
IPT gene under control of one of two functionally active fragments of the AtMYB32 promoter in white clover and canola. The transgenic plants displayed a significant delay of leaf senescence as compared to leaves from control plants 7 - 20 days following detachment.
Example 15
Production of transgenic plants for re-programming fructan biosynthesis in photosynthetic cells and for extended life of these photosynthetic cells
Using the methods outlined above transgenic plants have been generated that contain both, fructan biosynthetic genes (FT including FT fusion genes) under control of light-' regulated, photosynthetic promoters for re-programming fructan biosynthesis in photosynthetic cells and LXR® technology through co-expression of IPT gene driven by the AtMYB32 promoter for extending life of the photosynthetic cells.
Example 16 Use of the distinctive growth phenotype as a selection tool to identify transgenic plants in vitro
The superior growth phenotype of the transgenic FT fusion 1 or FT fusion 3 plants was observed in all plant types to which it was transformed (eg perennial ryegrass and wheat). In both ryegrass and wheat it was first observed during the early stages of plant regeneration conducted in plates. In the experiments conducted without antibiotic selection, strong shoot induction has been observed at the stage when after bombardment the calli have been kept in dark conditions for 8 weeks. (Figure 84 A-C). After transferring the plates to light conditions (7 days after transfer) strong shoot induction was observed in the transgenic plants and much lower level of shoot regeneration was detected in control plants (Figure 84 D-F).
Expression of the FT fusion under control of TaRbcS or other photosynthetic, sucrose- regulated or constitutive promoters could be used as a selection tool for the identification of transformed plants at the tissue culture stage. Expression of the FT fusion protein may be also driven by a set of promoters, which are active due to the high concentration of sucrose that exists in tissue culture medium, and much less active at the low sucrose levels present in soil-grown plants. This transgene may subsequently be segregated away from the transgenic plants in successive generations. The increased biomass of the transformed plants to be used as the selective agent should not require an antibiotic resistance marker for the selection process, enabling the production of a market ready product.
Analysis was carried out to assess the use of the distinctive growth phenotype to detect a positive transformation result in perennial ryegrass. Embryogenic perennial ryegrass calli FLP410-20 were bombarded with gold particles covered in TaRbcS FT fusion 1 alone, TaRbcS FT fusion 3 alone, AtMYB32::IPT (LXR®) alone, as well as TaRbcS FT fusion 1 plus LXR® vectors without any selectable marker. Control calli were bombarded just with golden particles.
Plants were regenerated without antibiotic selection and kept 2 weeks under dark conditions and then transferred to light conditions (16/8hr light/dark photo-period). The plant's growth was examined prior to transfer to light and weekly for five weeks under light conditions. Calli were kept under progressively starving conditions on the same plate for five weeks (Callus induction medium: MS full strength + 250mg/l_ L-asparagine +2.5mg/L 2.4-D+ 6% sucrose +0.7% agar).
Control plant growth was initiated during the first two to three weeks under light conditions but slowed significantly four and five weeks later (Figure 85). Some calli bombarded with TaRbcS FT fusion vectors showed more vigorous growth during the first two to three weeks and continued growing (with reduced rate) at weeks four and five (Figure 85). No obvious differences were observed for LXR® alone bombarded calli. Co-transformation with TaRbcS FT fusion 1 plus LXR® vectors showed an intermediate phenotype between the control and the TaRbcS FT fusion 1 vector alone (Figure 86).
Molecular analysis was undertaken to detect the presence of the TaRbcS FT fusion transgenes using qRT-PCR in putative transgenic lines. FT fusion transgenics showed between 60% and 70% transformation and selection efficiency without antibiotics. No LXR alone transgenic plants showed presence of the transgene. Co-transformation of TaRbcS FT fusion and LXR showed an 11% efficiency of co-transformation and selection (Figure 86).
A method of co-transformation of FT fusions and LXR® for positive selection to determine the co-transformation efficiency has been developed and is outlined below.
Initially, the co-transformation efficiency is determined for a variety of transformation events which include a vector containing an antibiotic selectable marker. These co- transformation events include:
1. FT fusion regulated by a photosynthetic promoter + hph selectable marker
2. LXR® plus hph selectable marker
3. FT fusion regulated by a photosynthetic promoter plus LXR® plus hph selectable marker
Selection on antibiotic media for transgenics takes place and the presence of the transgene for each double or triple co-transformation event is determined, generating a co-tranformation efficiency number for each event.
A second round of co-transformation events also takes place without an antibiotic selectable marker on selection free media. These co-transformation events include:
1. FT fusion regulated by a photosynthetic promoter + dsRED marker
2. LXR® plus dsRED marker
3. FT fusion regulated by a photosynthetic promoter plus LXR® plus dsRED marker Selection for increased growth rate of shoots and/or roots takes place and the presence of the transgene for each double or triple co-transformation event is determined. The presence of the dsRED marker gene is easily determined by visualisation of fluorescence and helps determine the co-tranformation efficiency for each of the transformation events. Comparison of the co-transformation efficiencies determined with and without selectable marker aids in establishing the efficacy of using a superior phenotype as a selection tool.
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Claims

1. A method of manipulating fructan biosynthesis in photosynthetic cells of a plant, said method including introducing into said plant an effective amount of a genetic construct including a promoter, or a functionally active fragment or variant thereof, operatively linked to nucleic acids encoding one or more fructan biosynthetic enzymes, or functionally active fragments or variants thereof.
2. A method according to claim 1 wherein said promoter is selected from the group consisting of constitutive, tissue specific and inducible promoters.
3. A method according to claim 2 wherein said promoter is a light regulated promoter.
4. A method according to claim 1 wherein said one or more fructan biosynthetic enzymes are selected from the group consisting of 1-SST, 1-FFT, 6-SFT and 6G- FFT.
5. A method according to claim 4 wherein said nucleic acids encoding one or more fructan biosynthetic enzymes encode a fusion protein of two or more of 1-SST, 1- FFT, 6-SFT and 6G-FFT.
6. A method of enhancing productivity of a biochemical pathway in a plant, said method including introducing into said plant an effective amount of a genetic construct including nucleic acids encoding two or more enzymes from said pathway, or functionally active fragments or variants thereof.
7. A method according to claim 6 wherein said nucleic acids are linked to form a fusion gene encoding a fusion protein of said two or more enzymes.
8. A method according to claim 7 wherein the biochemical pathway is a fructan biosynthetic pathway and the two or more enzymes from said pathway are selected from the group consisting of 1-SST, 1-FFT, 6-SFT and 6G-FFT.
9. A genetic construct capable of manipulating fructan biosynthesis in photosynthetic cells of a plant, said genetic construct including a promoter, or functionally active fragment or variant thereof, operatively linked to nucleic acids encoding one or more fructan biosynthetic enzymes, or functionally active fragments or variants thereof.
10. A genetic construct according to claim 9 wherein said promoter is selected from the group consisting of constitutive, tissue specific and inducible promoters.
11. A genetic construct according to claim 10 wherein said promoter is a light regulated promoter.
12. A genetic construct according to claim 9 wherein said one or more fructan biosynthetic enzymes are selected from the group consisting of 1-SST, 1-FFT, 6- SFT and 6G-FFT.
13. A genetic construct according to claim 12 wherein said nucleic acids encoding one or more fructan biosynthetic enzymes encode a FT fusion protein of two or more of 1-SST, 1-FFT, 6-SFT and 6G-FFT.
14. A genetic construct capable of enhancing productivity of a biochemical pathway in a plant said genetic construct including nucleic acids encoding two or more enzymes from said pathway, or functionally active fragments or variants thereof.
15. A genetic construct according to claim 14 wherein said nucleic acids are linked to form a fusion gene encoding a fusion protein of said two or more enzymes.
16. A genetic construct according to claim 15 wherein the biochemical pathway is a fructan biosynthetic pathway and the two or more enzymes from said pathway are selected from the group consisting of 1-SST, 1-FFT, 6-SFT and 6G-FFT.
17. A method of enhancing biomass in a plant, said method including introducing into said plant an effective amount of a genetic construct including a promoter, or a functionally active fragment or variant thereof, operatively linked to nucleic acids encoding one or more fructan biosynthetic enzymes, or functionally active fragments or variants thereof.
18. A method according to claim 17 wherein said promoter is selected from the group consisting of constitutive, tissue specific and inducible promoters.
19. A method according to claim 18 wherein said promoter is a light regulated promoter.
20. A method according to claim 17 wherein said one or more fructan biosynthetic enzymes are selected from the group consisting of 1-SST, 1-FFT, 6-SFT and 6G- FFT.
21. A method according to claim 20 wherein said nucleic acids encoding one or more fructan biosynthetic enzymes encode a FT fusion protein of two or more of 1 -SST, 1-FFT, 6-SFT and 6G-FFT.
22. A method of enhancing biomass in a plant, said method including introducing into said plant an effective amount of a genetic construct including nucleic acids encoding two or more enzymes from a biochemical pathway in said plant, or functionally active fragments or variants thereof.
23. A method according to claim 22 wherein said nucleic acids are linked to form a fusion gene encoding a fusion protein of said two or more enzymes.
24. A method according to claim 23 wherein the biochemical pathway is a fructan biosynthetic pathway and the two or more enzymes from said pathway are selected from the group consisting of 1-SST, 1-FFT, 6-SFT and 6G-FFT
25. A method of enhancing biomass in a plant, said method including introducing into said plant effective amounts of a genetic construct capable of manipulating fructan biosynthesis in photosynthetic cells of the plant and a genetic construct capable of manipulating senescence in the plant.
26. A method according to claim 25 wherein the genetic construct capable of manipulating fructan biosynthesis includes a promoter, or a functionally active fragment or variant thereof, operatively linked to nucleic acids encoding one or more fructan biosynthetic enzymes, or functionally active fragments or variants thereof.
27. A method according to claim 26 wherein the promoter is a light regulated promoter.
28. A method according to claim 26 wherein the genetic construct capable of manipulating senescence includes a MYB gene promoter or modified MYB gene promoter, or a functionally active fragment or variant thereof, operatively linked to a gene encoding an enzyme involved in biosynthesis of a cytokinin, or a functionally active fragment or variant thereof.
29. A method of selecting for transformed plants, said method including introducing into said plants an effective amount of a genetic construct including a promoter, or a functionally active fragment or variant thereof, operatively liked to nucleic acids encoding one or more fructan biosynthetic enzymes, or functionally active fragments or variants thereof and selecting plants with enhanced biomass.
30. A method according to claim 29 wherein said promoter is selected from the group consisting of constitutive, tissue specific and inducible promoters.
31. A method according to claim 30 wherein said promoter is a light regulated promoter.
32. A method according to claim 29 wherein said one or more fructan biosynthetic enzymes are selected from the group consisting of 1-SST, 1-FFT, 6-SFT and 6G- FFT.
33. A method according to claim 32 wherein said nucleic acids encoding one or more fructan biosynthetic enzymes encode a FT fusion protein of two or more of 1-SST, 1 -FFT, 6-SFT and 6G-FFT.
34. A transgenic plant cell, plant, plant seed or other plant part with modified fructan biosynthetic characteristics or enhanced biomass relative to an untransformed control plant.
35. A transgenic plant cell, plant, plant seed or other plant part according to claim 34 having increased biomass of at least approximately 10%, more preferably at least approximately 20%, more preferably at least approximately 30%, more preferably at least approximately 40% relative to an untransformed control plant.
36. A transgenic plant cell, plant, plant seed or other plant part according to claim 34 having increased soluble carbohydrate of least approximately 10%, more preferably at least approximately 20%, more preferably at least approximately 30%, more preferably at least approximately 40% relative to an untransformed control plant.
PCT/AU2009/001211 2008-09-15 2009-09-14 Modification of fructan biosynthesis, increasing plant biomass, and enhancing productivity of biochemical pathways in a plant WO2010028456A1 (en)

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JP2011526355A JP5805534B2 (en) 2008-09-15 2009-09-14 Methods for modifying fructan biosynthesis, increasing plant biomass, and enhancing biochemical pathway productivity in plants
EP09812549A EP2337848A4 (en) 2008-09-15 2009-09-14 Modification of fructan biosynthesis, increasing plant biomass, and enhancing productivity of biochemical pathways in a plant
CA2737059A CA2737059C (en) 2008-09-15 2009-09-14 Modification of fructan biosynthesis, increasing plant biomass, and enhancing productivity of biochemical pathways in a plant
US13/063,992 US9890387B2 (en) 2008-09-15 2009-09-14 Modification of fructan biosynthesis, increasing plant biomass, and enhancing productivity of biochemical pathways in a plant
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