WO2008129227A1 - Augmentation de la production d'huile dans les végétaux - Google Patents

Augmentation de la production d'huile dans les végétaux Download PDF

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WO2008129227A1
WO2008129227A1 PCT/GB2007/001461 GB2007001461W WO2008129227A1 WO 2008129227 A1 WO2008129227 A1 WO 2008129227A1 GB 2007001461 W GB2007001461 W GB 2007001461W WO 2008129227 A1 WO2008129227 A1 WO 2008129227A1
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mdar4
plant
plant cell
nucleic acid
seed
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PCT/GB2007/001461
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English (en)
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Peter J. Eastmond
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The University Of Warwick
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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
    • C12N15/8247—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 lipid metabolism, e.g. seed oil composition
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
    • C12N9/0036—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on NADH or NADPH (1.6)

Definitions

  • the present invention relates to plant oils and methods of increasing the amount of oil in plants, including plant parts, e.g. seeds.
  • the seeds of many plants contain oil that serves as an essential source of carbon to drive post-germinative growth and allow photosynthetic establishment (Hayashi et al., 1998).
  • the breakdown of this oil is accompanied by the generation of massive amounts OfH 2 O 2 within the peroxisome because H 2 O 2 is formed as a by-product of the acyl-CoA oxidase step of fatty acid ⁇ -oxidation (Graham and Eastmond, 2002).
  • TAG triacylglycerol
  • TAG lipase The free fatty acids released by TAG lipase are subsequently converted to sucrose via the sequential action of ⁇ -oxidation, the glyoxylate cycle and gluconeogenesis.
  • oil-bearing plant seeds including for example, Arabidopsis
  • Hydrogen peroxide H 2 O 2
  • WO2004/113543 there is disclosed plant lipase polypeptides which are neutral or acid lipases that have activity toward triacylglycerol. These enzymes are associated with oil bodies via a conserved membrane localisation domain.
  • WO2006/131750 discloses a lipase with activity towards triacylglycerol and which has no homology with the lipases disclosed in WO2004/113543.
  • the lipase gene is termed Reserve Deposition/Mobilisation 1 (RDM-I).
  • RDM-I mutants are unable to hydrolyze triacylglycerol indicating an essential role for this lipase in lipid metabolism.
  • the RDM- 1 lipase protein is located in the oil body membrane.
  • APX ascorbate peroxidase
  • MDAR monodehydroascorbate reductase
  • APX initiates electron transfer from two molecules of ascorbate to convert H 2 O 2 to water and the monodehydroascorbate is then recycled to reduced ascorbate by MDAR via electron transfer from NADH.
  • Monodehydroascorbate can also spontaneously disproportionate to ascorbate and dehydroascorbate and the latter can be reduced back to ascorbate by glutathione-dependent dehydroascorbate reductase (Jimenez et al., 1997; del Rio et al.,
  • catalase Although the importance of catalase is well established, the physiological requirement for a component of the APX/MDAR system has yet to be demonstrated. Although catalase is highly active in plant peroxisomes, it has a much lower affinity for H 2 O 2 than APX, suggesting that at low concentrations H 2 O 2 is likely to be preferentially scavenged by the APX/MDAR system (Bunkelmann and Trelease, 1996; Mullen and Trelease, 1996).
  • APX/MDAR membrane-association of APX/MDAR allows the system to protect membrane lipids and integral proteins from oxidative damage, and act as a cordon to limit the escape of H 2 O 2 into the cytosol (Yamaguchi et al., 1995; Mullen and Trelease, 1996; Karyotou and Donaldson, 2005). MDAR may also play a role in reductant balance within the peroxisome by recycling NAD + (Bowditch and Donaldson, 1990; Mullen and Trelease, 1996).
  • catalase in maintaining redox balance in plant peroxisomes has been demonstrated in several studies using mutants or antisense suppression (Kendall et al., 1983; Willekens et al., 1997; Takahashi et al., 1997; Vandenabeele et al., 2004).
  • the inventors have surprisingly found that a conditional seedling-lethal sugar- dependent! (sdp2) mutant of Arabidopsis thaliana is deficient in the peroxisomal membrane isoform of monodehydroascorbate reductase MDAR (MD AR4).
  • MDAR4 component of the ascorbate- dependent electron transfer system is responsible for detoxifying H 2 O 2 which escapes the peroxisome.
  • this function is necessary to protect oil bodies that are in close proximity to peroxisomes. Without protection from oxidative damage, the triacylglycerol lipase of the oil body membrane is inactivated and this cuts off the supply of carbon for seedling establishment.
  • the invention therefore provides a method of increasing the oil content of a plant cell comprising reducing or eliminating MDAR4 activity.
  • the method is therefore applicable to increasing the oil content of plant cells, whether in culture or in vivo in the form of plant tissues, whole plants, plant parts or seeds.
  • the reduction or elimination of MDAR4 activity preferably takes place in the peroxisome of the plant cell.
  • the reduction or elimination of MDAR4 activity preferably takes place during seed development and/or seed maturation, including desiccation of seeds. In plants where MDAR4 activity is reduced or eliminated, a greater accumulation of oil takes place than would otherwise be expected in the case of plants where MDAR4 activity is at a normal (unmodified) level.
  • Figure 1 shows a general pattern for levels of storage oil and other components during the plant life cycle where MDAR4 activity is not modified.
  • oil accumulates to a maximum level, but then generally falls back to a lesser level in the mature seed following desiccation. Breakdown of oil therefore starts to take place during desiccation of the seed and not just during germination.
  • the inventors believe that an increase in seed oil arising as a result of reducing or eliminating MDAR4 activity takes place because the rate of oil breakdown is reduced compared to when MDAR4 operates at normal levels of activity. In other words, the ratio of oil synthesis rate to oil breakdown rate during seed development and/or desiccation is greater (compared to normal) when MDAR4 activity is reduced or eliminated.
  • the reduction or elimination of MDAR4 activity may comprise supression of MDAR4 expression.
  • the plant is preferably transformed or transfected with a nucleic acid or vector capable of suppressing MDAR4 expression. Such transfection or transformation is preferably stable to the extent that the phenotype may be passed to the next generation.
  • the reduction or elimination of MDAR4 activity may comprise antisense suppression of MDAR4 expression.
  • the reduction or elimination of MDAR4 activity may comprise sense suppression of MDAR4 expression.
  • the method of the invention therefore includes the making of plant cells which are null for MDAR4.
  • Such cells and resultant plants and tissues include a non-functional copy of the nucleic acid sequence for MDAR4, wherein the activity of the polypeptide encoded by said nucleic acid is ablated.
  • Methods to provide such a cell are well known in the art and include the use of antisense genes to regulate the expression of specific targets; insertional mutagenesis using T-DNA; the introduction of point mutations and small deletions into open reading frames and regulatory sequences; and double stranded inhibitory RNA (RNAi).
  • RNAi double stranded inhibitory RNA
  • RNAi double stranded RNA
  • the RNAi molecule comprises two complementary strands of RNA (a sense strand and an antisense strand) annealed to each other to form a double stranded RNA molecule.
  • the RNAi molecule is typically derived from exonic or coding sequence of the gene which is to be ablated. Surprisingly, only a few molecules of RNAi are required to block gene expression that implies the mechanism is catalytic. The site of action appears to be nuclear as little if any RNAi is detectable in the cytoplasm of cells indicating that RNAi exerts its effect during mRNA synthesis or processing.
  • RNAi molecules are typically as small as 18 mers, although lengths in the range 16 mers to 50 mers are possible. Lengths of RNAi molecules include 17, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45 or 49 mers.
  • RNAi involves the synthesis of so called "stem loop RNAi" molecules that are synthesised from expression cassettes carried in vectors.
  • the DNA molecule encoding the stem-loop RNA is constructed in two parts, a first part that is derived from a gene the regulation of which is desired. The second part is provided with a DNA sequence that is complementary to the sequence of the first part.
  • the cassette is typically under the control of a promoter that transcribes the DNA into RNA.
  • the complementary nature of the first and second parts of the RNA molecule results in base pairing over at least part of the length of the RNA molecule to form a double stranded hairpin RNA structure or stem-loop.
  • the first and second parts can be provided with a linker sequence.
  • Stem loop RNAi has been successfully used in plants to ablate specific mRNAs and thereby affect the phenotype of the plant (for example, see Smith et al (2000) Nature 407, 319-320).
  • a cassette is provided with at least two promoters adapted to transcribe sense and antisense strands of a nucleic acid molecule encoding MDAR4.
  • Rapidly hybridizing RNA molecules can be used.
  • Rapidly hybridizing RNA molecules may be used.
  • the efficiency of antisense RNA molecules which have a size of more than 50 nucleotides will depend on the annealing kinetics in vitro. Thus, e.g., rapidly annealing antisense RNA molecules exhibit a greater inhibition of protein expression than slowly hybridizing RNA molecules (Wagner et al 1994) Annu. Rev. Microbiol., 48: 713-742; Rittner et al. (1993) Nucl. Acids Res. 21:1381-1387).
  • Such rapidly hybridizing antisense RNA molecules particularly comprise a large number of external bases (free ends and connecting sequences), a large number of structural subdomains (components) as well as a low degree of loops (Patzel et al (1998) Nature Biotechnology 16: 64-68) .
  • the hypothetical secondary structures of the antisense RNA molecule may, e.g., be determined by aid of a computer program, according to which a suitable antisense RNA DNA sequence is chosen.
  • DNA molecules used to transfect according to the invention include a sequence which comprises a deletion, insertion and/or substitution mutation of the MDAR.4 gene.
  • the number of mutant nucleotides is variable and varies from a single one to several deleted, inserted or substitutes nucleotides. The reading frame may be shifted by the mutation.
  • the site of the mutation is variable, as long as expression of an active protein is prevented.
  • the mutation is in the catalytic region of the protein.
  • the method of introducing mutations in DNA sequences are well known to the skilled person, as are the various possibilities of mutagenesis.
  • Coincidental mutageneses as well as, in particular, directed mutageses, e.g. the site-directed mutagenesis, oligonucleotide- controlled mutagenesis or mutageneses by aid of restriction enzymes may be employed.
  • the invention may also provide a DNA molecule which codes for a ribozyme which comprises two sequence portions of at least 10 to 15 base pairs each, which are complementary to sequence portions of an inventive DNA molecule as described above so that the ribozyme complexes and cleaves the mRNA which is transcribed from a natural MDAR4 DNA molecule.
  • the ribozyme will recognize the MRNA of the MDAR4 by complementary base pairing with the mRNA. Subsequently, the ribozyme will cleave and destroy the RNA in a sequence- specific manner, before the enzyme is translated. After dissociation from the cleaved substrate, the ribozyme will repeatedly hybridize with RNA molecules and act as specific endonuclease.
  • ribozymes may specifically be produced for inactivation of a certain mRNA, even if not the entire DNA sequence which codes for the protein is known. Ribozymes are particularly efficient if the ribosomes move slowly along the mRNA. hi that case it is easier for the ribozyme to find a ribosome-free site on the mRNA. For this reason, slow ribosome mutants are also suitable as a system for ribozymes (J. Burke, 1997, Nature Biotechnology; 15, 414-415). This DNA molecule is particularly advantageous for the downregulation and inhibition, respectively, of the expression of plant MDAR4. One possible way is also to use a varied form of a ribozyme, i.e. a minizyme.
  • Minizymes are efficient particularly for cleaving larger mRNA molecules.
  • a minizyme is a hammerhead ribozyme which has a short oligonucleotide linker instead of the stem/loop II. Dimer-minizymes are particularly efficient (Kuwabara et ah, 1998, Nature Biotechnology, 16; 961-965).
  • the activity of MDAR4 may be reduced or eliminated by an inhibitor or antagonist of MDAR4.
  • the MDAR4 inhibitor or antagonist is preferably expressed from an heterologous nucleic acid molecule or vector introduced into the plant or is an expression product of a gene endogenous to the genome of the plant.
  • nucleic acid When an heterologous nucleic acid molecule is introduced into the plant then it may integrated into the host plant genome.
  • the nucleic acid may integrate into a chromosomal location in the the plant genome by a process of homologous recombination.
  • a vector When a vector is introduced into the plant it may be an epigenetic element or an artificial chromosome.
  • the desired nucleic acid in a vector is operably linked to an appropriate promoter or other regulatory elements for transcription in the host cell.
  • Any workable promoter can be employed.
  • the vector may be a bi-functional expression vector which functions in multiple hosts. In the example of nucleic acids encoding polypeptides according to the invention this may contain its native promoter or other regulatory elements and in the case of cDNA this may be under the control of an appropriate promoter or other regulatory elements for expression in the host cell.
  • a promoter is the nucleotide sequence upstream from the transcriptional initiation site and which contains all the regulatory regions required for transcription. Suitable promoters include constitutive, tissue-specific, inducible, developmental or other promoters for expression in plant cells comprised in plants, depending on design. Such promoters include viral, fungal, bacterial, animal and plant-derived promoters capable of functioning in plant cells.
  • Constitutive promoters include, for example CaMV 35S promoter (Odell et al (1985) Nature 313, 9810-812); rice actin (McElroy et al (1990) Plant Cell 2: 163-171); ubiquitin
  • the nucleic acid or vector capable of suppressing MDAR4 expression may be a transposable element.
  • any suitable Arabidopsis transposable element may be used.
  • a preferred transposable element is Tilling.
  • an MDAR4 inhibitor or antagonist When employed MDAR4 inhibitor or antagonist it is preferably applied to the plant. Usually, such inhibitors or antagonists may be applied in aqueous solution in the form of a spray, dip or paste.
  • the treatment may be during or at a stage selected from flowering, fertilization, seed setting, desiccation of the seed or during seed storage.
  • the inhibition of MDAR4 is specific to MDAR4 and not any of the other MDAR4 isoforms.
  • Nucleic acids which suppress MDAR4 expression may consist of all or part of the unique peroxisomal membrane target motif.
  • the nucleic acids may target the 5' and/or 3' UTRs.
  • inhibition of just the MDAR4 isoform is desirable when increasing plant oil content, particularly with respect to seeds.
  • the nucleic acid preferably comprises: (a) a nucleotide sequence of SEQ ID NO: 1 or a sequence having at least 50% identity thereto,
  • the nucleotide sequence has at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 99.5% identity with SEQ ID NO: 1.
  • the nucleic acid preferably comprises a nucleotide sequence encoding:
  • the amino acid has a sequence of at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 99.5% identity with SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 , SEQ ID NO: 5 or SEQ ID NO:6.
  • the nucleic acids described above will usually comprise a first stand (as defined) and a second complementary strand.
  • the first and second strands being capable of hybridization with one another and other related strands, depending on the degree of sequence identity.
  • Hybridization of a nucleic acid molecule occurs when two complementary nucleic acid molecules undergo an amount of hydrogen bonding to each other.
  • the stringency of hybridization can vary according to the environmental conditions surrounding the nucleic acids, the nature of the hybridization method, and the composition and length of the nucleic acid molecules used. Calculations regarding hybridization conditions required for attaining particular degrees of stringency are discussed in Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001); and Tijssen, Laboratory Techniques in Biochemistry and Molecular Biology — Hybridization with Nucleic Acid Probes Part I, Chapter 2 (Elsevier, New York, 1993).
  • the T n is the temperature at which 50% of a given strand of a nucleic acid molecule is hybridized to its complementary strand. The following is an exemplary set of hybridization conditions and is not limiting:
  • Hybridization 5x SSC at 65 0 C for 16 hours
  • Hybridization 5x-6x SSC at 65°C-70°C for 16-20 hours
  • Hybridization 6x SSC at RT to 55°C for 16-20 hours
  • Tm Melting Temperature
  • T m (wA+xT) * 2 + (yG+zC) * 4 where w,x,y,z are the number of the bases A 5 T, G 5 C in the sequence, respectively (from Marmur, J., and Doty, P. (1962) JM?/ Biol 5: 109- 118).
  • T m 64.9 +41*(yG+zC-16.4)/(wA+xT+yG+zC)
  • the nucleic acid sequence may be of greater identity than 50% as described above.
  • the nucleic acid sequence may have an identity of at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or at least 99% identity with the nucleic acid sequence of SEQ ID NO:1, or the nucleic acid sequence encoding the protein of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6.
  • amino acid sequences of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6 also include amino acid sequences of at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or at least 99% identity therewith.
  • polypeptides encoded by SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6 may be modified by one or more substitutions, additions, deletions, truncations which may be present in any combination.
  • substitutions are those that vary from a reference polypeptide by conservative amino acid substitutions. Such substitutions are those that substitute a given amino acid by another amino acid of like characteristics.
  • amino acids are considered conservative replacements (similar): a) alanine, serine, and threonine; b) glutamic acid and aspartic acid; c) asparagine and glutamine d) arginine and lysine; e) isoleucine, leucine, methionine and valine and f) phenylalanine, tyrosine and tryptophan. Most highly preferred are variants which retain or enhance the same biological function and activity as the reference polypeptide from which it varies.
  • the nucleic acid sequence preferably encodes a deficient or at least partially inactive monodehydroascorbate reductase 4 (MD AR4).
  • a DNA construct or vector is introduced into a plant host is not critical to the invention.
  • Various methods for plant cell transformation include the use of Ti- or Ri- plasmids, microinjection, electroporation, DNA particle bombardment, liposome fusion, DNA bombardment or the like.
  • explants For transformation of plant cells using Agrobacterium, explants may be combined and incubated with the transformed Agrobacterium for sufficient time for transformation, the bacteria killed, and the plant cells cultured in an appropriate selective medium. Once callus forms, shoot formation can be encouraged by employing the appropriate plant hormones in accordance with known methods and the shoots transferred to rooting medium for regeneration of plants. The plants may then be grown to seed and the seed used to establish repetitive generations and for isolation of oils.
  • the germination of seeds deficient or reduced in MDAR4 activity may require exogenously supplied sucrose and/or other growth promoting substances to overcome the effect of a lack of oil breakdown and lack of seedling establishment.
  • transgenic plant which is capable of producing seed having increased amounts of oil compared to the unmodified plant
  • traditional plant breeding techniques including methods of mutagensis, may be employed to further manipulate the fatty acid composition.
  • Additional foreign fatty acid modifying DNA sequences may be introduced via genetic engineering to further manipulate the fatty acid composition.
  • the invention therefore provides the products of the method of the invention for increasing oil content of a plant cell; namely plant cells, plant tissues, plant organs and parts, as well as whole plants and their propagative materials, particularly seeds. All of the aforementioned plant cell characteristics described in connection with the method of the invention apply equally to the products of the invention.
  • the invention also provides a plant cell transformed or transfected with nucleic acid capable of reducing or eliminating monodehydroascorbate reductase (MD AR4) activity.
  • MDAR4 monodehydroascorbate reductase
  • the reduction or elimination of MDAR4 activity is preferably in the peroxisome.
  • the reduction or elimination of MDAR4 activity in the plants and plant cells preferably comprises supression of MDAR4 expression.
  • the plant cell is preferably transformed or transfected with a nucleic acid or vector capable of suppressing MDAR4 expression.
  • the vector preferably comprises a cell or tissue specific promoter.
  • the promoter is preferably an inducible promoter or a developmentally regulated promoter.
  • Chemical-regulated promoters may be used to modulate the expression of the desired nucleic acid sequence in a plant through the application of an exogenous chemical regulator.
  • the promoter may be a chemical-inducible promoter, where application of the chemical induced gene expression, or a chemical- repressible promoter, where application of the chemical represses gene expression.
  • Chemical-inducible promoters are known in the art and may include, but are not limited to, the maize In2-2 promoter, which is activated by benzenesulfonamide herbicide safeners, the maize GST promoter, which is activated by hydrophobic electrophilic compounds that are used as pre-emergent herbicides, and the tobacco PR- Ia promoter, which is activated by salicylic acid.
  • Other chemical-regulated promoters of interest include steroid-responsive promoters (see, for example, the glucocorticoid-inducible promoter in Schena et al (1991) Proc. Natl. Acad. Sci. USA 88: 10421-10425 and McNellie et al. (1998) Plant J.
  • tissue-specific promoters may be utilised.
  • Tissue-specific promoters include those described by Yamamoto et al. (1997) Plant J. 12(2): 255-265; Kawamata et al (1997) Plant Cell Physiol. 38(7): 792-803; Hansen et al (1997) MoI. Gen. Genet. 254(3): 337-343; Russell et al. (1997) Transgenic Res. 6(2): 157-168; Rinehart et al (1996) Plant Physiol. 112(3): 1331-1341; Van Camp et al (1996) Plant Physiol. 112(2): 525-535; Canevascni et al (1996) Plant Physiol.
  • tissue specific promoter is a promoter which is active during the accumulation of oil in developing oil seeds, (for example see Broun et al. (1998) Plant J. 13(2): 201-210).
  • nucleic acid sequence When a particular nucleic acid sequence is comprised in a vector or construct so as to be operably linked then it is linked and part of the same nucleic acid molecule, suitably positioned and oriented for transcription to be initiated from the promoter.
  • DNA operably linked . to a promoter is "under transcriptional initiation regulation" of the promoter.
  • vectors are nucleic acid constructs which operate as plant vectors. Specific procedures and vectors previously used with wide success upon plants are described by Guerineau and Mullineaux (1993) (Plant transformation and expression vectors. In: Plant Molecular Biology Labfax (Croy RRD ed) Oxford, BIOS Scientific Publishers, pp 121- 148. Suitable vectors may include plant viral-derived vectors (see e.g. EP-A-194809).
  • Vectors may also include selectable genetic marker such as those that confer selectable phenotypes such as resistance to herbicides (e.g. kanamycin, hygromycin, phosphinotricin, chlorsulfuron, methotrexate, gentamycin, spectinomycin, imidazolinones and glyphosate).
  • herbicides e.g. kanamycin, hygromycin, phosphinotricin, chlorsulfuron, methotrexate, gentamycin, spectinomycin, imidazolinones and glyphosate.
  • the methods of the invention may be used in relation to all kinds of plants including Gymnosperms, Pteridophytes and Bryophytes.
  • algae including both freshwater and marine algae, single or multicellular. Included within the scope of the invention are algal cells for the production of oils, whether in cell culture or in whole plant form.
  • the plant cell may be comprised in a plant selected from a monocot or a dicot.
  • the plant cell of all aspects of the invention may be comprised in a plant selected from the families Brassicaceae or Compositae, or as listed in table 5.
  • the cell, tissue or plant is selected from: corn ⁇ Zea mays), canola (Brassica napus, Brassica rapa ssp.), flax (Limirn usitatissimuni), alfalfa (Medicago sativ ⁇ ), rice (Oryza sativa), rye (Secale cerale), sorghum ⁇ Sorghum bicolor, Sorghum vulgare), sunflower (Helianthus annus), wheat ⁇ Tritium aestivum), soybean ⁇ Glycine max), tobacco ⁇ Nicotiana tabacum), potato ⁇ Solanum tuberosum), peanuts ⁇ Arachis hypogaea), cotton ⁇ Gossypium hirsutum), sweet potato (Iopmoea batatus), cassava (Man ⁇ hot esculentd), coffee (Cofea spp.), coconut (Cocos nucifer ⁇ ), pineapple (Anana comosus
  • the plants are crop plants (for example, cereals and pulses, maize, wheat, potatoes, tapioca, rice, sorghum, millet, cassava, barley, pea), and other root, tuber or seed crops.
  • Important seed crops are oil-seed rape, sugar beet, maize, sunflower, soybean, sorghum, and flax (linseed).
  • Horticultural plants to which the present invention may be applied may include lettuce, endive, and vegetable brassicas including cabbage, broccoli, and cauliflower.
  • the present invention may be applied in tobacco, cucurbits, carrot, strawberry, sunflower, tomato, pepper.
  • Grain plants that provide seeds of interest include oil-seed plants and leguminous plants.
  • Seeds of interest include grain seeds, such as corn, wheat, barley, rice, sorghum, rye, etc.
  • Oil seed plants include cotton, soybean, saffiower, sunflower, Brassica, maize, alfalfa, palm, coconut, etc.
  • Leguminous plants include beans and peas. Beans include guar, locust bean, fenugreek, soybean, garden beans, cowpea, mungbean, lima bean, fava been, lentils, chickpea, etc.
  • the invention also provides a plant, plant organ or plant tissue comprising a plant cell of the invention as herein defined, hi preferred embodiments, the invention provides a plant seed comprising a plant cell as defined herein.
  • the invention further provides a method of identifying an agent which increases the oil content of a seed, comprising contacting a plant deficient in monodehydroascorbate reductase (MD AR4), harvesting the mature or developing seed and determining the oil content of the seed.
  • the oil content of a seed is preferably determined by measuring fatty acid content of the seed.
  • the invention therefore provides a plant cell overexpressing monodehydroascorbate reductase (MDAR4).
  • MDAR4 monodehydroascorbate reductase
  • the invention includes plant tissue or a plant comprising a plant cell which overexpresses monodehydroascorbate reductase (MDAR.4).
  • the overexpression is measured in relation to a native, wild-type or non-transgenic plant of the same species, variety or cultivar.
  • the overexpression of MDAR4 may be least 2-fold above basal level expression, optionally at least 5-fold; 10-fold, 20-fold, 30-fold, 40-fold, or 50-fold.
  • the cell may overexpress the MDAR4 gene by at least 100-fold above basal level expression when compared to a non-transgenic cell of the same species.
  • a gene(s) may be placed under the control of a powerful promoter sequence or an inducible promoter sequence to elevate expression of mRNA encoded by said gene.
  • the modulation of mRNA stability is also a mechanism used to alter the steady state levels of an mRNA molecule, typically via alteration to the 5' or 3' untranslated regions of the mRNA.
  • the overexpression of MDAR4 may be engineered to take place during specific phases of seed development, e.g. post-fertilization and/or seed setting.
  • the invention also provides a method of identifying an agent which increases the oil content of a plant cell, tissue or seed, comprising contacting a plant tissue or plant which overexpresses monodehydroascorbate reductase (MDAR4) with a candidate agent and then determining the oil content of the cell, tissue or seed.
  • the oil content of a seed is preferably determined by measuring the fatty acid content of the seed.
  • Figure 1 shows in schematic form the patterns of storage oil synthesis and breakdown during the plant life cycle of Ar abidopsis.
  • Figure 2 shows post-germinative growth and fatty acid breakdown in sdp2.
  • Figure 3 shows the molecular characterization of SDP2.
  • A Mapping SDP2.
  • PCR based SSLP, CAPS and SNAP markers were used to map SDP2 to an 80 kb region on the top arm of Chromosome 3 near GLl.
  • the positions of markers a to f are denoted by bars and the number of recombination events / total number of chromosomes (380) are listed below each.
  • Figure 4 shows peroxisome clustering and proximity to oil bodies in sd ⁇ 2 seedlings.
  • Figure 5 shows ⁇ -oxidation-dependent oxidative damage to oil bodies in sdp2 seedlings.
  • Figure 6 shows carbonylation and inactivation of SDPl .
  • Figure 7 shows metabolite levels in germinating sdp2 seedlings.
  • A H 2 O 2 .
  • B total ascorbate (ascorbate + dehydroascorbate).
  • Figure 9 shows the effect of catalase deficiency on fatty acid breakdown during post- germinative growth.
  • A Total fatty acid.
  • Figure 10 shows a schematic diagram illustrating the proposed role of MDAR4 in storage oil breakdown in germinating Arabidopsis seeds.
  • ASC is ascorbate
  • MDA is monodehydroascorbate
  • SDPl is a lipase
  • PXAl is an ABC transporter
  • ACX is acyl-CoA oxidase
  • CAT is catalase
  • APX is ascorbate peroxidase
  • SDP2 or MDAR4 is monodehydroascorbate reductase.
  • Figure 11 is the cDNA sequence of SDP2/MDAR4 (At3g27820) SEQ ID NO:1
  • Figure 12 is the amino acid sequence of SDP2/MDAR4 (At3g27820) SEQ ID NO:2
  • Figure 13 is an amino acid sequence alignment of MDAR isoforms from Arabidopsis with SDP2/MDAR4 (SEQ ID NO:2) generated using ClustalX (version 1.83).
  • the isoform sequences are At5g03630 (SEQ ID N0:3); At3g09940 (SEQ ID N0:4); At3g52880 (SEQ ID NO: 5) and Alg63940 (SEQ ID NO: 6).
  • GIy 11 ' VaI 14 and GIy 386 from SDP2/MDAR4 (At3g27820) are marked in red.
  • Wild type Arabidopsis thaliana (ecotype Colombia 0 and Landsberg erecta) were obtained from the Nottingham Arabidopsis Stock Centre (University of Nottingham,
  • the sdp2-4 mutant (SALK_068667) was obtained from T-DNA express (Alonso et al., 2003).
  • the PTSl targeted GFP line A5 (Cutler et al., 2000), the pxal mutant
  • the fatty acid composition of seed and seedling lipids was measured by CG analysis after combined digestion and fatty acid methyl ester formation from frozen tissue using the method of Browse et al., (1986).
  • H 2 O 2 and ascorbate ascorbate and dehydroascorbate were extracted from O.lg FW of seedlings and measured spectophotometrically using the methods described by Huang et al., (2005).
  • the sdp2-l mutant was out-crossed to wild type ecotype Landsberg erecta. Fl plants were allowed to self fertilise and the F2 progeny were screened for the sugar-dependent phenotype. Genomic DNA was isolated from 190 F2 sdp2 ⁇ l lines using the Extract-N- Amp Plant PCR Kit (Sigma-Aldrich, Poole, Dorset, UK).
  • mapping was carried out using simple sequence polymorphisms (Bell and Ecker, 1994), cleaved amplified polymorphic sequences (Konieczny and Ausubel, 1993) and single-nucleotide amplified polymorphisms (Drenkard et al., 2000) utilizing information from the Monsanto Arabidopsis polymorphism collection (Jander et al., 2002). New markers used in this study are listed in Table 1 below. Candidate genes within the mapping interval were amplified from sdp2-l, sdp2-2 and sdp2-3 genomic DNA by PCR and sequenced to identify mutations.
  • DNAse-treated total RNA was isolated from Arabidopsis seedlings using the RNeasy kit from Qiagen Ltd. (Crawley, West Wales, UK). The synthesis of single stranded cDNA was carried out using SuperscriptTM II RNase H ' reverse transcriptase from Invitrogen Ltd. (Paisley, UK). SDP2 transcripts were detected by PCR using primers SDP2F (5'- CAAAGACGGGAGCCACTTAC-3'- SEQ ID NO: 16) and SDP2R (5'- CTGCTGACTCACAACCGTGT-3 - SEQ ID NO: 17').
  • TEM was carried out as described previously (Eastmond, 2006). Five d old Arabidopsis seedlings were fixed for 2 h in 2.5 % (v/v) glutaraldehyde, 4 % (v/v) formaldehyde in 100 mM phosphate buffer (pH 7.0), with a secondary fixation of 1 % (w/v) osmium tetroxide in phosphate buffer for 1 h. The tissue was embedded in Spurrs resin, sectioned and stained with uranyl acetate and Reynolds lead citrate. Confocal microscopy was performed using a Zeiss LSM 510 Meta on an Axioplan 2M, fitted with a 63 x PlanApo lens (NA 1.4). The sample was excited with a 488 nm Argon laser and GFP emission collected via a 505-530 nm BP filter. Bright field images were captured simultaneously with the transmission detector. Organelle purification, protein purification and measurement of oxidative damage
  • Oil bodies and oil body membranes were purified from 2 d old Arabidopsis seedlings, and recombinant N-terminal His 6 -tagged SDPl was expressed and purified using protocols that were described previously (Eastmond, 2006). Peroxisomal fractions were obtained from homogenates of 5 d old etiolated Arabidopsis seedlings using sucrose density gradient centrifugation as described previously (Eastmond et al., 2000b). The levels of LOOHs in purified oil body lipids were estimated using the FOX (ferrous oxidation xylenol orange) assay following the protocol described in Sattler et al., (2004).
  • FOX ferrous oxidation xylenol orange
  • the only modification to the method was that the lipids were extracted from oil bodies in 1.5 ml tubes without homogenization.
  • the levels of protein carbonyls in oil body membranes and purified recombinant SDPl was determined using the spectrophotometric quantification method described by Nguyen and Donaldson (2005).
  • SDPl was immunoprecipitated from oil body membranes using the IP 50 Protein G Immunoprecipitation Kit (Sigma) and carbonyl groups were detected using the OxyBlotTM Protein Oxidation Detection Kit (Millipore) as described by Nguyen and Donaldson (2005).
  • Triacylglycerol lipase activity was measured in purified oil body membranes and recombinant SDPl using an emulsion of [ 14 CJtriolein as described previously (Eastmond, 2006). Purified oil bodies were also used as a substrate for recombinant SDPl using the assay procedure described in Eastmond, (2006).
  • 2 d old seedlings grown on medium containing 1% (w/v) sucrose were ground in a pestle and mortar with 1 ml of buffer (150 mM Tris/HCl pH 7.5, 10 mM KCl, 1 mM EDTA, 10 mM FAD, 10 % (v/v) glycerol).
  • the extract was clarified by centrifugation at 15,000 g for 30 min at 4 0 C and the supernatant was desalted using a Sephadex G-50 spin column.
  • the extract was assayed spectrophotometrically for acyl-CoA oxidase, enoyl-CoA hydratase, L-3-hydroxyacyl-CoA dehydrogenase, 3-ketoacyl-CoA thiolase, isocitrate lyase, malate synthase, catalase and NADH-dependent glyceraldehyde-3 -phosphate dehydrogenase activitities according to methods described previously (Eastmond et al., 2000a; Rylott et al., 2006; Takahashi et al., 1997; Hancock et al., 2005).
  • a region of genomic DNA containing MDAR4 was amplified from Arabidopsis using primers 5'-atctcatgattgagtgggtgattggttg-3'- (SEQ ID NO: 18) and 5'- agcttcttcgagggttagggatgagatt-3' (SEQ ID NO: 19) and the product was cloned into the pCR2.1-TOPO vector from Invitrogen. Using Standard molecular-biology techniques, MDAR4 was excised and cloned into the pGREENII vector (Hellens et al., 2000).
  • the MDAR4 construct was transformed into Agrobacterium tumefaciens strain GV3101 containing the pSOUP vector (Hellens et al., 2000) by electroporation and into Arabidopsis sdp2-4 ecotype Columbia by the floral dip method (Clough and Bent, 1998). Transformants containing the T-DNA were selected by screening for loss of a sugar- dependent phenotype and the presence of wild type MDAR4 transcripts was confirmed by RT-PCR.
  • Example 1 - SDP2 is required for storage oil hydrolysis in germinating Arabidopsis seeds
  • the patatin domain TAG lipase encoded by SDPl has been characterized previously (Eastmond, 2006).
  • the sdp2 mutant germinates normally (see Table 2 below) but the cotyledons fail to expand or green and seedling growth arrests (see Figure 2A).
  • Example 2 - SDP2 encodes a component of the peroxisomal antioxidant system
  • the SDP2 locus was mapped to a region on Chromosome 3 near GLABRAl, and between PCR-based markers ciwl 1 and snap77 (see Figure 3A). Further mapping reduced the interval to a -80 kb region, containing 22 open reading frames. Sequencing candidate genes within this region revealed that three independent ethyl methanesulphonate (EMS) sdp2 alleles contained mutations in At3g27820 ( Figure 3B). This gene encodes an isoform of monodehydroascorbate reductase (MDAR4) that is associated with the peroxisomal membrane (Lisenbee et al., 2005).
  • MDAR4 monodehydroascorbate reductase
  • Example 4 - ⁇ -oxidation causes oxidative damage to oil bodies in sdp2 seedlings
  • acyl-CoA oxidase which is the first enzyme of peroxisomal fatty acid ⁇ -oxidation, is likely to be the major source Of H 2 O 2 production in the cell (Graham and Eastmond, 2002).
  • fatty acid ⁇ -oxidation inhibits oil body lipase activity in sdp2 seedlings the sdp2-4 mutant was crossed into the fatty acid catabolism deficient mutant pxal.
  • the PXA1/CTS/PED3 protein is an ABC transporter, which is required to import substrate (fatty acids or acyl- CoAs) into the peroxisome for ⁇ -oxidation (Zolman et al., 2001).
  • TAG lipase activity was almost undetectable in oil body membranes prepared from 2 d old sdp2-4 seedlings (Figure 5A). However, lipase activity was recovered to wild type levels in the sdp2-4 pxal double mutant ( Figure 5A).
  • FOX reactive oxidation xylenol orange
  • protein oxidation was monitored by detecting protein carbonyls (Levine et al., 1990).
  • Example 5 - SDPl is a target of oxidative damage in sdp2 seedlings
  • Example 7 Seedlings of sdp2 retain the capacity to ⁇ -oxidize 2,4- dichlorophenoxybutyric acid
  • the sdp2 mutant was grown on medium containing 2,4-dichlorophenoxybutyric acid (2,4-DB). This compound is converted to the herbicide 2,4-dichloro ⁇ henoxyacetic acid (2,4-D) by a single cycle of ⁇ -oxidation (Hayashi et al., 1998). Arabidopsis mutants in many genes that are either directly required for fatty acid ⁇ -oxidation or for peroxisome function in general exhibit a 2,4-DB resistant phenotype (see Baker et al., 2006).
  • Seeds were germinated on medium containing 1% (w/v) sucrose. Peroxisomal enzyme activities were measured in whole extracts from 2 d old seedlings.
  • Acyl-CoA oxidase (ACX) activity was assayed using palmitoyl-CoA, decanoyl-CoA and butyryl-CoA.
  • Enoyl-CoA hydratase (ECH) was assayed using crotonyl- CoA.
  • L-hydroxyacyl-CoA dehydrogenase (HAD) and 3-ketoacyl-CoA thiolase (KAT) activities were assayed using acetoacetyl-CoA.
  • ICL, MLS and CAT are isocitrate lyase, malate synthase and catalase, respectively.
  • Lipase activity was measured in purified oil body membranes using 10 mM [ i4 C]triolein as a substrate. Values are the mean ⁇ SE of measurements on three separate extracts. *Activity significantly different from WT (P ⁇ 0.001). nd, not determined.
  • NADH-dependent glyceraldehyde-3- phosphate dehydrogenase was also measured in 2 d old seedlings of sdp2-4 grown on medium containing sucrose (see Table 3 above). This enzyme is an indicator of oxidative damage caused by H 2 O 2 in Arabidopsis (Hancock et al., 2005, Job et al., 2005). The activity of GAPDH was not affected, suggesting that the deficiency in MDAR.4 is unlikely to have caused a general increase in oxidative damage to cytosolic proteins.
  • Example 8 - Catalase deficiency has a comparatively small effect on storage oil breakdown
  • mutants are described in Eastmond, (2006) Plant Cell 18, 665-675. Compared to wild type, the mutants show significantly higher seed oil content due to the suppression of MDAR4. Compared to the other mutants, sdp2 mutants have a surprisingly higher seed fatty acid content.
  • sdp2 lacks the APX/MDAR system and consequently some of the H 2 O 2 produced by acyl-CoA oxidase following seed germination escapes from the peroxisome and causes oxidative damage to oil bodies, inactivating SDPl ( Figure 10).
  • Analysis of sdp2 seedlings immediately following germination confirmed that they have elevated levels of H 2 O 2 and that their oil body proteins and lipids become oxidized.
  • H 2 O 2 levels, oxidative damage to oil bodies and loss of lipase activity were all suppressed.
  • SDPl The activity of SDPl can be inhibited by H 2 O 2 in vitro.
  • oxidised SDPl can be detected in oil bodies from sdp2 seedlings but not from wild type. Inactivation of SDPl is sufficient to account for much of the sugar-dependent phenotype of sdp2 (Eastmond, 2006). However, it cannot be discounted that additional proteins, which are necessary for oil hydrolysis and utilization might also be damaged. Unlike oil bodies, peroxisomes do not appear to depend so greatly on the APX/MDAR system for protection against H 2 O 2 . Seedlings of sdp2 are able to ⁇ -oxidize 2,4-DB.
  • peroxisomes and oil bodies cluster together in the cotyledon cells of living sdp2 seedlings and that this association persists as long as the oil bodies remain undegraded.
  • physical contact may play a role in storage oil breakdown in oilseeds by facilitating the transfer of fatty acids from oil bodies to peroxisomes so that they can be ⁇ -oxidized.
  • H 2 O 2 required to inhibit SDPl in vitro (-0.5 mM) is 16-fold higher than the estimated cytosolic concentration in 1 d old sdp2 seedlings (-0.03 mM). This concentration was calculated using the data from Figure 7A, assuming that the cytosol constitutes about 20% of the seedlings volume. However, H 2 O 2 concentrations are unlikely to be uniform and could be heightened at, or near, the peroxisomal membrane, particularly if acyl-CoA oxidases are situated there.
  • the sdp2 mutant is defective in the second enzyme in the APX/MDAR system (MD AR4).
  • Metabolite measurements suggest that in sdp2, the monodehydroascorbate produced at the peroxisomal membrane cannot be recycled efficiently causing the availability of ascorbate to dimmish and the APX/MDAR system to collapse.
  • Ascorbate could still be replenished for APX in the absence of MDAR4.
  • Arabidopsis peroxisomes also contain the soluble matrix isoform MDARl (Lisenbee et al. 2005).
  • Monodehydroascorbate can also disproportionate to ascorbate and dehydroascorbate and biochemical studies have suggested that glutathione-dependent dehydroascorbate reductase activity is present in plant peroxisomes and therefore could convert dehydroascorbate back to ascorbate (Jimenez et al., 1997; del Rio et al., 1998). Ascorbate and monodehydroascorbate are likely to be shuttled across the peroxisomal membrane since the catalytic site of MDAR4 is situated on the matrix side of the peroxisomal membrane, while the catalytic site of APX3 is on the cytosolic side (Lisenbee et al., 2005). The phenotype of sdp2 suggests that nothing can complement the function of MDAR4 in protecting oil bodies against ⁇ - oxidation-dependent oxidative damage.
  • APX3 The predominant APX isoform from the peroxisomal membranes of Arabidopsis is APX3 (At4g35000). Narendra et al., (2006) have recently reported that the APX3 gene is dispensable for growth and development. Therefore, a deficiency in APX activity might not give rise to the same phenotype as sdp2. Redundancy cannot be dismissed as an explanation for the apparent disparity considering that there are nine APX genes in the Arabidopsis genome (Lisenbee et al., 2003). Specifically there is a homologue of APX3 (APX5; At4g35970) with sequence similarity throughout the polypeptide sequence, including the C-terminal transmembrane domain and targeting motif.
  • APX5 is very low relative to APXS (Narendra et al., 2006).
  • the inventors do not preclude the possibility that MDAR4 functions independently of APX.
  • sucrose can partially relieve the post-germinative growth defect.
  • Tocopherols can scavenge lipid peroxy radicals yielding a tocopheroxyl radical that could be recycled by reacting with ascorbate to produce monodehydroascorbate (Liebler, 1993).
  • MDAR4 may function in a one-electron redox cycle that regenerates tocopherol from the tocopheroxyl radical at the peroxisomal membrane.
  • MDAR4 might be capable of recycling the oxidation products of other powerful antioxidants, such as phenolics (Sakihama et al., 2000). Indeed MDAR is unique in that it is the only enzyme known to use organic radicals as substrates (Hossain et al., 1984).
  • peroxisomal MDAR has also been implicated in fatty acid catabolism through the provision of NAD + cofactor for L-3- hydroxyacyl-CoA dehydrogenase and malate dehydrogenase (Bowditch and Donaldson, 1990; Mullen and Trelease, 1996). These enzymes are required for ⁇ -oxidation and glyoxylate cycle function, respectively and theoretically if H 2 O 2 was detoxified entirely by APX/MDAR the system could recycle sufficient NAD + for both pathways (Mullen and Trelease, 1996).
  • the photo-respiratory pathway In addition to fatty acid ⁇ -oxidation, the photo-respiratory pathway also generates large quantities of H 2 O 2 in plant peroxisomes as a result of the activity of glycolate oxidase (Willekens et al., 1997). Catalase has been shown to play a major role in detoxifying this H 2 O 2 . Anti-sense suppression of catalase results in oxidative damage and triggers cell death in tobacco and Arabidopsis plants that are subjected to high light treatment (Willekens et al., 1997; Vandenabeele et al., 2004).
  • Catalase and the APX/MDAR system are both important parts of the peroxisomal antioxidant machinery during the post-germinative growth of Arabidopsis seedlings.
  • the inventors have found that their roles are physiologically different and that neither can fully compensate for the loss of the other.
  • Catalase protects constituents of the peroxisomal matrix from oxidative damage while the main role of MDAR4 is proposed by the inventors to be the prevention OfH 2 O 2 from escaping beyond the outer surface of the peroxisomal -membrane.
  • the consequences of H 2 O 2 escape are fatal primarily because inactivation of triacylglycerol hydrolysis on closely associated oil bodies prevents the seedling from releasing the carbon skeletons and energy that it needs for initial post-germinative growth. Accession Numbers
  • GenBank accession number for an SDP2 ⁇ MDAR4 cDNA is AY039980.
  • Theaceae 80 Acoraceae Cyclanthaceae Theligonaceae Agavaceae Cymodoceaceae
  • Floral dip a simplified method for Agrobacterium- mediated transformation of Arabidopsis thaliana. Plant J. 16, 735-743.
  • Drenkard E., Richter, B.G., Rozen, S., Stutius, L.M., Angell, N.A., Mindrinos, M., Cho, RJ., Oefner, PJ., Davis, R. W. and Ausubel, F.M. (2000).
  • a simple procedure for the analysis of single nucleotide polymorphisms facilitates map-based cloning in Arabidopsis. Plant Physiol. 124, 1483-1492.
  • SUGAR-D EP ENDENTl encodes a patatin domain triacylglycerol lipase that initiates storage oil breakdown in germinating Arabidopsis seeds. Plant Cell 18, 665-675.
  • Peroxisomal ascorbate peroxidase resides within a subdomain of rough endoplasmic reticulum in wild-type Arabidopsis cells. Plant Physiol. 132, 870-882.
  • the Arabidopsis ascorbate peroxidase 3 is a peroxisomal membrane-bound antioxidant enzyme and is dispensable for Arabidopsis growth and development. J. Exp. Bot. 57, 3033-3042.
  • Vitamin E is essential for seed longevity and for preventing lipid peroxidation during germination. Plant Cell 16, 1419-1432.
  • Catalase is a sink for H 2 O 2 and is indispensable for stress defence in C 3 plants. EMBO J. 16, 4806-4816.
  • the Arabidopsis pxal mutant is defective in an ATP-binding cassette transporter-like protein required for peroxisomal fatty acid beta-oxidation. Plant Physiol. 127, 1266-1278.

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Abstract

Cette invention se rapporte à l'isoforme de la monodéhydroascorbate réductase (MDAR4) logée dans les peroxysomes qui s'avère jouer un rôle clé dans l'accumulation et la décomposition de l'huile dans les graines des végétaux. La MDAR4 forme une partie du système APX/MDAR membranaire qui protège les lipides membranaires et les protéines intrinsèques du stress oxydatif provoqué par H2O2. Le système APX/MDAR agit comme un cordon pour limiter la fuite de H2O2 du peroxysome. Des données ont aussi montré l'étroite association des corps lipidiques des graines avec les peroxysomes. Les plants transformés dans lesquels l'activité de la MDAR4 est supprimée, par exemple par mutation du gène codant la MDAR4 ou par suppression sens ou anti-sens, présentent une teneur en huile qui est supérieure à celle des plants sauvages ou non génétiquement modifiés. Les plants transfectés ou les cellules végétales transfectées qui surexpriment la MDAR4 sont utilisés pour rechercher les inhibiteurs ou les antagonistes de la MDAR4 qui, à leur tour, sont utilisés pour supprimer l'activité de la MDAR4 durant le développement des graines et générer une teneur en huile supérieure dans les graines.
PCT/GB2007/001461 2007-04-23 2007-04-23 Augmentation de la production d'huile dans les végétaux WO2008129227A1 (fr)

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CN112301044A (zh) * 2020-10-26 2021-02-02 扬州大学 一种本生烟NbAPX3基因多克隆抗体及其制备方法和应用
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110669744A (zh) * 2019-11-05 2020-01-10 海南大学 一种木薯抗坏血酸过氧化物酶基因及其原核表达载体的构建和应用
CN112301044A (zh) * 2020-10-26 2021-02-02 扬州大学 一种本生烟NbAPX3基因多克隆抗体及其制备方法和应用
CN116458429A (zh) * 2023-04-28 2023-07-21 广西壮族自治区南宁良凤江国家森林公园 小叶红叶藤种子与种胚组织培养繁殖的应用及其方法

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