WO2008129227A1 - Improved plant oil production - Google Patents

Abstract

The peroxisome located isoform of monodehydroascorbate reductase (MDAR4) is discovered to have a key role in the accumulation and breakdown of oil in plant seeds. MDAR4 forms part of the membrane-associated APX/MDAR system which protects membrane lipids and integral membrane proteins from oxidative damage by H2O2. The APX/MDAR system acts as a cordon to limit the escape of H2O2 from the peroxisome. Also discovered is the close association of oil bodies of seeds with peroxisomes. Transformed plants in which MDAR4 activity is suppressed, whether by mutation of the gene encoding MDAR4, or by sense or antisense suppression for example, have an oil content which is greater than unmodified or wild type plants. Transfected plants or plant cells which overexpress MDAR4 are useful for the screening of inhibitors or antagonists of MDAR4 which in turn are used to suppress MDAR4 activity during seed development and generated higher oil content in seeds.

Description

, IMPROVED PLANT OIL PRODUCTION

TECHNICAL FIELD

The present invention relates to plant oils and methods of increasing the amount of oil in plants, including plant parts, e.g. seeds.

BACKGROUND ART

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 OfH₂O₂ within the peroxisome because H₂O₂ is formed as a by-product of the acyl-CoA oxidase step of fatty acid β-oxidation (Graham and Eastmond, 2002).

The primary seed storage reserve of many higher plants is triacylglycerol (TAG), which is found in membrane-bound oil bodies. During germination, TAG reserves are broken down and the carbon skeletons used to support post-germinative growth. The initial step in the process is catalysed by a TAG lipase, which hydrolyses TAG at the oil/water interface to yield free fatty acids and glycerol. In most seeds TAG lipase activity is only detectable upon germination and increases concomitantly with the disappearance of TAG.

The free fatty acids released by TAG lipase are subsequently converted to sucrose via the sequential action of β-oxidation, the glyoxylate cycle and gluconeogenesis.

Following germination, oil-bearing plant seeds, including for example, Arabidopsis, rely on storage oil breakdown to supply carbon skeletons and energy for early seedling growth. Hydrogen peroxide (H₂O₂) is generated within the peroxisome as a by-product of fatty acid β-oxidation. In 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.

In eukaryotes the oxidative metabolism that takes place in peroxisomes generates H₂O₂, which must be detoxified in order to prevent damage to proteins, lipids and DNA (van den Bosch et al., 1992; Singh, 1996; Willekens et al., 1997). This important task is performed universally by catalase, which catalyses the decomposition of H₂O₂ into molecular oxygen and water within the peroxisomal matrix. Defects in catalase cause peroxisome dysfunction and are detrimental in mammals, plants and yeast (Sheikh et al., 1998; Willekens et al., 1997; Zhang et al., 1993; Horiguchi et al., 2001).

Uniquely therefore, in addition to catalase, higher plant peroxisomes possess a membrane bound ascorbate-dependent electron transfer system which is also able to remove H₂O₂

(Yamaguchi et al., 1995; Bunkelmann and Trelease, 1996; Mullen and Trelease, 1996;

Karyotou and Donaldson, 2005). This system is thought to rely on the cooperative action of ascorbate peroxidase (APX) and monodehydroascorbate reductase (MDAR). APX initiates electron transfer from two molecules of ascorbate to convert H₂O₂ 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.,

1998). 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 H₂O₂ than APX, suggesting that at low concentrations H₂O₂ is likely to be preferentially scavenged by the APX/MDAR system (Bunkelmann and Trelease, 1996; Mullen and Trelease, 1996). One hypothesis is that the 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 H₂O₂ 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).

The physiological importance of 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).

Over-expression of APXS, encoding a peroxisomal isoform from Arabidopsis thaliana, has been reported to increase protection against oxidative stress (Wang et al., 1999). However, Narendra et al., (2006) have shown that a null mutant in APX3 is healthy under normal growth conditions. The role of the peroxisomal APX/MDAR system in plant growth and development is unclear. There is also uncertainty about the role of the APX/MDAR system in relation to catalase.

It has previously been reported that during periods of rapid β-oxidation mammalian peroxisomes can leak H₂O₂ (Mueller et al., 2002).

A number of studies have previously presented electron micrographic evidence to suggest that associations exist in oilseeds cells between oil bodies and other sub-cellular compartments, principally peroxisomes (e.g. Wanner and Theimer, 1978; Hayashi et al., 2001). Chapman and Trelease (1991) provide biochemical evidence that in cotton seedlings neutral lipids are transferred directly from the oil body into the peroxisomal membrane. In yeast, a close association between oil bodies and peroxisomes has also recently been reported and evidence has been provided that proteins involved in β-oxidation (e.g. acyl- CoA oxidase) are localized close to the inner surface of the peroxisomal membrane at sites of contact with the oil body (B inns et al., 2006).

Proteomic analysis by Job et al., (2005) has established that in wild type Arabidopsis seeds and seedlings, proteins from many sub-cellular compartments exhibit a significant level of oxidation. However, no oxidised oil body proteins have been identified.

DISCLOSURE OF THE INVENTION

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). The inventors have also surprisingly discovered that the MDAR4 component of the ascorbate- dependent electron transfer system is responsible for detoxifying H₂O₂ which escapes the peroxisome. The inventors have found that 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. During the oil synthesis phase and prior to desiccation of the seed, 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. Without wishing to be bound by any particular theory, 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. In preferred aspects, 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. Alternatively or in addition, 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).

A number of techniques are well known to the average skilled person for specifically ablating genes and/or gene products. One is the introduction of double stranded RNA, also referred to as inhibitory RNA (RNAi), into a cell that results in the destruction of mRNA complementary to the sequence included in the RNAi molecule. 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.

Another embodiment of 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).

An effective suppression of MDAR4 expression is a combination of both the sense and the antisense technique. (See Waterhouse et al (1998) P.N.A.S. 95: 13959=13964).

In a preferred embodiment of the invention 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.

Different sequence regions of the DNA molecule may be inserted into the vector. One possibility consists, e.g., in inserting into the vector only that part which is responsible for ribosome annealing. Blocking in this region of the mRNA will suffice to stop the entire translation. A particularly high efficiency of the antisense molecules also results for the 5'- and 3'-nontranslated regions of the gene. Preferably the 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. In such "knock out" genes it is important that the expression of MDAR4 is disturbed, and the formation of an active, functional protein is prevented. In doing so, the site of the mutation is variable, as long as expression of an active protein is prevented. Preferably, 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. hi general, 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).

In other ways of operating the invention, 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.

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.

When a vector is introduced into the plant it may be an epigenetic element or an artificial chromosome.

Preferably, 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

(Christian et al . (1989) Plant MoI. Biol. 18 (675-689); pEMU (Last et al (1991) Theor

Appl. Genet. 81: 581-588); MAS (Velten et al (1984) EMBO J. 3. 2723-2730); ALS promoter (U.S. Application Seriel No. 08/409,297), and the like. Other constitutive promoters include those in U.S. Patent Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680, 5,268,463; and 5,608,142.

The nucleic acid or vector capable of suppressing MDAR4 expression may be a transposable element. Generally, any suitable Arabidopsis transposable element may be used. In crop plants, a preferred transposable element is Tilling.

When an MDAR4 inhibitor or antagonist is employed 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.

When the plant is treated with a nucleic acid or vector, or an inhibitor or an antagonist of MDAR4, then the treatment may be during or at a stage selected from flowering, fertilization, seed setting, desiccation of the seed or during seed storage. In particularly preferred embodiments 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. Also, the nucleic acids may target the 5' and/or 3' UTRs. Advantageously, 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,

(b) a fragment of at least 18 contiguous nucleic acids of (a), or (c) the complement of (a) or (b).

In preferred embodiments, 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:

(a) an amino acid sequence selected from SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6, or an amino acid sequence having at least 50% identity thereto,

(b) a polypeptide of at least 6 contiguous amino acids of (a), or a strand complementary to (a) or (b).

In preferred embodiments, 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:

Very High Stringency (allows sequences that share at least 90% identity to hybridize) Hybridization: 5x SSC at 65⁰C for 16 hours

Wash twice: 2x SSC at room temperature (RT) for 15 minutes each v' Wash twice: 0.5x SSC at 65°C for 20 minutes each

High Stringency (allows sequences that share at least 80% identity to hybridize) Hybridization: 5x-6x SSC at 65°C-70°C for 16-20 hours

Wash twice: 2x SSC at RT for 5-20 minutes each Wash twice: Ix SSC at 55°C-70°C for 30 minutes each

Low Stringency (allows sequences that share at least 50% identity to hybridize). Hybridization: 6x SSC at RT to 55°C for 16-20 hours

Wash at least twice: 2x-3x SSC at RT to 55°C for 20-30 minutes each.

There are a variety of equations and computer algorithms that can be used to calculate the Melting Temperature (Tm) of a nucleic acid duplex. This is another suitable measure for defining the range of nucleic acid sequences homologous to SEQ ID NO:1, or fragments of such sequences and their complements, that form part of the present invention.

For example, two standard approximation calculations can be used. For sequences less than 14 nucleotides the formula is:

T_m= (wA+xT) * 2 + (yG+zC) * 4 where w,x,y,z are the number of the bases A₅T, G₅C in the sequence, respectively (from Marmur, J., and Doty, P. (1962) JM?/ Biol 5: 109- 118).

For sequences longer than 13 nucleotides, the equation used is:

T_m= 64.9 +41*(yG+zC-16.4)/(wA+xT+yG+zC)

See Wallace, R.B., Shaffer, J., Murphy, R.F., Bonner, J., Hirose, T., and Itakura, K. (1979) Nucleic Acids Res 6:3543-3557 (Abstract) and Sambrook, J., and Russell, D.W. (2001) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press; Cold Spring Harbor, NY. (CHSL Press)

The assumptions made for both of the above equations is that the annealing occurs under the standard conditions of 50 nM nucleic acid, 50 mMNa⁺, and pH 7.0.

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.

The 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.

The 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. Among preferred variants 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. The following non-limiting list of 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).

The way in which 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. In many instances, it will be desirable to have the construct bordered on one or both sides by T-DNA, particularly having the left and right borders, more particularly the right border. This is particularly useful when the construct uses Agrobacterium tumefaciens or Agrobacterium rhizogenes as a mode for transformation, although the T-DNA borders may find use with other modes of transformation.

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.

Once a transgenic plant is obtained 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. In such a plant cell 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. Depending upon the objective, 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. 14(2): 247-257) and tetracycline-inducible and tetracycline-repressible promoters (see, for example, Gatz et al. (1991) MoI. Gen. Genet. 227: 229-237, and US Patent Nos. 5,814,618 and 5,789,156.

Where enhanced expression in particular tissues is desired, 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. 112(2): 513-524; Yamamoto et al (l994) Plant Cell Physiol. 35(5): 773-778; Lam (1994) Results Probl. Cell Differ. 20: 181-196; Orozco et al (1993) Plant MoI. Biol. 23(6): 1129-1138; Mutsuoka et al (1993) Proc. Natl. Acad. Sci. USA 90(20): 9586-9590; and Guevara- Garcia et al (1993) Plant J. 4(3): 495-50.

In a preferred embodiment of the invention said 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).

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.

Particular 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). The methods of the invention may be used in relation to all kinds of plants including Gymnosperms, Pteridophytes and Bryophytes. Of particular industrial utility are 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.

For all aspects of the invention, 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.

In preferred embodiments of all aspects of the invention, the cell, tissue or plant is selected from: corn {Zea mays), canola (Brassica napus, Brassica rapa ssp.), flax (Limirn usitatissimuni), alfalfa (Medicago sativά), 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 (Manϊhot esculentd), coffee (Cofea spp.), coconut (Cocos nuciferά), pineapple (Anana comosus), citris tree (Citrus spp.) cocoa (Theobroma cacao), tea (Camellia senensis), banana (Musa spp.), avacado (Persea americana), fig (Ficus casica), guava (Psidium guajava), mango (Mangifer indica), olive (Olea europaea), papaya (Carica papaya), cashew (Anacardium occidentale), macadamia (Macadamia inter grifolia), almond {Prunus amygdalus), sugar beets (Beta vulgaris), oats, barley, vegetables and ornamentals.

Preferably, 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).

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.

It will be apparent that means to increase the activity of a polypeptide encoded by a nucleic acid molecule are known to the skilled person. For example, and not by limitation, increasing the gene dosage by providing a cell with multiple copies of said gene. Alternatively or in addition, 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.

Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith.

The invention will now be described in more detail and by way of example having regard to the drawings in which:

BRIEF DESCRIPTION OF DRAWINGS

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.

(A) Images of 5 d old sdp2 and wild type seedlings grown on medium with and without 1% (w/v) sucrose. The scale bar is 1 cm. (B) Total fatty acid and (C) eicosenoic acid (20:1) content of sdρ2 and wild type seeds and 5 d old seedlings grown on medium with 1% (w/v) sucrose. Values are the mean ± SE of measurements on five batches of 20 seeds or seedlings.

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 (listed in Table 1) are denoted by bars and the number of recombination events / total number of chromosomes (380) are listed below each.

(B) Schematic diagram of the SDP2 locus showing mutations in At3g27820 (MDAR4). Black bars are exons and the white bar and arrow represent the 5' and

3' UTRs, respectively. Insertion / substitution positions are numbered relative to the ATG. A full-length cDNA clone of SDP2 has previously been published by the SSP consortium (GenBank accession number AY039980).

(C) Complementation of sdρ2-4 by transformation with a genomic copy of MDAR4. Images of 5 d old sdp2~4 (left) and sdp2-4 MDAR4 (right) seedlings grown on medium without sucrose. The scale bar is 1 cm.

(D) SDP2 transcript levels in 2 d old wild type and sdp2 seedlings detected using RT-PCR. The PCR primers amplify a 568 bp product from the 3' end of the cDNA. (E) SDP2 protein in peroxisomes purified from wild type and sdp2-4 seedlings detected using Western blotting. *The larger polypeptide band corresponding to MDAR4 is 54 kDa.

Figure 4 shows peroxisome clustering and proximity to oil bodies in sdρ2 seedlings. (A) Confocal images of epidermal cells.

(B) EM images of parenchyma cells of cotyledons from 5 d old sdp2-4 and wild type seedlings grown on medium containing 1% (w/v) sucrose. For confocal microscopy peroxisomes were visualized using a genetic background containing PTSl targeted GFP driven by the 35S promoter (Cutler et al., 2000). P is peroxisome, OB is oil body and C is chloroplast. In (A) and (B) scale bars are 10 μm and 1 μm, respectively.

Figure 5 shows β-oxidation-dependent oxidative damage to oil bodies in sdp2 seedlings.

(A) Rate of [¹⁴C]triolein hydrolysis. (B) lipid hydroperoxides (LOOHs) levels. (C) protein carbonyl levels in oil bodies purified from 2 d old sdp2-4,pxal, sdp2- 4pxal and wild type seedlings. The concentration of triolein was equivalent to 10 mM. Values are the mean ± SE of measurements on five separate preparations.

Figure 6 shows carbonylation and inactivation of SDPl .

(A) Effect of H₂O₂ concentration on recombinant SDPl activity and level of carbonylation. Purified recombinant SDPl was pre-incubated with H₂O₂ for 2 h at 22⁰C before the protein was assayed for either lipase activity or carbonyl levels. Lipase assays were performed using 10 mM [¹⁴C]triolein as a substrate. Prior to the addition of the substrate H₂O₂ was removed from the samples by adding 10 units ml^"1 of catalase and incubating for 10 min at 22⁰C. Catalase assays confirmed that essentially all the H₂O₂ is removed by this treatment. Values are the mean ± SE of measurements from four separate incubations.

(B) Activity of recombinant SDPl on oil bodies purified from 2 d old sdp2-4 and wild type seedlings. Values are the mean ± SE of measurements from four separate incubations.

(C) Detection using anti-DNP antibodies of carbonyl groups in SDPl inimunoprecipitated from oil body membranes of 2 d old sdp2-4 and wild type seedlings. (D) Detection of SDPl protein in purified oil body membranes from 2 d old sdp2-

4 and wild type seedlings, using anti-SDPl antibodies.

Figure 7 shows metabolite levels in germinating sdp2 seedlings. (A) H₂O₂. (B) total ascorbate (ascorbate + dehydroascorbate).

(C) reduced ascorbate as a % of total in 1 d old and 3 d old sdp2-4, pxal, sdp2-4 pxal and wild type seedlings germinated on medium containing 1% (w/v) sucrose, nd, not determined. Values are the mean ± SE of measurements on five separate extracts. Figure 8 shows β-Oxidation of 2,4-dichlorophenoxybutyric acid (2,4-DB) by sdp2 seedlings.

(A) Effect of 2,4-DB concentration.

(B) 2,4-D concentration on root length of 5 d old sdp2-4, pxal, sdp2-4 pxal and wild type seedlings grown on medium containing 1% (w/v) sucrose. Values are the mean ± SE of measurements on five batches of 20 seedlings.

Figure 9 shows the effect of catalase deficiency on fatty acid breakdown during post- germinative growth. (A) Total fatty acid.

(B) eicosenoic acid (20:1) content of seeds and 5 d old seedlings grown on medium with 1% (w/v) sucrose. Col4 is wild type, Pthw is empty vector control and CAT2AS, CAT2HP1 and CAT2HP2 are catalase deficient lines (Vandenabeele et al., 2004). Values are the mean ± SE of measurements on five batches of 20 seeds or seedlings.

(C) Catalase activities in extracts from 2 d old seedlings of Col4, Pthw, CAT2AS, CAT2HP1 and CAT2HP2. Values are the mean ± SE of measurements on three separate extracts.

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 and 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). GIy¹¹' VaI¹⁴ and GIy³⁸⁶ from SDP2/MDAR4 (At3g27820) are marked in red.

EXAMPLES

Plant material and growth conditions

Wild type Arabidopsis thaliana (ecotype Colombia 0 and Landsberg erecta) were obtained from the Nottingham Arabidopsis Stock Centre (University of Nottingham,

UK). 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

(Zolman et al., 2001) and catalase deficient lines (Vandenabeele et al., 2004) were kindly provided by Prof. Chris Somerville (Carnegie Institution, Stanford University, CA, USA), Prof. Bonnie Bartel (Rice University, Houston, Texas, USA) and Dr. Frank Van

Breusegem (Ghent University, VIB, Belgium), respectively. Seeds were surface sterilised, applied to agar plates containing ¹A strength MS salts (Sigma-Aldrich, Poole,

Dorset, UK) and imbibed in the dark for 4 d at 4⁰C. The plates were then transferred to a growth chamber set to 21⁰C (16h light/8h dark; PPFD = 150 μmol m^"2 s^"1). In some experiments 1% (w/v) sucrose, 2,4-dichlorophenoxybutyric acid (2,4-DB) or 2,4- dichlorophenoxyacetic acid (2,4-D) were added to the agar medium.

Metabolite analysis

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). H₂O₂ 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). Mapping

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.

Transcript and protein analysis

DNAse-treated total RNA was isolated from Arabidopsis seedlings using the RNeasy kit from Qiagen Ltd. (Crawley, West Sussex, UK). The synthesis of single stranded cDNA was carried out using Superscript™ 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'). Measurement of total protein levels, SDS-polyacrylamide gel electrophoresis and Western blot analysis were carried out as described previously (Eastmond, 2004). Rabbit anti-cucumber MDAR antiserum (Sano et al., 1995), anti-DNP antibody (Millipore) and anti-SDPl antibody were used as primary antibodies, at dilutions of 1 in 1000, 1 in 30 and 1 in 500, respectively. The SDPl antibody was raised against the SDPl specific peptide SSEDSGLQEPVSGSVC by Eurogentec (Belgium) and affinity purified.

Microscopy

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 His₆-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). 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 OxyBlot™ Protein Oxidation Detection Kit (Millipore) as described by Nguyen and Donaldson (2005).

Enzyme assays

Triacylglycerol lipase activity was measured in purified oil body membranes and recombinant SDPl using an emulsion of [¹⁴CJtriolein 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). For assays of peroxisomal enzymes 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 4⁰C 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).

Complementation of sdp2-4

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

In Arabidopsis seeds the breakdown of stored triacylglycerol (TAG) following germination is essential to drive the initial phase of seedling growth and allow photosynthetic establishment (Hayashi et al., 1998). To investigate this process, a forward genetic screen was used to isolate a collection of Arabidopsis mutants that require an alternate exogenous source of carbon (sucrose) to support post-germinative growth (Eastmond, 2006). Screening these sugar-dependent (sdp) mutants for defects in TAG hydrolysis revealed that sdpl, sdp2 and sdp3 are defective in the lipase activity that is associated with the oil body membranes. 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).

Seeds were sterilized and imbibed on agar plates containing ¹A MS (pH 5.7) for 4 d in the dark at 4⁰C and germinated in light (16h light/8h dark, PPFD = 150 μmol m^'2 s^"1) at 21⁰C. Germination was scored as radicle emergence and is expresses as a percentage of 500 seeds. DAI is days after imbibition.

Providing sucrose rescues the growth phenotype (Figure 2A) and once sdp2 seedlings establish photosynthetic competence they grow normally and complete their life cycle. Analysis of the fatty acid content of sdp2 seeds germinated in the presence of sucrose revealed that the mutant is severely impaired in its ability to breakdown TAG (see Figures 2B and 2C). Eicosinoic acid is specifically found in TAG in Arabidopsis (Lemieux et al., 1990) and therefore it can be used as a convenient marker to monitor TAG breakdown (Eastmond et al., 2000a). The levels of both total fatty acids and eicosinoic acid (20:1) decline by less than 20 % over the course of the first 5 d of post- germinative growth in sdp2, while in wild type seedlings they fall by 60% and 95%, respectively.

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).

In the sdp2-l mutant allele a T to C mutation causes a VaI to Ala amino acid substitution at position +14 in the polypeptide sequence (Figure 3B). In the sdp2-2 and sdp2-3 mutant alleles A to G mutations give rise to GIy to GIu amino acid substitutions in the polypeptide sequence at positions +11 and +386, respectively (Figure 3B). An amino acid sequence alignment suggests that the GIy residues at positions +11 and +386 in MDAR4 are conserved among all Arabidopsis MDAR isoforms (Obara et al., 2002). The VaI residue at position +14 is also conserved in MDARl, 2, 3 and 4, but not in MDAR5 and 6. These last two isoforms are encoded by Atlg63940 and they contain N-terminal targeting signals for either the chloroplast or the mitochondrion, depending on the transcriptional start site (Obara et al., 2002). A T-DNA allele of MDAR4 (sdp2-4) that contains an insertion in exon 6 (Figure 3B) was also obtained from the SALK collection (Alonso et al., 2003). This line exhibits the same phenotype as the EMS alleles (Figure 2A). To confirm that the phenotype of sdp2-4 is caused by a defect in MDAR4 the mutant was complemented by transformation with a construct carrying a genomic copy of the gene (Figure 3C).

RT-PCR performed on RNA from 2 d old seedlings showed that MDAR4 transcripts are present in wild type, sdp2-l, sdp2-2 and sdp2-3 but are absent from sdp2-4 (Figure 3D).

To investigate whether MDAR4 protein is present in plants having the sdp2-4 allele Western blots were carried out on peroxisome enriched fractions obtained by sucrose gradient centrifugation of crude seedling extracts (Eastmond et al., 2000b). A polyclonal antibody was used that was raised against a 47 kDa MDAR from cucumber (Sano et al., 1995). This antibody has been shown previously to recognize two polypeptides in purified Arabidopsis peroxisomes (Lisenbee et al., 2005). The 47 kDa band corresponds to the matrix isoform MDARl and the 54 fcDa band corresponds to the membrane isoform MDAR4. Both bands were present in peroxisomal fractions from wild type but the 54 kDa band was specifically absent in peroxisomal fractions from sdp2-4 (Figure 3E).

Example 3 - Peroxisomes and oil bodies cluster together in sdp2 seedlings

Confocal microscopy was used to investigate peroxisome morphology and proximity to oil bodies in living cells of sdp2 seedlings. In epidermal cells from the cotyledons of 5 d old sdp2-4 seedlings GFP- labelled peroxisomes formed large (5 to 10 μm) clusters that are associated with groups of transparent spherical un-degraded oil bodies (Figure 4A). In contrast, in wild type, essentially no oil bodies remained by this stage of development and the individual peroxisomes were dispersed throughout the cytosol (Figure 4A). EM images of sections through parenchyma cells from the cotyledons of 5 d old seedlings also suggest an association between oil bodies and peroxisomes (Figure 4B). The striking peroxisome clustering pheriotype in sdp2-4 was only observed in seedling tissues that contained un-degraded oil bodies and was not obvious in tissues from other stages in plant development under the growth conditions used in this study (data not shown).

Example 4 - β-oxidation causes oxidative damage to oil bodies in sdp2 seedlings

During the post-germinative growth of oilseeds acyl-CoA oxidase, which is the first enzyme of peroxisomal fatty acid β-oxidation, is likely to be the major source Of H₂O₂ production in the cell (Graham and Eastmond, 2002). In order to determine whether fatty acid β-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 β-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). To investigate whether oil bodies from sdp2 incur oxidative damage lipid peroxidation was estimated using the FOX (ferrous oxidation xylenol orange) assay (Griffiths et al., 2000) and protein oxidation was monitored by detecting protein carbonyls (Levine et al., 1990). Both lipid hydroperoxides (LOOHs) and protein carbonyl levels were significantly elevated in oil bodies purified from 2 d old sdp2-4 seedlings verses wild type and in both cases these increased levels were suppressed in the pxal background (Figure 5B,C).

Example 5 - SDPl is a target of oxidative damage in sdp2 seedlings

In vitro incubation of purified recombinant SDPl with H₂O₂ showed that the protein can be inactivated by concentrations greater than 0.5 mM (Figure 6A). The loss of catalytic activity caused by H₂O₂ was accompanied by oxidation of the protein, as indicated by a 20-fold increase in the level of carbonylation (Figure 6A). Enzyme assays showed that recombinant SDPl is still able to hydrolyse oil bodies purified from 2 d old sdp2 seedlings, but at a significantly lower rate than from wild type (Figure 6B). This suggests that peroxidation of substrate and/or oxidation of additional oil body proteins also forms a partial barrier to oil hydrolysis by SDPl.

- To investigate whether SDPl suffers oxidative damage in sdp2-4 seedlings the protein was purified from oil body membranes by immunoprecipitation using affinity purified polyclonal antibodies raised against an SDPl peptide. The purified protein was then reacted with 2,4-dintrophenylhydrazine (DNP) to derivatise carbonyl groups and the carbonyl groups were detected with anti-DNP antibodies. Carbonyls could only be detected in SDPl purified from sdp2-4 oil body proteins and not from wild type (Figure 6C). The polyclonal antibody raised against the SDPl peptide detected similar amounts of SDPl in oil body protein samples from sdp2-4 and wild type (Figure 6D). Together these data suggest that SDPl is transcribed, translated and targeted to oil bodies in sdp2 mutant seedlings, but that it is then inactivated by oxidative damage. Example 6 - An initial increase in H₂O₂ levels in sdp2 is likely to result from a failure to recycle ascorbate

H₂O₂, the total ascorbate pool (ascorbate + dehydroascorbate) and the percentage of the ascorbate pool that is reduced were all measured in 1 d old and 3 d old sdp2-4 seedlings grown on medium with sucrose (Huang et al., 2005). At d 1 the total ascorbate pool in sdp2-4 was similar to wild type but the percentage of reduced ascorbate was lower and the level OfH₂O₂ was increased (Figure 7). In contrast, at d 3 H₂O₂ levels in sdp2-4 were lower than those of wild type (Figure 7A). The production of H₂O₂ in sdp2-4 and wild type seedlings was strongly suppressed in apxal background at both d 1 and d 3 (Figure 7A). Without wishing to be bound by any particular theory, the inventors propose a model in which, immediately following sdp2 germination, the production of H₂O₂ by fatty acid β-oxidation leads to a negative feedback loop whereby the H₂O₂ inactivates SDPl and progressively cuts off the supply of substrate for β-oxidation, which in turn reduces H₂O₂ production.

Example 7 - Seedlings of sdp2 retain the capacity to β-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-dichloroρhenoxyacetic acid (2,4-D) by a single cycle of β-oxidation (Hayashi et al., 1998). Arabidopsis mutants in many genes that are either directly required for fatty acid β-oxidation or for peroxisome function in general exhibit a 2,4-DB resistant phenotype (see Baker et al., 2006). To date this list of mutants includes pxal/cts/pedB, acxl, acxS, acx4, aiml, pedl/kat2, cys2 cys3, chyl, ped2/pexl4, pex4, pex5 and pexό. However, unlike these mutants, root growth in sdp2-4 seedlings does not exhibit increased resistance to 2,4-DB (Figure 8), indicating that many peroxisomal membrane and matrix proteins remain at least partially functional. In vitro assays performed on extracts of 2 d old sdp2-4 seedlings also show that the activities of peroxisomal marker enzymes, acyl-CoA oxidase, enoyl-CoA hydratase, L-3- hydroxyacyl-CoA dehydrogenase, 3-ketoacyl-CoA thiolase, isocitrate lyase, malate synthase and catalase are all similar to wild type (see Table 3 below).

Table 3. Peroxisomal and oil body membrane enzyme activities in wild type, sdp2 and CAT2HP2 seedlings.

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 [ⁱ⁴C]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.

The activity of the cytosolic marker enzyme NADH-dependent glyceraldehyde-3- phosphate dehydrogenase (GAPDH) 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 H₂O₂ 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

A series of Arabidopsis CATALASE2 anti-sense lines (Vandenabeele et al., 2004) were screened for their ability to breakdown storage oil following germination. Surprisingly, unlike sdp2, none of these lines were strongly impaired in total fatty acid or eicosinoic acid breakdown when grown on medium containing sucrose (see Figure 9). Enzyme assays confirmed that the lines had reduced catalase activity following germination, with CAT2HP2 exhibiting the lowest level (only 10% of wild type; Figure 9C). Further analysis of CAT2HP2 seedlings revealed that the activities of specific peroxisomal enzymes (isocitrate lyase and malate synthase) are significantly reduced in comparison with wild type, while oil body membrane lipase activity is not affected (see Table 3 above). This indicates that a 90% reduction in catalase activity does impact negatively on peroxisome function albeit insufficiently to prevent fatty acid β-oxidation from occurring when seedlings are provided with sucrose.

Table 4 Total fatty acid content of seeds from Arabidopsis MDAR4 mutants

sdp3~l 8.71 ±0.27

SDP3-2 8.21±0.23 sdp3-2 8.70 ±0.19* (+6%)

Wild type and MDAR4 mutant plants were grown in the glasshouse in P24 trays containing F2 compost. Wild types are in capital letters and mutants in lower case. Table 4 shows the seed fatty acid content. The total fatty acid content of batches of seeds was measured by gas chromatography following direct extraction/methylation (see Browse et al (1986) Anal. Biochem. 152: 141). Values are the mean ±SE of measurements on seeds from 8 to 10 individual plants. *=significantly different from wild type (P < 0.05). The sdp2-4 and sdp2-5 alleles are SALK_068667 and SALK_030775 (Alonso et al., Science 301, 653-657. The other mutant alleles 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.

The inventors have found that the phenotype of sdp2 lacks the APX/MDAR system and consequently some of the H₂O₂ 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 H₂O₂ and that their oil body proteins and lipids become oxidized. Furthermore, when sdp2 was introduced into apxal background, which cannot β-oxidize fatty acids (Zolman et al., 2001), H₂O₂ levels, oxidative damage to oil bodies and loss of lipase activity were all suppressed. The activity of SDPl can be inhibited by H₂O₂ in vitro. Finally 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 H₂O₂. Seedlings of sdp2 are able to β-oxidize 2,4-DB. This capability implies that numerous proteins in the peroxisomal membrane and matrix retain at least part of their normal capacity (see Baker et al., 2006). The activities of seven peroxisomal marker enzymes are also not adversely affected in sdp2. It is possible that a defect in the APX/MDAR system is not severely detrimental to peroxisomes because catalase remains active within the matrix. However, the possibility exists that peroxisomes in sdp2 are damaged. Analysis of catalase anti-sense lines (Vandenabeele et al., 2004) showed that following Arabidopsis seed germination certain peroxisomal enzyme activities are reduced, while lipase activity on oil body membranes is unaffected. In particular the activity of the key glyoxylate cycle enzyme isocitrate lyase (Eastmond et al., 2000a) is reduced by 80% in the strongest catalase anti-sense line. This observation is consistent with in vitro data, which shows that isocitrate lyase from castor bean endosperm is readily inactivated by H₂O₂ and that this enzyme physically associates with catalase in the peroxisome (Yanik and Donaldson, 2005; Nguyen and Donaldson, 2005).

Using confocal microscopy the inventors have shown that 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. Without wishing to be bound by any particular theory, the inventors have found that 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 β-oxidized.

The concentration Of H₂O₂ 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, H₂O₂ concentrations are unlikely to be uniform and could be heightened at, or near, the peroxisomal membrane, particularly if acyl-CoA oxidases are situated there. Again, without wishing to be bound by any particular theory, the inventors observe that an association between oil bodies and peroxisomes explains at least in part why the deficiency in MDAR4 is so detrimental to storage oil hydrolysis since it would result in a close proximity between SDPl and acyl- CoA oxidases, which generate H₂O₂.

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. There are several alternative ways in which ascorbate could still be replenished for APX in the absence of MDAR4. In addition to 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 β- oxidation-dependent oxidative damage.

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. However, the level of expression of APX5 is very low relative to APXS (Narendra et al., 2006). The inventors do not preclude the possibility that MDAR4 functions independently of APX. Sattler et al., (2004) have shown that a deficiency in the lipid soluble antioxidants tocopherols (vitamin E) also leads to lipid peroxidation after the germination of Arabidopsis seeds and that this adversely effects storage oil breakdown and seedling growth. Furthermore, 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. However, it is unlikely that MDAR4 can operate solely by this mechanism since the sdp2 mutant has a more severe post- germinative growth arrest phenotype than the tocopherol deficient vte2 mutant under normal growth conditions (Sattler et al., 2004). It is also possible that ascorbate could scavenge reactive oxygen species directly, or that 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).

In addition to its role in H₂O₂-detoxification, 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 β-oxidation and glyoxylate cycle function, respectively and theoretically if H₂O₂ was detoxified entirely by APX/MDAR the system could recycle sufficient NAD⁺ for both pathways (Mullen and Trelease, 1996). However, Mettler and Beevers (1980) have proposed an alternative scheme in which peroxisomal malate dehydrogenase operates in 'reverse' to supply NAD⁺ for L-3-hydroxyacyl-CoA dehydrogenase via a metabolite shuttle, coupled to the mitochondrial electron transport chain. Malate dehydrogenase has since been shown to perform this role in yeast (van Roermund et al., 1995). Analysis of sdp2 suggests that MDAR4 is not essential for providing all the NAD⁺ in Arabidopsis peroxisomes since the mutant can β-oxidize 2,4-DB. In contrast, it has recently been reported that Arabidopsis seedlings that lack peroxisomal malate dehydrogenase activity are also impaired in oil breakdown and cannot β-oxidize 2,4-DB (see Baker et al., 2006). This provides evidence to suggest that the scheme proposed by Mettler and Beevers (1980) supplies the majority of NAD⁺ in vivo, although MDAR4 (and MDARl) activity must also contribute some NAD⁺.

In addition to fatty acid β-oxidation, the photo-respiratory pathway also generates large quantities of H₂O₂ 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 H₂O₂. 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). Although public micro array data shows that MDAR4 is expressed in leaves, sdp2 plants do not exhibit more obvious phenotypic symptoms of oxidative damage than wild type when they are subjected to high light (data not shown). Further studies will be required to determine if MDAR4 has a role associated with photo-respiration. Reactive oxygen species also play important signalling roles in plants (Hancock et al., 2005) and it is conceivable that inhibition of SDPl activity might be a significant mechanism in the regulation of oil hydrolysis during post-germinative growth.

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 OfH₂O₂ from escaping beyond the outer surface of the peroxisomal -membrane. The consequences of H₂O₂ 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

The GenBank accession number for an SDP2 {MDAR4) cDNA is AY039980.

Table 5

Acanthaceae Aristolochiaceae Bruniacβae

100 Aceraceae 55 Asclepiadaceae Buddlejaceae Achariaceae Asteropeiaceae Burseraceae Achatocarpaceae Aucubaceae 105 Buxaceae 60 Actinidiaceae Austrobaileyaceae Byblidaceae Adoxaceae Avicenniaceae Cabombaceae 110

Aextoxicaceae 65 Balanitaceae Cactaceae

Agdestidaceae Balanopaceae Callitrichaceae

Aizoaceae Balanophoraceae 115 Calycanthaceae 70 Akaniaceae Balsaminaceae Calyceraceae Alangiaceae Barbeuiaceae Campanulaceae 120

Alseuosmiaceae 75 Barbeyaceae Canellaceae

Alzateacβae Basellaceae Cannabaceae

Amaranthaceae Batacβae 125 Canotiaceae 80 Amborellaceae Begoniaceae Capparaceae Anacardiaceae Berberidaceae Caprifoliaceae 130

Ancistrocladaceae 85 Betulaceae Cardiopteridaceae

Anisophylleaceae Bignoniaceae Caricaceae

Annonaceae Bixaceae 135 Carlemanniaceae 90 Apocynaceae Bombacaceae Caryocaraceae Aquifoliaceae Boraginaceae Caryophyllaceae

140 Araliaceae 95 Bretschneideraceae Casuarinaceae

Aralidiaceae Brunelliaceae Cecropiaceae Celastraceae Cunoniaceae Eremolepidaceae

Cephalotaceae Cyclocheilaceae 115 Eremosynaceae

Uv/

5 Ceratophy! laceae Cynomoriaceae Ericaceae

Cercidiphyllaceae Cyrillaceae Ery th roxy I aceae

Chenopodiaceae 65 Daphniphyllaceae Escalloniaceae

1 i L0nU

Chloranthaceae Datiscaceaβ Eucommiaceae

Chrysobalanaceae Davidsoniaceae 125 Eucryphiaceae

70

1.5 Circaeasteraceae Degeneriaceae Euphorbiaceae

Cistaceae Dialypetalanthaceae Euphroniaceae

130

Clethraceae 75 Diapensiaceae Eupomatiaceae 0 Cneoraceae Dichapetalaceae Eupteleaceae

Cobaeaceae Didiereaceae 135 Fagaceae

80 5 Cochlospermaceae Didymelaceae Flacourtiaceae

Columelliaceae Diegodendraceae Fouquieriaceae

140

Combretaceae 85 Dilleniaceae Frankeniaceae 0 Compositae Dioncophyllaceae Garryaceae

Connaraceae Dipentodontaceae 145 Geissolomataceae

90 5 Convolvulaceae Dipsacaceae Gentianaceae

Coriariaceae Dipterocarpaceae Geraniaceae

150

Cornaceae 95 Droseraceae Gesneriaceae0 Corylaceae Duckeodendraceae Gisekiaceae

Corynocarpaceae Ebenaceae 155 Glaucidiaceae

100 5 Crassulaceae Elaeagnaceae Globulariaceae

Crossosomataceae Elaeocarpaceae Goetzeaceae

160

Cruciferae 105 Elatinaceae Gomortegaceae0 Crypteroniaceae Emblingiaceae Goodeniaceae

Ctenolophonaceae Empetraceae 165 Goupiaceae

110 5 Cucurbitaceae Epacridaceae Greyiaceae Griseliniaceae Juglandaceae

Malvaceae

Grossulariaceae Krameriaceae 115

60 IWarcgraviaceae

Grubbiaceae Labiatae Medusagynaceae

Gunneraceae Lacistemataceae 120 Medusandraceae

Guttiferae 65 Lactorrdaceae Melanophyilaceae

Gyrostemonaceae Lardizabalaceae

Melastomataceae

Halophytaceae Lauraceae 125

70 Meliaceae

Haloragaceae Lecythidaceae

Melianthaceaβ

Hamamelidaceae Lβeaceae 130 Meliosmaceae

Hectorellaceae 75 Leguminosae- caesalpinioideae Menispermaceae Helwingiaceae

Leguminosae- Menyanthaceae

Hernandiaceaβ mimosoideae 135

80 Misodendraceaβ

Hrmantandraceae Leguminosae- papilionoideae Molluginaceae

Hippocastanaceae

Leitneriaceae 140 Monimiaceae

Hippuridaceae 85

Lennoaceae Montiniaceae Hopiestigmataceae

Lentibulariaceae Moraceae

Huaceae 145

90 Lepidobotryaceae Morinaceae

Humiriaceae

Limnanthaceae Moringaceae

Hydnoraceae

Linaceae 150 Myoporaceae

Hydrangeaceae 95 Lissocarpaceae Myricaceae

Hydrophyllaceae

Loasaceae IWyristicaceae

Hydrostachyaceae 155

100 Loganiaceae Myrothamnaceae

(cacinaceae

Lophopyxidaceae Myrsinaceae ldiospermaceae

Loranthaceae 160 Myrtaceae lllecebraceae 105 Lythraceae Nelumbonaceae Kliciaceae

Magnoliaceae Nepenthaceae

Irvingiaceae 165

110 Malesherbiaceae Nesogenaceae

Ixonanthaceae

Malpighiaceae Neuradaceae Nyctaginaceae Pittosporaceae Rutaceae

115

Nymphaeaceae 60 Plagiopteraceae Sabiaceaβ Ochnaceae PIantaginaceae Saccifoliaceae

Olacaceae PIatanaceae 120 Salicaceae

65

OIeaceae Plumbaginaceae Salvadoraceae

Oliniaceae Podoaceae Santalaceaβ 125

Onagraceae 70 Podostemaceae Sapindaceae Oncothecaceae Polemoniaceae Sapotaceae

Opiliaceae Polygalaceae 130 Sarcolaenaceae

75

Oxalidaceae Polygonaceae Sargentodoxaceae

Paeoniaceae Port ul acaceae Sarraceniaceae 135

Pandaceae 80 Primulaceae Saururaceae Papaveraceae Proteaceae Saxifragaceae

Paracryphiaceae Ptaeroxylaceae 140 Schisandraceae

85

Parnassiaceae Pterostemonaceae Scrophulariaceae

Passifloraceae Quiinaceae Scyphostegiaceae 145

Pedaliaceae 90 Rafflesiaceae Scytopetalaceae Pellicieraceae Ranunculaceae Simaroubaceae

Penaeaceae Resedaceae 150 Simmondsiaceae

95

Pentaphragmataceae Retziaceae Solanaceae

Pentaphylacaceae Rhabdodendraceae Sphaerosepalaceae 155

Penthoraceae 100 Rhamnaceae Sphenostemonaceae Peridiscaceae Rhizophoraceae Stachyuraceae

Phellinaceae Rhoipteleaceae 160 Stackhousiaceae

105

Phrymaceae Rhynchocalycaceae Staphyleaceae

Physenaceae Roridulaceae Stegnospermataceae

165

Phytolaccaceae 110 Rosaceae Sterculiaceae

Piperaceae Rubiaceae Stilbaceae Strasburgeriaceae Umbelliferae Burmanniaceae

115

Stylidiaceae 60 Urticaceae Butomaceae Stylobasiaceae Vahliaceae Calectasiaceae

Styracaceae Valerianaceae 120 Cannacβae

65

Surianaceae Verbenaceae Centrolepidaceae

Symplocaceae Violaceae Colchicaceae 125

Tamaricaceae 70 Viscaceae Commelinaceae Tepuianthaceae Vitaceae Convallariaceae

Tetracentraceae Vochysiaceae 130 Corsiaceae

75

Tetrachondraceae Winteraceae Costaceae

Tetrameristaceae Zygophyllaceae Cyanastraceae 135

Theaceae 80 Acoraceae Cyclanthaceae Theligonaceae Agavaceae Cymodoceaceae

Theophrastaceae Alismataceae 140 Cyperaceae

85

Thymelaeaceae Alliaceae Dasypogonaceae

Ticodendraceae Aloaceae Dioscorβaceae 145

Tiliaceae 90 Alstroemeriaceae Doryanthaceaβ Torricelfiaceae Amaryllidaceae Dracaenaceae

Tovariaceae Anarthriaceae 150 Ecdeiocoleaceae

95

Trapaceae Anthericaceae Eriocaulaceae

Tremandraceae Aphyllanthaceae Eriospermaceae 155

Trigoniaceae 100 Aponogetonaceae Flagellariaceae Trimeniaceae Araceae Gramineae

Triplostegiaceae Asparagaceae 160 Haemodoraceae

105

Trochodendraceae Asphodelaceae Hanguanaceae

Tropaeolaceae Asteliaceae Heliconiaceae

165

Turneraceae 110 Blandfordiaceae Hemerocallidaceae

Ulmaceae Bromeliaceae Herreriaceae Marantaceae

Hostaceae Scheuchzeriaceae

Mayacaceae

Hyacinthaceae Smilacaceae

40 Λ/lelanthiaceae 75 Hydateilaceae Stemonaceae

Musaceae

Hydrocharitaceae Strelitziaceae

Orchidaceae

Hypoxidaceae 45 80 Taccaceae Palmae lridaceae Tecophilaeaceae

Pandanaceae

Ixioliriaceae Thurniaceae

50 Petermanniaceae 85 Joinvilleaceae Trichopodaceae

Philesiaceae

Juncaceae Trilliaceae

Philydraceae

Juncaginaceae 55 90 Triuridaceae Phormiaceae

Lanariaceae Typhaceae

Pontederiaceae

Lemnaceae Velloziaceae

60 Posidoniaceae 95 Lilaeaceae Xanthorrhoeaceae

Potamogetonaceae

Liliaceae Xyridaceae

Rapateaceae

Limnocharitaceae 65 100 Zannichelliaceae Restionaceae

Lomandraceae Zingiberaceae

Rhipogonaceae

Lowiaceae Zosteraceae

10 Ruscaceae

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Claims

1. A method of increasing the oil content of a plant cell comprising reducing or eliminating monodehydroascorbate reductase (MD AR4) activity.

2. A method as claimed in claim 1, wherein the reduction or elimination of MDAR4 activity is in the peroxisome.

3. A method as claimed in any of claims 1 to 3, wherein the reduction or elimination of MDAR4 activity takes place during seed development and/or maturation.

4. A method as claimed in any preceding claim, wherein the reduction or elimination of MDAR4 activity comprises supression of MDAR4 expression.

5. A method as claimed in claim 4, wherein the plant is transformed or transfected with a nucleic acid or vector capable of suppressing MDAR4 expression.

6. A method as claimed in any preceding claim, wherein the reduction or elimination of MDAR4 activity comprises antisense suppression of MDAR4 expression.

7. A method as claimed in any of claims 1 to 5, wherein the reduction or elimination of MDAR4 activity comprises sense suppression of MDAR4 expression.

8. A method as claimed in claimed in any preceding claim, wherein the activity of MDAR4 is reduced or eliminated by an inhibitor or antagonist of MDAR4.

9. A method as claimed in claim 8, wherein the MDAR4 inhibitor or antagonist is expressed from 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.

10. A method as claimed in claim 9, wherein the heterologous nucleic acid molecule introduced into the plant has integrated into the host plant genome.

11. A method as claimed in claim 9, wherein the vector introduced into the plant is an epigenetic element or an artificial chromosome.

12. A method as claimed in any of claims 5 or 8 to 11, wherein the nucleic acid or vector capable of suppressing MDAR4 expression is a transposable element.

13. A method as claimed in any preceding claim, wherein the MDAR4 inhibitor or antagonist is applied to the plant.

14. A method as claimed in any preceding claim, wherein the plant is treated with a nucleic acid or vector, an inhibitor or an antagonist of MDAR4, at or during a stage selected from flowering, fertilization, seed setting, seed desiccation or during seed storage.

15. A method as claimed in any preceding claim, wherein the nucleic acid comprises:

(a) a nucleotide sequence of SEQ ID NO: 1 or a sequence having at least 50% identity thereto,

(b) a fragment of at least 18 contiguous nucleic acids of (a), or

(c) the complement of (a) or (b).

16. A method as claimed in claims 1 to 14, wherein the nucleic acid comprises a nucleotide sequence encoding:

(a) an amino acid sequence selected from SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6, or an amino acid sequence having at least 50% identity thereto,

(b) a polypeptide of at least 6 contiguous amino acids of (a).

17. A method as claimed in claim 15 or claim 16, wherein the nucleic acid sequence encodes a monodehydroascorbate reductase 4 (MD AR4).

18. A method as claimed in any of claims 5 to 17, wherein the vector is Agrobacterium.

19. A method as claimed in any preceding claim, wherein the plant is a monocot or a dicot.

20. A method as claimed in any preceding claim, wherein the plant is a member of the Cruciferae or the Compositae.

21. A method as claimed in any preceding claim, wherein the plant is selected from corn (Zea mays), canola (Brassica napus, Brassica rapa ssp.), flax (Linum usitatissimum), alfalfa (Medicago sativd), rice (Oryza sativά), rye (Secale cerale), sorghum {Sorghum bicolor, Sorghum vulgare), sunflower (Helianthus annus), wheat (Tritium aestivum), soybean {Glycine max), tobacco {Nicotiana tabacuni), potato {Solarium tuberosum), peanuts {Arachis hypogaea), cotton (Gossypium hirsutum), sweet potato {Iopmoea batatus), cassava {Manihot esculenta), coffee (Cofea spp.), coconut (Cocos nucifera), pineapple (Anana comosus), citris tree (Citrus¹ spp.) cocoa {Theobroma cacao), tea (Camellia senensis), banana (Musa spp.), avacado (Per sea americana), fig (Ficus casica), guava (Psidium guajava), mango (Mangifer indica), olive (Olea europaea), papaya (Carica papaya), cashew (Anacardium occidentale), macadamia (Macadamia inter grifo lid), almond (Prunus amygdalus), sugar beets (Beta vulgaris), oats, barley, vegetables and ornamentals.

22. A plant cell transformed or transfected with nucleic acid capable of reducing or eliminating monodehydroascorbate reductase (MD AR4) activity.

23. A plant cell as claimed in claim 22, wherein the reduction or elimination of MDAR4 activity is in the peroxisome.

24. A plant cell as claimed in claim 22 or claim 23, wherein the reduction or elimination of MDAR4 activity comprises supression of MDAR4 expression.

25. A plant cell as claimed in any of claims 22 to 24 transformed or transfected with a nucleic acid or vector capable of suppressing MDAR4 expression.

26. A plant cell as claimed in claim 25, wherein the vector comprises a cell or tissue specific promoter.

27. A plant cell as claimed in claim 26, wherein the promoter is an inducible promoter or a developmentally regulated promoter.

28. A plant cell as claimed in any of claims 22 to 27, wherein the reduction or elimination of MDAR4 activity comprises antisense suppression of MDAR4 expression.

29. A plant cell as claimed in any of claims 22 to 28, wherein the reduction or elimination of MDAR4 activity comprises sense suppression of MDAR4 expression.

30. A plant cell as claimed in claimed in any of claims 22 to 29, wherein the activity of MDAR4 is reduced or eliminated by an inhibitor or antagonist of MDAR4 expressed by an heterologous nucleic acid molecules or vector, or an inhibitor or antagonist of MDAR4 expressed from a gene endogenous to the genome of the cell.

31. A plant cell as claimed in claim 30, wherein the heterologous nucleic acid molecule or vector is integrated into the host cell genome.

32. A plant cell as claimed in claim 30 or claim 31, wherein the heterologous nucleic acid or vector is an epigenetic element or an artificial chromosome.

33. A plant cell as claimed in any of claims 25 to 32, wherein the nucleic acid or vector capable of suppressing MDAR4 expression is a transposable element.

34. A plant cell as claimed in any of claims 22 to 33, wherein the nucleic acid comprises: (a) a nucleotide sequence of SEQ ID NO: 1 or a sequence having at least 50% identity thereto,

(b) a fragment of at least 18 contiguous nucleic acids of (a), or

(c) the complement of (a) or (b).

35. A plant cell as claimed in any of claims 22 to 34, wherein the nucleic acid comprises a nucleotide sequence encoding:

(a) an amino acid sequence selected from SEQ ID NO:2, SEQ ID NO:3, SEQ ID

NO:4, SEQ ID NO:5 or SEQ ID NO:6, or an amino acid sequence having at least 50% identity thereto, (b) a polypeptide of at least 6 contiguous amino acids of (a).

36. A plant cell as claimed in claim 34 or claim 35, wherein the nucleic acid sequence encodes monodehydroascorbate reductase 4 (MD AR4).

37. A cell as claimed in any of claims 22 to 36, wherein the vector is Agrobacterium.

38. A plant cell as claimed in any of claims 22 to 37 comprised in a plant selected from a monocot or a dicot.

39. A plant cell as claimed in any of claims 22 to 37 comprised in a plant selected from the Cruciferae or the Compositae.

40. A plant cell as claimed in any of claims 22 to 37 comprised in a plant selected from corn (Zea mays), canola (Brassica napus, Brassica rapa ssp.), flax (Linum usitatissimum), alfalfa (Medicago sativa), rice (Oryza sativd), 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 (Manihot esculenta), coffee (Cofea spp.), coconut (Cocos nucifera), pineapple (Anana comosus), citris tree (Citrus spp.) cocoa (Theobroma cacao), tea (Camellia senensis), banana (Musa spp.), avacado (Persea americana), fig (Ficus casica), guava (Psidium guajava), mango (Mangifer indica), olive (Oka europaea), papaya (Carica papaya), cashew (Anacardium occidentale), macadamia (Macadamia intergrifolia), almond (Prunus amygdalus), sugar beets (Beta vulgaris), oats, barley, vegetables and ornamentals.

41. A plant, plant organ or plant tissue comprising a plant cell as claimed in any of claims 23 to 40.

42. A plant seed comprising a plant cell as claimed in any of claims 23 to 40.

43. 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.

44. A method as claimed in claim 43, wherein the oil content of a seed is determined by measuring fatty acid content of the seed.

45. A plant cell overexpressing monodehydroascorbate reductase (MD AR4).

46. A plant tissue or plant comprising a plant cell of claim 45.

47. A method of identifying an agent which increases the oil content of a plant cell, tissue or seed, comprising contacting a plant cell of claim 45 or plant tissue or plant of claim 46 with a candidate agent and then determining the oil content of the cell, tissue or seed.

48. A method as claimed in claim 47, wherein the oil content of a seed is determined by measuring the fatty acid content of the seed.

49. An antibody specifically recative to MDAR4.

50. An antibody as claimed in claim 49 being a monoclonal antibody.