WO2009147409A2

WO2009147409A2 - Aquatic plant

Info

Publication number: WO2009147409A2
Application number: PCT/GB2009/001415
Authority: WO
Inventors: Ian Alexander Graham
Original assignee: The University Of York
Priority date: 2008-06-04
Filing date: 2009-06-03
Publication date: 2009-12-10
Also published as: GB0810111D0; WO2009147409A3

Abstract

We describe a transgenic aquatic plant and plant cell which is modified to enhance the production of plant-derived oil and including methods for the processing of plant derived biomass materials.

Description

Aquatic Plant

The invention relates to transgenic aquatic plant cells and transgenic aquatic plants that are modified to enhance the production of plant oils and fatty acids and including methods for the processing of plant derived biomass materials from said aquatic plants.

Plant derived products are currently being widely adopted both as industrial feedstock and as replacement fuels. So called first generation biofuels are either based on bioethanol or biodiesel. Bioethanol production relies on the process of fermentation using microbial organisms to produce ethanol. The feedstock for this microbial fermentation is typically sugar obtained from sugar cane or sugar beet or derived from starch obtained from cereal crops such as maize or wheat. Bioethanol production from sugarcane, sugar beet and cereal grains such as maize (corn), wheat and barley feedstock has been widely adopted. Biodiesel is an alternative biofuel to bioethanol. Crops used to produce feedstock for biodiesel production include soybean, castor bean, sunflower, rapeseed, Jatropha and palm.

Plant biomass is cheap and abundant and typically contains 25% lignin and 75% polysaccharides which represent a rich source of sugars. This biomass can be derived from agricultural residues (leftover material from crops, such as the stalks, leaves, and husks of corn plants), forestry wastes (chips and sawdust from lumber mills, dead trees, and tree branches), municipal solid waste (household garbage and paper products), food processing and other industrial wastes or so called ' Energy crops' (fast-growing trees and grasses) developed specifically for biomass. To utilize this biomass however requires much effort to release the sugars from lignocellulose in a process called saccharification.

In our co-pending application, WO2008/068498, we describe plants that are genetically engineered to enhance the production of fatty acids of mono- di- or triacylglycerols in non-seed tissues, for example foliar and vegetative tissues. This provides significant amounts of plant oils in vegetative tissues that can be used as an industrial feedstock or as a feedstock for biodiesel. The oil is extracted during processing leaving a biomass that can then be subjected to saccharification more readily. The disclosure in WO2008/068498 relates to food crops and crops typically used to produce feedstock for biodiesel production for example soybean, castor bean, sunflower, rapeseed, Jatropha and palm. This disclosure relates to the use of an alternative plant feed stock for the production of fatty acids for biodiesel and a plant biomass for saccharification that does not exploit food crop species.

"Duckweeds" are aquatic plants and refers to members of the family Lemnaceae. There are four known genera and 34 species of duckweed. Duckweed species grow rapidly; doubling times vary between species but can be as short as 20-24hrs. Duckweed growth is through vegetative budding of new fronds in nutrient rich (waste) water. It has a high protein content and very low lignin content thereby facilitating saccharification of the biomass to provide substrate for biofuel production. In addition it is transformable and is already used as a platform for biopharmaceutical production [see WO99/07210]. Alternatively, salt water aquatic species are also known, for example Spartina, which is used in estuarine regions to reclaim land for the production of animal feedstock.

We disclose the genetic engineering of aquatic plant species to enhance the production of fatty acids of mono- di- or triacylglycerols in non-seed tissues.

According to an aspect of the invention there is provided an aquatic transgenic plant cell the genome of which is modified by transfection with a nucleic acid molecule selected from the group consisting of: i) a nucleic acid molecule comprising an expression cassette which cassette comprises a nucleic acid sequence that encodes at least part of a gene that encodes a polypeptide that controls the synthesis, degradation or transport of fatty acids and/or fatty acyl CoAs and therefore synthesis and degradation of mono- di- or triacylglycerols wherein said cassette is adapted such that both sense and antisense nucleic acid molecules are transcribed from said cassette wherein the expression from said cassette produces an interfering RNA molecule that inhibits the expression of said gene; ii) a nucleic acid molecule comprising an expression cassette which cassette comprises a nucleic acid sequence that encodes at least part of a gene that encodes a polypeptide that controls the synthesis, degradation or transport of fatty acids and/or fatty acyl CoAs and therefore synthesis and degradation of mono- di- or triacylglycerols wherein said cassette is adapted such that an antisense nucleic acid molecule is transcribed from said cassette wherein the expression from said cassette produces an antisense RNA molecule that inhibits the expression of said gene; iii) a nucleic acid molecule that encodes a polypeptide that controls the synthesis, degradation or transport of fatty acids and/or fatty acyl CoAs and therefore synthesis and degradation of mono- di- or triacylglycerols in a plant cell which polypeptide is a variant polypeptide that varies from a native polypeptide sequence wherein said variant polypeptide is a dominant negative suppressor of the native polypeptide and inhibits the production of fatty acids and/or fatty acyl CoAs therefore synthesis and degradation of mono- di- or triacylglycerols, wherein said nucleic acid molecule in i), ii) and iii) is operably linked to a promoter sequence that is substantially a foliar inducible and/or senescence inducible promoter.

In a preferred embodiment of the invention said gene encodes a polypeptide involved in transport, actvation or degradation of fatty acids and/or fatty acyl Co As.

Methods to provide plants that are modified to down regulate or ablate genes are well known in the art and include the use of antisense genes to regulate the expression of specific targets; insertional mutagenesis using T-DNA; the introduction of point mutations and small deletions into open reading frames and regulatory sequences; and double stranded inhibitory RNA (RNAi). RNAi is a technique to specifically ablate gene function through the introduction of double stranded RNA 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. An alternative 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, see, Smith et al (2000) Nature 407, 319-320.

In a preferred embodiment of the invention said gene is encoded by a nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of:

i) a nucleic acid molecule comprising a nucleic acid sequence as represented in Figure 13a; ii) a nucleic acid molecule comprising a nucleic acid sequence that hybridises under stringent hybridisation conditions to a nucleic acid molecule in (i) and which encodes a fatty acid transporter polypeptide; iii) a nucleic acid molecule that encodes a variant polypeptide that varies from a polypeptide comprising the amino acid sequence as represented in

Figure 13b.

i) a nucleic acid molecule comprising a nucleic acid sequence as represented in Figure 14a or14c; ii) a nucleic acid molecule comprising a nucleic acid sequence that hybridises under stringent hybridisation conditions to a nucleic acid molecule in (i) and which encodes a long chain acyl Co A synthetase polypeptide; iii) a nucleic acid molecule that encodes a variant polypeptide that varies from a polypeptide comprising the amino acid sequence as represented in Figure 14b or 14d.

i) a nucleic acid molecule comprising a nucleic acid sequence as represented in Figure 15a, 15c, 15e, 15g, 15i or 15k, ; ii) a nucleic acid molecule comprising a nucleic acid sequence that hybridises under stringent hybridisation conditions to a nucleic acid molecule in (i) and which encodes an acyl CoA oxidase polypeptide; iv) a nucleic acid molecule that encodes a variant polypeptide that varies from a polypeptide comprising the amino acid sequence as represented in

Figure 15b, 15d, 15f, 15h, 15j or15l.

i) a nucleic acid molecule comprising a nucleic acid sequence as represented in Figure 16a; 16c,16e or 16g ii) a nucleic acid molecule comprising a nucleic acid sequence that hybridises under stringent hybridisation conditions to a nucleic acid molecule in (i) and which encodes a Keto-Acyl-CoA thiolase; iii) a nucleic acid molecule that encodes a variant polypeptide that varies from a polypeptide comprising the amino acid sequence as represented in

Figure 16b, 16d, 16f or 16h.

i) a nucleic acid molecule comprising a nucleic acid sequence as represented in Figure 17a or 17c ii) a nucleic acid molecule comprising a nucleic acid sequence that hybridises under stringent hybridisation conditions to a nucleic acid molecule in (i) and which encodes a multifunctional protein involved in peroxisomal β oxidation; iii) a nucleic acid molecule that encodes a variant polypeptide that varies from a polypeptide comprising the amino acid sequence as represented in Figure 17b or 17d.

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_m 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 65°C for 16 hours

Wash twice: 2x SSC at room temperature (RT) for 15 minutes each

Wash twice: 0.5x SSC at 65°C for 20 minutes each

High Stringency (allows seguences 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: 1 x 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.

In a further preferred embodiment of the invention said cassette adapted for expression of sense and antisense nucleic acid comprises a nucleic acid molecule wherein said molecule comprises a first part linked to a second part wherein said first and second parts are complementary over at least part of their sequence and further wherein transcription of said nucleic acid molecule produces an RNA molecule which forms a double stranded region by complementary base pairing of said first and second parts.

In a further preferred embodiment of the invention said promoter sequence is an inducible foliar specific promoter sequence.

In a further preferred embodiment of the invention said promoter sequence is a senescence inducible promoter sequence.

Foliar and/or senescence specific promoters are known in the art. For example, WO0070061; US2004025205; WO2006102559; US6, 359, 197; WO2006025664 the contents of which are incorporated by reference in their entirety, describe various plant promoters that become activated when senescence is induced. In addition US2002120955 and WO9800533, the contents of which are incorporated by reference, each describe a number of promoter sequences that have leaf or predominantly a leaf specific expression pattern. The present disclosure also describes two promoters that control the expression of genes involved in triacylglycerol metabolism. The genes that encode ACX 1 and KAT 2 are both induced during the induction of senescence and are therefore considered a least in part, senescence inducible.

In a preferred embodiment of the invention said nucleic acid molecule is part of a vector and is operably linked to a promoter.

"Operably linked" means joined as 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 a selectable genetic marker such as those that confer selectable phenotypes such as resistance to herbicides (e.g. kanamycin, hygromycin, phosphinotricin, chlorsulfuron, methotrexate, gentamycin, spectinomycin, imidazoliπones and glyphosate).

In a yet further preferred embodiment of the invention the genome of said aquatic transgenic plant cell is yet further modified by transfection with a nucleic acid selected from the group consisting of: i) a nucleic acid molecule comprising a nucleic acid sequence as represented in Figure 18a-18p; ii) a nucleic acid molecule comprising a nucleic acid sequence that hybridises under stringent hybridisation conditions to a nucleic acid molecule in (i) and which encodes a plant cell modifying polypeptide; iii) a nucleic acid molecule that encodes a polypeptide comprising the amino acid sequence as represented in Figure 18a-18p.

In a yet further preferred embodiment of the invention the genome of said transgenic plant cell is yet further modified by transfection with a nucleic acid selected from the group consisting of: i) a nucleic acid molecule comprising a nucleic acid sequence as represented in Figure 19a-19j; ii) a nucleic acid molecule comprising a nucleic acid sequence that hybridises under stringent hybridisation conditions to a nucleic acid molecule in (i) and which encodes an expansin polypeptide; iii) a nucleic acid molecule that encodes a polypeptide comprising the amino acid sequence as represented in Figure 19a-19j.

In a yet still further preferred embodiment of the invention the genome of said transgenic plant cell is yet further modified by transfection with a nucleic acid selected from the group consisting of: i) a nucleic acid molecule comprising a nucleic acid sequence as represented in Figure 20a-20p; ii) a nucleic acid molecule comprising a nucleic acid sequence that hybridises under stringent hybridisation conditions to a nucleic acid molecule in (i) and which encodes a plant cell wall hydrolase polypeptide; iii) a nucleic acid molecule that encodes a polypeptide comprising the amino acid sequence as represented in Figure 20a-20p. In tobacco and tomato (Solanum lycopersicum), both of which contain comparatively large endosperms in the mature seed, the endosperm is a major focus in efforts to understand the control of germination. In addition to its function as a storage tissue, in these species and others, the endosperm has been shown to exert control over germination by secreting cell wall loosening enzymes that weaken the mechanical resistance of the micropylar endosperm cap to radicle protrusion (reviewed in Bewley, 1997b). Importantly, the activity of endosperm-expressed cell wall loosening enzymes is controlled by both ABA and GA (Groot et al., 1988; Toorop et al., 2000).The controlled loosening of the micropylar endosperm cell walls to facilitate radicle emergence is achieved by the activity of multiple categories of cell wall-modifying enzymes, including β-mannanase, β-1 ,4-glucanase, expansins, xyloglucan endotransglycosidases, and polygalacturonases. A transcriptome study of Arabidopsis endosperm 24 hours after seed imbibition was performed and a number of genes associated with cell wall metabolism were identified (Penfield et al., 2006 and table 1).

Expression of these genes in aquatic biomass crops will result in cell wall loosening and cell wall breakdown which will be valuable for biomass utilisation by either making the cell walls more available to further breakdown to component sugars by additional enzymes or releasing sugars that can be used as feedstocks for fermentation directly.

In a preferred embodiment of the invention the genome of said transgenic plant cell is modified by transfection with a nucleic acid molecule that encodes a polypeptide the expression of which confers growth enhancing effects on said cell or a plant derived from said cell thereby increasing plant biomass.

In a preferred embodiment of the invention said nucleic acid molecule is over-expressed when compared to a non-transgenic reference plant cell of the same species.

"Plant biomass" refers to living plant tissue and lignocellulosic materials that comprise the plant and includes plant organs (e.g. stems, leaves, flowers, roots,) which may increase in size, number or quality to increase yield. Genes that encode proteins that enhance the growth characteristics of a plant are well known in the art. For example WO92/09685, the content of which is incorporated by reference, describes the plant homologue of the yeast cell-cycle control gene cdc2 referred to as p34Cd 2 and is an important regulator of cell proliferation, particularly in leaf tissue. WO2005/085452, the content of which is incorporated by reference, describes the shoot specific expression of cyclin D3, a cell growth regulator and the enhancement of plant yield. WO2004/087929, the content of which is incorporated by reference, describes the expression of the CCS52 gene, a gene that encodes a cell-cycle regulatory protein, and the enhancement of plant size and increased organ size and number. WO2005/059147, the content of which is incorporated by reference, describes a growth regulatory protein, GRUBX and the effect of over-expression on plant morphology. WO2005/083094 describes a D-type cyclin dependent kinase which when over-expressed results in increased seed yield, also see WOWO2005/085452, WO2005/061702 and WO2006/100112 each of which is incorporated by reference in their entirety.

In a preferred embodiment of the invention said nucleic acid molecule that encodes a polypeptide the expression of which confers growth enhancing effects is selected from the group consisting of: i) a nucleic acid molecule comprising a nucleic acid sequence as represented in Figure 21; ii) a nucleic acid molecule comprising a nucleic acid sequence that hybridises under stringent hybridisation conditions to a nucleic acid molecule in (i) and which encodes a transcription factor.

In our co-pending application (WO2007/063289) and which is incorporated by reference, we describe a transgenic plant that over-expresses a helix turn helix transcription factor referred to as Cesta. The phenotype of over-expressing plant lines is enhanced vegetative growth and an increase in leaf number.

In a further preferred embodiment of the invention said cell is transfected with a nucleic acid molecule selected from: i) a nucleic acid molecule comprising a nucleic acid sequence as represented in Figure 22b or 22d; ii) a nucleic acid molecule comprising a nucleic acid sequence that hybridises under stringent hybridisation conditions to a nucleic acid molecule in (i) and which encodes a oleate 12 -hydroxylase.

In a preferred embodiment of the invention said cell is transfected with a nucleic acid molecule selected from: i) a nucleic acid molecule comprising a nucleic acid sequence as represented in Figure 23a; ii) a nucleic acid molecule comprising a nucleic acid sequence that hybridises under stringent hybridisation conditions to a nucleic acid molecule in (i) and which encodes a cytochrome P450.

In a preferred embodiment of the invention said cell is transfected with a nucleic acid molecule selected from: i) a nucleic acid molecule comprising a nucleic acid sequence as represented in Figure 24a; ii) a nucleic acid molecule comprising a nucleic acid sequence that hybridises under stringent hybridisation conditions to a nucleic acid molecule in (i) and which encodes a delta 12 fatty acid acetylenase.

In a preferred embodiment of the invention said cell is transfected with a nucleic acid molecule selected from: i) a nucleic acid molecule comprising a nucleic acid sequence as represented in Figure 25b; ii) a nucleic acid molecule comprising a nucleic acid sequence that hybridises under stringent hybridisation conditions to a nucleic acid molecule in (i) and which encodes a delta 12 fatty acid desaturase.

In a preferred embodiment of the invention said cell is transfected with a nucleic acid molecule selected from: i) a nucleic acid molecule comprising a nucleic acid sequence as represented in Figure 26b; ii) a nucleic acid molecule comprising a nucleic acid sequence that hybridises under stringent hybridisation conditions to a nucleic acid molecule in (i) and which encodes a delta 12 fatty acid acetylenase.

In a preferred embodiment of the invention said cell is transfected with a nucleic acid molecule selected from: i) a nucleic acid molecule comprising a nucleic acid sequence as represented in Figure 27b; ii) a nucleic acid molecule comprising a nucleic acid sequence that hybridises under stringent hybridisation conditions to a nucleic acid molecule in (i) and which encodes a delta 6 fatty acid acetylenase. In a preferred embodiment of the invention said cell is transfected with a nucleic acid molecule selected from: i) a nucleic acid molecule comprising a nucleic acid sequence as represented in Figure 28b; ii) a nucleic acid molecule comprising a nucleic acid sequence that hybridises under stringent hybridisation conditions to a nucleic acid molecule in (i) and which encodes a delta 6 fatty acid desaturase.

In a preferred embodiment of the invention said cell is transfected with a nucleic acid molecule selected from: i) a nucleic acid molecule comprising a nucleic acid sequence as represented in Figure 29b; ii) a nucleic acid molecule comprising a nucleic acid sequence that hybridises under stringent hybridisation conditions to a nucleic acid molecule in (i) and which encodes a transcription factor.

In a preferred embodiment of the invention said cell is transfected with a nucleic acid molecule selected from: i) a nucleic acid molecule comprising a nucleic acid sequence as represented in Figure 30b; ii) a nucleic acid molecule comprising a nucleic acid sequence that hybridises under stringent hybridisation conditions to a nucleic acid molecule in (i) and which encodes a transcription factor.

In a preferred embodiment of the invention said cell is transfected with a nucleic acid molecule selected from: i) a nucleic acid molecule comprising a nucleic acid sequence as represented in Figure 31b; ii) a nucleic acid molecule comprising a nucleic acid sequence that hybridises under stringent hybridisation conditions to a nucleic acid molecule in (i) and which encodes a transcription factor.

In a preferred embodiment of the invention said cell is transfected with a nucleic acid molecule selected from: i) a nucleic acid molecule comprising a nucleic acid sequence as represented in Figure 32b; ii) a nucleic acid molecule comprising a nucleic acid sequence that hybridises under stringent hybridisation conditions to a nucleic acid molecule in (i) and which encodes a transcription factor.

In a preferred embodiment of the invention said cell is transfected with a nucleic acid molecule selected from: i) a nucleic acid molecule comprising a nucleic acid sequence as represented in Figure 33b; ii) a nucleic acid molecule comprising a nucleic acid sequence that hybridises under stringent hybridisation conditions to a nucleic acid molecule in (i) and which encodes a transcription factor.

In a preferred embodiment of the invention there is provided a transgenic aquatic plant comprising a cell according to the invention.

In a preferred embodiment of the invention said transgenic aquatic plant is of the family Lemnaceae.

In a preferred embodiment of the invention said aquatic transgenic plant is of the genus Lemna.

In a preferred embodiment of the invention said transgenic aquatic plant is a species selected from the group consisting of: Lemna aequinoctialis, L. disperma, L. ecuadoriensis, L. gibba, L. japonica, L. minor, L. miniscula, L. obscure, L. perpusilla, L. tenera, L. trisulca, L. turionifera and L. valdiviana.

In an alternative preferred embodment of the invention said transgenic aquatic plant is of the genus Spirodela.

In a preferred embodiment of the invention said transgenic aquatic plant is a species selected from the group consisting of: Spirodela intermedia, S. polyrrhiza and S. punctata.

In an alternative preferred embodiment of the invention said aquatic transgenic plant is of the genus Wolffia. In a preferred embodiment of the invention said transgenic aquatic plant is a species selected from the group consisting of: Wolffia angusta, Wa. arrhiza, Wa. australina, Wa. borealis, Wa. brasiliensis, Wa. columbiana, Wa. elongata, Wa. globosa, Wa. microscopica and Wa. neglecta.

In an alternative preferred embodiment of the invention said aquatic transgenic plant is of the genus Wolfiella.

In a preferred embodiment of the invention said transgenic aquatic plant is a species selected from the group consisting of: Wolfiella caudata, Wl. denticulate, Wl. gladiata, Wl. hyalina, Wl.lingulata, Wl. repunda, Wl. rotunda, and Wl. Neotropica.

Lemna species can be classified using the taxonomic scheme described by Landolt, Biosystematic Investigation on the Family of Duckweeds: The family of Lemnaceae - A Monograph Study. Geobatanischen lnstitut ETH, Stiftung Rubel, Zurich (1986)).

In an alternative preferred embodiment of the invention said aquatic plant is of the genus Spartina.

In a preferred embodiment of the invention said aquatic transgenic plant is selected from the group consisting of: Spartina alterniflora, S. anglica, S.bakeri, S.caespitosa, S. cynosuroides, S. densiflora, S. foliosa, S. gracilis, S. maritima, S. patens, S. pectinata, S S. spartinae and S. townsendii

According to a further aspect of the invention there is provided a seed comprising a plant cell according to the invention.

According to a further aspect of the invention there is provided a method to modulate and extract plant mono- di- or triacylglycerol fatty acids comprising the steps of: i) providing an aquatic transgenic plant the genome of which is modified by transfection with a nucleic acid molecule selected from the group consisting of: a) a nucleic acid molecule comprising an expression cassette which cassette comprises a nucleic acid sequence that encodes at least part of a gene that encodes a polypeptide that controls the synthesis, degradation or transport of fatty acids and/or fatty acyl CoAs and therefore synthesis and degradation of mono- di- or triacylglycerols, wherein said cassette is adapted such that both sense and antiseπse nucleic acid molecules are transcribed from said cassette wherein the expression from said cassette produces an interfering RNA molecule that inhibits the expression of said gene; b) a nucleic acid molecule comprising an expression cassette which cassette comprises a nucleic acid sequence that encodes at least part of a gene that encodes a polypeptide that controls the synthesis, degradation or transport of fatty acids and/or fatty acyl CoAs and therefore synthesis and degradation of mono- di- or triacylglycerols, wherein said cassette is adapted such that an antisense nucleic acid molecule is transcribed from said cassette wherein the expression from said cassette produces an antisense RNA molecule that inhibits the expression of said gene; c) a nucleic acid molecule that encodes a polypeptide that controls the synthesis, degradation or transport of fatty acids and/or fatty acyl CoAs and therefore synthesis and degradation of mono- di- or triacylglycerols, in a plant cell which polypeptide is a variant polypeptide that varies from a native polypeptide sequence wherein said variant polypeptide is a dominant negative suppressor of the native polypeptide and inhibits the production of mono- di- or triacylglycerol, wherein said nucleic acid molecule in a), b) or c) is operably linked to a promoter sequence; ii) inducing expression of at least one nucleic acid molecule according to the invention; iii) harvesting transgenic plant material; and optionally iv) extracting said harvested plant material to provide a mono- di- or triacylglycerol or free fatty acid fraction and an extracted plant material fraction.

In a preferred method of the invention the induction of expression of said nucleic acid molecules is by induction of senescence.

In a preferred method of the invention the induction of senescence is by growing said plant in reduced light conditions.

In an alternative preferred method of the invention the induction of senescence is by altered day-length. In a yet further method of the invention senescence is induced by chemical treatment.

In a yet further preferred method of the invention the genome of said transgenic plant cell is yet further modified by transfection with a nucleic acid selected from the group consisting of: i) a nucleic acid molecule comprising a nucleic acid sequence as represented in Figure 18a-18p; ii) a nucleic acid molecule comprising a nucleic acid sequence that hybridises under stringent hybridisation conditions to a nucleic acid molecule in (i) and which encodes a plant cell modifying polypeptide; iii) a nucleic acid molecule that encodes a polypeptide comprising the amino acid sequence as represented in Figure 18a-18p.

In a yet further preferred method of the invention the genome of said transgenic plant cell is yet further modified by transfection with a nucleic acid selected from the group consisting of: i) a nucleic acid molecule comprising a nucleic acid sequence as represented in Figure 19a-19j; ii) a nucleic acid molecule comprising a nucleic acid sequence that hybridises under stringent hybridisation conditions to a nucleic acid molecule in (i) and which encodes an expansin polypeptide; iii) a nucleic acid molecule that encodes a polypeptide comprising the amino acid sequence as represented in Figure 19a-19j.

In a yet still further preferred method of the invention the genome of said transgenic plant cell is yet further modified by transfection with a nucleic acid selected from the group consisting of: i) a nucleic acid molecule comprising a nucleic acid sequence as represented in Figure 20a-20p; ii) a nucleic acid molecule comprising a nucleic acid sequence that hybridises under stringent hybridisation conditions to a nucleic acid molecule in (i) and which encodes a plant cell wall hydrolase polypeptide; iii) a nucleic acid molecule that encodes a polypeptide comprising the amino acid sequence as represented in Figure 20a-20p. In a preferred method of the invention said extracted plant material is further processed by saccharification to sugar.

Saccharification is a process by which plant lignocellulosic materials (e.g., lignin, cellulose, hemicellulose) are hydrolysed to glucose through chemical and enzymic means. Typically this involves the pre-treatment of plant material with alkali to remove lignin followed by enzyme digestion of cellulose. This typically uses fungal cellulose, for example from the fungus Tichoderma reesei. The present invention utilises plant hydrolases in saccharification thereby simplifying the process.

In a further preferred method of the invention said sugar is used as a feedstock in the production of ethanol by microbial fermentation.

Microorganisms used in the process according to the invention are grown or cultured in the manner with which the skilled worker is familiar, depending on the host organism. As a rule, microorganisms are grown in a liquid medium comprising a carbon source (e.g. sugar as formed during the saccharification process), a nitrogen source, usually in the form of organic nitrogen sources such as yeast extract or salts such as ammonium sulfate, trace elements such as salts of iron, manganese and magnesium and, if appropriate, vitamins, at temperatures of between 0⁰C and 100⁰C, preferably between 10⁰C and 60⁰C, while gassing in oxygen.

The pH of the liquid medium can either be kept constant, that is to say regulated during the culturing period, or not. The cultures can be grown batchwise, semi-batchwise or continuously. Nutrients can be provided at the beginning of the fermentation or fed in semi-continuously or continuously. The products produced can be isolated from the organisms as described above by processes known to the skilled worker, for example by extraction or distillation. In this process, the pH value is advantageously kept between pH 4 and 12, preferably between pH 6 and 9, especially preferably between pH 7 and 8.

The culture medium to be used must suitably meet the requirements of the strains in question. Descriptions of culture media for various microorganisms can be found in the textbook "Manual of Methods for General Bacteriology" of the American Society for Bacteriology (Washington D.C., USA, 1981).

As described above, these media which can be employed in accordance with the invention usually comprise one or more, nitrogen sources, inorganic salts, vitamins and/or trace elements. Nitrogen sources are usually organic or inorganic nitrogen compounds or materials comprising these compounds. Examples of nitrogen sources comprise ammonia in liquid or gaseous form or ammonium salts such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate or ammonium nitrate, nitrates, urea, amino acids or complex nitrogen sources such as cornsteep liquor, soya meal, soya protein, yeast extract, meat extract and others. The nitrogen sources can be used individually or as a mixture.

Inorganic salt compounds which may be present in the media comprise the chloride, phosphorus and sulfate salts of calcium, magnesium, sodium, cobalt, molybdenum, potassium, manganese, zinc, copper and iron.

Inorganic sulfur-containing compounds such as, for example, sulfates, sulfites, dithionites, tetrathionates, thiosulfates, sulfides, or else organic sulfur compounds such as mercaptans and thiols may be used as sources of sulfur for the production of sulfur- containing fine chemicals, in particular of methionine.

Phosphoric acid, potassium dihydrogenphosphate or dipotassium hydrogenphosphate or the corresponding sodium-containing salts may be used as sources of phosphorus.

Chelating agents may be added to the medium in order to keep the metal ions in solution. Particularly suitable chelating agents comprise dihydroxyphenols such as catechol or protocatechuate and organic acids such as citric acid.

The fermentation media used according to the invention for culturing microorganisms usually also comprise other growth factors such as vitamins or growth promoters, which include, for example, biotin, riboflavin, thiamine, folic acid, nicotinic acid, pantothenate and pyridoxine. Growth factors and salts are frequently derived from complex media components such as yeast extract, molasses, cornsteep liquor and the like. It is moreover possible to add suitable precursors to the culture medium. The exact composition of the media compounds heavily depends on the particular experiment and is decided upon individually for each specific case. Information on the optimization of media can be found in the textbook "Applied Microbiol. Physiology, A Practical Approach" (Editors P.M. Rhodes, P.F. Stanbury, IRL Press (1997) pp. 53-73, ISBN 0 19 963577 3). Growth media can also be obtained from commercial suppliers, for example Standard 1 (Merck) or BHI (brain heart infusion, DIFCO) and the like.

All media components are sterilized, either by heat (20 min at 1.5 bar and 121⁰C) or by filter sterilization. The components may be sterilized either together or, if required, separately. All media components may be present at the start of the cultivation or added continuously or batch wise, as desired.

The culture temperature is normally between 15°C and 45°C, preferably at from 25°C to 40⁰C₁ and may be kept constant or may be altered during the experiment. The pH of the medium should be in the range from 5 to 8.5, preferably around 7.0. The pH for cultivation can be controlled during cultivation by adding basic compounds such as sodium hydroxide, potassium hydroxide, ammonia and aqueous ammonia or acidic compounds such as phosphoric acid or sulfuric acid. Foaming can be controlled by employing antifoams such as, for example, fatty acid polyglycol esters.

The fermentation broth can then be processed further. The biomass may, according to requirement, be removed completely or partially from the fermentation broth by separation methods such as, for example, centrifugation, filtration, decanting or a combination of these methods or be left completely in said broth.

According to a further aspect of the invention there is provided a composition comprising mono- di- or triacylglycerol formed by the method according to the invention.

In a preferred embodiment of the invention said composition is a biofuel.

In a further preferred embodiment of the invention said composition is a nutraceutical.

In a preferred embodiment of the invention said composition comprises elevated levels of galactolipids.

In a further preferred embodiment of the invention said composition comprises elevated levels of linolenic acid.

An additional method to regulate the expression of plant genes is by virus induced gene silencing (VIGS). A viral infection in a plant induces an RNA mediated defence response against the infecting virus that targets the viral genome and any foreign sequences cloned into the viral genome. The phenomenon is related to RNA interference and only requires a short region of foreign sequence to induce a specific degradation of the RNA that corresponds to the foreign nucleic acid. Advantageously, the method of VIGS does not require the stable genetic modification of the plant genome to effect an ablation effect on gene expression but simply the infection of a plant with a virus that is engineered to include a plant nucleic acid sequence the regulation of which is desired.

According to a further aspect of the invention there is provided a modified aquatic plant wherein said plant comprises a virus that includes a nucleic acid molecule wherein said nucleic acid molecule is at least part of a gene that encodes a protein that controls the synthesis, degradation or transport of fatty acids and/or fatty acyl CoAs and therefore the synthesis and degradation of mono- di- or triacylglycerols in a plant cell.

Figure 13b.

i) a nucleic acid molecule comprising a nucleic acid sequence as represented in Figure 14a or14c; ii) a nucleic acid molecule comprising a nucleic acid sequence that hybridises under stringent hybridisation conditions to a nucleic acid molecule in (i) and which encodes a long chain acyl Co A synthetase polypeptide; iii) a nucleic acid molecule that encodes a variant polypeptide that varies from a polypeptide comprising the amino acid sequence as represented in

Figure 14b or 14d.

In a preferred embodiment of the invention said gene is encoded by a nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of: i) a nucleic acid molecule comprising a nucleic acid sequence as represented in Figure 15a, 15c, 15e, 15g, 15i or 15k, ; ii) a nucleic acid molecule comprising a nucleic acid sequence that hybridises under stringent hybridisation conditions to a nucleic acid molecule in (i) and which encodes an acyl CoA oxidase polypeptide; iv) a nucleic acid molecule that encodes a variant polypeptide that varies from a polypeptide comprising the amino acid sequence as represented in

Figure 15b, 15d, 15f, 15h, 15j or15l.

Figure 16b, 16d, 16f or 16h.

i) a nucleic acid molecule comprising a nucleic acid sequence as represented in Figure 17a or 17c ii) a nucleic acid molecule comprising a nucleic acid sequence that hybridises under stringent hybridisation conditions to a nucleic acid molecule in (i) and which encodes a multifunctional protein involved in peroxisomal β oxidation; iii) a nucleic acid molecule that encodes a variant polypeptide that varies from a polypeptide comprising the amino acid sequence as represented in

Figure 17b or 17d. In a preferred embodiment of the invention said nucleic acid molecule is between 20-30 base pairs in length.

In a preferred embodiment of the invention said nucleic acid molecule consists of 21-24; pairs in length; preferably about 21 base pairs in length.

According to a further aspect of the invention there is provided a method to inhibit the expression of a plant gene comprising the steps of. i) contacting a plant with a viral vector that includes a nucleic acid molecule wherein said nucleic acid molecule is at least part of a gene that encodes a protein that controls the synthesis, degradation or transport of fatty acids and/or fatty acyl CoAs; and ii) cultivating the virally infected plant to allow viral induced gene silencing.

In a preferred method of the invention said infected plant material is harvested.

In a further preferred method of the invention said harvested plant material is extracted to provide a mono- di- or triacylglycerol or free fatty acid fraction.

Throughout the description and claims of this specification, the words "comprise" and "contain" and variations of the words, for example "comprising" and "comprises", means "including but not limited to", and is not intended to (and does not) exclude other moieties, additives, components, integers or steps.

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.

An embodiment of the invention will now be described by example only and with reference to the following figures: Figure 1 : The central role of the acyl CoA pool in plant lipid metabolism. Arrows represent directional fluxes of cytosolic acyl CoAs in a general model representing all plant tissues. Numbers refer to biochemical routes and genes referenced in the text;

Figure 2: Overview of the major metabolic pathways required for lipid reserve mobilisation in Arabidopsis seeds;

Figure 3: illustrates that Arabidopsis mutants disrupted in peroxisomal fatty acid beta- oxidation are sensitive to extended dark treatment;

Figure 4: illustrates the phenotype of pxal mutants after 48 hours extended dark compared with CoI-O wild types. Plants were grown in P40 trays for 4 weeks in a Sanyo growth cabinet with a 12h light/ 12 h dark cycle;

Figure 5: illustrates total fatty acids in Arabidopsis leaves kept in extended dark for up to 48 hours. -12h hours in the end of the day and Oh is the start of the extended dark period. Data is the average plus SD of 4 biological replicates;

Figure 6: illustrates acyl CoAs in Arabidopsis leaves kept in extended dark for up to 48 hours. -12h hours in the end of the day and Oh is the start of the extended dark period. Data is the average plus SD of 4 biological replicates;

Figure 7: illustrates the amounts of starch (A), sucrose (B), glucose (C), and fructose (D) in Arabidopsis leaves kept in extended dark for up to 48 hours. -12h hours in the end of the day and Oh are the start of the extended dark period. Data is the average plus SD of 4 biological replicates;

Figure 8 illustrates total fatty acids in Arabidopsis leaves kept in extended dark for up to 48 hours. Data is the average plus SD of 3 biological replicates;

Figure 9 illustrates non-free fatty acids in Arabidopsis leaves kept in extended dark for up to 48 hours, extracted using the base FAMEs method. Data is the average plus SD of 3 biological replicates; Figure 10 illustrates thin layer chromatography of total lipid extract from leaves from plants kept in 48h extended dark. Total lipids were extracted in 3:2 hexane: isopropanol using the standard lab lipid extraction method and developed in the solvent system: hexane: diethylether: acetic acid (70:30:1 v/v). Lipids were visualised by spraying with fluorescein and exposing to UV light;

Figure 11 illustrates total lipid analysis by LC-MS. A fraction of the total lipid extract from leaves as described in WO2006/018621, the content of which is incorporated by reference, was run on the LCQ using the TAG method developed in the lab. The fatty acid species present in the major galactolipid, DAG and TAG peaks were then identified based on the mass spectra obtained;

Figure 12 illustrates histochemical staining of leaves expressing various promoters: GUS constructs kept in extended dark for up to 48 hours;

Figure 13a is the DNA sequence of an ABC fatty acid transporter; Figure 13b is the amino acid sequence of the ABC fatty acid transporter;

Figure 14a is the DNA sequence of a long chain acyl Co A synthetase LACS 6; Figure 14b is the amino acid sequence of the long chain acyl Co A synthetase LACS 6; Figure 14c is the DNA sequence of the long chain acyl Co A synthetase LACS 7; Figure 14d is the amino acid sequence of the long chain acyl Co A synthetase LACS 7;

Figure 15a is the DNA sequence of a acyl oxidase ACX 1; Figure 15b is the amino acid sequence of the acyl oxidase ACX 1; Figure 15c is the DNA sequence of acyl oxidase ACX 2; Figure 15d is the amino acid sequence of acyl oxidase ACX 2 ; Figure 15e is the DNA sequence of the acyl oxidase ACX 3; Figure 15f is the amino acid sequence of the acyl oxidase ACX 3; Figure 15g is the DNA sequence of a acyl oxidase ACX 4; Figure 15h is the amino acid sequence of the acyl oxidase ACX 4; Figure 15i is the DNA sequence of a acyl oxidase ACX 5; Figure 15j is the amino acid sequence of the acyl oxidase ACX 5; Figure 15k is the DNA sequence of a acyl oxidase ACX 6; Figure 151 is the amino acid sequence of the acyl oxidase ACX 6;

Figure 16a is the DNA sequence of KAT 2; Figure 16b is the amino acid sequence of KAT 2; Figure 16c is the DNA sequence of KAT 1; Figure 16d is the amino acid sequence of KAT 1; Figure 16e is the DNA sequence of PKT2; Figure 16f is the amino acid sequence of PKT2; Figure 16g is the DNA sequence of PKT1; Figure 16h is the amino acid sequence of PKT1;

Figure 17a is the DNA sequence of MFP 2; Figure 17b is the amino acid sequence of MFP 2; Figure 17c is the DNA sequence of AIM 1; Figure 17d is the amino acid sequence ofAIM 1;

Figure 18a-Figure 18p represents the DNA and amino acid sequences of plant cell wall modifying enzymes;

Figure 19a-Figure 19j represents the DNA and amino acid sequences of plant expansin enzymes;

Figure 20a- Figure 2Op represents the DNA and amino acid sequences of plant cell wall hydrolase enzymes;

Figure 21 is the DNA sequence of transcription factor Cesta;

Figure 22a is the amino acid sequence of Ricinus communis oleate 12 -hydroxylase; Figuire 22b is the nucleic acid sequence of Ricinus communis oleate 12 -hydroxylase; Figure 22c is the nucleic acid sequence of Ricinus communis oleate 12 -hydroxylase isoform;

Figure 23a the nucleic acid sequence of a Euphorbia lagascae cytochrome P450;

Figure 24a is the amino acid sequence of a Crepis palaestina delta 12 fatty acid epoxygenase; Figure 24b is the nucleic acid sequence of a Crepis palaestina delta 12 fatty acid epoxygenase;

Figure 25a is the amino acid sequence of a Crepis palaestina delta 12 fatty acid desaturase; Figure 25b is the nucleic acid sequence of a Crepis palaestina delta 12 fatty acid epoxygenase;

Figure 26a is the amino acid sequence of a Crepis palaestina delta 12 fatty acid acetylenase; Figure 26b is the nucleic acid sequence of a Crepis palaestina delta 12 fatty acid acetylenase; Figure 27a is the amino acid sequence of a Ceratodon purpureus delta 6 fatty acid acetylenase; Figure 27b is the nucleic acid sequence of a Ceratodon purpureus delta 6 fatty acid acetylenase;

Figure 28a is the amino acid sequence of a Ceratodon purpureus delta 6 fatty acid desaturase; Figure 28b is the nucleic acid sequence of a Ceratodon purpureus delta 6 fatty acid desaturase;

Figure 29a is the amino acid sequence of the transcription factor LEC 2; Figure 29b is the nucleic acid sequence of the transcription factor LEC 2;

Figure 30a is the amino acid sequence of the transcription factor LEC 1 ; Figure 29b is the nucleic acid sequence of the transcription factor LEC 1;

Figure 31a is the amino acid sequence of the transcription factor FUS 3; Figure 31b is the nucleic acid sequence of the transcription factor FUS 3;

Figure 32a is the amino acid sequence of the transcription factor ABI 3; Figure 32b is the nucleic acid sequence of the transcription factor ABI3; and

Figure 33a is the amino acid sequence of the transcription factor WRH; Figure 33b is the nucleic acid sequence of the transcription factor WRH.

Materials and Methods

CoI-O, Ws₁ cfs2, pxaf and acx1acx2 plants were grown in P40 trays in a 12h light / 12h dark regime in a Sanyo growth cabinet with 150 μmol.m^"2.s^"1 light for 4 weeks (rosettes prior to bolting, between growth stages 3.70 and 3.90 according to Boyes et al 2001. Plant Cell 13, 1499), and then the lights were switched off. The following time points were used for material collection: minus 12h (end of the night before the extended dark period), Oh (start of day and of extended dark), 12h, 24h and 48h. In this experiment samples were collected for analysis of fatty acids and acyl CoAs (2 no.3 size leaf discs per each of 4 reps.), sugars, starch and amino acids (4 no.3 size leaf discs per each of 4 reps), and 2 outer, older leaves for RNA extraction. Samples were collected, weighed and immediately snap-frozen for subsequent analysis. Fatty acids and CoAs were extracted and analysed using standard lab methods. Amino acids and sugars were extracted from same sample using 80% ethanol and the remaining insoluble material was used to measure starch. Amino acids were derivatised and analysed on the LCQ. Soluble sugars (sucrose, glucose and fructose) and starch (after enzymatic conversion to glucose) were quantified spectrophotometrically, using a Boerhingher Mannheim kit from R-Biopharm Ltd.

The second dark experiment was set up exactly as the first, except the acx1acx2 mutant was not included. As well as repeating the dark experiment, mutant and wild type plants were placed under the following stresses: cold treatment (13^" C and 4^' C), salt and drought. In addition, all the available promoter-GUS lines were grown in the same conditions for subsequent analysis after dark treatment.

Leaf samples were collected from 4 week-old pxal, cts2, CoI-O and Ws plants kept in the dark for 48h. Samples were taken at the same time points as in the previous experiment for the analysis of total fatty acids (2 leaf discs), non-free fatty acids (2 leaf discs) and total lipid analysis by thin layer chromatography (2 leaves ~ 100mg tissue).

The data for total and non-free fatty acids (alkaline derivatisation) is presented in Figs 8 and 9. Total fatty acid data is consistent with experiment 1: after 48h extended dark, cts2 and pxal plants have considerably more total fatty acids than wild types. Remarkably, the levels of some of the major fatty acid species appear to increase in the mutants over the time course (e.g. 18:3n3) introducing the possibility that fatty acids are accumulating in a sink because they cannot be broken down.

The alkaline derivatisation method allows the quantification of non-free fatty acids. Therefore the data from this method can be compared with total fatty acid measurements from the same samples in order to establish the proportions of fatty acids that are free and not free. Fig. 9 shows the levels of non-free fatty acids in the mutants and wild types throughout the time course, and illustrates that a large proportion of the fatty acids in cts2 and pxal are not free.

In order to establish the location of the elevated fatty acids in cts2 and pxal, total lipids were extracted from 2 leaves using standard lipid extraction methods that use 3:2 hexane:isopropanol solvent extraction. The extracts were dried down and resuspended in a minimal volume of chloroform and spotted onto silica TLC plates. The plates were run in a hexane:diethylether:acetic acid (70:30:1 v/v) solvent system and visualised under UV light after spraying with fluorescein. The TLC plates illustrate that the cts2 and pxal mutants are accumulating significant amounts of mono- di- or triacylglycerols (TAGs) during the extended dark period, and that free fatty acid levels also increase (Fig.10).

A small aliquot of the total lipid extraction was run on the LC-MS in order to identify the lipids present in the mutant plants, (see WO2006/018621 the content of which is incorporated by reference), because this sample was prepared for TLC there is no internal standard present and so this data is qualitative rather than quantitative (Fig. 11). This analysis reveals that mutant plants are accumulating triacylglycerols and diacylglycerols. Interestingly, the cts2 mutant also has increased levels of galactolipids. Many of the DAGs and TAGs found in the mutants contain C16:3, which is an exclusively chloroplast fatty acid, not found in seed TAGs. Thus a triacylglycerol sink has been established to cope with fatty acids that are targeted for degradation but blocked due to the lesion in in a specific gene involved in breakdown of fatty acids (in this case either a fatty acid transporter or beta-oxidation gene). Any treatment (for example daylength, temperature) that induces fatty acid turnover in plant material that is unable to breakdown fatty acids is thus expected to result in an increase in triacylglycerol oil accumulation;

Examples

The dark-induced phenotype of pxa 1 is more severe than that of cts2, such that by 48h of extended dark, the older leaves have all collapsed and lost turgor (Fig. 4). The fatty acid, acyl CoA, starch and sugar data are presented in Figs 5, 6 and 7. The graphs in Fig 5 show that across the time course fatty acids decrease in wild types but not in mutants, particularly cts2 and pxal This is most marked by 48h of extended dark. The graphs in Fig. 6 show that acyl CoAs accumulate in mutants, particularly 18:3, 18:2 and 16:0 which are the major fatty acid species present in Arabidopsis leaves. In addition, isovaleryl CoA (i5:0), a branched chain amino acid derivative, appears after 12 hours of extended dark which is indicative of protein break down beginning to occur.

Fig. 7 illustrates the levels of soluble sugars and starch during the time course. Starch levels fall to undetectable levels over the night (Fig. 7A). Sucrose levels drop over the 12h night period, but in wild types sucrose does not disappear completely until 12h into the extended dark (Fig. 7B). In contrast all 3 mutants show a more rapid decrease in sucrose levels over the night, which is likely to result because fatty acid utilisation, which normally occurs during the night in wild types, cannot occur in the mutants. This indicates that substantial fatty acid turnover occurs during the normal night period in wild type plants and when this is blocked soluble sugars are more rapidly respired. Any treatment that increases fatty acid turnover during the night is therefore likely to increase the flux of carbon into the new triacylglycerol oil sink that is established when fatty acid breakdown is blocked.

The finding that cts2 and pxal mutants accumulate TAG is an important discovery. Blocking breakdown leads to accumulation of acyl CoAs and under conditions of dark induced starvation, that most probably also mimic natural senescence, the plants actually induce the process of TAG biosynthesis and divert fatty acids into DAGs and TAGs.

Beta oxidation gene promoter-GUS expression

Transgenic plants expressing several promoter-GUS lines were placed in extended dark over the same time course as the beta oxidation mutants, in order to investigate the effect of dark on the gene expression of ACX1, ACX2, ACX3, ACX4, KAT2, ICL and PEPCK1 (Fig. 12). Histochemical staining of dark-starved leaves over the time course suggests that ACX1 and KA 12 are induced by extended dark, while ACXZ is repressed. ACX2 and /CL are not expressed in leaves, and PEPCK1 expression does not change during the time course. The induction of ACX1 and KAT2 demonstrates that the dark treatment is leading to induction of fatty acid beta-oxidation genes. The dark treatment is therefore a convenient experimental treatment to induce fatty acid breakdown and analyse the impact of blocking this process in foliar tissue. This treatment therefore mimics other more physiological conditions such as aging and leaf senescence which would also be expected to result in TAG accumulation when fatty acid breakdown in blocked.

Claims

1. A transgenic aquatic plant cell the genome of which is modified by transfection with a nucleic acid molecule selected from the group consisting of: i) a nucleic acid molecule comprising an expression cassette which cassette comprises a nucleic acid sequence that encodes at least part of a gene that encodes a polypeptide that controls the synthesis, degradation or transport of fatty acids and/or fatty acyl CoAs and therefore synthesis and degradation of mono- di- or triacylglycerols wherein said cassette is adapted such that both sense and antisense nucleic acid molecules are transcribed from said cassette wherein the expression from said cassette produces an interfering RNA molecule that inhibits the expression of said gene; ii) a nucleic acid molecule comprising an expression cassette which cassette comprises a nucleic acid sequence that encodes at least part of a gene that encodes a polypeptide that controls the synthesis, degradation or transport of fatty acids and/or fatty acyl CoAs and therefore synthesis and degradation of mono- di- or triacylglycerols wherein said cassette is adapted such that an antisense nucleic acid molecule is transcribed from said cassette wherein the expression from said cassette produces an antisense RNA molecule that inhibits the expression of said gene; iii) a nucleic acid molecule that encodes a polypeptide that controls the synthesis, degradation or transport of fatty acids and/or fatty acyl CoAs and therefore synthesis and degradation of mono- di- or triacylglycerols in a plant cell which polypeptide is a variant polypeptide that varies from a native polypeptide sequence wherein said variant polypeptide is a dominant negative suppressor of the native polypeptide and inhibits the production of fatty acids and/or fatty acyl CoAs therefore synthesis and degradation of mono- di- or triacylglycerols, wherein said nucleic acid molecule in i), ii) and iii) is operably linked to a promoter sequence that is substantially a foliar inducible and/or senescence inducible promoter.

2. A cell according to claim 1 wherein said gene encodes a polypeptide involved in degradation of fatty acids and/or fatty acyl Co As.

3. A cell according to claim 1 wherein said gene is encoded by a nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of: i) a nucleic acid molecule comprising a nucleic acid sequence as represented in Figure 13a; ii) a nucleic acid molecule comprising a nucleic acid, sequence that hybridises under stringent hybridisation conditions to a nucleic acid molecule in (i) and which encodes a fatty acid transporter polypeptide; iii) a nucleic acid molecule that encodes a variant polypeptide that varies from a polypeptide comprising the amino acid sequence as represented in Figure

13b.

4. A cell according to claim 1 wherein said gene is encoded by a nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of: i) a nucleic acid molecule comprising a nucleic acid sequence as represented in Figure 14a or14c; ii) a nucleic acid molecule comprising a nucleic acid sequence that hybridises under stringent hybridisation conditions to a nucleic acid molecule in (i) and which encodes a long chain acyl Co A synthetase polypeptide; iii) a nucleic acid molecule that encodes a variant polypeptide that varies from a polypeptide comprising the amino acid sequence as represented in

Figure 14b or 14d.

5. A cell according to claim 1 wherein said gene is encoded by a nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of: i) a nucleic acid molecule comprising a nucleic acid sequence as represented in Figure 15a, 15c, 15e, 15g, 15i or 15k; ii) a nucleic acid molecule comprising a nucleic acid sequence that hybridises under stringent hybridisation conditions to a nucleic acid molecule in (i) and which encodes an acyl CoA oxidase polypeptide; iii) a nucleic acid molecule that encodes a variant polypeptide that varies from a polypeptide comprising the amino acid sequence as represented in Figure 15b, 15d, 15f, 15h, 15j or15l.

6. A cell according to claim 1 wherein said gene is encoded by a nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of: i) a nucleic acid molecule comprising a nucleic acid sequence as represented in Figure 16a; 16c,16e or 16g ii) a nucleic acid molecule comprising a nucleic acid sequence that hybridises under stringent hybridisation conditions to a nucleic acid molecule in (i) and which encodes a keto-acyl-CoA thiolase; iii) a nucleic acid molecule that encodes a variant polypeptide that varies from a polypeptide comprising the amino acid sequence as represented in

Figure 16b, 16d, 16f or 16h.

7. A cell according to claim 1 wherein said gene is encoded by a nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of: i) a nucleic acid molecule comprising a nucleic acid sequence as represented in Figure 17a or 17c ii) a nucleic acid molecule comprising a nucleic acid sequence that hybridises under stringent hybridisation conditions to a nucleic acid molecule in (i) and which encodes a multifunctional protein involved in peroxisomal β oxidation; iii) a nucleic acid molecule that encodes a variant polypeptide that varies from a polypeptide comprising the amino acid sequence as represented in

Figure 17b or 17d.

8. A cell according to any of claims 1-7 wherein said cassette adapted for expression of sense and antisense nucleic acid comprises a nucleic acid molecule wherein said molecule comprises a first part linked to a second part wherein said first and second parts are complementary over at least part of their sequence and further wherein transcription of said nucleic acid molecule produces an RNA molecule which forms a double stranded region by complementary base pairing of said first and second parts.

9. A cell according to any of claims 1-8 wherein said promoter sequence is an inducible foliar specific promoter sequence.

10. A cell according to any of claims 1-8 wherein said promoter sequence is a senescence inducible promoter sequence.

11. A cell according to any of claims 1-10 wherein said nucleic acid molecule is part of a vector and is operably linked to a promoter.

12. A cell according to any of claims 1-11 wherein the genome of said transgenic plant cell is yet further modified by transfection with at least one nucleic acid selected from the group consisting of: i) a nucleic acid molecule comprising a nucleic acid sequence as represented in Figure 18a-18p; ii) a nucleic acid molecule comprising a nucleic acid sequence that hybridises under stringent hybridisation conditions to a nucleic acid molecule in (i) and which encodes a plant cell wall modifying polypeptide; iii) a nucleic acid molecule that encodes a polypeptide comprising the amino acid sequence as represented in Figure 18a-18p.

13. A cell according to any of claims 1-12 wherein the genome of said transgenic plant cell is yet further modified by transfection with at least one nucleic acid selected from the group consisting of: i) a nucleic acid molecule comprising a nucleic acid sequence as represented in Figure 19a-19j; ii) a nucleic acid molecule comprising a nucleic acid sequence that hybridises under stringent hybridisation conditions to a nucleic acid molecule in (i) and which encodes an expansin polypeptide; iii) a nucleic acid molecule that encodes a polypeptide comprising the amino acid sequence as represented in Figure 19a-19j.

14. A cell according to any of claims 1-13 wherein the genome of said transgenic plant cell is yet further modified by transfection with at least one nucleic acid selected from the group consisting of: i) a nucleic acid molecule comprising a nucleic acid sequence as represented in Figure 20a-20p; ii) a nucleic acid molecule comprising a nucleic acid sequence that hybridises under stringent hybridisation conditions to a nucleic acid molecule in (i) and which encodes a plant cell wall hydrolase polypeptide; iii) a nucleic acid molecule that encodes a polypeptide comprising the amino acid sequence as represented in Figure 20a-20p.

15. A cell according to any of claims 1-14 wherein the genome of said transgenic plant cell is modified by transfection with a nucleic acid molecule that encodes a polypeptide the expression of which confers growth enhancing effects on said cell or a plant derived from said cell thereby increasing plant biomass.

16. A cell according to claim 15 wherein said nucleic acid molecule is over-expressed when compared to a non-transgenic reference plant cell of the same species.

17. A cell according to claim 15 or 16 wherein said nucleic acid molecule that encodes a polypeptide the expression of which confers growth enhancing effects is selected from the group consisting of: i) a nucleic acid molecule comprising a nucleic acid sequence as represented in Figure 21; ii) a nucleic acid molecule comprising a nucleic acid sequence that hybridises under stringent hybridisation conditions to a nucleic acid molecule in (i) and which encodes a transcription factor.

18. A transgenic aquatic plant comprising a cell according to any of claims 1-17.

19. A plant according to claim 18 wherein said transgenic aquatic plant is of the family Lemnaceae.

20. A plant according to claim 19 wherein said aquatic transgenic plant is of the genus Lemna.

21. A plant according to claim 20 wherein said transgenic aquatic plant is a species selected from the group consisting of: Lemna aequinoctialis, L. disperma, L. ecuadoriensis, L. gibba, L. japonica, L. minor, L. miniscula, L. obscure, L. perpusilla, L. tenera, L. trisulca, L. turionifera and L. valdiviana.

22. A plant according to claim 19 wherein said transgenic aquatic plant is of the genus Spirodela.

23. A plant according to claim 22 wherein said transgenic aquatic plant is a species selected from the group consisting of: Spirodela intermedia, S. polyrrhiza and S. punctata.

24. A plant according to claim 19 wherein said aquatic transgenic plant is of the genus Wolffia.

25. A plant according to claim 24 wherein said transgenic aquatic plant is a species selected from the group consisting of: Wolffia angusta, Wa. arrhiza, Wa. australina, Wa. borealis, Wa. brasiliensis, Wa. columbiana, Wa. elongata, Wa. globosa, Wa. Microscopica and Wa. neglecta.

26. A plant according to claim 19 wherein In an alternative preferred embodiment of the invention said aquatic transgenic plant is of the genus Wolfiella.

27. A plant according to claim 19 wherein said transgenic aquatic plant is a species selected from the group consisting of: Wolfiella caudata, Wl. denticulata, Wl. gladiata, Wl. hyalina, Wl.lingulata, Wl. repunda, Wl. rotunda, and Wl. Neotropica.

28. A plant according to claim 18 wherein said aquatic plant is of the genus Spartina.

29. A plant according to claim 28 wherein said aquatic transgenic plant is selected from the group consisting of: Spartina alterniflora, S. anglica, S.bakeri, S.caespitosa, S. cynosuroides, S. densiflora, S. foliosa, S. gracilis, S. maritima, S. patens, S. pectinata, S S. spartinae and S. townsendii

30. A seed comprising a plant cell according to any of claims 1-17.

31. A method to modulate and extract plant mono- di- or triacylglycerol fatty acids comprising the steps of: i) providing a transgenic aquatic plant the genome of which is modified by transfection with a nucleic acid molecule selected from the group consisting of: a) a nucleic acid molecule comprising an expression cassette which cassette comprises a nucleic acid sequence that encodes at least part of a gene that encodes a polypeptide that controls the synthesis, degradation or transport of fatty acids and/or fatty acyl CoAs and therefore synthesis and degradation of mono- di- or triacylglycerols, wherein said cassette is adapted such that both sense and antisense nucleic acid molecules are transcribed from said cassette wherein the expression from said cassette produces an interfering RNA molecule that inhibits the expression of said gene; b) a nucleic acid molecule comprising an expression cassette which cassette comprises a nucleic acid sequence that encodes at least part of a gene that encodes a polypeptide that controls the synthesis, degradation or transport of fatty acids and/or fatty acyl CoAs and therefore synthesis and degradation of mono- di- or triacylglycerols, wherein said cassette is adapted such that an antisense nucleic acid molecule is transcribed from said cassette wherein the expression from said cassette produces an antisense RNA molecule that inhibits the expression of said gene; c) a nucleic acid molecule that encodes a polypeptide that controls the synthesis, degradation or transport of fatty acids and/or fatty acyl CoAs and therefore synthesis and degradation of mono- di- or triacylglycerols, in a plant cell which polypeptide is a variant polypeptide that varies from a native polypeptide sequence wherein said variant polypeptide is a dominant negative suppressor of the native polypeptide and inhibits the production of mono- di- or triacylglycerol, wherein said nucleic acid molecule in a), b) or c) is operably linked to a promoter sequence; ii) inducing expression of at least one nucleic acid molecule according to the invention; iii) harvesting transgenic aquatic plant material; and optionally iv) extracting said harvested plant material to provide a mono- di- or triacylglycerol or free fatty acid fraction and an extracted plant material fraction.

32. A method according to claim 31 wherein the induction of expression of said nucleic acid molecules is by induction of senescence.

33. A method according to claim 32 wherein the induction of senescence is by growing said plant in reduced light conditions.

34. A method according to claim 32 wherein the induction of senescence is by altered day-length.

35. A method according to claim 32 wherein senescence is induced by chemical treatment.

36. A method according to any of claims 31-35 wherein the genome of said transgenic plant cell is yet further modified by transfection with a nucleic acid selected from the group consisting of: i) a nucleic acid molecule comprising a nucleic acid sequence as represented in Figure 18a-18p; ii) a nucleic acid molecule comprising a nucleic acid sequence that hybridises under stringent hybridisation conditions to a nucleic acid molecule in (i) and which encodes a plant cell modifying polypeptide; iii) a nucleic acid molecule that encodes a polypeptide comprising the amino acid sequence as represented in Figure 18a-18p.

37. A method according to any of claims 31-36 wherein the genome of said transgenic plant cell is yet further modified by transfection with a nucleic acid selected from the group consisting of: i) a nucleic acid molecule comprising a nucleic acid sequence as represented in Figure 19a-19j; ii) a nucleic acid molecule comprising a nucleic acid sequence that hybridises under stringent hybridisation conditions to a nucleic acid molecule in (i) and which encodes an expansin polypeptide; iii) a nucleic acid molecule that encodes a polypeptide comprising the amino acid sequence as represented in Figure 19a-19j.

38. A method according to any of claims 31-37 wherein the genome of said transgenic plant cell is yet further modified by transfection with a nucleic acid selected from the group consisting of: i) a nucleic acid molecule comprising a nucleic acid sequence as represented in Figure 20a-20p; ii) a nucleic acid molecule comprising a nucleic acid sequence that hybridises under stringent hybridisation conditions to a nucleic acid molecule in (i) and which encodes a plant cell wall hydrolase polypeptide; iii) a nucleic acid molecule that encodes a polypeptide comprising the amino acid sequence as represented in Figure 20a-20p.

39. A method according to any of claims 31-38 wherein said extracted plant material fraction is further processed by saccharification to sugar.

40. A method according to claim 39 wherein said sugar is used as a feedstock in the production of ethanol by microbial fermentation.

41. A composition comprising mono- di- or triacylglycerol formed by the method according to any of claims 31-40.

42. A composition according to claim 41 wherein said composition is a biofuel.

43. A composition according to claim 41 wherein said composition is a nutraceutical.

44. A composition according to claim 41 wherein said composition is an industrial feedstock.

45. A modified plant wherein said plant comprises a virus that includes a nucleic acid molecule wherein said nucleic acid molecule is at least part of a gene that encodes a protein that controls the synthesis, degradation or transport of fatty acids and/or fatty acyl CoAs and therefore synthesis and degradation of mono- di- or triacylglycerols in a plant cell.

46. A plant according to claim 45 wherein said gene is encoded by a nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of: i) a nucleic acid molecule comprising a nucleic acid sequence as represented in Figure 13a; ii) a nucleic acid molecule comprising a nucleic acid sequence that hybridises under stringent hybridisation conditions to a nucleic acid molecule in (i) and which encodes a fatty acid transporter polypeptide; iii) a nucleic acid molecule that encodes a variant polypeptide that varies from a polypeptide comprising the amino acid sequence as represented in Figure 13b.

47. A plant according to claim 45 or 46 wherein said gene is encoded by a nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of: i) a nucleic acid molecule comprising a nucleic acid sequence as represented in Figure 14a or14c; ii) a nucleic acid molecule comprising a nucleic acid sequence that hybridises under stringent hybridisation conditions to a nucleic acid molecule in (i) and which encodes a long chain acyl Co A synthetase polypeptide; iii) a nucleic acid molecule that encodes a variant polypeptide that varies from a polypeptide comprising the amino acid sequence as represented in

Figure 14b or 14d.

48. A plant according to any of claims 45-47 wherein said gene is encoded by a nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of:

i) a nucleic acid molecule comprising a nucleic acid sequence as represented in Figure 15a, 15c, 15e, 15g, 15i or 15k, ; ii) a nucleic acid molecule comprising a nucleic acid sequence that hybridises under stringent hybridisation conditions to a nucleic acid molecule in (i) and which encodes an acyl CoA oxidase polypeptide; iii) a nucleic acid molecule that encodes a variant polypeptide that varies from a polypeptide comprising the amino acid sequence as represented in Figure

15b, 15d, 15f, 15h, 15j or15l.

49. A plant according to any of claims 45-48 wherein said gene is encoded by a nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of: i) a nucleic acid molecule comprising a nucleic acid sequence as represented in Figure 16a; 16c,16e or 16g ii) a nucleic acid molecule comprising a nucleic acid sequence that hybridises under stringent hybridisation conditions to a nucleic acid molecule in (i) and which encodes a Keto-Acyl-CoA thiolase; iii) a nucleic acid molecule that encodes a variant polypeptide that varies from a polypeptide comprising the amino acid sequence as represented in Figure 16b, 16d, 16f or 16h.

50. A plant according to any of claims 45-49 wherein said gene is encoded by a nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of: i) a nucleic acid molecule comprising a nucleic acid sequence as represented in Figure 17a or 17c ii) a nucleic acid molecule comprising a nucleic acid sequence that hybridises under stringent hybridisation conditions to a nucleic acid molecule in (i) and which encodes a multifunctional protein involved in peroxisomal β oxidation; iii) a nucleic acid molecule that encodes a variant polypeptide that varies from a polypeptide comprising the amino acid sequence as represented in

Figure 17b or 17d.

51. A plant according to any of claims 45-50 wherein said nucleic acid molecule is between 20-30 base pairs in length.

52. A plant according to claim 51 wherein said nucleic acid molecule is of 21-24 base pairs in length..

53. A plant according to claim 52 wherein said nucleic acid molecule is about 21 base pairs in length.

54. A method to inhibit the expression of a plant gene comprising the steps of: i) contacting an aquatic plant with a viral vector that includes a nucleic acid molecule wherein said nucleic acid molecule is at least part of a gene that encodes a protein that controls the synthesis, degradation or transport of fatty acids and/or fatty acyl CoAs; and ii) cultivating the virally infected aquatic plant to allow viral induced gene silencing.

55. A method according to claim 54 wherein said infected plant material is harvested.

56. A method according to claim 54 or 55 wherein said harvested plant material is extracted to provide a mono- di- or triacylglycerol or free fatty acid fraction.

57. A plant cell according to any of claims 1-14 wherein said cell is transfected with a nucleic acid molecule selected from: i) a nucleic acid molecule comprising a nucleic acid sequence as represented in Figure 22b or 22c; ii) a nucleic acid molecule comprising a nucleic acid sequence that hybridises under stringent hybridisation conditions to a nucleic acid molecule in (i) and which encodes a oleate 12 -hydroxylase.

58. A plant cell according to any of claims 1-14 wherein said cell is transfected with a nucleic acid molecule selected from: i) a nucleic acid molecule comprising a nucleic acid sequence as represented in Figure 23a; ii) a nucleic acid molecule comprising a nucleic acid sequence that hybridises under stringent hybridisation conditions to a nucleic acid molecule in (i) and which encodes a cytochrome P450.

59. A plant cell according to any of claims 1-14 wherein said cell is transfected with a nucleic acid molecule selected from: i) a nucleic acid molecule comprising a nucleic acid sequence as represented in Figure 24b; ii) a nucleic acid molecule comprising a nucleic acid sequence that hybridises under stringent hybridisation conditions to a nucleic acid molecule in (i) and which encodes a delta 12 fatty acid acetylenase.

60. A plant cell according to any of claims 1-14 wherein said cell is transfected with a nucleic acid molecule selected from: i) a nucleic acid molecule comprising a nucleic acid sequence as represented in Figure 25b; ii) a nucleic acid molecule comprising a nucleic acid sequence that hybridises under stringent hybridisation conditions to a nucleic acid molecule in (i) and which encodes a delta 12 fatty acid desaturase.

61. A plant cell according to any of claims 1-14 wherein said cell is transfected with a nucleic acid molecule selected from: i) a nucleic acid molecule comprising a nucleic acid sequence as represented in Figure 26b; ii) a nucleic acid molecule comprising a nucleic acid sequence that hybridises under stringent hybridisation conditions to a nucleic acid molecule in (i) and which encodes a delta 12 fatty acid acetylenase.

62. A plant cell according to any of claims 1-14 wherein said cell is transfected with a nucleic acid molecule selected from: i) a nucleic acid molecule comprising a nucleic acid sequence as represented in Figure 27b; ii) a nucleic acid molecule comprising a nucleic acid sequence that hybridises under stringent hybridisation conditions to a nucleic acid molecule in (i) and which encodes a delta 6 fatty acid acetylenase.

63. A plant cell according to any of claims 1-14 wherein said cell is transfected with a nucleic acid molecule selected from: i) a nucleic acid molecule comprising a nucleic acid sequence as represented in Figure 28b; ii) a nucleic acid molecule comprising a nucleic acid sequence that hybridises under stringent hybridisation conditions to a nucleic acid molecule in (i) and which encodes a delta 6 fatty acid desaturase.

64. A plant cell according to any of claims 1-14 wherein said cell is transfected with a nucleic acid molecule selected from: i) a nucleic acid molecule comprising a nucleic acid sequence as represented in Figure 29b; ii) a nucleic acid molecule comprising a nucleic acid sequence that hybridises under stringent hybridisation conditions to a nucleic acid molecule in (i) and which encodes a transcription factor.

65. A plant cell according to any of claims 1-14 wherein said cell is transfected with a nucleic acid molecule selected from: i) a nucleic acid molecule comprising a nucleic acid sequence as represented in Figure 30b; ii) a nucleic acid molecule comprising a nucleic acid sequence that hybridises under stringent hybridisation conditions to a nucleic acid molecule in (i) and which encodes a transcription factor.

66. A plant cell according to any of claims 1-14 wherein said cell is transfected with a nucleic acid molecule selected from: i) a nucleic acid molecule comprising a nucleic acid sequence as represented in Figure 31b; ii) a nucleic acid molecule comprising a nucleic acid sequence that hybridises under stringent hybridisation conditions to a nucleic acid molecule in (i) and which encodes a transcription factor.

67. A plant cell according to any of claims 1-14 wherein said cell is transfected with a nucleic acid molecule selected from: i) a nucleic acid molecule comprising a nucleic acid sequence as represented in Figure 32b; ii) a nucleic acid molecule comprising a nucleic acid sequence that hybridises under stringent hybridisation conditions to a nucleic acid molecule in (i) and which encodes a transcription factor.

68. A plant cell according to any of claims 1-14 wherein said cell is transfected with a nucleic acid molecule selected from: i) a nucleic acid molecule comprising a nucleic acid sequence as represented in Figure 33b; ii) a nucleic acid molecule comprising a nucleic acid sequence that hybridises under stringent hybridisation conditions to a nucleic acid molecule in (i) and which encodes a transcription factor.

69. A transgenic plant comprising a cell according to any of claims 57-68.

70. A seed comprising a plant cell according to any of claims 57-68.