Plant tissue with an altered content of a flavonoid component

Field of the invention

The invention relates to a method for producing a plant tissue with an altered content of a flavonoid component and to a plant tissue obtained by such method.

Background of the invention Flavonoids are a large group of phenolic secondary metabolites that are widely present in plants and involved in several plant functions, including protection against UV irradiation, defense against pathogen attack, attractants to pollinating insects and as signal compounds for the initiation of symbiotic relationships (Parr AJ et al, (2000), J. of the Science of Food and Agriculture, 80: 985-1012).

As a dietary component, the flavonoids are thought to have health promoting properties. Their antioxidant activity is well established (Duthie G et al (2000), Current Opinion in

Lipidiology, 11:43-47) and a number of epidemiological studies have suggested associations between flavonoid intake and a lower risk of cardiovascular disease ( Hertog MGL, et al, (1997), The Lancet, 349: 699-) but also against cancer (Knekt P et al, (1997), American Journal of Epidemiology, 146: 223-230) and other age related diseases such as dementia (Commenges D. et al, European Journal of Epidemiology, 16: 357-363).

Consumers and food manufacturers have become interested in flavonoids for their medicinal properties, especially their potential role in the prevention of cancers and cardiova3cular disease. The beneficial effects of fruit, vegetables, and tea

or even red wine have been attributed to flavonoid compounds rather than to known nutrients and vitamins.

Several fruits such as citrus fruits, berries, onions, parsley, legumes are known to comprise flavonoids. Several genes involved in the production of flavonoids in tomato have already been identified (Willits M. G. et al, (2005), J. Agri . Food Chem., 53: 1231-1236) : chalcone synthase (CS), chalcone isomerase (CI), flavanone 3-hydroxylase (F3H) , flavanone 3', 5'- hydrolase, flavonol synthase.

Several methods have already been described for producing fruits having increased flavonoid contents. For example, WO 03/105568 describes the production of non-transgenic domesticated tomato that express flavonols in the flesh and peel of the tomato fruit using traditional breeding techniques. WO 99/37794 describes a method for manipulating the flavonoid production in plant by manipulating gene activity in the flavonoid biosynthetic pathway by expressing two or more genes encoding transcription factors for flavonoid biosynthesis.

The mechanism involved in the production of flavonoids are still relatively poorly understood.

Due to the interesting properties of flavonoids in human health, there is an increasing demand for food products or food ingredients comprising high amounts of flavonoids. However, the level of flavonoids in wild type fruit is rather low and the molecular mechanisms underlying flavonoid synthesis are still poorly understood. The present invention discloses an alternative method for producing a plant and/or a plant tissue such as a fruit having an altered, preferably increased content of a flavonoid.

Brief description of the drawings

Figure 1. T-DNA used to generate transgenic Arabidopsis plants expressing transcripts of TDR4 under the control of the CaMV 35S promoter. The Basta resistant construct was used to transform Arabidopsis and the kanamycin resistant construct was used to transform tomato.

Figure 2. (not shown) Figure 2 is a photographic image of the above soil portions of Arabidopsis plants. The plants were grown for 6 weeks under 16 hour days and constant temperature in M2 compost. Four groups of homozygous plants are grouped together in this image. From the left the CoI-O untransformed control is observed as a tall bunch of plants with a majority of green siliques. Three homozygous lines of the type CoI-O transformed with the TDR4 under the control of the CaMV 35S promoter, designated lines A, K and P, are also shown. In contrast to the control line, these lines all show a stature reduced to approximately one third of the wild type and distinctly red siliques and floral stems.

Figure 3. (not shown) Formalin-fixed, wax embedded, phloroglucinol-stained 8μm sections of Arabidopsis stage 17 siliques. Phloroglucinol staining of cross sections of Arabidopsis siliques showed that TDR4 under the control of the CaMV 35S promoter phenocopied that of FUL under the control of the same promoter with respect to both patterns of phloroglucionl stained lignification and the effect on separation layers.

Figure 4. (A) . This is a photographic image of the top of an Arabidopsis infloresence showing two sides of the infloresence . The left hand side image shows the side that was shaded from the light and the right hand side image shows the side that was

exposed to the light. The image shows that light induces the accumulation of anthocyanin in the carpels, replum and stems of these tissues (not shown) .

(B) . Figure 4B is a graph depicting the measurements of extracted anthocyanins in arabidopis siliques harvested from transgenic lines expressing TDR4 under the control of the CaMV 35S promoter and compared to the CoI-O siliques. Absorbance units at 530 nm per gram fresh weight are presented. The units were measured in acidic methanol extracts made from ground tissue. Five replicates were made from each test. Lines A, B, G and K all showed significant increases in anthocyanin accumulation compared to the control like. The significance was calculated using the students' T-Test at the p<0.001 level.

Figure 5. (not shown) Figure 5 is a composite image with three sections, A, B and C. Section A indicates the relative lengths of three stage 17 Arabidopsis siliques, showing from left to right the longest silique of the Ler wild type control, then the Ler ful-2 mutant line transformed with the TDR4 gene expressed under the control of the CaMV 35S promoter, where the silique is slightly longer than half the length of the wild type control silique. Finally Section A includes the Ler ful-2 mutant silique which is approximately one quarter of the length of the wild type silique. This indicates that TDR4 is capable of restoring some of the silique length lost by reduced expression of the Arabidopsis Fruitful gene.

Section B is an image a terminal flower of a Ler Arabidopsis inflorescence transformed with TDR4 under the control of the CaMV 35S promoter. The flowers all terminate together unlike wild type flowers, not shown. Section C is an image of 10 Ler Arabidopsis siliques from plans transformed with TDR4 under the control of the CaMV 35S promoter. These siliques show red colouring indicative of increased anthocyanin accumulation.

Detailed description

In a first aspect, the invention provides a plant tissue, wherein the expression of a nucleic acid sequence coding for a TDR4 homologue is altered compared to the wild type plant tissue it derives from and wherein the plant tissue has an altered content of a flavonoid component compared to the plant tissue it derives from.

As used herein, the term "plant tissue" refers to either a whole plant, including in general the class of higher plants amenable to transformation techniques, including both monocotyledonous and dicotyledonous plants or a part of a plant such as e.g. roots, stems, stalks, leaves, petals, fruits, seeds, tubers, pollen, meristems, callus, sepals, bulbs and flowers. The term plant as used herein further refers, without limitations, to plant cells in seeds, suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytic and sporophytic tissue, pollen, protoplasts and microspores. Furthermore, all plant tissues in all organs are included in the definition of the term plant as used herein. Plant tissues include, but are not limited to, differentiated and undifferentiated tissues of a plant, including pollen, pollen tubes, pollen grains, roots, shoots, shoot meristems, coleoptilar nodes, tassels, leaves, cotyledonous petals, ovules, tubers, seeds, kernels. Tissues of plants may be in planta, or in organ, tissue or cell culture. As used herein, monocotyledonous plant refers to a plant whose seeds have only one cotyledon, or organ of the embryo that stores and absorbs food. As used herein, dicotyledonous plant refers to a plant whose seeds have two cotyledons . Plants included in the invention are all plants amenable to transformation or by harnessing natural variation.

Preferably, the plant tissue is suitable for human and/or animal consumption. More preferably, the plant tissue is edible by human beings. Examples of such food plant include: vegetables such as spinach, a pea plant, broccoli, cauliflower, asparagus, carrots, onion and potato plant, fruit-bearing plants such as a plant bearing tomato, strawberry, apple, bilberry, melon, orange, lemon, or banana, oil producing plants such as sunflower, soybean and rape, or extractable plants such as tea plants. A list is given below to exemplify fruits and corresponding plants that could be encompassed by the present invention. Tomato (a Lycopersicon spp such as Solanum lycopersicum) , Apple (Malus spp.), Strawberry (Fragaria spp.), Bilberry (Vaccinium spp.), Melon (Cucumis melo) , Orange, (Citrus sinensis) I (Citrus aurantium) , Lemon (Citrus limon) , Banana (Musacea spp.), Acai (Euterpe oleracea) , Acerola (Malpighia glabra), African cherry orange (Citropsis schweinfurthii) , Akee (Blighia sapida or Cupania sapida) , Amazon Grape (Pourouma cecropiaefolia) , American grape (e.g., Vitis labrusca; Vitaceae and Vitis vinifera) , American Mayapple (Podophyllum peltatum) , American persimmon (Diospyros virginiana)

Angelica (Angelica spp.), Apricot (Prunus armeniaca or Armeniaca vulgaris) aprium, Araza (Eugenia Stipitata) , Arhat (Siraitia grosvenorii) , Avocado (Persea americana) , Babaco (Carica pentagona) , Bael (Aegle marmelos) , Bael (Aegle marmelos) , Barbados, Cherry (Malpighia glabra L.), Barberry (Berberis) , Bearberry (Arctostaphylos spp.), Bilimbi (Averrhoa bilimbi) , Black mulberry (Morus nigra), Blackberry, Blueberry (Vaccinium spp.), boysenberry, Breadfruit (Artocarpus altilis) ,

Buffaloberry (Shepherdia argenta) , Burmese grape (Baccaurea sapida), Butternut squash (Cucurbita moschata) , Calabash (Lagenaria siceraria) , CamuCamu (Myrciaria dubia) , Canistel

(Pouteria campechiana) , Carambola (Averrhoa carambola) , Cempedak (Artocarpus champeden) , Che (Cudrania tricuspidata) , Cherimoya (Annona cherimola) , Cherry, (Prunus avium, P. cerasus, and others) , Chocolate vine (Akebia quinata) , Chokeberry (Aronia) , Chokecherry (Prunus virginiana) , Citron (Citrus medica) , Clementine (Citrus reticulata var. Clementine), Cloudberry (Rubus chamaemorus) , Coconut (Cocos spp . ; Arecaceae) , Cocoplum (Chrysobalanus icaco; Chrysobalanaceae) , Cornelian cherry (Cornus mas; Cornaceae) , Cranberry (Vaccinium spp.) _/ Crowberry (Empetrum spp.), Currant (Ribes spp.), Cushaw squash (Cucurbita mixta), Custard apple (Annona reticulata) , Damson Plum (Chrysophyllum oliviforme) , Date palm (Phoenix dactylifera) , Date-plum (Diospyros lotus) dewberry Dragonfruit (Hylocereus spp.), Dragonfruit (Hylocereus undatus) , Durian (Durio spp.), Eggfruit (Pouteria campechiana) , Elderberry (Sambucus) , Elephant apple (Dillenia indica) , False- mastic (Mastichodendron foetidissimum) , Feij oa (Feijoa sellowiana) , Fig (Ficus spp.), Galendar (Darjeeling) , Gooseberry (Ribes spp.), Goumi (Elaeagnus multiflora ovata) , Grape (Vitis spp.), Grapefruit, Greengage, Ground Plum

(Astragalus caryocarpus) , Guarana (Paullinia cupana) , Guava (Psidium guajava) , Guavaberry (Myrciaria floribunda) , Hackberry (Celtis spp.), Hardy Kiwi (Actinidia arguta) , Hawthorn (Crataegus and Rhaphiolepis) , Honeysuckle (Lonicera spp.), Horned melon (Cucumis metuliferus) , Horned melon (Cucumis metuliferus) , Hubbard squash (Cucurbita maxima), Huckleberry (Vaccinium spp.), Ice Plant (Carpobrotus edulis) , Indian Prune (Flacourtia rukan) , Ivy (Hedera spp.), Jaboticaba (Myrciaria cauliflora) , Jackfruit (Artocarpus heterophyllus) , Jambul (Syzygium cumini) , Japanese wineberry (Rubus phoenicolasius) , Jatoba (Hymenae coubaril) , Jenipapo (Genipa americana) , Jujube (Ziziphus zizyphus) , Kaffir lime (Citrus hystix) , Kahikatea (Dacrycarpus dacrydioides) , Kandis (Garcinia forbesii) , Keppel

fruit (Stelechocarpus burakol) , Key Lime (Citrus aurantifolia) , Kiwifruit (Actinidia spp . ) , Kumquat (Fortunella spp . ) , Kundong (Garcinia sp.), Langsat (Lansium domesticum) , Lapsi (Choerospondias axillaris Roxb.), Limes, Linden (Tilia spp.) _/ Lingonberry (Vaccinium vitis-idaea) , Loganberry (Rubus loganobaccus) , Loganberry (Rubus) , Longan (Euphoria longan) , Loquat (Eryobotrya japonica) , Lύcuma (Pouteria lucuma) , Lychee (Litchi chinensis) , Mabolo, (Diospyros discolor) , Mamey sapote (Pouteria sapota) , Mamoncillo (Melicoccus bijugatus) , Mandarin (Citrus reticulata), Mango (Mangifera indica) , Mangosteen (Garcinia mangostana) , Manoao (Manoao colensoi) , Marang (Artocarpus odoratissima) , Mayapple (Podophyllum spp.), Medlar (Mespilus germanica) , Monstera (Monstera deliciosa) , Mulberry (Morus spp.), Murta (Ugni molinae Turcz . ) , Nageia (Nageia spp.), Nannyberry or sheepberry (Viburnum spp.), Naranjilla, LuIo (Solanum quitoense) , Nectarine (Prunus persica) , Olallieberry, Olive (Olea europea) , Orange (Citrus sinensis) / (Citrus aurantium) , Orangelo, Oregon grape (Mahonia aquifolium) , Osage-orange (Madura pomifera) , Papaya (Carica papaya), Passion fruit (Passiflora spp.), Pawpaw (Asimina triloba) , Peach, Peacotum, Peanut butter fruit (Bunchosia argentea) , Pear (Pyrus) , Pequi (Caryocar brasiliense) , Persian lime, Persimmon (Diospyros kaki) , Pigeon plum (Coccoloba diversifolia) , Pineapple (Ananas comosus or Ananas sativas) , Pitomba (Eugenia luschnathiana or Talisia esculenta) , Plantain, Plum, Pluot, Podocarpus (Podocarpus spp.), Poha or Cape Gooseberry (Physalis peruviana) , Poisonleaf (Dichapetalum cymosum) , Pomegranate (Punica granatum) , Pomelo (Citrus paradisi) , Pond-apple (Annona glabra) , Prickly pear (Opuntia spp.), Privet (Ligustrum spp.), Prumnopitys (Prumnopitys spp.), Prunes, Pumpkins (Cucurbita pepo) , Pupunha (Bactris gasipaes) , Quince (Cydonia oblonga and Chaenomeles) , Raisin tree (Hovenia dulcis) , Rambutan (Nephelium lappaceum) , Rangpur, Raspberry

(genus Rubus) , Red Mombin (Spondias purpurea) , Rhubarb (Rheum spp . ) , Riberry (Syzygium luehmannii) , Rimu (Dacrydium cupressinum) , Rose apple (Syzygium jambos) , Rose-hip, (Rosa) , Rowan (Sorbus) , Russian olive (Elaeagnus angustifolia) , Sageretia (Sageretia theezans) , Saguaro (Carnegiea gigantea) , Salak (Salacca edulis) , Salal berry (Gaultheria shallon) , Salmonberry (Rubus spectabilis) , Santol (Sandoricum koetjape) , Sapodilla (Achras/Manilkara zapota) , Saw Palmetto (Serenoa repens) , Sea Grape (Coccoloba uvifera) , Sea-buckthorn (Hippophae rhamnoides) , Service tree (Sorbus domestica) ,

Serviceberry (Amelanchier) , Shipova (* Sorbopyrus auricularis) , Silverbells (Halesia spp.), Snowberry (Symphoricarpos spp.), Soapberry (Sapindus spp.), Soursop (Annona muricata) , Star apple (Chrysophyllum cainito) , Strawberry (Fragaria) , Strawberry guava (Psidium litorale) , Strawberry Tree (Arbutus unedo) , Sugar apple (Annona squamosa) , Surinam Cherry (Eugenia uniflora) , Tamarillo (Cyphomandra betacea) , Tamarind (Tamarindus indica) , Tangelo, Tangerine, Taxus baccata (Yew), Texas persimmon (Diospyros texana) , Thimbleberry (Rubus parviflorus) , Toyon (Heteromeles arbutifolia) , UgIi fruit, Wahoo (Euonymus atropurpureus) , Watermelon (Citrullus vulgaris) , Wax apple (Syzygium samarangense) , Wineberry (Rubus phoenicolasius) , Wolfberry (Lycium spp.), Yangmei (Myrica rubra) .

The skilled person will understand that the invention encompasses both the plant tissue of the invention and any food product or ingredient comprising or being derived from or being based on the plant tissue of the invention. Preferred plant tissues include fruit, root, flower and leaf. A more preferred plant tissue is a fruit. Therefore, preferred plants used in the invention are plants known to be able to have fruits or known to be considered as vegetable suitable for human and/or

animal consumption as exemplified in the former list. In a preferred embodiment, a plant tissue of the invention possess and/or express a TDR4 homologue as defined herein. All angiosperms are known to fulfil this condition (Theissen G. et al et al (2000), Plant Molecular Biology, 42:115-149).

Preferred plants are those that are edible and/or are known to have a functional anthocyanin and/or flavonoid pathway. More preferred fruits are selected from the following list of fruits: tomato, apple, strawberry, bilberry, melon, orange, lemon, or banana. An even more preferred fruit is tomato.

Tomato plants belong to Lycopersicon spp. Preferred varieties from the cultivated tomato include Lycopersicon esculentum L./ Solanum lycopersicum L. Preferred vegetables include: spinach, pea, broccoli, cauliflower, asparagus, carrots, onion and potato.

In a preferred embodiment, the plant is not Arabidopsis .

A TDR4 homologue is herein preferably defined as being a polypeptide having an amino acid sequence with at least 50% identity with the amino acid sequence of SEQ ID NO:1. The activity of the TDR4 homologue is preferably assessed by over expressing the respective homologue in a plant model such as Arabidopsis and analyzing the flavonoid content of the produced plant. Any plant tissue may be analysed, preferably the Arabidopsis' ^' s siliques are analyzed, which are considered as a fruit's ancestor. When the Arabidopis's tissue produced by the transformed Arabidopsis comprises an altered, preferably increased content of a flavonoid compound as later defined herein, the TDR4 homologue, would be said to be active and functional. The skilled person knows how to assess the content of a flavonoid compound in a plant tissue. Preferably, the content of a flavonoid component is assessed by HPLC (see for more details example VII of WO 03/105568 or example 6 of WO

99/37794). Briefly, the potential flavonoid components are extracted from the plant tissue to be tested. Subsequently, an enzyme hydrolysis is optionally carried out. HPLC is performed on extracted flavonoid compounds and optionally hydrolysed compounds by methods known to the skilled person.

According to an even more preferred embodiment, the TDR4 homologue has at least 50%, 55%, 60%, 65%, 70%, 75%, 80% or 85%, even more preferably at least 90%, 92%, 95%, 97% , 98% or 99% identity with the amino acid sequence of SEQ ID NO:1. More preferably, the TDR4 homologue originates from an edible plant which is known to have a functional flavonoid and/or anthocyanin pathway. In a most preferred embodiment, the TDR4 homologue has the SEQ ID NO:1. This TDR4 polypeptide originates from the tomato fruit from Solanum lycopersicum obtained from the Tomato Genetics Resource Centre, at the University of California, Davis, accession number LA2838A or LA154. It was originally identified by Pnueli L et al . , (1991), The Plant Journal, 1:255-266. The nucleic acid sequence coding for the amino acid sequence of SEQ ID NO:1 is given in SEQ ID NO: 2.

Other known TDR4 homologues include SLMBP20 and LeMADS-MC whose amino acid sequences are given as SEQ ID NO: 3 and 5 respectively and whose nucleic acid sequences are given as SEQ ID NO: 4 and 6 respectively.

Percentage of identity is calculated as the number of identical amino acid residues between aligned sequences divided by the length of the aligned sequences minus the length of all the gaps. Multiple sequence alignment was performed using DNAman 4.0 optimal alignment program using default settings.

The skilled person will understand that the TDR4 homologues as defined in the present invention could be obtained from other

plants than Solanum lycopersicum, which has been used to clone the TDR4 of the invention as long as they have the required activity and identity. The TDR4 homologues as defined in the present invention could be obtained from any angiosperm. Preferred plants were already earlier defined herein. In a preferred embodiment, each homologue as identified above is obtained from other tomato species. In another preferred embodiment, TDR4 homologues are obtained from any edible plant. Within one single plant species (or variety) or several distinct plant species (or variety), several TDR4 homologues may be isolated fulfilling the above-given criteria of activity and identity.

According to another preferred embodiment, the TDR4 homologue as defined in the invention, is a variant polypeptide of a TDR4 homologue sequence as defined before. A variant polypeptide may be a non-naturally occurring form of the polypeptide. A polypeptide variant may differ in some engineered way from the polypeptide isolated from its native source. A variant may be made by site-directed mutagenesis starting from the amino acid sequence of SEQ ID NO:1, 3 or 5 or from the nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:1, 3 or 5 which is SEQ ID NO:2, 4 or 6 respectively. Preferably, the polypeptide variant contains mutations that do not alter the biological function of the encoded polypeptide. Biological function or activity of a TDR4 homologue has already been defined herein. In a preferred embodiment, the polypeptide variant has an increased activity compared to the polypeptide having SEQ ID NO:1 measured in the assay as earlier defined herein. Polypeptides with enhanced activities are very useful since when expressed in a plant tissue, this plant tissue is expected to possess an even more dramatically altered content of a flavonoid compound than a plant tissue expressing the polypeptide having SEQ ID NO:1. In

another preferred embodiment, the polypeptide variant has a decreased activity compared to the polypeptide having SEQ ID N0:l measured in the assay as earlier defined herein. Polypeptides with a decreased activity are also very useful since when expressed in a plant tissue, this plant tissue is expected to possess another altered content of a flavonoid compound than a plant tissue expressing the polypeptide having SEQ ID N0:l.

As previously indicated a TDR4 homologue is encoded by a nucleic acid sequence.

A nucleic acid sequence codes for a TDR4 homologue having an amino acid sequence with at least 50% identity with the amino acid sequence of SEQ ID NO:1. Preferably, the nucleic acid sequence codes for a TDR4 homologue having the amino acid sequence SEQ ID NO:1, and/or originates from a plant as earlier defined herein. More preferably, the plant is edible and is known to have a functional flavonoid and/or anthocyanin pathway. Even more preferably, the plant is a Lycopersicon, most preferably Solarium lycopersicum.

This nucleic acid sequence is preferably a nucleic acid sequence having at least 50% identity with the nucleic acid sequence of SEQ ID NO: 2. Preferably, the identity is of at least 55%, more preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, even more preferably at least 95%, even more preferably at least 98% and even more preferably at least 99%. Most preferably, the nucleic acid sequence has the nucleic acid sequence of SEQ ID NO: 2. SEQ ID NO : 2 has been attributed the following Genebank accession numbers: X60757, AY098733.

Percentage of identity was determined by calculating the ratio of the number of identical nucleotides in the sequence divided by the length of the total nucleotides minus the lengths of any gaps. DNA multiple sequence alignment was performed using DNAman version 4.0 using the Optimal Alignment (Full

Alignment) program. The minimal length of a relevant DNA sequence showing 50% or higher identity level should be 19 nucleotides or longer.

According to another preferred embodiment, a nucleic acid sequence as defined herein is a variant of any of the nucleic acid sequences as defined above. Nucleic acid sequence variants may be used for preparing polypeptide variants as defined earlier. A nucleic acid variant may be a fragment of any of the nucleic acid sequences as defined above. A nucleic acid variant may also be a nucleic acid sequence that differs from SEQ ID NO : 2 , 4 or 6 by virtue of the degeneracy of the genetic code. A nucleic acid variant may also be an allelic variant of SEQ ID NO:2, 4 or 6. An allelic variant denotes any of two or more alternative forms of a gene occupying the same chromosome locus. A preferred nucleic acid variant is a nucleic acid sequence, which contains silent mutation (s). Alternatively or in combination, a nucleic acid variant may also be obtained by introduction of nucleotide substitutions, which do not give rise to another amino acid sequence of the polypeptide encoded by the nucleic acid sequence, but which corresponds to the codon usage of the plant host intended for expression of the homologue as defined herein. According to a preferred embodiment, the nucleic acid variant encodes a polypeptide still exhibiting its biological function as earlier defined herein. More preferably, the nucleic acid sequence variant encodes a TDR4 homologue whose respective activity has already been defined herein. Nucleic acid

sequences encoding such a polypeptide may be isolated from any micro-organism. Preferably, such nucleic acid sequences are isolated from a plant as earlier defined herein. All these variants can be obtained using techniques known to the skilled person, such as screening of library by hybridisation (southern blotting procedures) under low to medium to high hybridisation conditions with for the nucleic acid sequence SEQ ID NO: 2, 4 or 6 or a variant thereof which can be used to design a probe. Low to medium to high stringency conditions means prehybridization and hybridization at 42 ⁰ C in 5X SSPE, 0.3% SDS, 200pg/ml sheared and denatured salmon sperm DNA, and either 25% 35% or 50% formamide for low to medium to high stringencies respectively. Subsequently, the hybridization reaction is washed three times for 30 minutes each using 2XSSC, 0.2%SDS and either 55 ⁰ C, 65 ⁰ C, or 75 ⁰ C for low to medium to high stringencies.

The sequence information as provided herein should not be so narrowly construed as to require inclusion of erroneously identified bases. The skilled person is capable of identifying such erroneously identified bases and knows how to correct for such errors .

In order to obtain a preferred plant tissue of the invention, a nucleic acid construct may be prepared comprising a nucleic acid sequence encoding a TDR4 homologue as earlier defined herein .

In a preferred embodiment, the alteration in the expression of the nucleic acid sequence coding for a TDR4 homologue is an increase. In this preferred embodiment, the nucleic acid sequence present in the nucleic acid construct is operably linked to one or more control sequences, which direct the expression of the encoded TDR4 homologue in a suitable plant tissue .

Operably linked is defined herein as a configuration in which a control sequence is appropriately placed at a position relative to the nucleic acid sequence coding for a TDR4 homologue such that the control sequence directs its expression in the chosen plant tissue.

Expression will be understood to include any step involved in the production of a polypeptide including, but not limited to transcription, post-transcriptional modification, translation, post-translational modification and secretion. Nucleic acid construct is defined as a nucleic acid molecule, which is isolated from a naturally occurring gene or which has been modified to contain segments of nucleic acid which are combined or juxtaposed in a manner which would not otherwise exist in nature. Control sequence is defined herein to include all components, which are necessary or advantageous for the expression of a polypeptide. At a minimum, the control sequences include a promoter and transcriptional and translational stop signals. In a more preferred embodiment, in order to obtain a plant tissue of the invention, an expression vector is prepared comprising a nucleic acid construct comprising a nucleic acid sequence encoding a TDR4 homologue as earlier defined herein. Preferably, the expression vector comprises said nucleic acid sequence, which is operably linked to one or more control sequences, which direct the production of the encoded TDR4 homologue in a suitable plant tissue. At a minimum control sequences include a promoter and transcriptional and translational stop signals. The expression vector may be seen as a recombinant expression vector. The expression vector may be any vector (e.g. plasmic, virus), which can be conveniently subjected to recombinant DNA procedures and can bring about the expression of the nucleic acid sequence encoding the TDR4 homologue. Depending on the identity of the plant tissue

wherein this expression vector will be introduced and on the origin of the nucleic acid sequence, the skilled person will know how to choose the most suited expression vector and control sequences. Most preferred plant tissues have already been presented herein.

In a preferred embodiment of the invention the promoter present in the expression vector is a plant promotor, i.e. a promoter capable of initiating transcription in plant cells. Plant promotors as used herein include tissue-specific, tissue-preferred, cell-type-specific, inducible and constitutive promotors. Tissue-specific promotors are promoters which initiate transcription only in certain tissues and refer to a sequence of DNA that provides recognition signals for RNA polymerase and/or other factors required for transcription to begin, and/or for controlling expression of the coding sequence precisely within certain tissues or within certain cells of that tissue. Expression in a tissue specific manner may be only in individual tissues or in combinations of tissues. More preferred promoters are fruit specific promoters. More preferred fruit specific promoters include: the 2A11 promoter (Pear JR, et al, Plant MoI. Biol., (1989), 13: 639-651), the E8 promoter (Holdsworth MJ et al, (1988), Plant MoI Biol., 11:81-88), and the PG promoter (Nicholass F.J., et al (1995), Plant Molecular biology, 28: 423-435). In another preferred embodiment, the expression of a TDR4 homologue is rendered inducible by fusing its encoding nucleic acid to an inducible promoter suitable for inducible level protein expression in the selected organism. A preferred inducible promoter is the jasmonate promoter (WO 01/41568) .

In another preferred embodiment, the alteration in the expression of the nucleic acid sequence coding for a TDR4 homologue is a decrease. In this preferred embodiment, the

nucleic acid construct is preferably named an inactivation construct. The skilled person knows how to prepare such a construct. It is also encompassed by the present invention that the decrease of the expression of a TDR4 homologue is achieved by using a variant of a TDR4 homologue having a decreased TDR4 activity, and/or by operably linking a nucleic acid sequence coding for a TDR4 homologue to a weak promoter. A promoter is preferably said to be weak in this context when its activity is weaker than the activity of the endogenous TDR4 promoter. The skilled person knows how to assess the activity of a promoter. An example of a preferred plant weak promoter has already been described in Comai L et al (Comai L et al, (1990), Plant MoI. Biol., 15: 372-381).

Alternatively or in combination with former preferred embodiments relating to an increase or a decrease of the expression of a TDR4 homologue in a plant tissue, transposon mutagenesis (McClintock B et al, (1950), Proc. Natl. Acad, of Sci., 36: 344-355, and Tissier AF, et al, (1999), Plant cell, 11: 1841-1852) and/or site directed mutagenesis (Kempin et al (1997), Nature, 389: 802-803) are preferably carried out in a plant cell or tissue. Transposon mutagenesis and/or site directed mutagenesis are attractive ways of obtaining the plant tissue of the invention since using a TILLING approach (Mc Callum CM. et al, (2000) Nature Biotechnol., 18:455-457), it is possible to mutate the TDR4's promoter to obtain a stronger or a weaker TDR4 promoter and/or to knock out a gene inactivating or up-regulating TDR4. In this context, a promoter is preferably said to be weaker or stronger by comparison with the promoter activity of the endogenous TDR4 promoter. Similarly, by methylation- and acetylation-induced mutagenesis using chemicals such as 5-azacytidine and trichostatin A respectively, it is possible to mutate TDR4 and

its regulators to obtain variations in TDR4 function and expression (Kanazawa A et al, (2007), Plant Cell Physiol., 2007 Feb, E pub ahead of print) . The developmentally regulated tomato gene LeSPL-Cnr, identified at the Cnr locus, is proposed as the genetic factor affected by methylation differences discovered in its promoter revealed through a map based cloning approach (Manning K et al, (2006) . Nature Genetics, 38:948-952). LeSPL-Cnr is a SQUAMOSA promoter binding protein class of transcription factor and as such is expected to be able to bind to the promoters of the SQUAMOSA clade transcription factors. The tomato Colourless non- rlpenlng mutant accumulates fewer TDR4 transcripts in tomato fruit and indicates that TDR4 expression is enhanced by development, methylation and the genetic factor responsible for this mutation (Eriksson E. M., et al, (2004), Plant Physiol., 136: 4184-4197).

In the context of the invention, a nucleic acid construct, preferably an expression vector when introduced into a plant cell or tissue will lead to a plant cell or tissue having an altered, preferably increased expression level of the nucleic acid sequence present in the expression vector, and/or an altered, preferably increased expression level of the homologue encoded by the nucleic acid sequence present in the expression vector and/or an altered, preferably increased activity level of the homologue encoded by the nucleic acid sequence present in the expression vector. In this context, the alteration, preferably increase is assessed by comparison with a plant cell or tissue which does not comprise said expression vector and/or with a plant cell or tissue which does not comprise an endogenous homologue having at least 50% identity with SEQ ID NO:1.

Accordingly in a preferred embodiment, the plant tissue of the invention is such that the expression of a nucleic acid sequence encoding a TDR4 homologue is transiently and/or inducibly altered, preferably transiently and/or transiently increased or decreased.

Accordingly, the invention provides a plant cell or tissue comprising the nucleic acid construct, preferably the expression vector as earlier defined herein. The choice of the plant cell or tissue will to a large extent depend upon the source of the nucleic acid sequence of the invention. Depending on the identity of the plant cell or tissue, the skilled person would know how to transform it with the construct or vector of the invention. Preferred plant cells or tissues have been earlier defined herein.

Accordingly, the invention relates to a plant that is genetically modified, preferably by the method of the invention, in that the plant comprises a nucleic acid construct as herein defined above. The nucleic acid construct preferably is a construct containing nucleic acid sequences that are manipulated or modified in vitro or at least ex planta. As such, the nucleic acid construct preferably provides the plant with a combination of nucleic acid sequences which is not found in nature. The nucleic acid construct preferably is stably maintained, either as a autonomously replicating element, or, more preferably, the nucleic acid construct is integrated into the plant's genome, in which case the construct is usually integrated at random positions in the plant's genome, for instance by non-homologuous recombination. Stably transformed (transgenic) plants or plant cells are produced by known methods. The term stable transformation refers to exposing plants, tissues or cells thereof to methods to transfer and incorporate foreign DNA into the plant genome. These methods

include, but are not limited to, Agrobacterium tumefaciens- mediated gene transfer, transfer of purified DNA via microparticle bombardment, electroporation of protoplasts and microinjection or use of silicon fibers to facilitate penetration and transfer of DNA into the plant cell.

Dicotyledonous plants are most frequently transformed by Agrobacterium-mediated gene transfer such as for instance by co-culture of regenerating plant protoplasts or cell cultures with Agrobacterium tumefaciens . In general, when Agrobacterium tumefaciens is used for transformation, the transformation vectors are preferably cointigrating vectors or binary vectors. Dicotyledonous plants can furthermore be transformed by transformation of leaf discs, by protoplast transformation by polyethylene glycol-induced DNA transfer, electroporation, sonication or microinjection as well as transformation of intact cells or tissues by micro- or macroinjection into tissues or embryos, tissue electroporation, incubation of dry embryos in DNA-containing solution, vacuum infiltration of seed and biolistic gene transfer. Monocotyledonous plants are transformed via for example particle bombardment, electrically or chemically induced DNA incorporation into protoplasts, electroporation of partially permeabilized cells, macroinjection of DNA into inflorescences, microinjection of DNA into microspores and pro-embryos, the introduction of DNA into germinating pollen and DNA integration into embryos by swelling.

An alternative method to express a protein or polypeptide of interest in plants relies on transient expression from virus- based vectors. It is known that viruses replicate with high efficiency and, in some cases, can infect the entire host plant, creating the potential to express a protein or polypeptide of interest in large amounts. Vectors that can be

used in this alternative method are tobamovirus, potexvirus, Potato Virus X mediated VIGS and potyvirus. A preferred virus is the Potato Virus X mediated VIGS.

When a transformed tissue or cell (e. g., pieces of leaf, stem segments, roots, but also protoplasts or plant cells cultivated by suspension) is obtained with the method according to the invention, whole plants can be regenerated from said transformed tissue or cell in a suitable medium, which optionally may contain antibiotics or biocides known in the art for the selection of transformed cells.

Resulting transformed plants are preferably identified by means of selection. The nucleic acid construct according to the invention therefore preferably also comprises a marker gene which can provide selection or screening capability in a treated plant. Selectable markers are generally preferred for plant transformation events, but are not available for all plant species. Suitable selectable markers can be antibiotic or herbicide resistant genes which, when inserted in some cells of a plant in culture, would confer on those cells the ability to withstand exposure to an antibiotic or a herbicide. Another type of marker gene is one that can be screened by histochemical or biochemical assay, even though the gene cannot be selected for. A suitable marker gene found useful in such plant transformation experience is the GUS gene (Jefferson et al., EMBO J., 6: 3901-3907 (1987)). Another example of a marker gene is luciferase. An advantage of this marker is the nondestructive procedure of application of the substrate and the subsequent detection. The transformed plants can also be identified by expression of a nucleic acid sequence present on the expression vector as defined herein and/or by the flavonoid content of the plant as later defined herein.

According to a preferred embodiment, the plant cell or tissue hence obtained has an increased expression level of the nucleic acid sequence present in the nucleic acid construct, preferably the expression vector, and/or has an increased expression level of a TDR4 homologue encoded by the nucleic acid sequence present in the nucleic acid construct, preferably the expression vector and/or has an increased activity level of the TDR4 homologue encoded by the nucleic acid sequence present in the nucleic acid construct, preferably the expression vector. In this embodiment, the nucleic acid sequence present in the nucleic acid construct, preferably the expression construct codes for a TDR4 homologue having at least 50% identity with SEQ ID NO:1. In this context, the increase is assessed by comparison with the plant cell or tissue which does not comprise said nucleic acid construct, preferably said expression vector and/or with the host cell which does not comprise an endogenous TDR4 homologue having at least 50% identity with SEQ ID NO:1 when both cultivated and/or assayed under the same conditions.

"Increased expression level of a TDR4 homologue" is herein preferably defined as producing more of a TDR4 homologue as earlier defined than what the parental plant cell or tissue the transformed plant cell or tissue derives from will produce when both types of cells or tissues (parental and transformed plant cells or tissues) are cultured under the same conditions. Preferably, the plant cell or tissue of the invention produces at least 3%, 6%, 10% or 15% more of a TDR4 homologue having at least 50% identity with SEQ ID NO:1 than the parental plant cell or tissue the transformed plant cell or tissue derives from will produce when both types of plant cells or tissues (parental and transformed plant cells or tissues) are cultured under the same conditions. Also plant

cells or tissues which produce at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or 150% more of said TDR4 homologue than the parental plant cell or tissue are preferred. According to another preferred embodiment, the production level of a TDR4 homologue of the plant cell or tissue of the invention is compared to the production level of the TDR4 homologue of Solanum lycopersicum which is taken as control.

The assessment of the production level of a TDR4 homologue may be performed at the mRNA level by carrying out a Northern Blot, a quantitative real time PCR or a Microarray analysis and/or at the polypeptide level by carrying out a Western blot. All these methods are well known to the skilled person. A qualitative assessment of TDR4 expression may be carried out as follows: specific primers given as SEQ ID NO: 7 and SEQ ID NO: 8 were designed to amplify the TDR4 transcript. They detected the presence of the expressed TDR4 sequence following reverse transcriptase PCR in the transgenic lines and the products were analysed by size separation through an agarose gel by electrophoresis.

"Increase in a TDR4 activity" is herein defined as exhibiting a higher TDR4 activity than the one of the parental plant cell or tissue the transformed plant cell or tissue derives from using an assay specific for said activity. Preferably, the assay is a gel shift assay, wherein the binding of TDR4 to its specific binding site is assessed (Gustafson T. A., et al, (1989), Proc. Natl. Acad. Sci. USA, 86: 2162-2166). Alternatively, in another preferred embodiment, the TDR4 activity is measured by assessing the content of a flavonoid compound present in a given plant tissue. Preferably, the plant cell or tissue of the invention exhibits at least 3%, 6%, 10% or 15% higher TDR4 activity (more DNA binding and/or

increase of the content of a flavonoid compound) than the parental plant cell or tissue the transformed plant cell or tissue derives from will exhibit as assayed using a specific assay for said activity, which is preferably one of the two assays as mentioned above. Also plant cell or tissue which exhibits at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or 150% more of said activity than the parental cell or tissue are preferred. According to another preferred embodiment, the level of TDR4 activity of the plant cell or tissue of the invention is compared to the corresponding activity of a Lycopersicon species, preferably a Solanum lycopersicum as defined before, which is taken as control.

The increase in polypeptide expression and/or activity may have been achieved by conventional methods known in the art, such as by introducing more copies of the nucleic acid sequence encoding the TDR4 homologue into the plant cell or tissue, be it on a carrier or in the chromosome, than naturally present. Alternatively or in combination with former embodiment, a variant of a TDR4 homologue with increased activity may be expressed in the plant cell of the invention. Alternatively, the nucleic acid sequence encoding the TDR4 homologue may be over expressed by fusing it to highly expressed or strong promoter suitable for high level protein expression in the selected organism, or combination of the two former approaches. The skilled person will know which strong promoter is the most appropriate depending on the identity of the host cell. A preferred strong promoter is the CaMV 35S promoter (Benfey P.N. et al, (1990), Science, 250: 959-966).

According to another preferred embodiment, the plant cell or tissue hence obtained has a decreased expression level of the

nucleic acid sequence present in the nucleic acid construct, and/or has a decreased expression level of a TDR4 homologue encoded by the nucleic acid sequence present in the nucleic acid construct and/or has a decreased activity level of the TDR4 homologue encoded by the nucleic acid sequence present in the nucleic acid construct. In this embodiment, the nucleic acid sequence present in the nucleic acid construct codes for a TDR4 homologue having at least 50% identity with SEQ ID NO:1. In this context, the decrease is assessed by comparison with the plant cell or tissue which does not comprise said nucleic acid construct and/or with the host cell which does not comprise an endogenous TDR4 homologue having at least 50% identity with SEQ ID NO:1 when both cultivated and/or assayed under the same conditions. "Decreased expression level of a TDR4 homologue" is herein preferably defined as producing less of a TDR4 homologue as earlier defined than what the parental plant cell or tissue the transformed plant cell or tissue derives from will produce when both types of cells or tissues (parental and transformed plant cells or tissues) are cultured under the same conditions. Preferably, the plant cell or tissue of the invention produces at least 3%, 6%, 10% or 15% less of a TDR4 homologue having at least 50% identity with SEQ ID NO:1 than the parental plant cell or tissue the transformed plant cell or tissue derives from will produce when both types of plant cells or tissues (parental and transformed plant cells or tissues) are cultured under the same conditions. Also plant cells or tissues which produce at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or 150% less of said TDR4 homologue than the parental plant cell or tissue are preferred.

According to another preferred embodiment, the production level of a TDR4 homologue of the plant cell or tissue of the

invention is compared to the production level of the TDR4 homologue of Solanum lycopersicum which is taken as control. The assessment of the production level of a TDR4 homologue has already be defined earlier herein. "Decrease in a TDR4 activity" is herein defined as exhibiting a lower TDR4 activity than the one of the parental plant cell or tissue the transformed plant cell or tissue derives from using an assay specific for said activity. Preferably, the assay is a gel shift assay, wherein the binding of TDR4 to its specific binding site is assessed (Gustafson T. A., et al, (1989), Proc. Natl. Acad. Sci. USA, 86: 2162-2166). Alternatively, in another preferred embodiment, the TDR4 activity is measured by assessing the content of a flavonoid compound present in a given plant tissue. Preferably, the plant cell or tissue of the invention exhibits at least 3%, 6%, 10% or 15% lower TDR4 activity (less DNA binding and/or decrease of the content of a flavonoid compound) than the parental plant cell or tissue the transformed plant cell or tissue derives from will exhibit as assayed using a specific assay for said activity, which is preferably one of the two assays as mentioned above. Also plant cell or tissue which exhibits at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or 150% less of said activity than the parental cell or tissue are preferred. According to another preferred embodiment, the level of TDR4 activity of the plant cell or tissue of the invention is compared to the corresponding activity of a Lycopersicon species, preferably a Solanum lycopersicum as defined before, which is taken as control.

Surprisingly, the plant cell or tissue of the invention has attractive properties, which renders it very attractive to be used as or incorporated into food products. The plant tissue has an altered content of a flavonoid component compared to the

content of a flavonoid component of the plant tissue it derives from. "Altered content of a flavonoid component" preferably means that the total content of flavonoid components is altered, preferably increased. Alternatively, it preferably means that the content of a specific flavonoid component is altered, preferably increased whereas the content of other flavonoid components remains unaltered or has been decreased. Alternatively, it preferably means that the content of at least two, at least three, at least four or more of flavonoid compounds has been altered, preferably increased, whereas the content of other flavonoid components remains unaltered or has been decreased.

In a preferred embodiment, the altered content of a flavonoid component of such plant tissue has been altered in at least part of the plant or plant tissue. For example, for vegetables it is preferred that the content of a flavonoid compound has been increased in those parts of the vegetable that are normally eaten. For example, for plants of which the leaves are normally eaten or used in food products such as spinach or tea, it is advantageous that the content of a flavonoid compound has been increased in the leaves. For fruit-bearing plants such as tomato, strawberry, it is advantageous the content of a flavonoid compound has been increased in the fruit. For plant with edible flowers, e.g. broccoli and cauliflower, it is advantageous that the content of a flavonoid compound has been increased in the flower, for plant with edible stems, such as asparagus, it is advantageous that the content of a flavonoid compound has been increased in the stem, for plant with edible seeds, such as peas, sunflower seeds or rapeseed, it is advantageous that the content of a flavonoid compound has been increased in the seed. For oil producing plants, it is preferred that the content of a flavonoid compound has been

increased in the oil producing parts, e.g. the sunflower seed or the soy bean.

The skilled person knows which type of regulatory sequences may be used in order to target the expression of the expression construct of the invention in the desired plant tissue.

In the context of the invention, a flavonoid compound include a known plant flavonoid such as a Chalcone, a Flavone (among other Apigenin, Luteolin) , a Flavonol (among other citol, Quercetin, Rutin, Kaempferol, Myricetin) , a Flavanone (among other Naringenin) , an Anthocyanin, an Isoflavonoid, an Neoflavonoid or a Flavono Lignans and their derivatives. In one preferred embodiment, the plant tissue of the invention has a flavonoid content of at least 0.5μg/mgdwt (mg dry weight) in the fruit flesh of the plant, more preferably at least l.μg/mgdwt, even more preferably at least 1.5μg/mgdwt, even more preferably at least 2μg/mgdwt.

In another preferred embodiment, the plant tissue of the invention has a flavonoid content of at least 5μg/mgdwt in the fruit peel, more preferably at least lOμg/mgdwt, even more preferably at least 20μg/mgdwt.

Fruit peel is preferably obtained when peeling the fruit with a knife. The peel is preferably 2 mm thick. Flesh tissue or fruit flesh preferably corresponds to the internal fruit tissues: seeds, locular gel, pericarp and placental tissue.

In another preferred embodiment, the altered, preferably increased content of a flavonoid component is an altered, preferably increased content of a Chalcone and/or a Flavone (among other Apigenin, Luteolin) , and/or a Flavonol (among other Quercitol, Quercetin, Rutin, Kaempferol, Myricetin) , and/or a Flavanone (among other Naringenin) , and/or an Anthocyanin, and/or an Isoflavonoid, and/or a Neoflavonoid and/or a Flavono Lignans and/or their derivatives.

The content of a flavonoid component is preferably expressed in mg per kg fresh weight and assessed by HPLC as earlier defined herein .

"Altered content of a flavonoid component", preferably means "Increased content of a flavonoid component". "Increased content of a flavonoid component" is herein preferably further defined as comprising more of a flavonoid component than what the wild type plant tissue the plant tissue of the invention derives from comprises when both types of plant tissues (wild type plant tissue and plant tissue of the invention) are cultivated under the same conditions. Preferably, the plant tissue of the invention comprises at least 3%, 6%, 10% or 15% more of a flavonoid compound than the wild type plant tissue of the invention derives from comprises when both types of plant tissues (wild type plant tissue and plant tissue of the invention) are cultivated under the same conditions. Also a plant tissue which comprises at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or 150% more or twice or third times or four times or more of a flavonoid component than the wild type plant tissue it derives from is preferred. In another preferred embodiment, the plant tissue of the invention comprises at least 3%, 6%, 10% or 15% or more of two or more specific flavonoid compounds than the wild type plant tissue of the invention derives from comprises when both types of plant tissues (wild type plant tissue and plant tissue of the invention) are cultivated under the same conditions. The flavonoid compound is selected from the group consisting of a Chalcone, a Flavone (among other Apigenin, Luteolin) , a Flavonol (among other Quercitol, Quercetin, Rutin, Kaempferol, Myricetin) , a Flavanone (among other Naringenin) , an

Anthocyanin, an Isoflavonoid, an Neoflavonoid or a Flavono Lignans and their derivatives. Also a plant tissue which comprises at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%

or 150% more or twice or third times or four times or more of two or more flavonoid components than the wild type plant tissue it derives from is preferred.

In another preferred embodiment, the content of a specific flavonoid component is altered, preferably increased in the plant tissue of the invention, whereas the content of other flavonoid components remains unaltered or has been decreased. Alternatively, it preferably means that the content of at least two, at least three, at least four or more of flavonoid compounds has been altered, preferably increased in the plant tissue of the invention, whereas the content of other flavonoid components remains unaltered or has been decreased. According to another preferred embodiment, the content of a flavonoid compound is compared to the content of a flavonoid compound of a control plant tissue. More preferably, when the plant tissue is a fruit, even more preferably a tomato, the content of a flavonoid compound of the tomato of the invention is compared to the content of a flavonoid compound of the tomato from a Lycopersicon species, preferably Solanum lycopersicum which is taken as control.

In a further aspect, the invention provides a method for obtaining a plant tissue having an altered preferably increased content of a flavonoid component, wherein the method comprises the following steps: a. providing a plant tissue, b. altering, preferably increasing the expression of a nucleic acid sequence coding for a TDR4 homologue in the plant tissue of a) compared to the wild type plant tissue it derives from, c. optionally breeding the obtained plant tissue and optionally select for plant tissue having an altered, preferably increased content of a flavonoid component

compared to the wild type plant tissue it derives from.

All features of this method have already been defined herein. Accordingly, the invention in a further aspect relates to a plant tissue or a plant obtainable by the method of the invention .

Accordingly, in a further aspect, the invention provides a food product or ingredient consisting of, comprising or being based on or derived from the plant or plant tissue of the invention.

In yet a further aspect, the invention provides the use of a nucleic acid sequence coding for a TDR4 homologue for obtaining a plant tissue having an altered, preferably increased content of a flavonoid component as earlier defined herein. All features of this use have already been defined herein.

In this document and in its claims, the verb "to comprise" and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one".

The present invention is now further illustrated by the following examples which should not be construed as limiting the scope of the present invention.

Examples

Example 1: TDR4 tomato orthologue of the Arabidopsis dehiscence regulatory gene FRUITFUL (FUL)

Objective: Determine if TDR4, the likely tomato orthologues of the Arabidopsis dehiscence regulatory gene FRUITFULL (FUL) , can rescue Arabidopsis silique ful

1.1. Generation of 35S: TDR4 lines in wild type Arabidopsis Arabidopsis CoI-O plants were transformed with the TDR4 coding sequence under the control of the CaMV 35S promoter. The mRNA coding region for the TDR4 protein sequence was amplified from Solanum lycopersicum cultivar "Ailsa Craig" pericarp cDNA using primers designed to include the methionine and translation stop codons of TDR4 (SGN, Unigene sequence U144041) . The primers were gateway adapted to allow cloning via pDONR221 (Invitrogen) into the pK7WG2 plant expression vector. The T-DNA generated (Fig.l), was designed to transcribe TDR4 under the control of the CaMV 35S promoter away from the right border.

The construct was sequenced to confirm the correct insertion into the expression vector and the recovered plasmid was transformed into Agrobacterium tumefaciens cv "GV3101". Arabidopsis thaliana lines were transformed by the floral dip method. The selection of transformants was carried out by germinating the recovered seeds in compost soaked with the herbicide "Basta". To obtain homozygous lines, twenty Basta resistant individuals were selected and seventeen of these were confirmed to contain the sequence conferring Basta resistance by PCR. Eight homozygous lines were identified by Basta selection and these were named C435-A, C435-B, C435-C, C435-G, C435-K, C435-P, C435-R and C435-S. The lines C435-C, C435-R and C435-S did not display an obvious phenotype.

35S: TDR4 Arabidopsis general plant and silique phenotypes - Lines C435-A, C435-B, C435-G, C435-K and C435-P displayed a set of phenotypes which included a short stature with an abundance of secondary inflorescences. The final plant height was reduced for each of the five lines as demonstrated for lines C435-A, K and P in Fig 2, A. The transcript accumulation of TDR4 in these lines was confirmed by reverse-transcriptase PCR (data not shown) . It was immediately obvious that the lines C435-A, B, G, K and P flowered early. We confirmed that early flowering, as a function of leaf number, indicates changes in overall plant development and these effects were also observed in the FUL transformants under the control of the same promoter (see table D •

Description of the experiment leading to the data of table 1 : 20 arabidopsis seedlings were grown for each line and they were scored on a daily basis for the emergence of an infloresence bolt. This is a frequency table indicating the absolute flowering time with respect to the germination of the plant. The wild type line CoI-O flowers approximately 24 days post germination whilst the ful-2 mutant is delayed and the line over expressing FUL flowers early, indicating that FUL is both necessary and sufficient to affect flowering time. Additionally, the data shows that over expression of TDR4 in 5 independent lines is also sufficient to shorten flowering time indicating that both FUL and TDR4 share this function. The data shows that the CoI-O control and the ful-2 mutant display a flowering time that is predominatly centered at 9 true leaves. In contrast the lines containing FUL and TDR4 under the control of the CaMV 35S promoter all displayed a flowering time that predominatly centered around 4 true leaves.

This shows that TDR4 and FUL act in the same way to promote flowering time with respect to the development of the plant.

Table 1 : Absolute flowering time with respect to the germination of the plant

The dates that each measurement was made with respect to flowering time revealed that the development rate of the Arabidopsis plant was accelerated by the expression of TDR4 as can be observed from Table 2. Clearly in Arabidopsis, this is a function directly attributable to FUL as the gene function is both necessary and sufficient to modify the developmental status of the plant.

Description of the experiment leading to the data of table 2 : This is a frequency table indicating the number of true leaves that develop before the infloresence emerges from the plant and hence indicates the flowering time with respect to the development of the plant. The data shows that both the CoI-O wild type and ful-2 mutant flower after approximately 9 true leaves, the lines over expressing the genes FUL and TDR4 all flower immediately following 3-5 true leaves. The frequency is plotted against the days post germination where the emergence of a flower bolt was recorded on a daily basis. The measurements show that the CoI-O control produced the flower bolt predominately 24 days post germination, whilst the TDR4 and FUL lines under the control of the CaMV 35S promoter produced flower bolts more quickly and predominately around 19 days post germination. Interestingly, the ful-2 mutant produced the majority of its bolts at 28 days post germination. This indicates that FUL is both necessary and sufficient to control the rate of development in Arabidopsis and shows that TDR4 is also sufficient to control developmental rates.

Table 2 : Flowering time with respect to the development of the plant .

The CoI-O Arabidopsis plants produce dry siliques that contained a dehiscence zone (DZ) held under tension by lignifications . In addition, a separation layer (SL) developed that allowed explosive dehiscence of the siliques when they are touched. The SL is evident as indentations due to cell separation at the junctions between the carpels and the replum as seen in the wild type image in Fig. 3. and the lignified cells (LC) are adjacent to this SL. The pattern of phloroglucinol staining in the 35S: : TDR4 transformed lines mirrored that in the 35S:: FUL lines. Furthermore, in the 35S: :TDR4 transformed lines (Fig. 3) neither a separation layer nor the lignified cells were evident, which showed the absence

of a dehiscence zone. This is the same phenotype as 35S:: FUL (Fig. 3) .

Unexpectedly, the TDR4 transformed lines accumulated anthocyanin in their siliques, in response to light, fig. 4 A. We extracted and measured the accumulation of anthocyanins in stage 17 siliques from both CoI-O control and transformed Arabidopsis lines, displayed graphically in Fig. 4 B. All four lines tested displayed a significant increase (P<0.001) in anthocyanin content from the control line when assessed by the student's t-test.

We observed that the terminal florescence of the 35S: : TDR4 lines (Fig. 4 A), terminated early and there was a decrease in the stature of the plant (Fig 2) . In addition, we observed that the time of plant development of the transgenic lines was more similar to each other to the non-transgenic controls. The appearance of anthocyanins in the TDR4 over expressing lines prompted us to undertake a microarray experiment to investigate the profile of gene expression in their siliques. Total RNA was extracted from a pooled sample of siliques at 16 days post anthesis prior to senescence and dehiscence of the fruits in order to obtain sufficient intact RNA for the array experiments. The RNA concentration was measured by nano-drop spectrophotometeric analysis and was subjected to quality control, labelling and hybridisation to the Affymetrix ATHl whole genome Arabidopsis GeneChip at the Nottingham Arabiodpsis Stock Centre. The data from this single microarray hybridisation was compared to the average of three biological replicates of both Arabidopsis siliques at a similar stage of development (Schmid et al . , 2005) . Initial analysis was carried out by calculating the fold ratio of the signal value between C435K (the TDR4 line) and 78 (the analysis for wild type material) . The values were painted onto the AraCyc pathway

analysis program version 3.5 release 06/02/2007 (data not shown, Paley and Karp, 2006) .

The TDR4 transgenic Arabidopsis plants showed accumulation of anthocyanins in response to light. The array data indicated that transcripts for the Arabidopsis gene At5G17040, a homologue of UDPG: flavonoid 3-O-glucosyltransferase were up regulated 3.2 fold in response to the over expression of TDR4. In grape, this gene is involved in the glycosylation of anthocyanins allowing their stabilisation and transport into the vacuole. With respect to flavonol biosynthesis, all the genes in the pathway were up-regulated in the TDR4 over- expressing lines.

The most important observation with respect to anthocyanin production in response to TDR4 over expression was the effect on the R2R3 MYB transcription factor AtPAP2 (AtlG66390; MYB90) . AtPAP2 transcript accumulation was upregulated 9.8 fold in our microarray experiment and is represented in the list of the top 20 upregulated genes (data not shown) . The role of AtPAP2 homologues such as ZmC, LeANTl, PhAN2 in flavonoid and anthocyanin biosynthesis has been well documented and include the control of glucosyl transferases (Lloyd et al . , 1992, Quattrocchio et al . , 1999, Bovy et al, 2002, Verhoeyen 2002, Matthews et al., 2003) . The current model for the regulation of flavonoid biosynthetic pathways assumes a 1:1:1 ratio of MYB : WD-40 : and HLH proteins to activate transcription efficiently (Koes et al . , 2005). Interestingly, AT5G26900, an uncharacterised WD-40 repeat protein displays up regulated transcript accumulation (8.5 fold) in response to TDR4 over expression as determined by the microarray experiment (data not shown) . WD-40 repeat proteins play roles in cell cycle control, but the closest homologue of AT5G26900 from carrot is regulated independently of cell cycle control although it is ubiquitously expressed (Lou et al . , 1997). Although the highest expression

level of a HLH domain protein annotated on the microarray chip was 4.5 fold flavonol synthase (AT5G63590) is upregulated 7 fold. Our data indicate a convincing role for TDR4 and probably closely related genes such as LeFUL2 in altering antioxidant balance in fruits and suggest a major control point in tomato.

1.2. Generation of 35S:TDR4 lines in the Arabidopsis ful mutant To test if TDR4 could act as an orthologue of the Arabidopsis FUL gene we transformed plants of the Ler ful mutant with the construct described in experiment 1.1. The plants showed short siliques which failed to develop a dehiscence zone. Twenty two lines were selected by their resistance to the herbicide "Basta". Homozygous lines were selected for three of the lines displaying elongated siliques; LF435-K, -Q and -R. The homozygous line LF435-Q also displayed a shortened stature and bunching of the terminal inflorescence and anthocyanin accumulation. A similar phenotype to that observed in section 1.1 of this document. The elongated silique phenotype, compacted flowers and anthocyanin accumulation are shown in Fig. 5.

The ability of the TDR4 gene to partially rescue the ful mutant phenocopies the published effects of over-expressing 35S: FUL in the ful mutant. The data support the original hypothesis that TDR4 is a tomato ortholog of FUL. They also provide additional data for an effect of TDR4 on anthocyanin biosynthesis (discussed in 1.1).

Conclusion, The heterologous expression experiments indicate that the tomato gene TDR4 can act as a functional equivalent of FUL. They also suggest that a major role for TDR4 is modulation of the flavonoid biosynthetic pathway.

1.3 Increase of flavonoids

Table 3 shows the relative increase in quantities of a Flavonol (Quercetin and derivatives thereof) identified by HPLC analysis from stage 17 fruit collected from plants growing on compost (mean of 3 biological replicates, CoI-O =100%) . CoI-O is the Arabidopsis base line control, whilst C435-P, K and G lines are independently transformed Arabidopsis lines and expressing TDR4 under the control of the CaMV 35S promoter.

Table 3. TDR4 induced increase in flavonoids (Quercetin and derivatives thereof)

Conclusion : The data shows that TDR4 is sufficient to induce the increased accumulation of flavonoids in plant tissue.

Method of flavonoids identification.

50 mg of freeze-dried powder was extracted in ImL of methanol containing salicylic acid (20μg) as internal standard for HPLC analysis. The extract was incubated at 90 ⁰ C for one hour and then cooled on ice. The extract was centrifuged at 3000 g for 5 minutes before the supernatant was removed and filtered through a 0.2μm filter. A HPLC solvent delivery system (Dionex Gynkotek) was used to separate phenylpropanoids and the flavonoids on a reverse-phase C18 column (Hichrom) . The mobile phase consisted of two components to generate a gradient. (A) 2% water in methanol, acidified with 0.015%HCl by volume and (B) acetonitrile . The gradient conditions used were 95% A and 5% B for 10 min, followed by a linear gradient to 50% B over 30 min. An online photodiode array detector enabled detection and identification from characteristic UV/Vis spectra. Standards were used to confirm the identity of the phenylpropanoids and

the flavonoids. Quantification was performed by comparison of integrated peak areas with the internal standard at the maximum wavelength of the phenylpropanoids and the flavonoids detected.

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