WO2006067219A1 - Methods and means to increase the amounts of carbohydrates in plants - Google Patents

Abstract

The present invention relates generally to methods for genetically altering higher plant materials and changing their metabolism in regard to increased carbohydrate synthesis. More particularly it relates to affecting the amounts of soluble and insoluble amounts of carbohydrates in plants (and on the fibre length of plants).

Description

Methods and means to increase the amounts of carbohydrates in plants

Field of the invention

The present invention relates generally to methods for genetically altering higher plant materials and changing their metabolism in regard to increased carbohydrate synthesis. More particularly it relates to affecting the amounts of soluble and insoluble amounts of carbohydrates in plants and on the length of plant fibres.

Introduction to the invention

In plants, carbohydrates are the ultimate products of photosynthesis and they are utilized in biochemical reactions for the synthesis of many different molecules. Carbohydrates are also used as reserves in seeds to facilitate germination and are stored in over-wintering organs - like roots of sugar beets - to be mobilized during next spring when plants start to flower to feed the outgrowing inflorescence. Plant biomass is mainly build up from carbohydrates, from which bio-fuels, chemicals and on fibre based complex products can be manufactured. In contrast to limited fossil fuels, bio-fuels can be produced by fermentation out of carbohydrates, which are a renewable energy source. In a sustainable agriculture renewable energy technologies will have the potential for a significant energy contribution in the future. Up to 10% of ethanol is already widely used in the US as a gasoline replacement and it has been shown that ethanol based fuels can significantly reduce greenhouse gas emissions. For example, in Brazil cars are build that can either use gasoline, pure ethanol or mixes of both for combustion. However, on a worldwide scale bio-fuels are still too costly to compete with petroleum based oil products. Therefore, strategies that will contribute to a significant reduction of costs will provide a basis for a competitive bio-fuel production out of biomass. One way to realize cheaper bio-fuels is to increase the amount of soluble and insoluble carbohydrates in plants. In addition, a higher yield of carbohydrates can also be used to manufacture bio-plastics which are, due to their biodegradable nature, more environmental friendly. In addition, a higher amount of carbohydrates also leads to an increased accumulation of cellulose microfibrils in plant cell walls. After enzymatic or chemical hydrolysis to mono-saccharides, these can subsequently also be used for bio-fuel or bio-plastic production. Cellulose micro fibrils are incorporated into wood fibres, which are the main ground material for the paper production process while longer fibres are also desired in the clothing industry. Due to their longer fibres, which increase the breaking strength of paper, coniferous trees are mainly used for paper production. However, also Populus and Eucalyptus trees are used in certain regions of the world, but especially Eucalyptus trees contain shorter fibres, which are less suitable for the production of high quality paper. In summary, the accumulation of higher amounts of carbohydrates in plants ensures high seed quality, sweeter fruits, higher amounts of sucrose that can be extracted from sugarcanes and sugar beets and longer fibres desired for the paper and clothing industries. Previous research has mostly used traditional breeding practices to select for fruit varieties with enhanced sweetness. This has been achieved in many different varieties, e.g. super-sweet corn and melons. In all cases, the increase in sweetness has been achieved by selecting for varieties with a higher concentration of total soluble sugars. However, in other fruit, such as the tomato, increases in the concentration of total soluble sugars is associate with a decline in total yield. Modern varieties, which have been selected to be high yielding, tend to have lower total sugar levels and reduced sweetness, relative to other cultivars. Carbohydrate metabolism and carbohydrate composition has also been modified in transgenic plants by increasing starch levels or by altering the relative levels of sucrose (see for example in JP2002204623, US6031154, WO9742333 and US5498831).Thus, the prior art fails to provide a cost-effective means of producing plants with increased carbohydrate levels without undesired traits. The present invention satisfies these needs and is based on the development of transgenic plants that over-express the FLOWERING PROMOTING FACTOR 1 (FPF1) gene and related genes. Said over-expression results in higher amounts of soluble and insoluble carbohydrates in the transgenic plants. In addition we have found that the seeds of the resulting transgenic lines germinate earlier and show an increased longevity compared to corresponding wild type plants.

Figure

Figure 1 : The fibre length of 1 cm middle sections of intemodes from the main stem of

Nicotiana sylvestris\NT (A) and 35S::NtFPF1 plants has been analysed.

Aims and detailed description of the invention

The present invention provides methods and means for increasing the amounts of soluble and insoluble carbohydrates in plants and plant cells. The methodology is based on the over- expression of the flower promoting factor FPFI, a homologue or functional fragment thereof. FPF1 and homologues form a small gene family in plants and the first gene was been identified in an approach to understand the molecular basis of floral induction. Floral induction is a key developmental switch from a vegetative to a reproductive phase. This switch is in many plants often influenced by environmental cues like the temperature or the photoperiod superimposed by endogenous programs that are triggered by the developmental stage of the plants. Arabidopsis thaliana is a facultative long-day plant that flowers earlier under long-day than short-day photoperiods and in many Arabidopsis accessions flowering is also promoted by cold temperatures for a certain time (vernalization). Using a molecular screening approach the FPF1 gene of mustard and Arabidopsis plants was identified (Melzer et al., 1990, Kania et al., 1997). The FPFI gene is a member of a small gene family in Arabidopsis, which encodes a small protein of about 12.5 kDa that shows no structural similarities to other known proteins. By RNA expression analysis it was shown that the FPF1 gene is expressed early after floral induction in the peripheral zone of apical meristems and later on also in roots and leaves, whereas the two related genes FPF LIKE 1 and 2 (FPL1 and FPL2) are expressed throughout development in roots, leaves and also in floral organs. Constitutive expression of all three genes leads to identical phenotypes of earlier flowering in Arabidopsis (Kania et al., 1997; Melzer et al., 1999). In the present invention we have found that transgenic plants that over- express FPFI, a homologue (such as FPL1 and FPL2), or a functional fragment thereof have elevated carbohydrate levels in their tissues or organs.

Accordingly, the present invention provides in one embodiment a method for inducing (or "enhancing" or "stimulating") the production of carbohydrates in plants or plant cells by transformation of said plants or plant cells with an expression vector comprising an expression cassette that further comprises a gene coding for a flowering promoting factor or a homologue of FPF1 or a functional fragment of FPF1 or a functional fragment of a homologue of FPFl In another embodiment the invention provides a method for enhancing ("increasing" or "stimulating" are equivalent terms) the sweetness in tissues of a transgenic plant, the method comprising the introduction into the plant an expression cassette comprising a promoter operably linked to FPF1 or functional fragment or homologue thereof.

With the term "carbohydrates" it is meant soluble sugars such as sucrose, glucose, fructose, xylose, related (isomeric) structures of said sugars and insoluble sugars such as polymers of sugars (e.g. starch) and cellulose. In the present invention we artificially distinguish between soluble sugars and insoluble sugars. The increase of soluble sugars leads for example to fruit varieties with enhanced sweetness, higher amounts of sucrose in plants that can for example serve for extraction from sugarcanes and sugar beets and higher quality of seeds. The over- expression of FPF1 also leads to the formation of longer fibres in plants which is for example desired in the paper and clothing industries. Therefore FPF1, homologues and fragments can be used to generate transgenic plants that possess longer fibres. Examples of "FPF1 related proteins" are protein depicted in SEQ ID NO 2, 4, 6 and 8. The corresponding nucleic acid sequences are depicted in SEQ ID NO 1,3, 5 and 7. Still other examples of flowering promoting factors are:

CV092055 Malus x domestica cDNA

AW 186361 Glycine max cDNA

CF588437 Alstroemeria peruviana cDNA

BM816685 Hordeum vulgare cDNA

AF179223 Oryza sativa, genomic clone RGPF1

BE034410 Mesembryanthemum crystallinum cDNA AW739268 Moss EST library, Physcomitrella patens cDNA

AW699964 Moss EST library, Physcomitrella patens cDNA

CV243797 Populus balsamifera subsp. Trichocarpa cDNA

AJ808916 Antirrhinum majus

CA824169 Populus balsamifera cDNA

CN 188812 C/fπ/s sinensis cDNA

CB827941 Lotus japonicus

CO238816 P/^'cea g/at/ca cDNA

CO223310 Picea sitchensis cDNA

CA922293 Medicago truncatula cDNA

AY110727 Zea mays

The left column refers to the accession numbers which can be found in the National Center for

Biotechnology Information (http://www.ncbi.nlm.nih.gov/) .

The present invention also envisages the use of homologues of FPF1 and related proteins, examples of such homologues are provided as protein sequences in SEQ ID NO 10 and 12.

The corresponding nucleic acid sequences are depicted in SEQ ID NO 9 and 11.

The invention also envisages the use of functional fragments of FPF1 and related proteins.

Examples of functional fragments of FPF1 and related proteins are fragments derived from the amino acid sequence of SEQ ID NO 2, 4, 6, 8, 10 and 12. A functional fragment can be a deletion of the amino- (NH2) - terminal end of FPF1 or a homologue thereof (such as for example SEQ ID NO 2, 4, 6, 8, 10 and 12) with 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15,

16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30 or more amino acids. A functional fragment can also be a deletion a the carboxy- (COOH) - terminal end of FPF 1 or a homologue thereof (such as for example SEQ ID NO 2, 4, 6, 8, 10 and 12) with 1, 2, 3, 4, 5, 6,

7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more amino acids. A functional fragment can also be a combination of deletions between the herein before described amino-terminal and carboxy-terminal fragments. The term "functional fragment" herein means a fragment capable of inducing the carbohydrate content in plant or plant cells when overexpressed in said plant or plant cells. It is understood that a person skilled in the art can fish for homologues of FPF1 of particular interest in the genome or in

(commercial) databases. FPFI, a homologue thereof or a functional fragment thereof can be heterologous or homologous to the plant or plant cell. The wording "enhancing the production of carbohydrates in plants" refers to the fact that untransformed plants are compared with transformed plants with an over-expressed FPF1 gene, homologue or functional fragment thereof with respect to the amount of carbohydrates.

By the term "enhanced production" it is meant that the level of one or more soluble and/or insoluble carbohydrates may be enhanced by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or at least 100% relative to the untransformed plant cell which was used to transform with an expression vector comprising an expression cassette further comprising a gene coding for FPF1, a homologue or functional fragment thereof. An enhanced production of carbohydrates can result in a detection of a higher level of soluble carbohydrates of the plant or in a particular plant organ (e.g. root) or in a particular plant tissue. Alternatively, a higher level of carbohydrates can result from higher levels of insoluble carbohydrates that can be hydrolysed to soluble carbohydrates. The over-expression also leads to the formation of longer fibres. Fibres comprise predominantly cellulose as insoluble sugar.

As used herein, the word "polynucleotide" may be interpreted to mean the DNA and cDNA sequence as detailed by Yoshikai et al. (1990) Gene 87:257, with or without a promoter DNA sequence as described by Salbaum et al. (1988) EMBO J. 7(9):2807. The terms 'identical' or percent 'identity' in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e. 70% identity over a specified region), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using sequence comparison algorithms or by manual alignment and visual inspection. Preferably, the identity exists over a region that is at least about 25 amino acids or nucleotides in length, or more preferably over a region that is 50- 100 amino acids or nucleotides or even more in length. Examples of useful algorithms are PILEUP (Higgins & Sharp, CABIOS 5:151 (1989), BLAST and BLAST 2.0 (Altschul et al. J. MoI. Biol. 215: 403 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www/ncbi.nlm.nih.gov/). "Expression cassettes", of the present invention are generally DNA constructs preferably including (5' to 3' in the direction of transcription): a promoter region, a gene encoding for FPF1, a homologue or functional fragment thereof operatively linked with the transcription initiation region, and a termination sequence including a stop signal for RNA polymerase and a polyadenylation signal. It is understood that all of these regions should be capable of operating in the plant or plant cells to be transformed. The promoter region comprising the transcription initiation region, which preferably includes the RNA polymerase binding site, and the polyadenylation signal may be native to the biological cell to be transformed or may be derived from an alternative source, where the region is functional in the plant or plant cell. The FPF1 gene, a homologue or functional fragment thereof of this invention may be expressed in for example a plant cell under the control of a promoter that directs constitutive expression or regulated expression. Regulated expression comprises temporally or spatially regulated expression and any other form of inducible or repressible expression. Temporally means that the expression is induced at a certain time point, for instance, when a certain growth rate of the plant cell culture is obtained (e.g. the promoter is induced only in the stationary phase or at a certain stage of development). Spatially means that the promoter is only active in specific organs, tissues, or cells (e.g. only in roots, leaves, epidermis, guard cells or the like. Other examples of regulated expression comprise promoters whose activity is induced or repressed by adding chemical or physical stimuli to the plant cell. In a particular embodiment the expression of the FPFI gene, a homologue or functional fragment thereof is under control of environmental, hormonal, chemical, and/or developmental signals, also can be used for expression of FPF1, a homologue or functional fragment thereof in plant cells, including promoters regulated by (1) heat, (2) light, (3) hormones, such as abscisic acid and methyl jasmonate (4) wounding or (5) chemicals such as salicylic acid, chitosans or metals. Alternatively, theFPFI gene, a homologue or functional fragment thereof can be placed under the control of a constitutive promoter. A constitutive promoter directs expression in a wide range of cells under a wide range of conditions. Examples of constitutive plant promoters useful for expressing heterologous polypeptides in plant cells include, but are not limited to, the cauliflower mosaic virus (CaMV) 35S promoter, which confers constitutive, high-level expression in most plant tissues including monocots; the nopaline synthase promoter and the octopine synthase promoter are other examples. The expression cassette is usually provided in a DNA or RNA construct which is typically called an "expression vector" which is any genetic element, e.g., a plasmid, a chromosome, a virus, behaving either as an autonomous unit of polynucleotide replication within a cell (i.e. capable of replication under its own control) or being rendered capable of replication by insertion into a host cell chromosome, having attached to it another polynucleotide segment, so as to bring about the replication and/or expression of the attached segment. Suitable vectors include, but are not limited to, plasmids, bacteriophages, cosmids, plant viruses and artificial chromosomes. The expression cassette may be provided in a DNA construct which also has at least one replication system. In addition to the replication system, there will frequently be at least one marker present, which may be useful in one or more hosts, or different markers for individual hosts. The markers may a) code for protection against a biocide, such as antibiotics, toxins, heavy metals, certain sugars or the like; b) provide complementation, by imparting prototrophy to an auxotrophic host: or c) provide a visible phenotype through the production of a novel compound in the plant. Exemplary genes which may be employed include neomycin phosphotransferase (NPTII), hygromycin phosphotransferase (HPT), chloramphenicol acetyltransferase (CAT), nitrilase, and the gentamicin resistance gene. For plant host selection, non-limiting examples of suitable markers are β-glucuronidase, providing indigo production, luciferase, providing visible light production, Green Fluorescent Protein and variants thereof, NPTII, providing kanamycin resistance or G418 resistance, HPT, providing hygromycin resistance, and the mutated aroA gene, providing glyphosate resistance. The term "promoter activity" refers to the extent of transcription of a gene that is operably linked to the promoter whose promoter activity is being measured. The promoter activity may be measured directly by measuring the amount of RNA transcript produced, for example by Northern blot or indirectly by measuring the product coded for by the RNA transcript, such as when a reporter gene is linked to the promoter. The term "operably linked" refers to linkage of a DNA segment to another DNA segment in such a way as to allow the segments to function in their intended manners. A DNA sequence encoding a gene product is operably linked to a regulatory sequence when it is ligated to the regulatory sequence, such as, for example a promoter, in a manner which allows modulation of transcription of the DNA sequence, directly or indirectly. For example, a DNA sequence is operably linked to a promoter when it is ligated to the promoter downstream with respect to the transcription initiation site of the promoter and allows transcription elongation to proceed through the DNA sequence. A DNA for a signal sequence is operably linked to DNA coding for a polypeptide if it is expressed as a pre-protein that participates in the transport of the polypeptide. Linkage of DNA sequences to regulatory sequences is typically accomplished by ligation at suitable restriction sites or adapters or linkers inserted in lieu thereof using restriction endonucleases known to one of skill in the art. The term "heterologous DNA" or "heterologous RNA" refers to DNA or RNA that does not occur naturally as part of the genome or DNA or RNA sequence in which it is present, or that is found in a cell or location in the genome or DNA or RNA sequence that differs from that which is found in nature. Heterologous DNA and RNA (in contrast to homologous DNA and RNA) are not endogenous to the cell into which it is introduced, but has been obtained from another cell or synthetically or recombinantly produced. An example is a human gene, encoding a human protein, operably linked to a non-human promoter. Another example is a gene isolated from one plant species operably linked to a promoter isolated from another plant species. Generally, though not necessarily, such DNA encodes RNA and proteins that are not normally produced by the cell in which the DNA is transcribed or expressed. Similarly exogenous RNA encodes for proteins not normally expressed in the cell in which the exogenous RNA is present. Heterologous DNA or RNA may also refer to as foreign DNA or RNA. Any DNA or RNA that one of skill in the art would recognize as heterologous or foreign to the cell in which it is expressed is herein encompassed by the term heterologous DNA or heterologous RNA. Examples of heterologous DNA include, but are not limited to, DNA that encodes proteins, polypeptides, receptors, reporter genes, transcriptional and translational regulatory sequences, selectable or traceable marker proteins, such as a protein that confers drug resistance, RNA including mRNA and antisense RNA and ribozymes.

In a preferred embodiment the FPF1 gene, a homologue or functional fragment thereof is expressed under control of a promoter which is less active (has an undetectable expression) in apical meristems. Examples of such promoters are the promoter from the plant enzyme Rubisco. The expression of FPF1, a homologue or functional fragment thereof under control of such a promoter uncouples the early flowering effect from the enhanced carbohydrate effect. In some cases the early flowering effect can be an unwanted side effect. In a more preferred embodiment the promoter is the strict non-meristematic promoter of the glycine and proline rich cell wall protein. An example of such a promoter sequence is depicted in SEQ ID NO: 13. The present invention can for example be practiced with any plant variety for which cells of the plant can be transformed with an expression cassette of the current invention and for which transformed cells can be cultured in vitro. Suspension culture, callus culture, hairy root culture, shoot culture or other conventional plant cell culture methods may be used (as described in: Drugs of Natural Origin, G. Samuelsson, 1999, ISBN 9186274813).

By "plant cells" it is understood any cell which is derived from a plant and can be subsequently propagated as callus, plant cells in suspension, organized tissue and organs (e.g. hairy roots). Tissue cultures derived from the plant tissue of interest can be established. Methods for establishing and maintaining plant tissue cultures are well known in the art (see, e.g. Trigiano R.N. and Gray D.J. (1999), "Plant Tissue Culture Concepts and Laboratory Exercises", ISBN: 0-8493-2029-1 ; Herman E.B. (2000), "Regeneration and Micropropagation: Techniques, Systems and Media 1997-1999", Agricell Report). Typically, the plant material is surface- sterilized prior to introducing it to the culture medium. Any conventional sterilization technique, such as chlorinated bleach treatment can be used. In addition, antimicrobial agents may be included in the growth medium. Under appropriate conditions plant tissue cells form callus tissue, which may be grown either as solid tissue on solidified medium or as a cell suspension in a liquid medium.

A number of suitable culture media for callus induction and subsequent growth on aqueous or solidified media are known. Exemplary media include standard growth media, many of which are commercially available (e.g., Sigma Chemical Co., St. Louis, Mo.). Examples include Schenk-Hildebrandt (SH) medium, Linsmaier-Skoog (LS) medium, Murashige and Skoog (MS) medium, Gamborg's B5 medium, Nitsch & Nitsch medium, White's medium, and other variations and supplements well known to those of skill in the art (see, e.g., Plant Cell Culture, Dixon, ed. IRL Press, Ltd. Oxford (1985) and George et al., Plant Culture Media, VoI 1, Formulations and Uses Exegetics Ltd. Wilts, UK, (1987)). For the growth of conifer cells, particularly suitable media include 1/2 MS, 1/2 L. P., DCR, Woody Plant Medium (WPM), Gamborg's B5 and its modifications, DV (Durzan and Ventimiglia, In Vitro Cell Dev. Biol. 30:219-227 (1994)), SH, and White's medium.

The term "plant" as used herein refers to vascular plants (e.g. gymnosperms and angiosperms). The method comprises transforming a plant cell with an expression cassette of the present invention and regenerating such plant cell into a transgenic plant. Such plants can be propagated vegetatively or reproductively. The transforming step may be carried out by any suitable means, including by Agrobacterium-med\ated transformation and non-Agrobacterium- mediated transformation, as discussed in detail below. Plants can be regenerated from the transformed cell (or cells) by techniques known to those skilled in the art. Where chimeric plants are produced by the process, plants in which all cells are transformed may be regenerated from chimeric plants having transformed germ cells, as is known in the art. Methods that can be used to transform plant cells or tissue with expression vectors of the present invention include both Agrobacterium and non-Agrobacterium vectors. Agrobacterium- mediated gene transfer exploits the natural ability of Agrobacterium tumefaciens to transfer DNA into plant chromosomes and is described in detail in Gheysen, G., Angenon, G. and Van Montagu, M. 1998. Agrobacterium-med\ated plant transformation: a scientifically intriguing story with significant applications. In K. Lindsey (Ed.), Transgenic Plant Research. Harwood Academic Publishers, Amsterdam, pp. 1-33 and in Stafford, H.A. (2000) Botanical Review 66: 99-118. A second group of transformation methods is the non-Agrobacterium mediated transformation and these methods are known as direct gene transfer methods. An overview is brought by Barcelo, P. and Lazzeri, P.A. (1998) Direct gene transfer: chemical, electrical and physical methods. In K. Lindsey (Ed.), Transgenic Plant Research, Harwood Academic Publishers, Amsterdam, pp.35-55. Hairy root cultures can be obtained by transformation with virulent strains of Agrobacterium rhizogenes, and they can produce high contents of secondary metabolites characteristic to the mother plant. Protocols used for establishing of hairy root cultures vary, as well as the susceptibility of plant species to infection by Agrobacterium (Toivunen L. (1993) Biotechnol. Prog. 9, 12; Vanhala L. et al. (1995) Plant Cell Rep. 14, 236). It is known that the Agrobacterium strain used for transformation has a great influence on root morphology and the degree of secondary metabolite accumulation in hairy root cultures. It is possible that by systematic clone selection e.g. via protoplasts, to find high yielding, stable, and from single cell derived-hairy root clones. This is possible because the hairy root cultures possess a great somaclonal variation. Another possibility of transformation is the use of viral vectors (Turpen TH (1999) Philos Trans R Soc Lond B Biol Sci 354(1383): 665-73). Any plant tissue or plant cells capable of subsequent clonal propagation, whether by organogenesis or embryogenesis, may be transformed with an expression vector of the present invention. The term 'organogenesis' means a process by which shoots and roots are developed sequentially from meristematic centers; the term 'embryogenesis' means a process by which shoots and roots develop together in a concerted fashion (not sequentially), whether from somatic cells or gametes. The particular tissue chosen will vary depending on the clonal propagation systems available for, and best suited to, the particular species being transformed. Exemplary tissue targets include protoplasts, leaf disks, pollen, embryos, cotyledons, hypocotyls, megagametophytes, callus tissue, existing meristematic tissue (e.g. apical meristenns, axillary buds, and root meristems), and induced meristem tissue (e.g., cotyledon meristem and hypocotyls meristem).

These plants may include, but not limited to, plants or plant cells of agronomically important crops, such as plants, including species from the genera Antirrhinum, Arabidopsis, Asparagus, Atropa, Avena, Brassica, Bromus, Browaalia, Cannabis, Citrus, Citrullus, Citrus, Capsicum, Ciophorium, Cichorium, Cucumis, Cucurbita, Datura, Daucus, Digitalis, Eucalyptus, Fragaria, Glycine, Geranium, Gossypium (e.g. Gossypium hirsutum), Helianthus, Hererocallis, Hordeum, Hyoscyamus, Lactuca, Linum, Lolium, Lotus, Lycopersicum, Malus, Manihot, Majorana, Medicago, Nemesia, Nicotiana, Onobrychis, Oryza, Panicum, Pelargonium, Pennisetum, Persea, Petunia, Pisum, Populus, Pyrus, Prunus, Ranunculus, Raphanus, Salpiglossis, Sinapis, Secale, Senecio, Solanum, Sorghum, Trifolium, Triticum, Trigonella, Urtica, Vitis, Vigna, and Tea. One of skill in the art will recognize that after the expression is stably introduced in transgenic plants and confirmed to be operable, it can be introduced into other plants by sexual crossing. Any number of standard breeding techniques can be used, depending upon the species to be crossed.

Examples

1) Isolation of FPF1 and FPF1 like genes from Arabidopsis and Nicotiana tabacum A FPF1 cDNA was initially identified after a subtractive hybridisation enrichment of flowering specific cDNAs from mustard apical meristems that had been florally induced by long day conditions (pSFD 5.04, Melzer et al., 1990). To isolate an Arabidopsis FPF1 cDNA a cDNA library from apices of Arabidopsis plants that were induced to flowering was screened with a radiolabeled mustard FPF1 cDNA fragment. The hybridisation was performed at 62°C in 6 x SSC (1 x SSC is 0.15 M NaCI and 0.015 M sodium citrate), 5 X Denhardt's solution (0.1% Ficoll, 0.1% PVP, and 0.1% BSA), 0.1% SDS, and 100 pg/mL herring sperm DNA. Low stringency washes were done three times at 40°C for 10 min. After two rounds of rescreening, phage inserts were subcloned into pBluescript Il SK+ (Stratagene), and both strands were sequenced (Kania et al., 1997). A PCR approach was used to amplify FPF1-\\ke genes from tobacco. PCR reactions were performed with cDNAs from RNA of florally induced apices from N. tabacum by using two degenerated primers (5'FPFdeg CGGAATTCATGTCNGGNGTNTG- GGTNTTC and 3'FPFdeg CGGAATTCCTTGACNACNATNTCGTACATGT), which were designed based on mustard and Arabidopsis FPF1 and FPL genes. Standard PCR reactions were performed with 40 cycles (denaturation at 94°C, annealing at 45°C and extension at 72°C). According to the primer design, expected PCR products of about 300 bp were digested with EcoR I, cloned into the pBluescript Il vector (Stratagene) and sequenced. For the isolation of a NtFPFI gene, five hundred thousand pfu of a genomic library from Nicotiana tabacum cv Samsun in λ EMBL3 were screened with a NtFPFI PCR fragment according to standard protocols (Smykal et al., 2004). PCR products encoding proteins homologous to FPF1 from Arabidopsis and mustard were used to screen a genomic library of N. tabacum cv Samsun. Three genomic clones that contained identical genomic fragments were identified and from one clone a 3.5 kb EcoRI fragment was subcloned and fully sequenced. An open reading frame within this EcoRI fragment encodes a peptide of 102 amino acids with a calculated molecular weight of 12 kDa. Since this NtFPFI protein showed 65% amino acid identity to AtFPFI, and a lower degree of amino acid identities to two closely related proteins in Arabidopsis, as well as an expression pattern that was similar to that of the Arabidopsis and mustard FPF1 genes, the tobacco gene was named NtFPFL (Accession numbers: AtFPFI: Y11988; AtFPUΛtδg 10630; >4.FPL2:AT4G31380; SaFPFlY11987; NtFPFI :AY496934). The FPF1 proteins from Arabidopsis and Nicotiana are short peptides of 110 amino acids with a high similarity, but with no conserved structures to any proteins with a known function and are unique for plants. The Arabidopsis sequencing project has revealed that two closely related proteins exists in Arabidopsis plants. Since these two peptides showed a high sequence homology to FPF1 from Arabidopsis we named these genes FPL1 and FPL2 (FPFY-like 1 and 2). These two proteins are 92% (FPL1) or 80% (FPL2) identical to FPF1 , respectively. The NtFPFI protein showed 65% amino acid identity to FPF1 from Arabidopsis, and a lower degree of amino acid identities to two closely related proteins in Arabidopsis FPL1 and FPL2. All proteins share a high sequence similarity at the N-terminus, as well as several short conserved stretches in other parts of the proteins. However, these plant-specific proteins have no conserved motifs in common with other known proteins.

2. Expression pattern of FPF1 gene family members in Arabidopsis and Nicotiana varieties Using in situ hybridizations it could be shown that FPFI is expressed in apical meristems during the transition to flowering in Arabidopsis plants, whereas FPL1 and FPL2 are already expressed during vegetative stages in whole seedlings and in older plants in roots as well as in mature leaves. It could subsequently be shown by promoter analyses with a GUS reporter gene, that FPL1 and FPL2 are expressed in phloem and phloem parenchyma cells of leaves, stems and roots. FPFI showed the strongest expression in apical meristems during the transition to flowering, but was also expressed in the veins of leaves and roots in later phases during development. Since the NtFPFI gene was expressed at a very low level in tobacco plants a semi-quantitative RT-PCR analysis with RNAs from different tissue samples with a primer combination from the coding region and from the 3' untranslated region was performed. The NtFPFI gene was expressed at a low level in seeds and was up regulated following germination in seedlings. Transcripts were also detectable in young developing leaves, but not in mature leaves and stems and were detectable to a higher extent also in roots. In contrast to FPFI expression in Arabidopsis, transcripts of NtFPFI were already detectable in apices of vegetative tobacco plants, but were present in higher amounts in apices of florally induced plants. Since NtFPFI might be developmentally regulated, RNA accumulation in apical buds of different tobacco varieties grown under long- and short-days was analysed by RT-PCR. Apical buds were harvested 3, 6, 9, and 12 weeks after sowing. All tobacco plants remained vegetative until 9 weeks after sowing, but 12 weeks after sowing, all plants grown under inductive conditions had undergone the transition to flowering. NtFPFI was expressed in apical buds of day-neutral tobacco in short days in higher amounts during the floral transition in 12 weeks old plants than in vegetative plants. Under long-days, high amounts of FPFI transcripts were observed in apical buds of 3 weeks old seedlings. A similar observation was made in one week old whole seedlings. In 6 and 9 week old plants, the amount of NtFPFI transcripts decreased and again increased during the 12^th week after sowing, when the plants already had visible floral buds. In short-day cv Hicks MM plants we detected lower NtFPFI mRNA amounts in vegetative stages under short and long days and an increase in NtFPFI transcript levels under inductive short-days at the time of the floral transition. In long-day N. sylvestris plants no NtFPFI transcripts were observed in 3 week old seedlings under short- days. NtFPFI transcripts accumulate from the 6^th week until the 12^th week after sowing in short days, during which the plants were still in a rosette stage and non-flowering. However, under long days a continuous increase to high NtFPFI transcript levels was observed in apical buds of plants that had started to flower 12 weeks after sowing.

3. Constitutive Expression of AtFPFl AtFPU, AtFPL2 and NtFPFI

The possible functions of FPFI, FPLI and FPL2 gene were first analysed by constitutively expressing the cDNAs in transgenic Arabidopsis plants. To this end, the coding regions of FPF1, FPLI and FPL2 were amplified via PCR by using primer combinations that introduced restriction sites at the ends of the amplification products. The primer AtFPFI-S (5'-GCAGGATCCA- CCATGGCAGGCGTGTGGGTGTC-3'), AtFPLI -5' (δ'-GCAGGATCCACCATGGCAGGAG- TTTGGGTGTT-3') and AtFPLl-S (5'-GCAGGATCCACCATGGCTGGTGTGTGGGTATT-S') of the 5' ends of the coding regions contained a Λ/co1 site at the ATG start codon and a SamHI site before the Λ/col site. The 3' end primers AtFPFI-Z' (5'- ATGCGGATCCATG- GGAGTCTCGGAC-3'), AtFPLI-Z' (5'-ATGCGGATCCTCAATTGAAATCGCGG-S') and AtFPLl-Z' (5'-ATGCGGATCCCTACATGTCACGGAC-S') introduced SamHI sites after the stop codons. The amplified coding regions were introduced via Λ/co1 and SamHI into the vector pSH9 that contains a cauliflower mosaic virus 35S promoter with a Ω element as a translational enhancer adjacent to the Λ/col site and a translational terminator with a polyadenylation site adjacent to the SamHI site (Holtorf et al., 1995). From the recombinant vectors pSH9-FPF1, the expression cassette was ligated as a H/ndlll fragment into the binary plant transformation vector pBIN19 (Bevan, 1984) to create the recombinant plant transformation vectors pBIN19-FPFl The recombinant plasmids were introduced into Agrobacterium tumefaciens C58C1 by a standard transformation procedure (Hόfgen and Willmitzer, 1988). The Arabidopsis ecotypes Columbia (CoI) and Landsberg erecta (Ler) were transformed with the vacuum infiltration method (Bechtold et al., 1993). Plant seeds were harvested 4 to 5 weeks after infiltration, sterilized, and plated on germination medium containing 500 mg/L timenten to suppress agrobacterial growth and 50 mg/L kanamycin for selection of transformants. We obtained six CoI lines and eight Ler lines expressing the AtFPFI cDNA. Plants were grown under LD (16-hr light and 8-hr darkness) or SD (8-hr light and 16-hr darkness) conditions. The coding region of NtFPFI was also amplified by standard PCR reactions by using the primer pair NtFPFI-S (δ'-GCAGGATCCATGTCTGGAGTTTGGGTA-S') and NtFPFI-Z' (S'-GCAGGATCCTCATATGTCTCTAACTTC-a'), which created EcoRI sites before the start and after the stop codon. The PCR fragments were cut with EcoR\ and were fused to the CaMV 35S promoter at the EcoRI site of the pRT101 vector (Tόpfer et al., 1987). The expression cassette of the pRT101 vector was introduced as a Hind III fragment into pRD400 (Datla et al., 1992) and the binary vector was transformed into the Agrobacterium tumefaciens strain C58C1. For Agrobacterium mediated leaf disc transformation (Horsch et al., 1985), day neutral N. tabacum cv. Hicks, N. tabacum cv Hicks MM and N. sylvestris plants were grown in vitro. Regenerating plants were selected on agar plates containing MS media supplemented with 500 mg/l Timenten and 200 mg/l kanamycin. Seeds from T1 and T2 plants were tested for homozygosity on kanamycin plates and T3 or T4 homozygous lines were used for subsequent experiments, in which we used ten independently transformed lines from each tobacco variety. Transgenic lines were assayed for transgenic expression by Northern blot analysis. Therefore, total RNA was isolated and 15 μg of total RNA was separated on formaldehyde agarose gels, transferred to nylon membranes and hybridized with a AtFPFI or NtFPFI probe, respectively, at 65°C according to standard protocols.

4. Over-expression of AtFPFI and NtFPFI promotes flowering in transgenic plants In the facultative long-day plant Arabidopsis all transgenic 35S::AtFPF1 lines flowered earlier and produced fewer leaves than the control plants under both long and short days. The organization of the flowers, the fertility, and the seed set were the same as in wild-type control plants. However, the architecture of the transgenic plant had changed. Whereas, on the inflorescence stem of wild-type plants, cauline leaves without petioles were inserted, the transgenic plant had rosette like leaves with petioles at the same positions. This was because of an internode elongation between rosette leaves, which caused bolting of the plants before rosette leaf production was terminated. Compared with the wild-type plants, the transgenic plants had fewer rosette leaves and the number of cauline leaves was increased. However, the overall leaf number was reduced under both day-length conditions (Kania, et al. 1997). The same results have been obtained by over-expressing FPLI and FPL2 in Arabidopsis, indicating that the encoded proteins have the same functions, but obtain their specific function by a tissue specific expression. The tobacco NtFPFI transgene promoted flowering in each transgenic Nicotiana line under inductive photoperiods. Wild type plants of day-neutral cv Hicks flowered after the production of 27 leaves under long-days, whereas the different transgenic lines flowered after forming 15-23 leaves. A comparable shortening of the vegetative phase was also observed under short-day conditions. Transgenic N. sylvestris plants flowered also earlier under inductive long-days by reducing the leaf number from 23 in wild type plants to 12 leaves in the earliest transgenic line. All transgenic N. sylvestris lines showed no initial rosette stage under long-days. This was also obvious under short-days under which wild-type plants remained for up to 9 months in the rosette stage before the onset of senescence. N. tabacum cv Hicks MM wild-type plants also exhibit a short initial rosette stage under inductive short days and flowered after forming 24 leaves, whereas the transgenic lines showed no initial rosette stage and flowered after forming 13 to 17 leaves. N. tabacum cv Hicks MM wild type plants never flowered in our experimental conditions in long days. However, all transgenic lines showed no initial rosette stage and flowered in this otherwise non-inductive photoperiod after the formation of 27 to 33 leaves (Smykal et al., 2004). In all transgenic lines analyzed, the intemodes were elongated, resulting in plants which were 20-50 cm taller compared to wild type plants. It was also obvious that the appearance of the leaves was altered in the transgenic plants. The shapes were irregular and intercostal fields were enlarged, giving the leaves a more wrinkled appearance. This was most obvious on the later formed leaves of the transgenic lines in all varieties.

5. Identification and characterization of FPFI and FPL mutants

A PCR screening has been used to identify transposon insertion mutants from a population of plants that contain the En 1 transposon from maize randomly integrated in the genome (Baumann et al, 1998). Three transposon lines with En1 insertions around the FPFI gene have been obtained. One insertion was 321 bp upstream of the ATG in the promoter region and the other two 253 bp and 1179 bp behind the stop codon. None of the insertions has lead to an inactivation of the FPF1 gene. In the promoter insertion line were still RNA transcripts at a normal level detectable by quantitative RT-PCR. Therefore, we have used the three tagged lines for a transposition of the transposon into the coding region. From the three insertion lines 1600 plants were grown from which leaves from 16 plants each were pooled. DNA was extracted from the 100 pools and PCR was performed by using the f470 primer with either En205 or En8130. Depending on the initial insertion we expected no bands for the insertions downstream of the FPF1 coding region and a band of 1100bp for the initial insertion in the promoter. A transposition into the coding region should give an additional band of a size from 140 to 470 bp. In seven out of the 100 pools we could detect smaller bands. From these seven pools DNA was isolated from single plants and these were again tested by PCR with the primer combination that gave a positive result. From five plants we could confirm the initial transposition. By sequencing it turned out that four of them were transpositions into the coding region. To test whether the transpositions in these plants were somatic or heritable insertions, other leaves from these plants were harvested and it was confirmed that two transpositions were also present in other leaves. Only one of the transpositions segregated 3:1 in the following generation. The new insertion, located 61 bp behind the start codon has led to a null mutation of the FPF1 gene. The mutation caused a later flowering phenotype of the mutant, indicating that the FPFI gene is involved in flowering time control. A similar PCR screen was used to get transposon insertions for both FPL1 genes. Thereby, one transposon insertion line for FPL1 and one for FPL2 have been obtained. Both mutants did not show any phenotype under normal physiological conditions. Out of the three single fpf1 and fpH and fpl2 mutants double mutants and a triple mutant have been established by genetic crosses.

6. Increased levels of soluble and insoluble carbohydrates in transgenic Arabidopsis and Nicotiana lines

For carbohydrate analyses about 500 mg leaves from Arabidopsis and Nicotiana tabacum WT and 35S::FPF1, 35S::FPL1 and 35S::FPL2 transgenic plants as well as from fpf1 and triple fpf1/fpl1/fpl2 Arabidopsis mutants were harvested and frozen in liquid N₂. 1 ml of frozen 10% HCLO₄ was added and the tissues were grounded with a mortar and a pistil. The thawed extracts were then collected in E. tubes and the mortars were washed with 300 μl 1% HCLO₄. Insoluble debris was precipitated by centrifugation and the supematants were neutralized with 5 M K₂CO₃ (pH7) and stored at -80° until they were used for sugar measurements. In addition, the pellets from the thawed extracts were used for starch measurements. Therefore, the pellets were first extracted with 10 ml of 80% acetone o/n at 4°C. After centrifugation the cleared pellets were washed 3 times with 0,5 M MES buffer (ph 4,5) and resuspended in 500 μl 0,5 M MES buffer (pH 4,5). For starch digestion 14 U amyloglucosidase and 4 U α-amylase were added and incubated overnight at RT. After vortexing the digestion mix was centrifuged and the supernatant was taken to measure glucose. Soluble carbohydrates and from starch digestion derived glucose were determined by HPAE-PAD (high-performance anion-exchange chromatography with pulsed amperometric detection) using the ED50 Electrochemical Detector from Dionex (HPLC-Dionex ICS-2500). The sugar determination is based on the measurement of current resulting from oxidation of carbohydrates at the surface of a gold electrode. Soluble carbohydrates and glucose from starch digestions were analyzed from Arabidopsis WT and 35S::FPF1, 35S::FPL1 and 35S::FPL2 transgenic plants as well as from fpf1 and triple fpf1/fpl1/fpl2 mutants and from Nicotiana tabacum WT and 35S::NtFPF1 plants. The samples were harvested at 16:00 from plants that had been grown under long day conditions (light from 8:00 to 00:00). Table 1 shows a comparison of soluble carbohydrates (added amounts of glucose, fructose and sucrose), including starch-derived glucose, from two different experiments.

Table 1 : Comparison of soluble carbohydrates between different transgenic plants of Arabidopsis thaliana (A. th.), wild type plants and mutants as well as transgenic Nicotiana tabacum (N. t.) and wild type plants.

A. th. A. th. A. th. A. th. A. th. A. th. N. t. N. t. genotype WT 35S::- 35S::- 35S:: fpf1 fpfilfpl WT 35S::-

FPF1 F PU -FPL 1/fpl2 NtFPFI

2 μM soluble 4,0 7-11 6,8-12 6,5- 3,4 2,5 4,2 7,8-12 sugars per g 11 fresh weight

7) Increased carbohydrate contents in fruits and seeds

Maize plants contain several FPF1 like genes in their genome. However, the homologue to FPFI in Arabidopsis has not yet been identified (Pokutta, 2002). Over-expression of SaFPFI has not caused earlier flowering in transgenic maize plants, but has also led to higher carbohydrate accumulation. This is especially also true for seeds and the whole corn cop, indicating that FPF1 and FPF1 like transgenes can be used to increase carbohydrates in seeds and also fruits.

Z. Z. mays

Genotype mays 35SwFPF 1 μM soluble sugars 22,0 28,3 per g fresh weight

8. Uncoupling the early flowering effect form the enhanced carbohydrate production The method of the invention leading to enhanced carbohydrate production inherently also leads to an early flowering phenotype of the plants. In some occasions the latter phenotype might be undesirable and it might be necessary to uncouple these two effects. A solution for this is to express the FPFI gene under the control of a promoter that is not expressed in apical meristems. Such a suitable promoter has been identified in the subtractive hybridisation approach (described in example 1) were a gene encoding a glycine and proline rich cell wall protein has been identified (SFD2.43, Melzer et al. 1990). The promoter of SFD2.43 is depicted in SEQ ID NO: 13. The SFD2.43 gene is strongly expressed in whole plants, but never in meristematic tissues. Transgenic approaches to over-express the SFD2.43 gene under the control of a constitutive promoter have always failed to produce any transgenic plant, indicated that it might be crucial that the gene is not expressed in meristems. Therefore, the very tightly regulated SFD2.43 promoter is a suitable promoter to over-express genes at high levels in plants, but ensures that the genes are not expressed in meristems. Since the over- expression of FPFI leads to early flowering, which is forced by the expression of FPFI in meristems, an over-expression under the control of a SFD2.43 promoter will not alter the flowering time of transgenic plants, but will have the benefit of higher carbohydrate contents.

9. Altered fibre length in transgenic Nicotiana lines over-expressing FPFI Fibres are narrow, elongated cells, which are normally associated with vascular tissues. They provide mechanical support in plant tissues that are not longer elongating. For the production of high quality paper a certain fibre length is necessary. Therefore, breeding strategies for better and longer fibres are still desired for trees that are used by the paper industries. For instance in Brazil, paper is produced mainly from Eucalyptus trees that are grown in big plantations. Eucalyptus fibres are shorter than those of Picea or Populus species, which are mainly used in Europe for paper production. Therefore, strategies have been developed to cope with the shorter Eucalyptus fibres. However, for paper of high quality it is still necessary to search for plants with longer fibres, but also for other trees that are used for paper production longer fibres are still a breeding goal. To analyse fibre length 1 cm pieces of Nicotiana stems have been used. Therefore, the cortex and phloem was peeled away from isolated stem fragments and the 1 cm stem fragments were cut into 1 cm to 4 mm² pieces. Maceration was performed for 20 h in a boiling solution of 3% hydrogen peroxide and 50% glacial acetic acid. After maceration the samples were washed several times in water and were neutralized with a saturated solution of sodium bicarbonate. After neutralization the sodium bicarbonate was decanted and the samples were again washed three times with water and were finally resuspended in 1 ml of water. The samples were vigorously vortexed to separate the cells from the xylem. Individual fibres were examined on a Leitz microscope under dark field illumination. Figure 1 shows a representative example of relative fibres length from Nicotiana sylvestris\NJ (A) and 35S/.FPF1 transgenic plants (B).

Photographs have been taken from different samples to measure the relative length of the fibres on printed figures (Table 2). This example clearly shows that the transgenic plants contain longer fibres in stem segments than the untransformed control plants. Table 2

N. sylvestris N. sylvestris N. tabacum N. tabacum

Genotype WT 35S::NtFPF1 WT 35S::NtFPF1 relative fibre 3,1 5,2 4,1 5,3 length

10. Constitutive expression of FPF1 prolongs seed longevity and earlier and homogenous germination of seeds

The quality of seeds plays an immense role for the longevity and for a homogenous germination of seeds. Since seed longevity is heavily affected by environmental conditions during seed formation, harvest and storage, it is not much known about the genetic basis of differences in seed quality. It is well known that the phytohormone abcisic acid plays an essential role for the observed seed dormancy after the ripening of the fruits in many plant species. Germination is only possible after breaking dormancy either by cold treatments, by prolonged storage of seeds or by treatments of seeds with the phytohormone gibberellin, which allows immediate germination of dormant seeds. On the contrary gibberellin deficient mutants in Arabidopsis show much lower germination rates due to leaky mutations or do not germinate if the mutation leads to a complete shut down of gibberellin biosynthesis. The ga2-1 mutation is a leaky gibberellin biosynthesis mutant that still germinates in the absence of exogenously applied gibberellins. However, the seeds of the ga2-1 mutant are less viable after a longer time period and germination frequencies were dropping down faster than those of the corresponding wild type backgrounds. In a set of experiments with different four-year-old seed batches, which were all stored at room temperature, it was analyzed whether the FPFI, FPLI and FPL2 genes play a role for germination. Sterilized seeds of the Arabidopsis Columbia wild type, of fpf1 and fpf1/fpH/fpl2 triple mutants, of 35S::FPF1 transgenic plants, of the gibberellin biosynthesis mutant ga2-1 and the ga2-1 mutant over-expressing FPF1 were sown on 4 independent replica plates each (Y₂ MS medium pH5.7 with 2% sucrose and 0,8% agar) and the germination frequency was calculated after five days. It was obvious that seeds from transgenic Arabidopsis plants over-expressing FPFI in different genetic backgrounds germinated earlier and were more viable after a prolonged storage time, at which wild type seeds showed a dramatic drop of germination. After four years of storage seeds of the ga1-2 mutant had lost the ability to germinate, whereas the transgenic ga1-2 mutant over-expressing FPF1 still germinated. Table 3 shows that the germination frequencies are higher if FPFI is over- expressed, indicating that the constitutive expression of FPF1 leads to a prolonged longevity of Arabidopsis seeds. The same phenotype has also been observed by over-expressing FPLI and FPL2 in transgenic Arabidopsis plants. Table 3

A. th. A. th. A. th. A. th. ga1-2 ga1-2

Genotype (4 years) CoI 35S/.FPF fpf1 fpf1lfpl1lfpl2 35S/.FPF1

WT 1 germination 40 % 80% 42% 38% 0% 40% frequencies

Materials and methods

The cultivation of Arabidopsis and Nicotiana plants

Arabidopsis seeds of the ecotypes Landsberg erecta and Columbia were obtained from the

Nottingham Arabidopsis stock centre. Seeds were sown on soil and seedlings were singled out in pots after 14 days. Arabidopsis plants were grown in phytotrons, either under short- (8h light) or long-day conditions (16h light) at 20⁰C under fluorescent tubes emitting a photon flux density of 150 μmol m^"2sec^"1.

Seeds from N. tabacum cv Hicks, N. tabacum cv Hicks MM and N. sylvestris plants were sown on soil and seedlings were singled out in 16 cm pots after 21 days. Plants were grown either in phytotrons under short- (8h light) or long-day conditions (16h light) under fluorescent tubes emitting a photon flux density of 150 μmol m^"2sec^"1 at 20°C or in greenhouses at 22°C during the day and 18°C during the night. In greenhouses daylight was supplemented with additional light for 16 h long-day conditions.

References

Baumann, E., Lewald, J., Saedler, H., Schulz, B., and Wisman, E. (1998). Successful

PCR-based reverse genetic screens using an En-7-mutagenised Arabidopsis thaliana population generated via single-seed descent. Theor Appl Genet. 97, 729-734 Bechtold, N., Ellis, J., and Pelletier, G. (1993). In planta Agrobacterium-mediated gene transfer by infiltration of adult Arabidopsis thaliana plants. C. R. Acad. Sci.

Paris 316, 1194- 1199. Bevan, M.W. (1984). Binary Agrobacterium vectors for plant transformation. Nucl.

Acids Res. 12, 8711-8721.

Chaffey, N., Cholewa, E., Regan, S., and Sundberg, B. (2002). Secondary xylem development in Arabidopsis: a model for wood formation. Physiol. Plant. 114, 594-600. Datla, R.S.S., Hammerlindl, J.K., Panchuk, B., Pelcher, L.E. and Keller, W. (1992)

Modified binary plant transformation vectors with the wild-type gene encoding NPT

II. Gene 211, 383-384. Franklin, G. L. (1945). Preparation of thin sections of synthetic resins and wood-resin composites, and a new macerating method for wood. Nature 155, 51. Hόfgen, R., and Willmitzer, L. (1988). Storage of competent cells for Agrobacterium transformation. Nucl. Acids Res. 16, 9877. Holtorf S, Apel K, and Bohlmann H. (1995). Comparison of different constitutive and inducible promoters for the overexpression of transgenes in Arabidopsis thaliana. Plant

MoI Biol. 29, 637-46. Kamm, B., and Kamm, M. (2004). Principles of biorefineries. Appl. Microbial. Biotechnol.

64, 137-145. Kania, T., Russenberger, D., Peng, S., Apel, K., and Melzer, S. (1997). FPF1 promotes flowering in Arabidopsis. Plant Cell 9, 1327-1338. Melzer, S., Majewski, D.M., and Apel, K. (1990). Early changes in gene expression during the transition from vegetative to generative growth in the long-day plant

Sinapis alba. Plant Cell 2, 953-961. Melzer, S., Kampmann, G., Chandler, J. and Apel, K.(1999) FPF1 modulates the competence to flowering in Arabidopsis. Plant J. 18, 394-405. Pokutta, L. (2002). lsolierung und Charakterisierung von FPF1 (FLOWERING PRO

MOTING FACTOR 1) - homologen Genen aus Mais (Zea mays L.). Dissertation,

Universitat Hamburg.

Smykal, P., Gleissner, R., Corbesier, L. Apel, K. and Melzer, S. (2004) Modulation of flowering responses in different Nicotiana varieties. Plant MoI. Biol. 55, 253-262. Sun, Y., and Cheng, J. (2002). Hydrolysis of lignocellulosic materials for ethanol pro- duction: a review. Bioresource Technology 83, 1-11.

Tόpfer, R., Matzeit, V., Gronenborn, B., Schell, J. and Steinbiss, H.-H. (1987). A set of plant expression vectors for transcriptional and translational fusions. Nucl. Acids Res. 15, 5890.

Claims

Claims

1. Use of an expression cassette comprising a gene FPF1 , encoding a flowering promoting factor, or a homologue of FPF1 , or a functional fragment of FPF1 , or a functional fragment of a homologue of FPF 1 to enhance the production of carbohydrates in plants and plant cells comprising a) transforming said plant or plant cells with an expression vector comprising said expression cassette, b) selecting transformed plants or plant cells with an enhanced production of carbohydrates, and c) propagating such selected plants or plant cells.

2. Use according to claim 1 wherein said gene present in said expression cassette is SEQ ID NO: 1, 3, 5, 7, 9, 11 or a functional fragment thereof.

3. Use according to claims 1 and 2 wherein said carbohydrates are soluble.

4. Use according to claims 1 and 2 wherein said carbohydrates are insoluble.

5. Use according to claims 1-4 wherein said expression cassette is under control of promoter that has no detectable expression in plant apical meristems.

6. Use according to claim 5 wherein said promoter is depicted in SEQ ID NO: 13.