# Patent

## Title
Means and methods for controlling flowering in plants

## Abstract

Described are means and methods for controlling flowering in plants. In particular, described are nucleic acid molecules which, when expressed in sense orientation or in antisense orientation, respectively, in plants lead to a prevention of flowering. Moreover, a method for controlling flowering in plants is provided which comprises the inducible restoration of flowering in plants in which flowering is prevented.

## Background
The present invention relates to nucleic acid molecules involved in the control of flowering in plants as well as to methods for controlling flowering in plants.
It is known that the production of culm (stem) and seed head (inflorescence) formation may decrease the feeding value of various kinds of fodder plants such as forage grasses. In forage grasses the leaf blades are more digestible, richer in crude protein and poorer in cell-wall constituents than sheaths and culms (Deinum and Dirvan, Neth. J. Agri. Sci. 23 (1975) 69-82; Wilman et al., J. Br. Grassl. Soc. 31 (1976), 73-79). The ageing of grasses (development towards flowering and seed setting) is associated with an increase in lignification and a decrease in digestibility, which is markedly higher for the stems than for the leaves (Delagarde et al., Anim. Feed. Sci. Technol. 84 (2000), 49-68). In consequence, the digestibility of grasses becomes markedly reduced during the course of the growth season. This reduction is largely caused by an increase in the content of poorly digestible cell wall structural components, mainly lignins. In parallel, there is a decrease in content of soluble carbohydrates. Poorly digestible structural components create an imbalance between carbohydrate and protein levels during ruminant fermentation, leading to a loss of nitrogen (ammonia) to the environment. Grass varieties with an increased level of soluble carbohydrates and increased digestibility will lead to a more efficient uptake of proteins in ruminants and, thus, an enhanced milk and meat production. Feeding trials on cows have documented that increasing the digestibility of forage grass is directly associated with a daily increase in feed uptake and milk production (Oba and Allen, J. Dairy Sci. 82 (1999), 589-596). Secondly, flowering in many plants is associated with an uncontrollable gene flow from cultivated to wild relatives via the active spread of pollen. Systems to control flowering will provide a means to avoid spread of pollen, e.g. in the grass field, and thus provide systems for biological containment of transgenes. Thirdly, flowering in many perennial plants is also associated with an exposure to pollen allergens, such as grass pollen allergens. A cultivar, in particular a grass cultivar, with an extended vegetative growth associated with decreased or even eliminated inflorescence production would thus be attractive to agriculture and society.
It would be desirable to have methods of controlling plant life cycles and growth phases, e.g. the transition from the vegetative to the reproductive stage, flowering processes, and inflorescence and flower development in plants, including dicots and monocots and in particular including grass species such as ryegrasses (Lolium species) and fescues (Festuca species). This would facilitate the production of, for example, pasture and turf grasses with enhanced or shortened or modified life cycles, enhanced or reduced or otherwise modified inflorescence and flower development, male and female sterility, inhibited flowering (e.g. non-flowering), modified flowering architecture (e.g. indeterminate and determinate), earlier or delayed flowering, enhanced or modified number of leaves, enhanced or reduced or otherwise modified number of reproductive shoots, enhanced persistence and improved herbage quality, enhanced seed and leaf yield, altered growth and development, leading to improved seed production, improved biomass production, improved pasture production, and improved pasture quality.
The life cycle of flowering plants in general can be divided into three growth phases: vegetative, inflorescence, and floral (Poethig, Science 250 (1990), 923-930). In the vegetative phase, the shoot apical meristem (SAM) generates leaves that will later ensure the resources necessary to produce fertile offspring. Upon receiving the appropriate environmental and developmental signals, the plant switches to floral, or reproductive, growth and the SAM enters the inflorescence phase (I1) and gives rise to an inflorescence with flower primordia. During this phase, the fate of the SAM and the secondary shoots that arise in the axils of the leaves is determined by a set of meristem identity genes, some of which prevent and some of which promote the development of floral meristems.
The regulation of meristem identity and floral transition has been investigated in a number of dicotyledonous plants including Arabidopsis, Antirrhinum, tomato, and tobacco. However, in agronomically important seed crops such as wheat, barley, rice, forage grasses, and other monocotyledonous plants, information on the genetic regulation of floral transition is still limited.
Perennial ryegrass will not flower unless it receives a vernalisation period. This cold treatment is required to alleviate a natural flowering “roadblock” that ensures that flowering occurs in the spring. In growth chamber conditions, flowering in perennial ryegrass is induced by a vernalization period of 12 to 14 weeks below 5° C. followed by secondary induction with long-day photoperiods (generally, more daylight hours than dark hours and, more specifically, LD, 16 hours of light, 8 hours of darkness) and temperatures above 15 to 20° C.
The TERMINAL FLOWER 1 (TFL1) gene from Arabidopsis thaliana has been identified to specify an indeterminate identity of inflorescence meristems. Mutations in TFL1 result in the conversion of the inflorescence into a terminal flower (Shannon and Meeks-Wagner, Plant Cell 3 (1991), 877-892), and in addition, TFL1 has been found to extend the vegetative growth phase of Arabidopsis (Shanon and Meeks-Wagner, loc. cit.; Ratcliff et al., Development 126 (1998), 1109-1120). TFL1 proteins have sequence similarity with mammalian phosphatidylethanolamine-binding proteins (PEBPs).
Previously, Arabidopsis thaliana, red fescue (Festuca rubra L.), and perennial ryegrass (Lolium perenne L) were transformed with LpTFL1 and it was shown that overexpression of LpTFL1 results in a dramatic extension of the vegetative growth phase correlating to the level of gene expression with the highest expressing lines remaining non-flowering (Jensen et al., Plant Physiol. 125 (2001), 1517-1528; Jensen et al., Mol. Breeding 13 (2004), 37-48; P11792US-20030226). In addition, it was shown that LpTFL1 is capable of preventing flowering in red fescue and perennial ryegrass in subsequent years.
FLC/FLF represents a major floral inhibitor in Arabidopsis integrating several of the floral inductive pathways and was recently identified as a MADS box protein (Michaels and Amasino, Plant Cell 11 (1999), 949-956; Sheldon et al., Plant Cell 11 (1999), 445-458). The use of FLC/FLF to alter the flowering time/behavior in plants has been described in WO 00/50615 and WO 00/32780.
The INDETERMINATE1 (ID1) gene of maize controls the transition of flowering in this species by encoding a transcriptional regulator of the floral transition (Colasanti et al., Cell 93 (1998), 513-603). In this study, ID1 was identified by a mutation with the phenotype of dramatic reduction in the ability of maize to undergo the transition to reproductive growth. Homozygous id1 maize mutant plants produced many more leaves than did wild-type maize plants, and remained in a prolonged vegetative growth state. The use of the maize ID1 gene as a method for producing plants with altered time of floral transition has been suggested, but not demonstrated, in WO 96/34088. In WO 02/38768, a method of using ID1 homologues isolated from perennial ryegrass to modify plant life cycles and/or growth phases has been suggested, in which the use of 3 different polynucleotide sequences and their corresponding polypeptides isolated from perennial ryegrass is described. The described peptides belong to the group of Zink Finger proteins characterized by the presence of two conserved so called Zink Finger domains, as does the maize ID1.
The CONSTANS gene (CO) encodes a polypeptide belonging to the group of Zink Finger proteins and has been shown to represent the major regulator of the photoperiodic floral pathway in Arabidopsis (Putterill et al., Cell 80 (1995), 847-857). The use of CO to influence the flowering characteristics of plants has been described in WO 96/14414.
FT, The FLOWERING LOCUS T (FT) gene belongs to the family of plant PEBP genes as does TFL1, but has been shown to play a role opposite to TFL1 in mediating flower inducing signals in Arabidopsis (Kardailsky et al., Science 286 (1999), 1962-1965; Kobayashi et al., Science 286 (1999), 1960-1962). A method of modulating flowering time in a plant by the use of FT has been suggested in U.S. Pat. No. 6,225,530.
LEAFY (LFY) is a unique gene with little homology to other gene classes whereas APETALA1 (AP1) belongs to the MADS box family. Together they represent members of the group of ‘meristem identity genes’, specifying floral meristem identity (Weigel et al., Cell 69 (1992), 843-859; Mandel et al., Nature 360 (1992), 273-277). The use of LFY to control floral meristem development and enhance or delay flowering in a plant has been suggested in WO 96/19105 and U.S. Pat. No. 5,844,119. The use of AP1 to manipulate flowering time in a plant has been suggested in WO 97/46078 and U.S. Pat. No. 5,844,119. The use of MADS box proteins to manipulate flowering and plant architecture of Lolium or Festuca plant species has been suggested in WO 02/33091.
The technical problem underlying the present invention is the provision of means and methods allowing to control flowering in plants, preferably in forage grasses such as ryegrasses and fescues.
This technical problem is solved by the provision of the embodiments as characterized in the claims.
Accordingly, in a first aspect the present invention relates to polynucleotides which, when expressed in sense orientation in plants lead to a prevention of flowering, selected from the group consisting of

    (a) polynucleotides comprising a nucleotide sequence encoding a polypeptide with the amino acid sequence of SEQ ID NO:2;
    (b) polynucleotides comprising the coding region of the nucleotide sequence shown in SEQ ID NO:1;
    (c) polynucleotides comprising a nucleotide sequence encoding a fragment of the polypeptide encoded by a polynucleotide of (a) or (b), wherein said nucleotide sequence when expressed in sense orientation in plants leads to a prevention of flowering;
    (d) polynucleotides comprising a nucleotide sequence having a sequence identity of at least 50% with a polynucleotide of any one of (a) to (c) and which when expressed in sense orientation in plants leads to a prevention of flowering;
    (e) polynucleotides comprising a nucleotide sequence the complementary strand of which hybridizes to the polynucleotide of any one of (a) to (c), wherein said nucleotide sequence when expressed in sense orientation in plants leads to a prevention of flowering; and
    (f) polynucleotides comprising a nucleotide sequence that deviates from the nucleotide sequence defined in (e) by the degeneracy of the genetic code.


Thus, the present invention relates in a first aspect to a polynucleotide which when expressed in sense orientation in plants leads to a prevention of flowering. Preferably, such a polynucleotide comprises the coding region of the nucleotide sequence shown in SEQ ID NO:1 or encode a polypeptide comprising the amino acid sequence shown in SEQ ID NO:2.
The present invention is based on the identification of a gene from Lolium perenne which leads to a prevention of flowering in plants when expressed in sense orientation.
This gene, of which the cDNA sequence is shown in SEQ ID NO:1 and the derived amino acid sequence is shown in SEQ ID NO:2, will be referred to in the following as LpFT-like gene and protein, respectively. This gene was identified by PCR amplification based on a Lolium perenne cDNA library and by using primers based on known FT-like genes (see Example 1).
The present invention in particular relates to polynucleotides containing the nucleotide sequence indicated under SEQ ID NO:1, encoding the amino acid sequence shown under SEQ ID NO:2 or a part thereof which, when expressed in sense orientation in plants leads to a prevention of flowering.
The term “prevention of flowering” means the reduction, delay or complete inhibition of flowering. “Flowering” means the development of female and/or male floral and/or reproductive organs, such as floral meristems, inflorescences, spikelets, sepals, petals, carpels, stamens, embryos, pollen, seeds, etc. A reduction of flowering” means the lack of a complete set of the above-mentioned female and/or male floral and/or reproductive organs. The term “delay of flowering” means that flowering occurs at a later time point in comparison to wild-type, i.e. not genetically-modified, plants grown under the same conditions. A “later time point” preferably means at least 20 days later, more preferably 40 days later, even more preferably 60 days later, particularly preferred at least 100 days later, especially preferred at least 200 days later and most preferably at least 600 days later.
The term “complete inhibition of flowering” means that no organs or tissue associated with sexual reproduction are developed while the plant continues to produce vegetative tissues.
Moreover, the present invention relates to polynucleotides the complementary strand of which hybridizes with a polynucleotide mentioned in sections (a) to (c), above, and which when expressed in sense orientation in plants, lead to a prevention of flowering.
The present invention also relates to polynucleotides which encode a polypeptide, which has a homology, that is to say a sequence identity, of at least 60%, preferably of at least 70%, more preferably of at least 75%, even more preferably of at least 80% and particularly preferred of at least 85%, especially preferred of at least 90% and even more preferred of at least 95, 96, 97, 98 or 99% to the entire amino acid sequence indicated in SEQ ID NO: 2, the polypeptide leading to a prevention of flowering when expressed in a plant.
Moreover, the present invention relates to polynucleotides the nucleotide sequence of which has a homology, that is to say a sequence identity, of at least 50%, preferably of at least 60%, more preferably of at least 70%, even more preferably of more than 80%, in particular of at least 85%, especially preferred of at least 90%, in particular of at least 95% and even more preferred of at least 98% when compared to the coding region of the sequence shown in SEQ ID NO: 1, and which when expressed in sense orientation in plants, lead to a prevention of flowering.
The present invention also relates to polynucleotides the sequence of which deviates from the nucleotide sequences of the above-described polynucleotides due to the degeneracy of the genetic code.
The invention also relates to polynucleotides comprising a nucleotide sequence which is complementary to the whole or a part of one of the above-mentioned sequences.
In the context of the present invention the term “hybridization” means hybridization under conventional hybridization conditions, preferably under stringent conditions, as for instance described in Sambrook and Russell (2001), Molecular Cloning: A Laboratory Manual, CSH Press, Cold Spring Harbor, N.Y., USA. In an especially preferred embodiment, the term “hybridization” means that hybridization occurs under the following conditions:
Hybridization buffer: 2×SSC; 10×Denhardt solution (Fikoll 400+PEG+BSA; ratio 1:1:1); 0.1% SDS; 5 mM EDTA; 50 mM Na2HPO4;

    
    
        250 μg/ml of herring sperm DNA; 50 μg/ml of tRNA; or
        0.25 M of sodium phosphate buffer, pH 7.2;
        1 mM EDTA
        7% SDS
    
    


Hybridization temperature T=60° C.
Washing buffer: 2×SSC; 0.1% SDS
Washing temperature T=60° C.
In another preferred embodiment, stringent conditions are selected to be about 5° C. to 20° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched sequence (probe). Preferably, the Tm values of the sequences, i.e. the sequences according to the invention described above and the hybridizing sequences, are within 10° C. of each other if they are mixed together and denatured simultaneously. More preferably hybridization may be performed under stringent conditions, e.g., for a specified period of time at a temperature of between 50 and 70° C. in double strength SSC (2×NaCl 17.5 g/l and sodium citrate (SC) at 8.8 g/l) buffered saline containing 0.1% sodium dodecyl sulphate (SDS) followed by washing at the same temperature but with a buffer having a reduced SSC concentration. Depending upon the degree of stringency required, and thus the degree of similarity of the sequences, such reduced concentration buffers are typically single strength SSC containing 0.1% SDS, half strength SSC containing 0.1% SDS and one tenth strength SSC containing 0.1% SDS. In a preferred embodiment hybridization is carried out with one of the sequences being fixed to a support, e.g., a filter or Nylon membrane.
Sequences having the highest degree of similarity are those the hybridization of which is least affected by washing in buffers of reduced concentration. It is most preferred that the hybridizing sequences are so similar to the above described sequences according to the invention that the hybridization between them is substantially unaffected by washing or incubation at high stringency, for example, in one tenth strength sodium citrate buffer containing 0.1% SDS.
Polynucleotides which hybridize with the polynucleotides disclosed in connection with the invention can for instance be isolated from genomic libraries or cDNA libraries of plants, in particular from the family of Poaceae, preferably from a grass species such as from Phleum spp., Dactylis spp., Lolium spp., Festulolium spp., Festuca spp., Poa spp., Bromus spp., Agrostis spp., Arrhenatherum spp., Phalaris spp., Brachypodium ssp. and Trisetum spp., for example, Phleum pratense, Phleum bertolonii, Dactylis glomerata, Lolium perenne, Lolium multiflorum, Lolium multiflorum westervoldicum, Festulolium braunii, Festulolium loliaceum, Festulolium holmbergii, Festulolium pabulare, Festuca pratensis, Festuca rubra, Festuca rubra rubra, Festuca rubra commutata, Festuca rubra trichophylla, Festuca duriuscula, Festuca ovina, Festuca arundinacea, Poa trivialis, Poa pratensis, Poa palustris, Bromus catharticus, Bromus sitchensis, Bromus inermis, Deschampsia caespitose, Agrostis capilaris, Agrostis stolonifera, Arrhenatherum elatius, Phalaris arundinacea, Brachypodium distachyon and Trisetum flavescens. Most preferably the polynucleotide according to the invention is from Lolium perenne. 
Such hybridizing polynucleotides may be identified and isolated by using the polynucleotides described hereinabove or parts or reverse complements thereof, for instance by hybridization according to standard methods (see for instance Sambrook and Russell (2001), Molecular Cloning: A Laboratory Manual, CSH Press, Cold Spring Harbor, N.Y., USA). Polynucleotides comprising the same or substantially the same nucleotide sequence as indicated in SEQ ID NO: 1 or parts thereof can, for instance, be used as hybridization probes. The fragments used as hybridization probes can also be synthetic fragments which are prepared by usual synthesis techniques, and the sequence of which is substantially identical with that of a polynucleotide according to the invention.
The molecules hybridizing with the polynucleotides of the invention also comprise fragments, derivatives and allelic variants of the above-described polynucleotides having the same function. Function means with respect to SEQ ID NO: 1 that it leads to a prevention of flowering when expressed in sense orientation in plants.
Herein, fragments are understood to mean parts of the polynucleotides which are long enough to show the same function. In this context, the term derivative means that the sequences of these molecules differ from the sequences of the above-described polynucleotides in one or more positions and show a high degree of homology to these sequences, preferably within sequence ranges that are essential for their function.
Preferably, the degree of homology is determined by comparing the respective sequence with the nucleotide sequence of the coding region of SEQ ID NO: 1. When the sequences which are compared do not have the same length, the degree of homology preferably refers to the percentage of nucleotide residues in the shorter sequence which are identical to nucleotide residues in the longer sequence. The degree of homology can be determined conventionally using known computer programs such as the DNASTAR program with the ClustalW analysis. This program can be obtained from DNASTAR, Inc., 1228 South Park Street, Madison, Wis. 53715 or from DNASTAR, Ltd., Abacus House, West Ealing, London W13 0AS UK (support@dnastar.com) and is accessible at the server of the EMBL outstation.
When using the Clustal analysis method to determine whether a particular sequence is, for instance, 80% identical to a reference sequence the settings are preferably as follows: Matrix: blosum 30; Open gap penalty: 10.0; Extend gap penalty: 0.05; Delay divergent: 40; Gap separation distance: 8 for comparisons of amino acid sequences. For nucleotide sequence comparisons, the Extend gap penalty is preferably set to 5.0.
Alternative programs which are used for database searching and sequence alignment and comparison, for example, from the Wisconsin Package Version 10.2, such as BLAST, FASTA, PILEUP, FINDPATTERNS or the like (GCG, Madison, Wis.) or public available sequence databases such as GenBank, EMBL, Swiss-Prot and PIR or private sequence databases such as PhytoSeq (Incyte Pharmaceuticals, Palo Alto, Calif.) may be used to determine sequence identity. The Alignment for sequence comparison may be conducted by the local homology algorithm of Smith and Waterman (Adv. Appl. Math. 2 (1981), 482), by the homology alignment algorithm of Needleman and Wunsch (J. Mol. Biol. 48 (1970), 443), by the search for similarity method of Pearson and Lipman (Proc. Natl. Acad. Sci. USA. 85 (1988) 2444), by computerized implementations of these algorithms.
Preferably, the degree of homology of the hybridizing polynucleotide is calculated over the complete length of its coding sequence. It is furthermore preferred that such a hybridizing polynucleotide, and in particular the coding sequence comprised therein, has a length of at least 200 nucleotides, preferably at least 400 nucleotides, more preferably of at least 600 nucleotides, even more preferably of at least 800 nucleotides and most preferably of at least 1000 nucleotides.
Preferably, sequences hybridizing to a polynucleotide according to the invention comprise a region of homology of at least 90%, preferably of at least 93%, more preferably of at least 95%, still more preferably of at least 98% and particularly preferred of at least 99% identity to an above-described polynucleotide, wherein this region of homology has a length of at least 400 nucleotides, more preferably of at least 600 nucleotides, even more preferably of at least 800 nucleotides and most preferably of at least 1000 nucleotides.
Homology, moreover, means that there is a functional and/or structural equivalence between the corresponding polynucleotides or the polypeptides encoded thereby. Polynucleotides which are homologous to the above-described molecules and represent derivatives of these molecules are normally variations of these molecules which represent modifications having the same biological function. They may be either naturally occurring variations, preferably orthologs of a polynucleotide comprising the nucleotide sequence of SEQ ID NO:1, for instance sequences from other alleles, ecotypes, varieties, species, etc., or mutations, and said mutations may have formed naturally or may have been produced by deliberate mutagenesis. The variants, for instance allelic variants, may be naturally occurring variants or variants produced by chemical synthesis or variants produced by recombinant DNA techniques or combinations thereof. Deviations from the above-described polynucleotides may have been produced, e.g., by deletion, substitution, insertion and/or recombination.
The polypeptides encoded by the different variants of the polynucleotides of the invention possess certain characteristics they have in common with the polypeptide comprising the amino acid sequence of SEQ ID NO:2. These include for instance biological activity, molecular weight, immunological reactivity, conformation, etc., and physical properties, such as for instance the migration behavior in gel electrophoreses, chromatographic behavior, sedimentation coefficients, solubility, spectroscopic properties, stability, pH optimum, temperature optimum etc.
The polynucleotides of the invention can be DNA molecules, in particular genomic DNA or cDNA. Moreover, the polynucleotides of the invention may be RNA molecules. The polynucleotides of the invention can be obtained for instance from natural sources or may be produced synthetically or by recombinant techniques, such as PCR.
In a further aspect, the present invention relates to recombinant nucleic acid molecules comprising a polynucleotide of the invention described above. The term “recombinant nucleic acid molecule” refers to a nucleic acid molecule which contains in addition to a polynucleotide of the invention as described above at least one further heterologous coding or non-coding nucleotide sequence. The term “heterologous” means that said nucleotide sequence originates from a different species or from the same species, however, from a different location in the genome than said polynucleotide to which it is added. The term “recombinant” implies that nucleotide sequences are combined into one nucleic acid molecule by the aid of human intervention. The recombinant nucleic acid molecule of the invention can be used alone or as part of a vector.
For instance, the recombinant nucleic acid molecule may encode the polypeptide encoded by a polynucleotide according to the invention fused to a marker sequence, such as a peptide which facilitates purification of the fused polypeptide. The marker sequence may for example be a hexa-histidine peptide, such as the tag provided in a pQE vector (Qiagen, Inc.) which provides for convenient purification of the fusion polypeptide. Another suitable marker sequence may be the HA tag which corresponds to an epitope derived from influenza hemagglutinin polypeptide (Wilson, Cell 37 (1984), 767). As a further example, the marker sequence may be glutathione-S-transferase (GST) which, apart from providing a purification tag, enhances polypeptide stability, for instance, in bacterial expression systems. If it furthermore preferred that the marker sequence contains a protease cleavage site such as the thrombin cleavage site allowing to remove the marker sequence or a part of it from the expressed polypeptide.
In a preferred embodiment, the recombinant nucleic acid molecules further comprises expression control sequences operably linked to the polynucleotide comprised by the recombinant nucleic acid molecule, more preferably these recombinant nucleic acid molecules are expression cassettes. The term “operably linked” (or “operatively linked”), as used throughout the present description, refers to a linkage between one or more expression control sequences and the coding region or parts of the polynucleotide to be expressed in such a way that expression is achieved under conditions compatible with the expression control sequence(s).
Expression comprises transcription of the heterologous DNA sequence into an RNA sequence, which may be a translatable or a non-translatable RNA sequence. Examples for non-translatable RNA molecules are antisense molecules, cosuppression molecules, ribozymes or RNAi molecules. These embodiments are described in more detail below in connection with the transgenic plant cells according to the invention. Preferably expression means transcription into a translatable mRNA. Regulatory elements ensuring expression in prokaryotic as well as in eukaryotic cells, preferably in plant cells, are well known to those skilled in the art. They encompass promoters, enhancers, termination signals, targeting signals and the like. Examples are given further below in connection with explanations concerning vectors. In the case of eukaryotic cells, expression control sequences may comprise poly-A signals ensuring termination of transcription and stabilization of the transcript, for example, those of the 35S RNA from Cauliflower Mosaic Virus (CaMV) or the nopaline synthase gene from Agrobacterium tumefaciens. Additional regulatory elements may include transcriptional as well as translational enhancers. A plant translational enhancer often used is the CaMV omega sequence. Similarly, the inclusion of an intron (e.g. intron-1 from the shrunken gene of maize) has been shown to increase expression levels by up to 100-fold (Mait, Transgenic Research 6 (1997), 143-156; Ni, Plant Journal 7 (1995), 661-676).
Moreover, the invention relates to vectors, in particular plasmids, cosmids, viruses, bacteriophages and other vectors commonly used in genetic engineering, which contain a polynucleotide or recombinant nucleic acid molecule of the invention as described above. In a preferred embodiment of the invention, the vectors are suitable for the transformation of bacterial cells, yeast cells, fungal cells, animal cells or, in particular, plant cells. In a particularly preferred embodiment such vectors are suitable for stable transformation of plants.
In a preferred embodiment, the vectors further comprise expression control sequences operably linked to said polynucleotides contained in the vectors. These expression control sequences may be suited to ensure transcription and synthesis of a translatable RNA in prokaryotic or eukaryotic cells.
The expression of the polynucleotides of the invention in prokaryotic or eukaryotic cells, for instance in Escherichia coli, is interesting because it permits a more precise characterization of the biological activities of the encoded polypeptide. In addition, it is possible to insert different mutations into the polynucleotides encoding the polypeptide by methods usual in molecular biology (see for instance Sambrook and Russell (2001), Molecular Cloning: A Laboratory Manual, CSH Press, Cold Spring Harbor, N.Y., USA), leading to the synthesis of polypeptides possibly having modified biological properties. In this regard, it is on the one hand possible to produce deletion mutants in which polynucleotides are produced by progressive deletions from the 5′ or 3′ end of the coding DNA sequence, and said polynucleotides lead to the synthesis of correspondingly shortened polypeptides.
Furthermore, the introduction of point mutations is also conceivable at positions at which a modification of the amino acid sequence for instance influences the biological activity or the regulation of the polypeptide.
In the case of expression in plants, plant tissue or plant cells, the introduction of mutations into the polynucleotides of the invention allows the gene expression rate and/or the activity of the polypeptides encoded by the polynucleotides of the invention to be reduced or increased.
For genetic engineering in prokaryotic cells, the polynucleotides of the invention or parts of these molecules can be introduced into plasmids which permit mutagenesis or sequence modification by recombination of DNA sequences. Standard methods (see Sambrook and Russell (2001), Molecular Cloning: A Laboratory Manual, CSH Press, Cold Spring Harbor, N.Y., USA) allow base exchanges to be performed or natural or synthetic sequences to be added. DNA fragments can be connected to each other by applying adapters and linkers to the fragments. Moreover, engineering measures which provide suitable restriction sites or remove surplus DNA or restriction sites can be used. In those cases, in which insertions, deletions or substitutions are possible, in vitro mutagenesis, “primer repair”, restriction or ligation can be used. In general, a sequence analysis, restriction analysis and other methods of biochemistry and molecular biology are carried out as analysis methods.
Additionally, the present invention relates to, a method for producing genetically engineered host cells comprising introducing the above-described polynucleotides, recombinant nucleic acid molecules or vectors of the invention into a host cell.
Another embodiment of the invention relates to host cells, in particular prokaryotic or eukaryotic cells, genetically engineered with the above-described polynucleotides, recombinant nucleic acid molecules or vectors of the invention or obtainable by the above-mentioned method for producing genetically engineered host cells, and to cells derived from such transformed cells and containing a polynucleotide, recombinant nucleic acid molecule or vector of the invention. In a preferred embodiment the host cell is genetically modified in such a way that it contains said polynucleotide stably integrated into the genome. The term “genetically modified” implies that the polynucleotide of the invention contained in the host cell is “heterologous” (or as used synonymously herein “foreign”) with respect to the host cell. This means that said polynucleotide does not occur naturally in the host cell or that it is present in the host cell at a location in the genome different from the location of the corresponding naturally occurring polynucleotide, if present. Preferentially, the host cell of the invention is a bacterial, yeast, fungus, plant or animal (e.g. insect or vertebrate such as mammalian) cell. In a preferred embodiment, the host cell of the invention is a plant cell which may include any conceivable type of plant cell, such as cultured or non-cultured cells, protoplasts, suspension culture cells, callus cells, meristem cells, cells being part of a plant tissue, plant organ and/or plant.
More preferably the polynucleotide can be expressed so as to lead to the production of a polypeptide. An overview of different expression systems is for instance contained in Methods in Enzymology 153 (1987), 385-516, in Bitter et al. (Methods in Enzymology 153 (1987), 516-544) and in Sawers et al. (Applied Microbiology and Biotechnology 46 (1996), 1-9), Billman-Jacobe (Current Opinion in Biotechnology 7 (1996), 500-4), Hockney (Trends in Biotechnology 12 (1994), 456-463), Griffiths et al., (Methods in Molecular Biology 75 (1997), 427-440). An overview of yeast expression systems is for instance given by Hensing et al. (Antonie van Leuwenhoek 67 (1995), 261-279), Bussineau et al. (Developments in Biological Standardization 83 (1994), 13-19), Gellissen et al. (Antonie van Leuwenhoek 62 (1992), 79-93, Fleer (Current Opinion in Biotechnology 3 (1992), 486-496), Vedvick (Current Opinion in Biotechnology 2 (1991), 742-745) and Buckholz (Bio/Technology 9 (1991), 1067-1072).
Expression vectors have been widely described in the literature. As a rule, they contain not only a selection marker gene and a replication-origin ensuring replication in the host selected, but also a bacterial or viral promoter, and in most cases a termination signal for transcription. Between the promoter and the termination signal there is in general at least one restriction site or a polylinker which enables the insertion of a coding DNA sequence. The DNA sequence naturally controlling the transcription of the corresponding gene can be used as the promoter sequence, if it is active in the host organism used. However, this sequence can also be exchanged for other promoter sequences. It is possible to use promoters ensuring constitutive expression of the gene and inducible promoters which permit a deliberate control of the expression of the gene. Bacterial and viral promoter sequences possessing these properties are described in detail in the literature. Regulatory sequences for the expression in microorganisms (for instance E. coli, S. cerevisiae) are sufficiently described in the literature. Promoters permitting a particularly high expression of a downstream sequence are for instance the T7 promoter (Studier et al., Methods in Enzymology 185 (1990), 60-89), lacUV5, trp, trp-lacUV5 (DeBoer et al., in Rodriguez and Chamberlin (Eds), Promoters, Structure and Function; Praeger, N.Y., (1982), 462-481; DeBoer et al., Proc. Natl. Acad. Sci. USA (1983), 21-25), Ip1, rac (Boros et al., Gene 42 (1986), 97-100). Inducible promoters are preferably used for the synthesis of polypeptides. These promoters often lead to higher polypeptide yields than do constitutive promoters. In order to obtain an optimum amount of polypeptide, a two-stage process is often used. First, the host cells are cultured under optimum conditions up to a relatively high cell density. In the second step, transcription is induced depending on the type of promoter used. In this regard, a tac promoter is particularly suitable which can be induced by lactose or IPTG (=isopropyl-β-D-thiogalactopyranoside) (deBoer et al., Proc. Natl. Acad. Sci. USA 80 (1983), 21-25). Termination signals for transcription are also described in the literature.
In another preferred embodiment the polynucleotide according to the invention can be expressed so as to lead to the production of a non-translatable RNA. Examples for non-translatable RNA molecules are antisense molecules, cosuppression molecules, ribozymes or RNAi molecules. These embodiments are described in more detail below in connection with the transgenic plants and plant cells according to the invention.
The transformation of the host cell with a polynucleotide, recombinant nucleic acid molecule or vector according to the invention can be carried out by standard methods, as for instance described in Sambrook and Russell (2001), Molecular Cloning: A Laboratory Manual, CSH Press, Cold Spring Harbor, N.Y., USA; Methods in Yeast Genetics, A Laboratory Course Manual, Cold Spring Harbor Laboratory Press, 1990. The host cell is cultured in nutrient media meeting the requirements of the particular host cell used, in particular in respect of the pH value, temperature, salt concentration, aeration, antibiotics, vitamins, trace elements etc. The polypeptide according to the present invention can be recovered and purified from recombinant cell cultures by methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Polypeptide refolding steps can be used, as necessary, in completing configuration of the polypeptide. Finally, high performance liquid chromatography (HPLC) can be employed for final purification steps.
Accordingly, the present invention also relates to a method for the production of a polypeptide encoded by a polynucleotide of the invention as described above in which the above-mentioned host cell is cultivated under conditions allowing for the expression of the polypeptide and in which the polypeptide is isolated from the cells and/or the culture medium.
Moreover, the invention relates to a polypeptide which is encoded by a polynucleotide according to the invention or obtainable by the above-mentioned method for the production of a polypeptide encoded by a polynucleotide of the invention.
The polypeptide of the present invention may, e.g., be a naturally purified product or a product of chemical synthetic procedures or produced by recombinant techniques from a prokaryotic or eukaryotic host (for example, by bacterial, yeast, higher plant, insect or mammalian cells in culture). Depending upon the host employed in a recombinant production procedure, the polypeptide of the present invention may be glycosylated or may be non-glycosylated. The polypeptide of the invention may also include an initial methionine amino acid residue. The polypeptide according to the invention may be further modified to contain additional chemical moieties normally not being part of the polypeptide. Those derivatized moieties may, e.g., improve the stability, solubility, the biological half life or absorption of the polypeptide. The moieties may also reduce or eliminate any undesirable side effects of the polypeptide and the like. An overview for these moieties can be found, e.g., in Remington's Pharmaceutical Sciences (18th ed., Mack Publishing Co., Easton, Pa. (1990)). Polyethylene glycol (PEG) is an example for such a chemical moiety which has been used for the preparation of therapeutic polypeptides. The attachment of PEG to polypeptides has been shown to protect them against proteolysis (Sada et al., J. Fermentation Bioengineering 71 (1991), 137-139). Various methods are available for the attachment of certain PEG moieties to polypeptides (for review see: Abuchowski et al., in “Enzymes as Drugs”; Holcerberg and Roberts, eds. (1981), 367-383). Generally, PEG molecules or other additional moieties are connected to the polypeptide via a reactive group found on the polypeptide. Amino groups, e.g. on lysines or the amino terminus of the polypeptide are convenient for this attachment among others.
Furthermore, the present invention also relates to an antibody specifically recognizing a polypeptide according to the invention. The antibody can be monoclonal or polyclonal and can be prepared according to methods well known in the art. The term “antibody” also comprises fragments of an antibody which still retain the binding specificity.
The polypeptide according to the invention, its fragments or other derivatives thereof, or cells expressing them can be used as an immunogen to produce antibodies thereto. The present invention in particular also includes chimeric, single chain, and humanized antibodies, as well as Fab fragments, or the product of an Fab expression library. Various procedures known in the art may be used for the production of such antibodies and fragments.
Antibodies directed against a polypeptide according to the present invention can be obtained, e.g., by direct injection of the polypeptide into an animal or by administering the polypeptide to an animal, preferably a non-human animal. The antibody so obtained will then bind the polypeptide itself. In this manner, even a sequence encoding only a fragment of the polypeptide can be used to generate antibodies binding the whole native polypeptide. Such antibodies can then, e.g., be used to isolate the polypeptide from tissue expressing that polypeptide or to detect it in a probe. For the preparation of monoclonal antibodies, any technique which provides antibodies produced by continuous cell line cultures can be used. Examples for such techniques include the hybridoma technique (Köhler and Milstein, Nature 256 (1975), 495-497), the trioma technique, the human B-cell hybridoma technique (Kozbor et al., Immunology Today 4 (1983), 72) and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. (1985), 77-96). Techniques describing the production of single chain antibodies (e.g., U.S. Pat. No. 4,946,778) can be adapted to produce single chain antibodies to immunogenic polypeptides according to the present invention. Furthermore, transgenic mice may be used to express humanized antibodies directed against immunogenic polypeptides of the present invention.
In a further preferred embodiment, the invention relates to nucleic acid molecules specifically hybridizing with a polynucleotide of the invention or with a complementary strand of such a polynucleotide.
Such hybridizing nucleic acid molecules may be oligonucleotides having a length preferably of at least 10, in particular at least 15, and particularly preferably of at least 50 nucleotides. Advantageously, their length does not exceed a length of 1000, preferably 500, more preferably 200, still more preferably 100 and most preferably 50 nucleotides. They are characterized in that they specifically hybridize to the polynucleotides of the invention, that is to say that they only to a very minor extent and preferably not at all hybridize to polynucleotides encoding another polypeptide. The hybridizing nucleic acid molecules according to this embodiment can be used for instance as primers for amplification techniques such as PCR or as a hybridization probe for instance in order to isolate related genes. The hybridization conditions and homology values described above in connection with the polynucleotides of the invention may likewise apply in connection with the specifically hybridizing nucleic acid molecules mentioned herein.
Furthermore, the invention relates to a method for producing a transgenic plant comprising the steps of

    (a) introducing at least one of the above-described polynucleotides, recombinant nucleic acid molecules or vectors of the invention into the genome of a plant cell; and
    (b) regenerating the cell of (a) to a transgenic plant.


Optionally, the method may further comprise step (c) producing progeny from the plants produced in step (b).
In a further aspect, the invention relates to transgenic plants or plant tissue comprising plant cells which are genetically engineered with a polynucleotide of the invention and/or which contain the recombinant nucleic acid molecule or the vector of the invention and to transgenic plants obtainable by the method mentioned above.
Preferably, in the transgenic plant of the invention, the polynucleotide of the invention is expressed at least in one part, i.e. organ, tissue or cell type, of the plant.
The transgenic plants containing the polynucleotides according to the present invention related to SEQ ID NO: 1 or a recombinant nucleic acid molecule or vector containing such a polynucleotide show preferably an altered amount of the corresponding encoded polypeptides and, as a consequence, an altered flowering behaviour. The amount of the protein may be increased or reduced depending on whether a translatable or non-translatable RNA is expressed.
Preferably, the transgenic plants, plant tissue or plant cells are characterized by an increase of the amount of transcript corresponding to the polynucleotide of the invention by at least 20%, preferably at least 50% and more preferably at least 100% as compared to the corresponding wild-type plant, plant tissue or plant cell. Likewise, it is preferred that transgenic plants, plant tissues or plant cells are characterized by an increase of the protein amount of the polypeptide of the invention by at least 20%, preferably at least 50% and more preferably at least 100% as compared to the corresponding wild-type plant, plant tissues or plant cells.
Alternatively, the transgenic plants, plant tissues or plant cells are characterized by a reduction of the amount of transcript corresponding to the polynucleotide of the invention by at least 20%, preferably by at least 50% and more preferably by at least 80% as compared to the corresponding wild-type plant, plant tissue or plant cell. Likewise, it is preferred that transgenic plants, plant tissues or plant cells are characterized by a decrease of the protein amount of the polypeptide of the invention by at least 20%, preferably at least 50% and more preferably at least 80% as compared to the corresponding wild-type plant, plant tissues or plant cells.
According to the provisions of the invention, transgenic plants can be prepared by introducing a polynucleotide into plant cells and regenerating the transformed cells to plants by methods well known to the person skilled in the art.
Methods for the introduction of foreign genes into plants are also well known in the art. These include, for example, the transformation of plant cells or tissues with T-DNA using Agrobacterium tumefaciens or Agrobacterium rhizogenes, the fusion of protoplasts, direct gene transfer (see, e.g., EP-A 164 575), injection, electroporation, vacuum infiltration, biolistic methods like particle bombardment, pollen-mediated transformation, plant RNA virus-mediated transformation, liposome-mediated transformation, transformation using wounded or enzyme-degraded immature embryos, or wounded or enzyme-degraded embryogenic callus and other methods known in the art. The vectors used in the method of the invention may contain further functional elements, for example “left border”- and “right border”-sequences of the T-DNA of Agrobacterium which allow stable integration into the plant genome. Furthermore, methods and vectors are known to the person skilled in the art which permit the generation of marker free transgenic plants, i.e. the selectable or scorable marker gene is lost at a certain stage of plant development or plant breeding. This can be achieved by, for example co-transformation (Lyznik, Plant Mol. Biol. 13 (1989), 151-161; Peng, Plant Mol. Biol. 27 (1995), 91-104) and/or by using systems which utilize enzymes capable of promoting homologous recombination in plants (see, e.g., WO97/08331; Bayley, Plant Mol. Biol. 18 (1992), 353-361); Lloyd, Mol. Gen. Genet. 242 (1994), 653-657; Maeser, Mol. Gen. Genet. 230 (1991), 170-176; Onouchi, Nucl. Acids Res. 19 (1991), 6373-6378). Methods for the preparation of appropriate vectors are described by, e.g., Sambrook and Russell (2001), Molecular Cloning: A Laboratory Manual, CSH Press, Cold Spring Harbor, N.Y., USA.
Suitable strains of Agrobacterium tumefaciens and vectors as well as transformation of Agrobacteria and appropriate growth and selection media are well known to those skilled in the art and are described in the prior art (GV3101 (pMK90RK), Koncz, Mol. Gen. Genet. 204 (1986), 383-396; C58C1 (pGV 3850kan), Deblaere, Nucl. Acid Res. 13 (1985), 4777; Bevan, Nucleic. Acid Res. 12(1984), 8711; Koncz, Proc. Natl. Acad. Sci. USA 86 (1989), 8467-8471; Koncz, Plant Mol. Biol. 20 (1992), 963-976; Koncz, Specialized vectors for gene tagging and expression studies. In: Plant Molecular Biology Manual Vol 2, Gelvin and Schilperoort (Eds.), Dordrecht, The Netherlands: Kluwer Academic Publ. (1994), 1-22; EP-A-120 516; Hoekema: The Binary Plant Vector System, Offsetdrukkerij Kanters B. V., Alblasserdam (1985), Chapter V, Fraley, Crit. Rev. Plant. Sci., 4, 1-46; An, EMBO J. 4 (1985), 277-287). Although the use of Agrobacterium tumefaciens is preferred in the method of the invention, other Agrobacterium strains, such as Agrobacterium rhizogenes, may be used, for example if a phenotype conferred by said strain is desired.
Methods for the transformation using biolistic methods are well known to the person skilled in the art; see, e.g., Wan, Plant Physiol. 104 (1994), 37-48; Vasil, Bio/Technology 11 (1993), 1553-1558 and Christou (1996) Trends in Plant Science 1, 423-431. Microinjection can be performed as described in Potrykus and Spangenberg (eds.), Gene Transfer To Plants. Springer Verlag, Berlin, N.Y. (1995).
The transformation of most dicotyledonous plants is possible with the methods described above. But also for the transformation of monocotyledonous plants several successful transformation techniques have been developed. These include the transformation using biolistic methods as, e.g., described above as well as protoplast transformation, electroporation of partially permeabilized cells, introduction of DNA using glass fibers, etc. Also, the transformation of monocotyledonous plants by means of Agrobacterium-based vectors has been described (Chan et al., Plant Mol. Biol. 22 (1993), 491-506; Hiei et al., Plant J. 6 (1994) 271-282; Deng et al, Science in China 33 (1990), 28-34; Wilmink et al, Plant Cell Reports 11 (1992), 76-80; May et al., Bio/Technology 13 (1995), 486-492; Conner and Dormisse, Int. J. Plant Sci. 153 (1992), 550-555; Ritchie et al. Transgenic Res. 2 (1993), 252-265). An alternative system for transforming monocotyledonous plants is the transformation by the biolistic approach (Wan and Lemaux, Plant Physiol. 104 (1994), 37-48; Vasil et al., Bio/Technology 11 (1993), 1553-1558; Ritala et al., Plant Mol. Biol. 24 (1994) 317-325; Spencer et al., Theor. Appl. Genet. 79 (1990), 625-631). The transformation of maize in particular has been repeatedly described in the literature (see for instance WO 95/06128, EP 0 513 849, EP 0 465 875, EP 29 24 35; Fromm et al, Biotechnology 8, (1990), 833-844; Gordon-Kamm et al., Plant Cell 2, (1990), 603-618; Koziel et al., Biotechnology 11 (1993), 194-200; Moroc et al., Theor. Appl. Genet. 80, (1990), 721-726). The successful transformation of other types of cereals has also been described for instance of barley (Wan and Lemaux, supra; Ritala et al., supra, Krens et al., Nature 296 (1982), 72-74), wheat (Nehra et al., Plant J. 5 (1994), 285-297) and rice. Methods for transforming Lolium, in particular Lolium perenne, and Brachypodium, in particular Brachypodium distachyon, are described in the attached Examples. Methods for transformation of plants of the Poaceae family have been published, e.g., in Altpeter et al. (Mol. Breeding 6 (2000), 519-528 for Lolium perenne), Altpeter and Xu (J. Plant Physiol. 157 (2000), 441-448 for Festuca rubra), Dalton et al. (Plant Cell Reports 18 (1999), 721-726 for Lolium perenne, Lolium multiflorum and Lolium temulentum), Dalton et al. (Plant Science 132 (1998), 31-43 for Lolium multiflorum, Lolium perenne, Festuca arundinacea and Agrostis stolonifera), Foiling et al. (Plant Science 139 (1998), 29-40 for Lolium), Spangenberg et al. (J. Plant Physiol. 145 (1995), 693-701 for Festuca arundinacea and Festuca rubra) and Wang et al. (J. Plant Physiol. 151 (1997), 83-90 for Lolium perenne and Lolium multiflorum).
The resulting transformed plant cell can then be used to regenerate a transformed plant in a manner known by a skilled person.
The present invention likewise refers to mutant plants showing a prevention of flowering, whereby the definition of the term “prevention of flowering” explained above with regard to the polynucleotides of the present invention accordingly applies to mutant plants. The term “mutant plant” (or “plant mutant”), refers to plants the genotype of which is modified compared to the corresponding source plants, preferably by other means than genetic engineering, i.e. the introduction of an exogenous nucleic acid molecule into plant cells. Such “mutant plants” may be provided by methods known in the art, e.g. produced under the influence of a suitable dose of ionizing radiation (e.g. x-rays, gamma or neutron radiation) or by the effect of suitable mutagens (e.g. EMS, MMS, etc.). Furthermore encompassed are mutant plants wherein the mutation occurs naturally. Mutant plants showing the desired trait, i.e. a prevention of flowering, may be screened out of a pool of mutant plants generated according to standard methods. The selection may be performed for altered flowering in samples taken from these plants. Preferably, selection may be carried out utilizing the knowledge of the nucleotide sequences as provided by the present invention. Consequently, it is possible to screen for a genetic trait being indicative for an altered flowering behaviour. Such a screening approach may involve the application of conventional nucleic acid amplification (e.g. PCR) and/or hybridization techniques.
The transgenic plants of the invention may, in principle, be plants of any plant species. They may be both monocotyledonous and dicotyledonous plants. Preferably, the plants are useful plants, i.e. commercially important plants, cultivated by man for nutrition or for technical, in particular industrial, purposes. They may be sugar storing and/or starch-storing plants, especially cereal species (rye, barley, oat, wheat, rice, maize, millet, sago etc.), pea, marrow pea, cassava, sugar cane, sugar beet and potato; tomato, rape, soybean, hemp, flax, sunflower, cow pea or arrowroot, fiber-forming plants (e.g. flax, hemp, cotton), oil-storing plants (e.g. rape, sunflower, soybean) and protein-storing plants (e.g. legumes, cereals, soybeans). The plants within the scope of the invention also include fruit trees, palms and other trees or wooden plants being of economical value such as in forestry. Moreover, the plants of the invention may be to forage plants (e.g. forage and pasture grasses, such as alfalfa, clover, ryegrass) and vegetable plants (e.g. tomato, lettuce, chicory) or ornamental plants (e.g. roses, tulips, hyacinths). Preferably, the plant belongs to the Poaceae, such as Phleum spp., Dactylis spp., Lolium spp., Festulolium spp., Festuca spp., Poa spp., Bromus spp., Agrostis spp., Arrhenatherum spp., Phalaris spp., and Trisetum spp., for example, Phleum pratense, Phleum bertolonii, Dactylis glomerata, Lolium perenne, Lolium multiflorum, Lolium multiflorum westervoldicum, Festulolium braunii, Festulolium loliaceum, Festulolium holmbergii, Festulolium pabulare, Festuca pratensis, Festuca rubra, Festuca rubra rubra, Festuca rubra commutata, Festuca rubra trichophylla, Festuca duriuscula, Festuca ovina, Festuca arundinacea, Poa trivialis, Poa pratensis, Poa palustris, Bromus catharticus, Bromus sitchensis, Bromus inermis, Deschampsia caespitose, Agrostis capilaris, Agrostis stolonifera, Arrhenatherum elatius, Phalaris arundinacea, and Trisetum flavescens. 
In a preferred embodiment, the present invention relates to transgenic or mutant plants which show an increase in the amount of the polypeptide encoded by the polynucleotide of the invention compared to a corresponding wild-type plant.
In the transgenic plants according to this embodiment, the increased amount of the corresponding protein is caused by the presence of a suitable foreign nucleic acid molecule in the genome of said plants.
The term “presence of a suitable foreign nucleic acid molecule” as used herein refers to any foreign nucleic acid molecule that is present in cells of said transgenic plant but absent from the cells of the corresponding source plant. Thereby encompassed are nucleic acid molecules, e.g. gene sequences, which differ from a corresponding nucleic acid molecule in the source plant cell by at least one mutation (substitution, insertion, deletion, etc. of at least one nucleotide). Furthermore encompassed by the term “foreign” are nucleic acid molecules which are homologous with respect to the source plant cell but are situated in a different chromosomal location or differ, e.g., by way of a reversed orientation for instance with respect to the promoter.
In principle, the nucleic acid molecule to be introduced in accordance with the present embodiment may be of any conceivable origin. It may be from any organism which comprises such molecules. Furthermore, it may be synthetic or derived from naturally occurring molecules by, e.g., modification of its sequence, i.e. it may be a variant or derivative of a naturally occurring molecule. Such variants and derivatives include but are not limited to molecules derived from naturally occurring molecules by addition, deletion, mutation of one or more nucleotides or by recombination. It is, e.g., possible to change the sequence of a naturally occurring molecule so as to match the preferred codon usage of plants, in particular of those plants in which the nucleic acid molecule shall be expressed.
Preferably, the increase of the amount of the polypeptide in the transgenic plant is caused by the expression of a polynucleotide of the invention which is present in cells of the transgenic plant due to genetic engineering.
The polynucleotide introduced into the transgenic plant can in principle be expressed in all or substantially all cells of the plant. However, it is also possible that it is only expressed in certain parts, organs, cell types, tissues etc. Preferred parts are, e.g., leaves. Moreover, it is possible that expression of the polynucleotide only takes place upon induction or at a certain developmental stage. In a preferred embodiment, the polynucleotide is expressed in those parts of the plant that are involved in flowering, most preferably in the apical meristem.
In order to be expressed, the polynucleotide that is introduced into a plant cell is preferably operatively linked to one or more expression control sequences, e.g. a promoter, active in this plant cell.
The promoter may be homologous or heterologous with regard to its origin and/or with regard to the gene to be expressed. Suitable promoters are for instance the promoter of the 35S RNA of the Cauliflower Mosaic Virus (see for instance U.S. Pat. No. 5,352,605), the ubiquitin-promoter (see for instance U.S. Pat. No. 5,614,399) and the rice actin promoter (U.S. Pat. No. 5,641,876) which lend themselves to constitutive expression, the patatin gene promoter B33 (Rocha-Sosa et al., EMBO J. 8 (1989), 23-29) which lends itself to a tuber-specific expression in potatoes or a promoter ensuring expression in photosynthetically active tissues only, for instance the ST-LS1 promoter (Stockhaus et al., Proc. Natl. Acad. Sci. USA 84 (1987), 7943-7947; Stockhaus et al., EMBO, J. 8 (1989) 2445-2451), the Ca/b-promoter (see for instance U.S. Pat. No. 5,656,496, U.S. Pat. No. 5,639,952, Bansal et al., Proc. Natl. Acad. Sci. USA 89 (1992), 3654-3658) and the Rubisco SSU promoter (see for instance U.S. Pat. No. 5,034,322; U.S. Pat. No. 4,962,028) or the glutelin promoter from wheat which lends itself to endosperm-specific expression (HMW promoter) (Anderson, Theoretical and Applied Genetics 96, (1998), 568-576, Thomas, Plant Cell 2 (12), (1990), 1171-1180), the glutelin promoter from rice (Takaiwa, Plant Mol. Biol. 30(6) (1996), 1207-1221, Yoshihara, FEBS Lett. 383 (1996), 213-218, Yoshihara, Plant and Cell Physiology 37 (1996), 107-111), the shrunken promoter from maize (Maas, EMBO J. 8 (11) (1990), 3447-3452, Werr, Mol. Gen. Genet. 202(3) (1986), 471-475, Werr, Mol. Gen. Genet. 212(2), (1988), 342-350), the USP promoter, the phaseolin promoter (Sengupta-Gopalan, Proc. Natl. Acad. Sci. USA 82 (1985), 3320-3324, Bustos, Plant Cell 1 (9) (1989), 839-853) or promoters of zein genes from maize (Pedersen et al., Cell 29 (1982), 1015-1026; Quatroccio et al., Plant Mol. Biol. 15 (1990), 81-93). However, promoters which are only activated at a point in time determined by external influences can also be used (see for instance WO 93/07279). In this connection, promoters of heat shock proteins which permit simple induction may be of particular interest. Likewise, artificial and/or chemically inducible promoters may be used in this context. Moreover, seed-specific promoters such as the USP promoter from Vicia faba which ensures a seed-specific expression in Vicia faba and other plants may be used (Fiedler et al., Plant Mol. Biol. 22 (1993), 669-679; Baumlein et al., Mol. Gen. Genet. 225 (1991), 459-467). Moreover, fruit-specific promoters, such as described in WO 91/01373 may be used too. In one embodiment, promoters which ensure constitutive expression are preferred. However, in another preferred embodiment, the polynucleotide may be operatively linked to a promoter which is inducible. For ensuring expression specifically in the apical meristem of plants it is, e.g., possible to use the promoter of the cen gene (see, e.g., WO 97/10339). The promoters described in PCT/EP03/11038 (WO 04/35797) can be used to drive expression in apical/floral/inflorescence meristems. These promotes are derived from MADS box genes.
Moreover, the polynucleotide may be linked to a termination sequence which serves to terminate transcription correctly and to add a poly-A-tail to the transcript which is believed to have a function in the stabilization of the transcripts. Such elements are described in the literature (see for instance Gielen et al., EMBO J. 8 (1989), 23-29) and can be replaced at will. The termination sequence may be from the same gene as the promoter sequence or from a different gene. It may be homologous or heterologous with respect to the gene to be expressed. Particularly suitable terminators are polyadenylation signals, such as the CaMVpolyA signal or the termination signals from the nopaline synthase (nos), the octopine synthase (ocs) or the rbcS genes.
Furthermore, if needed, polypeptide expression can in principle be targeted to any sub-localization of plant cells (e.g. cytosol, plastids, vacuole, mitochondria) or the plant (e.g. apoplast). In order to achieve the localization in a particular compartment, the coding region to be expressed may be linked to DNA sequences encoding a signal sequence (also called “transit peptide”) ensuring localization in the respective compartment. It is evident that these DNA sequences are to be arranged in the same reading frame as the coding region to be expressed. Preferably, the proteins of the present invention are localized in the nucleus or the cytosol.
In order to ensure the location in the plastids, it is conceivable to use one of the following transit peptides: of the plastidic Ferredoxin: NADP+ oxidoreductase (FNR) of spinach which is enclosed in Jansen et al. (Current Genetics 13 (1988), 517-522). In particular, the sequence ranging from nucleotides −171 to 165 of the cDNA sequence disclosed therein can be used which comprises the 5′ non-translated region as well as the sequence encoding the transit peptide. Another example is the transit peptide of the waxy protein of maize including the first 34 amino acid residues of the mature waxy protein (Klösgen et al., Mol. Gen. Genet. 217 (1989), 155-161). It is also possible to use this transit peptide without the first 34 amino acids of the mature protein. Furthermore, the signal peptides of the ribulose bisphosphate carboxylase small subunit (Wolter et al., Proc. Natl. Acad. Sci. USA 85 (1988), 846-850; Nawrath et al., Proc. Natl. Acad. Sci. USA 91 (1994), 12760-12764), of the NADP malat dehydrogenase (Gallardo et al., Planta 197 (1995), 324-332), of the glutathione reductase (Creissen et al., Plant J. 8 (1995), 167-175) or of the R1 protein (Lorberth et al. Nature Biotechnology 16, (1998), 473-477) can be used. In order to ensure the location in the vacuole, it is conceivable to use one of the following transit peptides: the N-terminal sequence (146 amino acids) of the patatin protein (Sonnewald et al., Plant J. 1 (1991), 95-106) or the signal sequences described by Matsuoka and Neuhaus (Journal of Experimental Botany 50 (1999), 165-174); Chrispeels and Raikhel (Cell 68 (1992), 613-616); Matsuoka and Nakamura (Proc. Natl. Acad. Sci. USA 88 (1991), 834-838); Bednarek and Raikhel (Plant Cell 3 (1991), 1195-1206); and Nakamura and Matsuoka (Plant Phys. 101 (1993), 1-5).
In order to ensure the localization in the mitochondria, it is for example conceivable to use the transit peptide described by Braun (EMBO J. 11, (1992), 3219-3227).
In order to ensure the localization in the apoplast, it is conceivable to use one of the following transit peptides: signal sequence of the proteinase inhibitor II-gene (Keil et al., Nucleic Acid Res. 14 (1986), 5641-5650; von Schaewen et al., EMBO J. 9 (1990), 30-33), of the levansucrase gene from Erwinia amylovora (Geier and Geider, Phys. Mol. Plant Pathol. 42 (1993), 387-404), of a fragment of the patatin gene B33 from Solanum tuberosum, which encodes the first 33 amino acids (Rosahl et al., Mol Gen. Genet. 203 (1986), 214-220) or of the one described by Oshima et al. (Nucleic Acid Res. 18 (1990), 181).
In addition to expressing a polynucleotide of the invention that is present in a plant cell due to genetic engineering, an increase of the amount of the corresponding polypeptide in transgenic plants of the invention may also be achieved by other methods known to a skilled person.
For example, the endogenous gene corresponding to a polynucleotide of the invention may be modified at its natural location to cause an increase in the amount of the protein, e.g. by homologous recombination. In particular, the promoter of this gene can for instance be altered in a way that promoter activity is enhanced. In the alternative, other regulatory elements of the gene influencing for instance mRNA stability, translation or post-translational processing or the coding region of the gene can be modified so that the encoded polypeptide shows an increased activity, e.g. by specifically substituting amino acid residues in the catalytically active domain of the polypeptide. Applicable homologous recombination techniques (also known as “in vivo mutagenesis”) are known to the person skilled in the art and are described in the literature. One such technique involves the use of a hybrid RNA-DNA oligonucleotide (“chimeroplast”) which is introduced into cells by transformation (TIBTECH 15 (1997), 441-447; WO95/15972; Kren, Hepatology 25 (1997), 1462-1468; Cole-Strauss, Science 273 (1996), 1386-1389). Thereby, part of the DNA component of the RNA-DNA oligonucleotide is homologous with the target gene sequence, however, displays in comparison to this sequence a mutation or a heterologous region which is surrounded by the homologous regions. The term “heterologous region” refers to any sequence that can be introduced and which is different from that to be modified. By means of base pairing of the homologous regions with the target sequence followed by a homologous recombination, the mutation or the heterologous region contained in the DNA component of the RNA-DNA oligonucleotide can be transferred to the corresponding gene. By means of in vivo mutagenesis, any part of the gene encoding the polypeptide of the invention can be modified as long as it results in an increase of the biological activity of this protein. Alternatively, the expression or the amount of a protein according to the invention in a cell can also be increased by modulating the expression of genes known to influence/regulate the expression of the gene in question. Thus, if it is, e.g., known that a certain gene represses transcription of the gene in question, the reduction of expression of said gene leads to a higher expression of the gene in question.
Transgenic plants which show an increased amount of the polypeptide according to the invention encoded by a polynucleotide according to the invention show preferably a prevention of flowering as defined above in connection with the polynucleotides according to the invention.
Moreover, the present invention relates in a further preferred embodiment to transgenic or mutant plants which show a reduced amount of a polypeptide encoded by a polynucleotide of the invention compared to a corresponding wild-type plant.
The transgenic plants according to this embodiment show a reduced amount of a polypeptide of the invention due to the presence of a suitable foreign nucleic acid molecule in the genome of its cells.
The above explanations concerning techniques for producing transgenic plants and plant cells as well as suitable transformation techniques and vectors mentioned in connection with the transgenic plants having an increased amount of a polypeptide of the present invention may be likewise applied in the present embodiment.
Methods for specifically reducing the amount of a protein in plant cells by the introduction of nucleic acid molecules are exhaustively and widely described in the literature and are known to the person skilled in the art. These include but are not limited to antisense inhibition, ribozyme inhibition, co-suppression, RNA interference, expression of dominant negative mutants, antibody expression and in vitro mutagenesis approaches.
It is particularly preferred that the nucleic acid molecule introduced into a plant cell in accordance with the present embodiment has to be expressed in the transgenic plant in order to exert the reducing effect upon the amount of the protein. The term “expressed” means for such a nucleic acid molecule that it is at least transcribed, and for some embodiments also translated into a protein, in at least some of the cells of the plant. Preferred examples of such nucleic acid molecules relate to those embodiments of the transgenic plants of the invention wherein said reduced amount of the protein is achieved by an antisense, co-suppression, ribozyme or RNA interference effect or by the expression of antibodies or other suitable (poly)peptides capable of specifically reducing said activity or by the expression of a dominant-negative mutant. These methods are further explained in the following.
Accordingly, the use of nucleic acid molecules encoding an antisense RNA which is complementary to transcripts of a gene of the present invention is a preferred embodiment of the present invention. Thereby, complementarity does not signify that the encoded RNA has to be 100% complementary. A low degree of complementarity may be sufficient as long as it is high enough to inhibit the expression of such protein upon expression of said RNA in plant cells. The transcribed RNA is preferably at least 90% and most preferably at least 95% complementary to the polynucleotide of the invention. In order to cause an antisense effect during the transcription in plant cells such RNA molecules have a length of at least 15 bp, preferably a length of more than 100 bp and most preferably a length or more than 500 bp, however, usually less than 1600 bp, preferably shorter than 1200 bp. Exemplary methods for achieving an antisense effect in plants are for instance described by Müller-Röber (EMBO J. 11 (1992), 1229-1238), Landschütze (EMBO J. 14 (1995), 660-666), D'Aoust (Plant Cell 11 (1999), 2407-2418) and Keller (Plant J. 19 (1999), 131-141) and are herewith incorporated in the description of the present invention. Likewise, an antisense effect may also be achieved by applying a triple-helix approach, whereby a nucleic acid molecule complementary to a region of the gene, encoding the relevant protein, designed according to the principles for instance laid down in Lee (Nucl. Acids Res. 6 (1979), 3073); Cooney (Science 241 (1998), 456) or Dervan (Science 251 (1991), 1360) may inhibit its transcription.
A similar effect as with antisense techniques can be achieved by producing transgenic plants expressing suitable constructs in order to mediate an RNA interference (RNAi) effect. Thereby, the formation of double-stranded RNA leads to an inhibition of gene expression in a sequence-specific fashion. More specifically, in RNAi constructs, a sense portion comprising the coding region of the gene to be inactivated (or a part thereof, with or without non-translated region) is followed by a corresponding antisense sequence portion. Between both portions, an intron not necessarily originating from the same gene may be inserted. After transcription, RNAi constructs form typical hairpin structures. In accordance with the teachings of the present invention, the RNAi technique may be carried out as described by Smith (Nature 407 (2000), 319-320) or Marx (Science 288 (2000), 1370-1372).
Also DNA molecules can be employed which, during expression in plant cells, lead to the synthesis of an RNA which reduces the expression of the gene encoding the polypeptide of the invention in the plant cells due to a co-suppression effect. The principle of co-suppression as well as the production of corresponding DNA sequences is precisely described, for example, in WO 90/12084. Such DNA molecules preferably encode an RNA having a high degree of homology to transcripts of the target gene. It is, however, not absolutely necessary that the coding RNA is translatable into a protein. The principle of the co-suppression effect is known to the person skilled in the art and is, for example, described in Jorgensen, Trends Biotechnol. 8 (1990), 340-344; Niebel, Curr. Top. Microbiol. Immunol. 197 (1995), 91-103; Flavell, Curr. Top. Microbiol. Immunol. 197 (1995), 43-36; Palaqui and Vaucheret, Plant. Mol. Biol. 29 (1995), 149-159; Vaucheret, Mol. Gen. Genet. 248 (1995), 311-317; de Borne, Mol. Gen. Genet. 243 (1994), 613-621 and in other sources.
Likewise, DNA molecules encoding an RNA molecule with ribozyme activity which specifically cleaves transcripts of a gene encoding the relevant protein can be used. Ribozymes are catalytically active RNA molecules capable of cleaving RNA molecules and specific target sequences. By means of recombinant DNA techniques, it is possible to alter the specificity of ribozymes. There are various classes of ribozymes. For practical applications aiming at the specific cleavage of the transcript of a certain gene, use is preferably made of representatives of the group of ribozymes belonging to the group I intron ribozyme type or of those ribozymes exhibiting the so-called “hammerhead” motif as a characteristic feature. The specific recognition of the target RNA molecule may be modified by altering the sequences flanking this motif. By base pairing with sequences in the target molecule, these sequences determine the position at which the catalytic reaction and therefore the cleavage of the target molecule takes place. Since the sequence requirements for an efficient cleavage are low, it is in principle possible to develop specific ribozymes for practically each desired RNA molecule. In order to produce DNA molecules encoding a ribozyme which specifically cleaves transcripts of a gene encoding the relevant protein, for example a DNA sequence encoding a catalytic domain of a ribozyme is bilaterally linked with DNA sequences which are complementary to sequences encoding the target protein. Sequences encoding the catalytic domain may for example be the catalytic domain of the satellite DNA of the SCMO virus (Davies, Virology 177 (1990), 216-224 and Steinecke, EMBO J. 11 (1992), 1525-1530) or that of the satellite DNA of the TobR virus (Haseloff and Gerlach, Nature 334 (1988), 585-591). The expression of ribozymes in order to decrease the activity of certain proteins in cells is known to the person skilled in the art and is, for example, described in EP-B10 321 201. The expression of ribozymes in plant cells is for example described in Feyter (Mol. Gen. Genet. 250 (1996), 329-338).
Furthermore, nucleic acid molecules encoding antibodies specifically recognizing the relevant protein in a plant, i.e. specific fragments or epitopes of such a protein, can be used for inhibiting the activity of this protein. These antibodies can be monoclonal antibodies, polyclonal antibodies or synthetic antibodies as well as fragments of antibodies, such as Fab, Fv or scFv fragments etc. Monoclonal antibodies can be prepared, for example, by the techniques as originally described in Köhler and Milstein (Nature 256 (1975), 495) and Galfré (Meth. Enzymol. 73 (1981) 3), which comprise the fusion of mouse myeloma cells to spleen cells derived from immunized mammals. Furthermore, antibodies or fragments thereof to the aforementioned peptides can be obtained by using methods which are described, e.g., in Harlow and Lane “Antibodies, A Laboratory Manual”, CSH Press, Cold Spring Harbor, 1988. Expression of antibodies or antibody-like molecules in plants can be achieved by methods well known in the art, for example, full-size antibodies (Düring, Plant. Mol. Biol. 15 (1990), 281-293; Hiatt, Nature 342 (1989), 469-470; Voss, Mol. Breeding 1 (1995), 39-50), Fab-fragments (De Neve, Transgenic Res. 2 (1993), 227-237), scFvs (Owen, Bio/Technology 10 (1992), 790-794; Zimmermann, Mol. Breeding 4 (1998), 369-379; Tavladoraki, Nature 366 (1993), 469-472; Artsaenko, Plant J. 8 (1995), 745-750) and variable heavy chain domains (Benvenuto, Plant Mol. Biol. 17 (1991), 865-874) have been successfully expressed in tobacco, potato (Schouten, FEBS Lett. 415 (1997), 235-241) or Arabidopsis, reaching expression levels as high as 6.8% of the total protein (Fiedler, Immunotechnology 3 (1997), 205-216).
In addition, nucleic acid molecules encoding a mutant form of the relevant protein can be used to interfere with the activity of the wild-type protein. Such a mutant form preferably has lost its biological activity and may be derived from the corresponding wild-type protein by way of amino acid deletion(s), substitution(s), and/or additions in the amino acid sequence of the protein. These mutant forms may be naturally occurring or, as preferred, genetically engineered mutants.
In another preferred embodiment, the nucleic acid molecule, the presence of which in the genome of a plant cell leads to a reduction of the amount of the protein, does not require its expression to exert its effect. Correspondingly, preferred examples relate to methods wherein said reduced amount is achieved by in vivo mutagenesis or by the insertion of a heterologous DNA sequence in the corresponding gene.
The term “in vivo mutagenesis”, relates to methods where the sequence of the gene encoding the relevant protein is modified at its natural chromosomal location such as for instance by techniques applying homologous recombination. This may be achieved by using a hybrid RNA-DNA oligonucleotide (“chimeroplast”) as it is already described supra. For the purpose of reducing the amount of a certain endogenous protein, in vivo mutagenesis can in particular be directed to the promoter, e.g. the RNA polymerase binding site, as well as the coding region, in particular those parts relevant for the activity or a signal sequence directing the protein to the appropriate cellular compartment.
Reduction of the amount of protein may furthermore be achieved by knocking out the corresponding endogenous gene by way of inserting a heterologous DNA sequence into said gene. The term “heterologous DNA sequence” refers to any DNA sequences which can be inserted into the target gene via appropriate techniques other than those described above in connection with in vivo mutagenesis. The insertion of such a heterologous DNA sequence may be accompanied by other mutations in the target gene such as the deletion, inversion or rearrangement of the sequences flanking the insertion site. This embodiment of the invention includes that the introduction of a nucleic acid molecule leads to the generation of a pool, i.e. a plurality, of transgenic plants in the genome of which the nucleic acid molecule, i.e. the heterologous DNA sequence, is randomly spread over various chromosomal locations, and that this generation of transgenic plants is followed by selecting those transgenic plants out of the pool which show the desired genotype, i.e. an inactivating insertion in the relevant gene and/or the desired phenotype, i.e. a reduced amount of the protein and/or other phenotypic traits correlating with a reduced amount, i.e. alterations in flowering behaviour.
Suitable heterologous DNA sequences that can be taken for such an approach are described in the literature and include, for instance, vector sequences capable of self-integration into the host genome or mobile genetic elements. Particularly preferred in this regard are T-DNA or transposons which are well-known to the person skilled in the art from so-called tagging experiments used for randomly knocking out genes in plants. The production of such pools of transgenic plants can for example be carried out as described in Jeon (Plant J. 22 (2000), 561-570) or Parinov (Curr. Op. Biotechnol. 11 (2000), 157-161).
Another example of insertional mutations that may result in gene silencing includes the duplication of promoter sequences which may lead to a methylation and thereby an inactivation of the promoter (Morel, Current Biology 10 (2000), 1591-1594).
Furthermore, it is immediately evident to the person skilled in the art that the above-described approaches, such as antisense, ribozyme, co-suppression, in-vivo mutagenesis, RNAi, expression of antibodies, other suitable peptides or polypeptides or dominant-negative mutants and the insertion of heterologous DNA sequences, can also be used for the reduction of the expression of genes that encode a regulatory protein such as a transcription factor, that controls the expression of the relevant protein or, e.g., proteins that are necessary for the protein to become active. It is also evident from the disclosure of the present invention that any combination of the above-identified approaches can be used for the generation of transgenic plants, which, due to the presence of one or more of the above-described nucleic acid molecules in their cells, display a reduced amount of the relevant protein compared to corresponding source plants. Such combinations can be made, e.g., by (co-) transformation of corresponding nucleic acid molecules into the plant cell, plant tissue or plant or by crossing transgenic or mutant plants that have been generated according to different techniques. Likewise, the transgenic plants of the present invention showing a reduced amount of the relevant protein can be crossed with plants, e.g. transgenic plants, having other desired traits.
The invention also relates to propagation material of the transgenic plants of the invention comprising plant cells according to the invention. The term “propagation material” comprises those components or parts of the plant which are suitable to produce offspring vegetatively or generatively. Suitable means for vegetative propagation are for instance cuttings, callus cultures, rhizomes or tubers. Other propagation material includes for instance fruits, seeds, seedlings, protoplasts, cell cultures etc. The preferred propagation materials are tubers and seeds.
The invention also relates to harvestable parts of the plants of the invention such as, for instance, fruits, seeds, tubers, rootstocks, leaves or flowers.
Corresponding to the above explanations, the invention furthermore relates to a method for preventing flowering in a plant comprising the step of providing a transgenic or mutant plant in which the amount, preferably the expression of a polypeptide encoded by the above-described polynucleotide of the invention is increased compared to a corresponding wild-type plant.
In another aspect the present invention relates to a method of controlling flowering in a plant by providing an inducible restoration of flowering in plants in which flowering is prevented characterized in that

    (a) the prevention of flowering of the plant is the result of the genetic modification of the plant which leads
    
        (i) either to the increase of one or more floral inhibitors; or
        (ii) to the reduction of one or more floral enhancers
    
    
    and
    (b) the inducible restoration of the flowering is achieved by
    
        (iii) either reducing the expression of the floral inhibitor(s) mentioned in (i), above, by induced expression of a corresponding nucleic acid molecule; or
        (iv) increasing the expression of the floral enhancer(s) mentioned in (ii), above, by induced expression of a corresponding nucleic acid molecule, or
        (v) induced expression of one or more floral enhancers different from the floral enhancer(s) mentioned in (ii), above, which is capable of overcoming the floral inhibition caused by the expression of the floral inhibitor of (a)(i) or the reduced expression of the floral enhancer of (a)(ii).
    
    


It was found that it is possible to establish a system for controlling flowering in plants in which plants are first genetically modified so as to show a prevention of flowering and flowering is then inducibly restored by inducing expression of nucleic acid molecules which act against the effect of the genetic modification leading to the prevention of flowering.
The term “prevention of flowering” has the same meaning as set forth above in connection with the polynucleotides according to the invention.
The genetic modification which leads to the prevention of flowering is a modification which either leads to an increase of a floral inhibitor in comparison to wild-type plants or to the reduction of a floral enhancer in comparison to wild-type plants.
A “floral inhibitor” is a polynucleotide or polypeptide which reduces, delays or inhibits the formation of sexual reproductive tissues/organs such as floral meristems, inflorescences, spikelets, sepals, petals, carpels, stamens, embryos, pollen, seeds etc. With respect to the meaning of the terms “reduction”, “delay” and “inhibition” the same applies as has been set forth above in connection with the nucleic acid molecules according to the invention.
An example of a floral inhibitor is the above-described polypeptide according to the present invention and the corresponding above-described polynucleotide. Thus, in a preferred embodiment of the method according to the invention the amount of a protein according to the invention is increased in comparison to wild-type plants and the plants consequently show a prevention of flowering.
Another example for a floral inhibitor is the TERMINAL FLOWER1 (TFL1) gene known, e.g., from Arabidopsis thaliana (WO 97/10339) and from Lolium perenne (Jensen et al., Plant Physiol. 125 (2001), 1517-1528; GeneBank accession number AF316419).
Functionally active fragments, derivatives and homologues of LpTFL1 are described in, e.g., P11792US-2003 0226. The use of TFL1 polynucleotides/polypeptides for preventing flowering in plants has already been described in WO 97/10339 and in Jensen et al. (2001; loc. cit.). In principle, any TFL1 polynucleotide/polypeptide from any plant species can be used in the method according to the invention as well as any homolog of TFL1 which may have a different name in other plant species, for example, the cen gene from Antirrhinum disclosed in WO 97/10339. “Homolog” means that the corresponding polynucleotide/polypeptide has a certain degree of homology, that is to say sequence identity (preferably at least 40%, more preferably at least 60%, even more preferably at least 65%, particularly preferred at least 66%, 68%, 70%, 75%, 80%, 86%, 88%, 90%, 92%, 95%, 97% or 99%). With respect to the terms “homology” and “sequence identity” the same applies which had been set forth above in connection with the polynucleotides of the present invention. “Homolog” furthermore means that the function is equivalent to the function of TFL1. Preferably this function is the property to prevent flowering when overexpressed in plants.
Furthermore, also sequences hybridizing to known TFL1 sequences can be used as long as they effect the prevention of flowering. With respect to “hybridizing” or “hybridisation” the same applies which has been set forth above in connection with the polynucleotides according to the invention.
Moreover, any part of a TFL1 polynucleotide/polypeptide, of a homolog or of a hybridizing sequence can be used in the method according to the invention as long as the part is long enough to effect the prevention of flowering.
A further example for a floral inhibitor is the FLC/FLF protein (the terms FLC/FLF are synonyms for the type of protein). FCL has already been described in Arabidopsis thaliana (WO 00/50615); Michaels and Amasino. Plant Cell 11 (1999), 949-956; Sheldon et al., Plant Cell 11 (1999), 445-458; GeneBank accession numbers AF537203 or AF116527). WO 00/50615 describes three FLC genes from Arabidopsis thaliana and two FLC genes from Brassica rapa. Moreover, this document describes the characteristics of FLC genes and the encoded proteins and methods for identifying FLC genes from other plant species. This document also describes the use of FLC for preventing flowering in plants. FLF from A. thaliana has also been described in WO 00/32780 as well as its use for preventing flowering in plants and its use to isolate homologous sequences from other plant species such as Brassica napus. In principle, any FLC/FLF polynucleotide/polypeptide from any plant species can be used in the method according to the invention as well as any homolog of FLC/FLF which may have a different name in other plant species. FLC/FLF proteins have, e.g. also been described for Brassica oleracea (GenBank accession number AY 273161) and Raphanus sativurn (GenBank accession number AY 273160), Brassica napus (GenBank accession numbers AY 036888=BnFLC1, AY 036889=BnFLC2; AY 036890=BnFLC3; AY 036891=BnFLC4 and AY 036892=BnFLC5). “Homolog” means that the corresponding polynucleotide/polypeptide has a certain degree of homology, that is to say sequence identity (preferably at least 40%, more preferably at least 60%, even more preferably at least 65%, particularly preferred at least 66%, 68%, 70%, 75%, 80%, 86%, 88%, 90%, 92%, 95%, 97% or 99%). With respect to the terms “homology” and “sequence identity” the same applies which had been set forth above in connection with the polynucleotides of the present invention. “Homolog” furthermore means that the function is equivalent to the function of FLC/FLF. Preferably this function is the property to prevent flowering when overexpressed in plants. FLC/FLF proteins are characterized as being MADS box proteins. They are classified as FLF/FLC proteins by sequence homology to the Arabidopsis thaliana FLC/FLF locus. Preferably, they are functionally classified as FLC/FLF proteins by their ability to complement the Arabidopsis FLC/FLF mutant (Sheldon et al., Plant Cell 11 (1999), 445-458).
Furthermore, also sequences hybridizing to known FLC/FLF sequences can be used as long as they effect the prevention of flowering. With respect to “hybridizing” or “hybridisation” the same applies which has been set forth above in connection with the polynucleotides according to the invention.
Moreover, any part of a FLC/FLF polynucleotide/polypeptide, of a homolog or of a hybridizing sequence can be used in the method according to the invention as long as the part is long enough to effect the prevention of flowering.
A further example for a floral inhibitor is the SVP (short vegetative period) protein. This protein belongs to the MADS box family and was identified in Arabidopsis as an early flowering mutation. The SVP protein defines a separate class of MADS box proteins and functions as an inhibitor of flowering (Hartmann et al., Plant J. 21 (2000), 351-360). In principle, any SVP protein from any plant species can be used in the method according to the invention as well as any homolog of SVP which may have a different name in other plant species. In a preferred embodiment SVP from A. thaliana is used. In the context of the present invention the term SVP protein also includes SVP-like proteins like the LpMADS 10, LpMADS 14 and LpMADS 16 proteins. The cloning of the nucleotide sequences encoding these proteins is described in the Examples. The nucleotide sequences are shown in SEQ ID NOs:3, 5 and 7, respectively. The corresponding amino acid sequences are shown in SEQ ID NOs: 4, 6 and 8, respectively. Thus, in another preferred embodiment the SVP protein is a protein comprising the amino acid sequence as shown in any one of SEQ ID NOs:4, 6 or 8 or a homolog thereof. “Homolog” means that the corresponding polynucleotide/polypeptide has a certain degree of homology, that is to say sequence identity (preferably at least 40%, more preferably 60%, even more preferably at least 65%, particularly preferred at least 66%, 68%, 70%, 75%, 80%, 86%, 88%, 90%, 92%, 95%, 97% or 99%). With respect to the terms “homology” and “sequence identity” the same applies which had been set forth above in connection with the polynucleotides of the present invention. “Homolog” furthermore, means that the function is equivalent to the function of the SVP. Preferably this function is the property to prevent flowering when overexpressed in plants.
The increase of the floral inhibitor in the plants can be achieved by methods well known to the person skilled in the art. In this respect, the same possibilities exist as have been described in detail above in connection with the plants according to the invention which show an increased amount of a protein according to the invention. In a preferred embodiment, the increase of the floral inhibitor is achieved by expressing a corresponding nucleic acid molecule in the plant. In this respect, the same possibilities exist as described above in connection with the expression of the polynucleotides of the present invention in plant cells. Preferably, the expression of the corresponding nucleic acid molecule may be under the control of a promoter which ensures constitutive, tissue specific or developmental specific expression.
A “floral enhancer” is a polynucleotide or polypeptide which accelerates or increases the formation of tissues/organs for sexual reproduction such as floral meristems, inflorescences, spikelets, sepals, petals, carpels, stamens, embryos, pollen, seeds etc.
The term “accelerates” means that when the amount of the floral enhancer is increased flowering occurs at an earlier time point when compared to wild-type plants grown under the same conditions. An “earlier time point” preferably means at least 7 days earlier, even more preferably at least 30 days earlier, particularly preferred at least 60 days earlier and most preferably at least 120 days earlier. The term “increases flowering” means that more organs/tissues for sexual reproduction are formed.
An example for a floral enhancer is the INDETERMINATE1 (ID1) gene. It has, e.g., been described for maize (see, e.g., WO 96/34088). This document also discloses the use of ID1 polynucleotides/polypeptides for preventing flowering. The ID1 cDNA from Lolium perenne, LpID1, is disclosed in the present application (see polynucleotides relating to SEQ ID NO:9 and the corresponding amino acid sequence SEQ ID NO:10). In principle, any ID1 polynucleotide/polypeptide from any plant species can be used in the method according to the invention as well as any homolog of ID1 which may have a different name in other plant species. In a preferred embodiment the ID1 protein is from Zea mays (GenBank accession number AF058757). “Homolog” means that the corresponding polynucleotide/polypeptide has a certain degree of homology, that is to say sequence identity (preferably at least 40%, more preferably at least 60%, even more preferably at least 65%, particularly preferred at least 66%, 68%, 70%, 75%, 80%, 86%, 88%, 90%, 92%, 95%, 97% or 99%). With respect to the terms “homology” and “sequence identity” the same applies which had been set forth above in connection with the polynucleotides of the present invention. “Homolog” furthermore means that the function is equivalent to the function of ID1. Preferably this function is the property to enhance flowering when overexpressed in plants. ID1 proteins are characterized as belonging to the C2H2 type family of the zinc finger proteins. It is a transcriptional regulator of the floral transition.
Furthermore, also sequences hybridizing to known ID1 sequences can be used as long as they effect the enhancement of flowering. With respect to “hybridizing” or “hybridisation” the same applies which has been set forth above in connection with the polynucleotides according to the invention.
Moreover, any part of a ID1 polynucleotide/polypeptide, of a homolog or of a hybridizing sequence can be used in the method according to the invention as long as the part is long enough to effect the enhancement of flowering.
The reduction of the expression of the floral inhibitor according to step (b)(iii) of the method according to the invention can be achieved by means and methods known to the person skilled in the art. Suitable means and methods which can be used to reduce expression of a given sequence are known to the skilled person and have been listed above in connection with the plant cells according to the invention in which the expression/amount of a protein according to the invention is reduced. This comprises, e.g., induced expression of corresponding nucleic acid molecules coding for antisense molecules, cosuppression molecules, RNAi or ribozymes, molecules coding for dominant negative mutants, molecules coding for antibodies etc.
The increase of the floral enhancer according to step (b)(iv) of the method according to the invention can be achieved by methods well-known to the person skilled in the art. As mentioned above, the increase is achieved by the induced expression of a corresponding nucleic acid molecule encoding the floral enhancer. In this respect the same applies which had been said above in connection with the possibilities of increasing the expression/amount of a polypeptide according to the invention in a plant cell.
The term “induced expression” refers to a situation where gene expression is obtained or increased by a physical treatment, treatment with a chemical compound, exposure to environmental stimuli, etc.
The floral enhancer mentioned in step (b)(v) of the method according to the invention may be any floral enhancer which is capable of overcoming the floral inhibition resulting from steps (a)(i) or (a)(ii) of the method according to the invention.
One example for such a floral enhancer is the ID1 gene/protein mentioned above.
Another example is the CONSTANS gene (CO) which encodes a polypeptide belonging to the group of zinc finger proteins. The sequences of the CONSTANS genes from Arabidopsis thaliana and from Brassica napus are, e.g., disclosed in WO 96/14414. The sequence of the CO gene from Lolium perenne is shown in SEQ ID NO:11. The corresponding amino acid sequence is shown in SEQ ID NO:12. A multitude of sequences coding for CO proteins from other plant species are accessible in data bases. In principle, any CO polynucleotide/polypeptide from any plant species can be used in the method according to the invention as well as any homolog of CO which may have a different name in other plant species. One preferred embodiment are CO proteins from Arabidopsis thaliana (GenBank accession numbers X94937 and S77098. “Homolog” means that the corresponding polynucleotide/polypeptide has a certain degree of homology, that is to say sequence identity (preferably at least 40%, more preferably at least 60%, even more preferably at least 65%, particularly preferred at least 66%, 68%, 70%, 75%, 80%, 86%, 88%, 90%, 92%, 95%, 97% or 99%). With respect to the terms “homology” and “sequence identity” the same applies which had been set forth above in connection with the polynucleotides of the present invention. “Homolog” furthermore means that the function is equivalent to the function of CO. Preferably the function of the CO protein is the property to enhance flowering in plants. A characteristic property of CO is that it is a transcription factor with one zinc finger region being composed of two 3-box domains and a C-terminal CCT domain. In Arabidopsis and rice the CO protein has been shown to mediate floral stimuli from the photoperiodic pathway.
Furthermore, also sequences hybridizing to known CO sequences can be used as long as they effect the enhancement of flowering. With respect to “hybridizing” or “hybridisation” the same applies which has been set forth above in connection with the polynucleotides according to the invention.
Moreover, any part of a CO polynucleotide/polypeptide, of a homolog or of a hybridizing sequence can be used in the method according to the invention as long as the part is long enough to effect the enhancement of flowering.
A further example for a floral enhancer to be used in step (b)(v) of the method is the LEAFY gene (LFY).
The sequence of the LEAFY gene from Lolium perenne is shown in SEQ ID NO:13. The corresponding amino acid sequence is shown in SEQ ID NO:14. The use of LEAFY sequences for enhancing flowering has been disclosed in WO 96/19105. In principle, any LEAFY polynucleotide/polypeptide from any plant species can be used in the method according to the invention as well as any homolog of LEAFY which may have a different name in other plant species. The LEAFY gene has, for example, also been described for Arabidopsis (Weigel et al., Cell 69 (1992), 843-859), in tobacco (Kelly et al., Plant Cell 7, (1995), 225-234, Sinapis alba (Bonhomme et al., Plant Mol. Biol. 34 (1997), 573-582, where it is called SaMADS D), Petunia (Souer et al., Development 125 (1998), 733-742), Eucalyptus (Southerton et al., Plant Mol. Biol. 37 (1998), 897-910), Pinus radiata (Mouradov et al., Proc. Natl. Acad. Sci. USA 95 (1998), 6537-6542), Impatiens (Pouteau et al., Plant J. 14 (1998), 235-246) and maize (Bomblies et al., Development 130 (2003), 2385-2395). In a preferred embodiment the LEAFY sequence used in the method according to the invention is the sequence from Arabidopsis thaliana as shown in GenBank Accession number M91208.
“Homolog” means that the corresponding polynucleotide/polypeptide has a certain degree of homology, that is to say sequence identity (preferably at least 40%, more preferably at least 60%, even more preferably at least 65%, particularly preferred at least 66%, 68%, 70%, 75%, 80%, 86%, 88%, 90%, 92%, 95%, 97% or 99%). With respect to the terms “homology” and “sequence identity” the same applies which had been set forth above in connection with the polynucleotides of the present invention. “Homolog” furthermore means that the function is equivalent to the function of LEAFY Preferably this function is the property to enhance flowering in plants. LEAFY proteins belong to the group of so-called “meristem identity genes” which specify vegetative or floral identity of the shoot apical meristem.
Furthermore, also sequences hybridizing to known LEAFY sequences can be used as long as they effect the enhancement of flowering. With respect to “hybridizing” or “hybridisation” the same applies which has been set forth above in connection with the polynucleotides according to the invention.
Moreover, any part of a LEAFY polynucleotide/polypeptide, of a homolog or of a hybridizing sequence can be used in the method according to the invention as long as the part is long enough to effect the enhancement of flowering.
Further examples of floral enhancers to be used in step (b)(v) of the method according to the invention are APETALA-1 (AP-1) proteins. These are MADS box proteins and also belong to the group of “meristem identity genes”. AP-1 was first isolated from A. thaliana (Mandel et al., Nature 360 (1992), 273-277). Preferably, the AP-1 protein is a MADS1, MADS2 or MADS3 protein. These are AP-1 homologs isolated from Lolium perenne. LpMADS1 is the closest homolog to the major vernalization locus in wheat, VRN1 (Yan et al., Proc. Natl. Acad. Sci. USA 100 (2003), 6263-6268). VNR1 (TmAP1) is a close AP-1 homolog and specifies vernalization requirement in wheat. Spring varieties which do not require vernalization show a basal expression of TmAP1 whereas winter types which require vernalization in order to flower only show TmAP1 expression in response to vernalization. Similarly, it has been shown that LpMADS1, -2, -3 are up regulated by vernalization in L. perenne (Petersen et al., J. Plant Physiol. 161 (2004), 439-447).
The sequences of MADS1, 2 and 3 of Lolium perenne are shown in SEQ ID NOs:15, 17 and 19, respectively. Homologs to MADS 1, 2 and 3 of L. perenne are known, e.g., from Lolium temulentum and other cereals, such as wheat. The use of AP-1 to manipulate flowering time in plant has been suggested in WO 97/46078 and U.S. Pat. No. 5,844,119. The use of MADS box proteins to manipulate flowering in Lolium and Festuca plant species has been suggested in WO 02/33091.
In principle, any AP-1 and in particular any MADS1, 2 or 3 polynucleotide/polypeptide from any plant species can be used in the method according to the invention as well as any homolog of AP-1 or MADS1, 2, 3 which may have different names in other plant species. In a preferred embodiment the AP-1 protein is from Arabidopsis thaliana (see GenBank accession number Z16421). “Homolog” means that the corresponding polynucleotide/polypeptide has a certain degree of homology, that is to say sequence identity (preferably at least 40%, more preferably at least 60%, even more preferably at least 65%, particularly preferred at least 66%, 68%, 70%, 75%, 80%, 86%, 88%, 90%, 92%, 95%, 97% or 99%). With respect to the terms “homology” and “sequence identity” the same applies which had been set forth above in connection with the polynucleotides of the present invention. “Homolog” furthermore means that the function is equivalent to the function of MADS1, 2, 3 or AP-1. Preferably this function is the property to enhance flowering in plants.
Furthermore, also sequences hybridizing to known AP-1, MADS1, 2 or 3 sequences can be used as long as they effect the enhancement of flowering. With respect to “hybridizing” or “hybridisation” the same applies which has been set forth above in connection with the polynucleotides according to the invention.
Moreover, any part of an AP-1 or of a MADS1, 2 or 3 polynucleotide/polypeptide, of a homolog or of a hybridizing sequence can be used in the method according to the invention as long as the part is long enough to effect the enhancement of flowering.
A further example for a floral enhancer is the SOC-1 (suppressor of overexpression of CO-1) protein (also known as AGL20). Mutations of SOC-1 partially suppress the effect of 35 S::CO and SOC-1 integrates signals from the photoperiod, vernalization and gibberelin floral promotive pathways (Borner et al., Plant J. 24 (2000), 591-599; Lee et al., Genes Dev. 14 (2000), 2366-2376; Samach et al., Science 288 (2000), 1613-1616). SOC-1 expression gradually increases during development and is up-regulated by vernalization and GA application (Borner et al., loc. cit.). The photoperiodic pathway gene CO and the vernalization pathway gene FLC regulate SOC-1 expression thereby modulating flowering time. CO does this largely by increasing activity of SOC-1, whereas FLC delays flowering, at least in part, by repressing the expression of SOC-1 (Samach et al., loc. cit.). SOC-1 homologs have been isolated from different plant species, e.g., Arabiodopsis (NM 130128), rice (AB003328) and maize (AF112150). In principle, any SOC-1 protein from any plant species can be used in the method according to the invention as well as any homolog of SOC-1 which may have a different name in other plant species. In a preferred embodiment SOC-1 from A. thaliana is used. “Homolog” means that the corresponding polynucleotide/polypeptide has a certain degree of homology, that is to say sequence identity (preferably at least 40%, more preferably 60%, even more preferably 65%, particularly preferred at least 66%, 68%, 70%, 75%, 80%, 86%, 88%, 90%, 92%, 95%, 97% or 99%). With respect to the terms “homology” and “sequence identity” the same applies which had been set forth above in connection with the polynucleotides of the present invention. “Homology” furthermore means that the function is equivalent to the function of SOC-1. Preferably this function is the property to enhance flowering in plants.
A further example for a floral enhancer is the FT protein. This protein belongs to the family of PEBP proteins and has been shown to play a role opposite to TFL1 in mediating flower inducing signals in Arabidopsis (Kardailsky et al., Science 286 (1999), 1962-1965). In principle, any FT protein from any plant species can be used in the method according to the invention as well as any homolog of FT which may have a different name in other plant species. In a preferred embodiment FT from A. thaliana is used. “Homolog” means that the corresponding polynucleotide/polypeptide has a certain degree of homology, that is to say sequence identity (preferably at least 40%, more preferably 60%, even more preferably 65%, particularly preferred at least 66%, 68%, 70%, 75%, 80%, 86%, 88%, 90%, 92%, 95%, 97% or 99%). With respect to the terms “homology” and “sequence identity” the same applies which had been set forth above in connection with the polynucleotides of the present invention. “Homology” furthermore means that the function is equivalent to the function of FT. Preferably this function is the property to enhance flowering in plants.
A variety of inducible systems, well known to those skilled in the art, may be employed for controlled restoration of flowering, e.g. the tetracycline repressor (TetR)-based tetracycline inducible system, the glucocorticoid receptor-based, steroid-inducible system, the estrogen receptor-based, steroid-inducible system, the ecdysone receptor-based, insecticide-inducble system, the ACEI-based, copper-inducible system, or other promoters that are responsive to growth regulators, metabolic signals, nutrients, elicitors, wound signals, herbicide safeners and chemicals that induce genes for systemic acquired resistance, e.g. the In2-2 (Inducible gene s-s) promoter or the benzothiadiazole (BTH)-inducible PR-1a promoter.
In a preferred embodiment of the method according to the present invention the induced restoration of flowering is achieved by ethanol inducible expression, most preferably by the use of the ethanol-inducible promoter (AlcA) in combination with the ethanol-regulated transcription factor AlcR from Aspergillus nidulans. This system is described in detail in the appended Examples and its use in model and crop plant species has already been shown in Sweetman et al. (Plant Physiol. 129 (2002), 943-948). A preferred variant of the ethanol inducible system includes an inducible self-maintaining loop based on one construct containing an artificial fusion of the alcA-minimal35S promoter to −434 operator sequences controlling the expression of the 434/VP16 activator protein together with a constitutive promoter controlling the expression of the AlcR transcription factor. An ethanol pulse will lead to the transient expression of the 434/VP16 activator protein that in turn will activate its own stable expression via the 434 operator sequences introduced into the alcA-minimal35S promoter. This stable expression of the 434/VP16 activator protein is then used to stably express the floral restorer polypeptide on a separate gene cassette, controlled by the alcA-minimal35S promoter with  ̃434 operator elements. To assure that the self-maintaining loop is not initiated caused by a certain leakiness of the artificial alcA-minimal35S promoter with 434 operator elements yet another construct containing a constitutive promoter controlling the expression of the 434-repressor protein is introduced repressing any leaky expression of the 434/VP16 activator protein. The combination of the different gene cassettes as illustrated in FIG. 13 ensures a repressed state of the loop without ethanol (via repression of transcription by the 434-repressor), an inducible expression by ethanol induction and a stably maintained expression by the 343/VP16 activator protein.
The method according to the present invention can in principle be applied to any plant which shows flowering.
Preferably, the plant is a dicotyledonous (dicot) or monocotyledonous (monocot) perennial or biennial plant. More preferably, the plant belongs to the monocots, such as Poaceae, such as Phleum spp., Dactylis spp., Lolium spp., Festulolium spp., Festuca spp., Poa spp., Bromus spp., Agrostis spp., Arrhenatherum spp., Phalaris spp., and Trisetum spp., for example, Phleum pratense, Phleum bertolonii, Dactylis glomerata, Lolium perenne, Lolium multiflorum, Lolium multiflorum westervoldicum, Festulolium braunii, Festulolium loliaceum, Festulolium holmbergii, Festulolium pabulare, Festuca pratensis, Festuca rubra, Festuca rubra rubra, Festuca rubra commutata, Festuca rubra trichophylla, Festuca duriuscula, Festuca ovina, Festuca arundinacea, Poa trivialis, Poa pratensis, Poa palustris, Bromus catharticus, Bromus sitchensis, Bromus inermis, Deschampsia caespitosa, Agrostis capilaris, Agrostis stolonifera, Arrhenatherum elatius, Phalaris arundinacea, and Trisetum flavescens. 
In a further aspect the present invention relates to a system for controlling expression of a gene of interest in plant cells comprising the following elements:

    (a) an expression cassette in which the gene of interest is placed under the control of the Alc A promoter which comprises a 434 operator sequence;
    (b) an expression cassette in which the coding sequence encoding the Alc Regulator (AlcR) is placed under the control of a promoter active in plant cells; and
    (c) an expression cassette in which a coding sequence encoding an artificial 434/VP16 transcription factor is placed under the control of the AlcA promoter containing a 434 operator sequence.


In a preferred embodiment the system according to the invention furthermore comprises:

    (d) an expression cassette in which a coding region encoding a 434-repressor protein is placed under the control of a promoter active in plant cells.


The system for controlling expression of a gene of interest in plant cells according to the invention is an “ethanol inducible self-maintaining loop system”. The AlcA promoter upon administration of ethanol is activated by the AlcR protein. In the system, according to the invention the ethanol induction is not only used directly to control expression of a gene of interest, instead it is also used to induce an artificial transcription factor (434/VP16). This transcription factor activates in a second step the expression of the gene of interest from an artificial promoter (alcA-plant promoter with 434 operator sequences). In order to establish the self-maintaining loop a further gene cassette (cassette (c)) is introduced expressing the 434/VP16 transcription factor itself from an artificial promoter (alcA-plant promoter with 434 operator sequences). One ethanol pulse will produce the first 434/VP16 transcription factor molecules, which in turn will produce itself in a self-maintaining loop from gene cassette (c) and in turn further activate the expression of the gene of interest. The self-maintaining loop will reset during meiosis and seed production so that in the next generation the loop is inactivated and the gene of interest is not expressed. In order to exclude leakiness of the self-maintaining loop in the un-induced state gene cassette (d) may be introduced constitutively expressing the 434-repressor protein. The 434-repressor secures the tightness of the artificial promoter (alcA-plant promoter with 434 operator sequences) driving the expression of the artificial activator (434/VP16) and the gene of interest. Only an ethanol-induced over-expression of the 434/VP16 activator will overcome the repression of the alcA-plant promoter with 434 operator sequences by the 434-repressor. For a better understanding the system is schematically drawn in FIG. 12.
The gene of interest expression of which is controlled in the system according to the invention can be any gene intended to be expressed in plant cells. It may, e.g. encode a polypeptide or an RNA intended to repress expression of a gene, e.g. an antisense RNA, an RNAi, a ribozyme, a cosupression RNA etc.
The AlcA promoter is the strong ethanol inducible alcohol dehydrogenase promoter from the ethanol utilization regulon from Aspergillus nidulans (Lockingon et al., Gene 33 (1985), 137-149). The AlcA promoter and expression systems using it have already been described in, e.g., Felenbok (J. Biotechnol. 17 (1991), 11-17) and the use of it in plants has already been described, e.g., by Caddick et al. (Nature Biotechnol. 16 (1998), 177-180), Salter et al. (Plant J. 16 (1998), 127-132), Roslan et al. (Plant J. 28 (2001), 225-235) and Sweetman et al. (Plant Physiol. 129 (2002), 943-948). The AlcA promoter used in the system according to the present invention is preferably a promoter as described in one of the systems of the references cited above or as described in Kulmburg et al. (J. Biol. Chem. 267 (1992), 21146-21153). The AlcA promoter in expression cassettes (a) and (c) of the system according to the invention comprises a 434 operator sequence, i.e. the sequence of the right operator OR2 of bacteriophage 434 (see, e.g., Bushman (J. Mol. Biol. 230 (1993), 28-40)). The corresponding sequence is shown in FIG. 15.
The AlcR encoded by expression cassette (b) is the trans-active regulatory protein in the ethanol utilization regulon of Aspergillus nidulans as described in Felenbok (loc. cit.). Its use for controlling expression of genes in plants has already been described in e.g. Caddick et al. (loc. cit.), Salter et al. (loc. cit.), Roslan et al. (loc. cit.), Sweetman et al. (loc. cit.), and Devenaux et al. (Plant J. 36 (2003), 918-930). Preferably, the AlcR is the protein encoded by the sequence disclosed in Felenbok et al. (Gene 73 (1988), 385-396) or in Kulmburg et al. (J. Biol. Chem. 267 (1992), 21146-21153).
The AlcR in cassette (b) is placed under the control of a promoter active in plant cells. This can be any promoter active in plant cells. Examples have been listed in connection with the polynucleotides according to the invention. Preferably, the plant promoter is a tissue specific promoter. Most preferably, the plant promoter ensures constitutive expression. Examples for promoters ensuring constitutive expression in plant cells are the ubiqutin promoter, the CaMV 35S promoter or the rice actin promoter.
The artificial transcription factor 434/VP16 in expression cassette (c) is a fusion of the 434 and VP16 activator proteins (see Wilde et al., Plant Mol. Biol. 24 (1994), 381-388). Such an artificial transcription factor has, e.g., also already been disclosed in Storgaard et al. (Transgenic Research 11 (2002), 151-159).
The expression of the artificial 434/VP16 transcription factor is placed under the control of the AlcA promoter which contains a 434 operator sequence (Kulmburg et al., J. Biol. Chem. 267 (1992), 21146-21153).
The promoter driving expression of the gene of interest and of the 434/VP16 transcription factor, apart from the AlcA promoter, preferably also comprises part of a plant promoter required for a minimal transcriptional activity. An example is the minimal CaMV 35S promoter (Gallie et al., Nucl. Acids Res. 15 (1987), 3257-3273).
The expression cassette (d) contains a coding sequence encoding a 434-repressor protein. The term “434 repressor” refers to repressor of temperate phages, such as 434 and lambda, which control transcription by binding a set of DNA operator sites. The different affinity of the repressors for each of these sites ensures efficient regulation. The repressor recognizes its operators by its complementary to a particular DNA conformation as well as by a direct interaction with base pairs in the major groove (Andersen et al., Nature 326 (1987), 846-852; Koudelka, Nucl. Acids Res. 26 (1998), 669-675). The use of the operator site in combination with the receptor protein in other systems confers transcriptional repression (Webster and Bramma, Microbiology-UK 141 (1995), 2191-2200; Part 9). Expression of the 434-repressor protein is driven by a promoter active in plant cells. In this respect, the same applies as has been set forth supra in connection with the promoter controlling expression of AlcR.
The system according to the invention has the advantage that multiple treatments with ethanol can be avoided due to the self-maintaining loop.
The present invention also relates to plant cells or plants comprising a system according to the invention. These can, in principle, be plants of any type, e.g. monocotyledonous or dicotyledonous plants, preferably perennial or biennial plants. More preferably, the plant belongs to the monocots, such as Poaceae, such as Phleum spp., Dactylis spp., Lolium spp., Festulolium spp., Festuca spp., Poa spp., Bromus spp., Agrostis spp., Arrhenatherum spp., Phalaris spp., and Trisetum spp., for example, Phleum pratense, Phleum bertolonii, Dactylis glomerata, Lolium perenne, Lolium multiflorum, Lolium multiflorum westervoldicum, Festulolium braunii, Festulolium loliaceum, Festulolium holmbergii, Festulolium pabulare, Festuca pretensis, Festuca rubra, Festuca rubra rubra, Festuca rubra commutata, Festuca rubra trichophylla, Festuca duriuscula, Festuca ovina, Festuca arundinacea, Poa trivialis, Poa pratensis, Poa palustris, Bromus catharticus, Bromus sitchensis, Bromus inermis, Deschampsia caespitosa, Agrostis capilaris, Agrostis stolonifera, Arrhenatherum elatius, Phalaris arundinacea, and Trisetum flavescens. 
The present invention also relates to a method for controlling expression of a gene of interest in a plant cell or plant which method comprises the use of a system according to the present invention. In a particularly preferred embodiment the gene of interest is a nucleic acid molecule the induced expression of which leads to a restoration of flowering in plants in which flowering is prevented. Most preferably such a nucleic acid molecule is a molecule as defined in anyone of step (b)(iii) to (v) of the method of controlling flowering in a plant according to the invention described above.
These and other embodiments are disclosed and encompassed by the description and examples of the present invention. The disclosure of all literature cited herein is incorporated into the description of the present invention by reference. Further literature concerning any one of the methods, uses and compounds to be employed in accordance with the present invention may be retrieved from public libraries, using for example electronic devices. For example the public database “Medline” may be utilized which is available on the Internet, for example under www.ncbi.nlm.nih.gov/PubMed/medline.html. Further databases and addresses, such as www:ncbi.nlm.nih.gov/, www.infobiogen.fr/, www.fmi.ch/biology/research_tools.html, www.tigr.org/, are known to the person skilled in the art and can also be obtained using, e.g., www.google.de. An overview of patent information in biotechnology and a survey of relevant sources of patent information useful for retrospective searching and for current awareness is given in Berks, TIBTECH 12 (1994), 352-364.
Furthermore, the term “and/or” when occurring herein includes the meaning of “and”, “or” and “all or any other combination of the elements connected by said term”.



The present invention will now be more fully described with reference to the accompanying examples and drawings. It should be understood, however, that the following description is illustrative only and should not be taken in any way as a restriction on the generality of the invention described above.
FIG. 1: illustrates the expression profile in perennial ryegrass of LpMADS1, LpMADS2 and LpMADS3 during the floral transition.

    
    
        Transcript levels of LpMADS genes were tested using real-time PCR and data is calculated with the Q-Gene software tool (Muller et al., Biotechniques 32 (2002), 1372). Samples were tested in triplicate and normalized to LpGAPDH and LpACTIN1 (light grey or dark grey bars, respectively), and the mean±SE is shown. Two scales are provided on the y-axis, responding to relative expression level to LpGAPDH (left) and LpACTIN1 (right). The transcript levels were tested on RNA extracted from shoot apex harvest at 3 stages, non-induced (veg), 6 (vern1) and 12 (vern2) weeks vernalized at short day and 4° C., from inflorescence at 6 stages at long day and 20° C. (LD1-LD6), from leaf at non-induced (veg), 12 weeks vernalized (vern2) and long day stage 5 (LD5), from stem, node and root.
    
    


FIG. 2: illustrates the expression profile in perennial ryegrass of LpMADS10, LpMADS14 and LpMADS16 during the floral transition.

    
    
        Transcript levels of LpMADS genes were tested using real-time PCR and data is calculated with the Q-Gene software tool (Muller et al., loc. cit.). Samples were tested in triplicate and normalized to LpGAPDH and LpACTIN1 (light grey or dark grey bars, respectively). Two scales are provided on the y-axis, responding to relative expression level to LpGAPDH (left) and LpACTIN1 (right). The transcript levels were tested on RNA extracted from shoot apex harvest at 3 stages, non-induced (veg), 6 (vern1) and 12 (vern2) weeks vernalized at short day and 4° C., from inflorescence at 6 stages at long day and 20° C. (LD1-LD6), from leaf at non-induced (veg), 12 weeks vernalized (vern2) and long day stage 5 (LD5), from stem, node and root.
    
    


FIG. 3: illustrates the phylogenetic relationship of LpFT-like and other Phosphatidyl Ethanolamine Binding Proteins (PEBS) including the LpTFL1 polypeptide.
FIG. 4: illustrates the late flowering phenotype of Arabidopsis thaliana plants (T2-generation) expressing the LpFT-like cDNA under the control of the 35S promoter. Pictures and drawing shows leaf-like structures produced in place of normal floral structures. Drawing illustrates the determinate highly branched growth pattern of the LpFT-like expressing lines very similar to the growth pattern observed by expression of the LpTFL1 transgene. Plants were verified for the presence of the intact transgene by PCR and for expression of transgene by northern blot analysis. The highest expressing lines were extremely late flowering and in some cases completely non-flowering.
FIG. 5: illustrates the additive late flowering effect of LpTFL1 and LpFT-like. Late flowering Arabidopsis plants homozygous for either the LpTFL1 or the LpFT-like ORF under the control of the constitutive 35S promoter were crossed and the offspring scored for flowering phenotype. The offspring carrying both the 35S::LpTFL1 and the 35S::LpFT-like constructs showed an additive lateness in flowering time compared to wild-type plants and plants carrying any of the LpTFL1 or the LpFT-like transgenes alone.
FIG. 6: illustrates the conserved functionality of the LpCO polypeptide, as displayed by functional complementation of the Arabidopsis thaliana co-2 mutant. Arabidopsis co-2 mutant plants were transformed by the “floral dip” method with the LpCO cDNA under the control of the constitutive 35S promoter. Plants were verified for the presence of the intact transgene by PCR and for expression of the LpCO transgene by northern blot analysis. Plants were phenotypic scored at the T2-generation.
FIG. 7: illustrates ethanol inducible GUS expression in Festuca rubra plants transformed with a construct including the maize ubiquitin promoter controlling the AlcR regulator protein and a chimeric AlcA-35S-minimal promoter controlling GUS expression. 3 independent transgenic lines are shown before (−) and after (+) ethanol induction followed by GUS-staining. The principle in the ethanol inducible AlcA/R system: Without ethanol induction AlcR will not bind to the AlcA box. Upon induction with ethanol (or other compounds) AlcR will bind to the AlcA box in the chimeric AlcA/35S-minimal promoter and induce expression of the GUS reporter gene. Transgenic plants verified by PCR and real time PCR were induced with ethanol as follows: Two tillers were cut in pieces and placed in tubes with water. The water volume was doubled with a 4% ETOH solution to give 2% ETOH in the tubes. A beaker with tissue cloth and 4% ETOH was placed in a plastic bag and sealed followed by incubation in LD chamber for 2 days. The induced tillers were cut into X-Gluc reaction buffer and incubated at 37 degree Celsius over the weekend. Then bleached in 96% ETOH over night.
FIG. 8: illustrates progression through flowering stages for control plants and transgenic plants of L. perenne constitutively expressing LpFT1 Transgenic L. perenne plants expressing LpFT1 under control of the rice actin1 promoter were produced and characterised for transgene expression by RT-PCR. Control plants (wt or Act1::GUS transgene) and transgenic plants (with detectable transgene expression, yet unrespective of expression level) were vernalized and stage progression through flowering (0=non flowering, 1=elongating stem, 2=leaf sheath, 3=flower emerged, 4=anthesis) was monitored upon shift to LD conditions.
FIG. 9: illustrates the number of days after germination (DAG) (or leaves produced) to bolting (A) and flowering (B) of the PTGS lines, the LpTFL1 background lines and the wildtype under SD conditions.
FIG. 10: illustrates the number of days after germination (DAG) (or leaves produced) to bolting (A) and flowering (B) of the PTGS lines, the LpTFL1 background lines and the wildtype under LD conditions.
FIG. 11: illustrates an RNA gel blot analysis of the PTGS lines, the LpTFL1 background lines and the wild-type. 2.5 μg of poly-A+ mRNA each line were blotted and probed with a 250 bp LpTFL1 or a 330 bp AtGAPDH cDNA probe. The top graph illustrates the levels of LpTFL1 mRNA relative to the level of AtGAPDH, and the highest detected value was set to 100 (line LpTFL1-3).
FIG. 12: illustrates a DNA gel blot hybridization analysis of genomic DNA isolated from the PTGS lines, the LpTFL1 background lines and the wildtype. DNA samples of 5 μg were restricted with BamHI and BamHI in combination with NcoI, (A), or with EcoRI and EcoRI in combination with HinDIII (B). Blot A was probed with a 950 bp fragment containing the RNAi intron, LpTFL1-PTGS, and part of the 35S terminator. Blot B was probed with a 0.4-kb fragment containing the 3′-end of the ubiquitin intron and the 5′-end of the LpTFL1 coding region. BamHI together with NcoI releases a 2.4-kb fragment containing the entire 35S::LpTFL1-PTGS cassette (arrow). BamHI has a single restriction site within the T-DNA borders of the 35S::LpTFL1-PTGS cassette. EcoRI together with HinDIII release a 2.8-kb fragment containing the entire LpTFL1 cassette (arrow). EcoRI has a single restriction site within the T-DNA borders of the UBI::LpTFL1 cassette.
FIG. 13: illustrates the ethanol inducible self-maintaining loop.

    
    
        Schematic drawing of the different elements of the ethanol inducible self-maintaining loop. A: The loop is inactive, because the artificial alcA-minimal35S promoter with 434 operator sequences is repressed by the constitutive expression of the 434-repressor protein. The AlcR regulator protein is inactive without ethanol. B: An ethanol pulse activates the AlcR transcription factor, which binds to the alcA promoter and overcomes repression by the 434-repressor resulting in the production of 434/VP16 activator protein and the production of the gene of interest. C: The produced 434/VP16 activator protein in part B under ethanol induction binds to the 434 operator sequences in the alcA-minimal35S promoter with 434 operator sequences and activates its own expression in a self-maintaining loop. Part of the activator will activate the cassette with the gene of interest. The self-maintaining loop will be stopped during meiosis and in the gametophytes the loop is still shut down. The loop gets activated again by a second round of ethanol induction.
    
    


FIG. 14: illustrates Brachypodium distacyon transformed with plasmid G10 and G12. Shown is leaf material from two independent transgenic Brachypodium lines expressing the plasmid G10 (minimal 35S promoter with one 434 operator element fused to GUS) or two independent lines transformed with G12 (like G10 plus a gene cassette expressing the 434/VP16 activator under the control of the rice actin promoter).

    
    
        No blue GUS staining is visible in the G10 transformed lines, because the 434/VP16 activator is missing and the minimal promoter with 434 operator elements is not leaky.
        However, if 434/VP16 activator protein is present like in transgenic lines transformed with G12, GUS expression is visualized by blue staining.
    
    


FIG. 15: shows the alcA promoter sequence with the two 434 OR2 operator sequences (bold and underlined).
FIG. 16: illustrates the number of days to flowering of the P05 lines, the LpTFL1-6 background lines and the wild-type (white bars) in response to ethanol vapour induction. Gray bars indicate plants, which were PCR-positive only for the UBI::LpTFL1 cassette and black bars indicate plants, which were PCR-positive for both the UBI::LpTFL1 cassette and the P05 construct.
FIG. 17: illustrates the phenotypes of P05 line 18 (A), the LpTFL1-6 background line (B), and the wild-type (C) in response to ethanol vapour induction.
FIG. 18: illustrates the results of the PCR test for presence of the UBI::LpTFL1 cassette and the P05 construct in the ethanol induced (A) and un-induced plants (B).
FIG. 19: illustrates the average number of days to flowering of the P05 lines, the LpTFL1-6 background lines and the wild-type in response to ethanol vapour induction. Gray bars indicate un-induced plants and black bars indicate ethanol-induced plants.
FIG. 20: illustrates two examples of floral revertance in ethanol-induced P05 plants.
FIG. 21: illustrates the correlation between LpFT1 transgene expression and heading date in transgenic T1 offspring of B. distachyon. The figure shows the comparison between heading date and LpFT1-transgene expression in 14 transgenic offspring plants of one of the lines with highest LpFT1 transgene expression. Strong transgene expression resulted in substantial delay in heading date
FIG. 21: illustrates the correlation between LpFT1 transgene expression and heading date in transgenic T1 offspring of B. distachyon. The figure shows the comparison between heading date and LpFT1-transgene expression in 14 transgenic offspring plants of one of the lines with highest LpFT1 transgene expression. Strong transgene expression resulted in substantial delay in heading date
FIG. 22: illustrates the phenotypic difference between wt control plants of B. distachyon and transgenic plants constitutively expressing LpFT. Transgenic plants display a substantial delay in heading date and extensive branching.



MATERIALS AND METHODS
The following materials and methods were used in the Examples:
1. RNA Extraction and mRNA Purification
Lolium perenne L. (Tetramax variety) were grown as described earlier (Jensen et. al., Plant Physiol. 125 (2001), 1517-1528). Plants were vernalization at short day (8 hours of light) below 5° C. for at least 12 weeks. Following vernalization, plants were grown under 16 hours of light at 22° C. and 18° C., day and night temperature, respectively, for secondary induction. RNA was extracted from various tissues using the FastRNA Green Kit supplied by BIO101, Inc. (Vista, Calif., USA) according to the manufactorer's recommendation. Total RNA samples were treated with RNase-free DNaseI to remove residual DNA, and mRNA was purified from total RNA using Dynabeads Oligo (dT)25 from DynaI (N-0212 Oslo, Norway).
2. Sequencing
Isolated cDNA clones were sequenced using the Big Dye Terminator Cycle Sequencing Ready Reaction Kit (Perkin-Elmner Applied Biosystems, Foster City, Calif., USA) and an ABI PRISM 377 DNA sequencer (Perkin-Elmner Applied Biosystems). Upon sequencing, the isolated by comparison with nucleotide sequences in the National Center for Biotechnology Information (www.ncbi.nim.nih.gov) database with the BLASTN search program were used as probes to screen a shoot apex cDNA library in order to obtain full-length clones.
3. Quantitative RT-PCR Analysis
Single-strand cDNA was transcribed from mRNA isolated from 5 μg total DNA-free RNA using Superscript II reverse transcriptase (Gibco-BRL) according to the manufacturer's instructions. An aliquot of 1/50 of the RT reactions was applied for PCR amplifications performed in a quantitative Rotorgene 2000 system (Corbett Research, Sydney, Australia).
SYBR Green I was used as a fluorogenic intercalating dye to quantify the PCR amplification according to the manufacturer's protocol. Each 20 μl reaction contain 3.5 mM MgCl2, 1×PCR buffer, 0.5 μM of each primer, 10 μM dNTPs, 0.5×SYBR Green I, 0.2 U Taq polymerase (Life Technology) and 1/50 of template cDNA. No template controls (NTC) were run to determine contamination and level of primer dimer formation. PCR parameters were: an initial denaturing step at 94° C. for 60 sec, followed by 40 cycles of 94° C. for 15 sec, 55° C. for 20 sec, and 72° C. for 30 sec. The following primers were used:











LpMADS1-fwd:





5′-CAGCTCGCACGGTGCTTC-3′
(SEQ ID NO: 24)



 



LpMADS1-rev:



5′-GAAACTGAGCAGAACAGA-3′
(SEQ ID NO: 25)



 



LpMADS2-fwd:



5′-CTTCATGATGAGGGATCA-3′
(SEQ ID NO: 26)



 



LpMADS2-rev:



5′-AGGTACGTACACCAGCAT-3′
(SEQ ID NO: 27)



 



LpMADS3-fwd:



5′-GAGCAGACGAATGGAGCA-3′
(SEQ ID NO: 28)



 



LpMADS3-rev:



5′-ACTGATGGTGCGGAGCAT-3′
(SEQ ID NO: 29)



 



LpMADS10fwd:



5′-ATTACCCTGCAGTGCGT-3′
(SEQ ID NO: 30)



 



LpMADS10rev:



5′-AGTACCATAGGTACATGGA-3′
(SEQ ID NO: 31)



 



LpMADS14fwd:



5′-ATGGCGGGGAAGAGGGAGA-3′
(SEQ ID NO: 32)



 



LpMADS14rev:



5′-TCACTTTGAGTTGAAAAGTG-3′
(SEQ ID NO: 33)



 



LpMADS16fwd:



5′-CAATGACGACGGTTCTGA-3′
(SEQ ID NO: 34)



 



LpMADS16rev:



5′-GCAGACTTAACGATGACA-3′
(SEQ ID NO: 35)



 



LpGAPDH-fwd:



5′-CAGGACTGGAGAGGTGG-3′
(SEQ ID NO: 36)



 



LpGAPDH-rev:



5′-TTCACTCGTTGTCGTACC-3′
(SEQ ID NO: 37)



 



LpACTIN1-fwd:



5′-GAGAAGATGACCCARATC-3′
(SEQ ID NO: 38)



 



LpACTIN1-rev:



5′-CACTTCATGATGGAGTTGT-3′
(SEQ ID NO: 39)



 



LpFT1fwd1:



5′-AGCATCAACAGATGATAGCT-3′
(SEQ ID NO: 75)



 



LpFT1rev:



5′-TGATACAGCACCAGCACGA-3′
(SEQ ID NO: 76)



 



LpFT1fwd2:



5′-TCGTGCTGGTGCTGTATCA-3′
(SEQ ID NO: 77)



 



rbs rev:



5′-AAGGTGGGAGACATCATCGA-3′
(SEQ ID NO: 78)






For each set of primers the reading temperature was determined by melting analyses and fluorescence data were acquired at 87° C. Standard curves were generated for each primer set with plasmid DNA harbouring the corresponding cDNA template. Four 100-fold serial dilutions covering a range from 1 ng to 1 fg of the plasmids were used to determine the standard curves. PCR reactions were performed in triplicate and normalized relative to the initial template amount in each sample estimated by the expression levels of the housekeeping genes LpGAPDH or LpACTIN1, and the level of the MADS-box fragments are presented relative to the level of the LpGAPDH or LpACTIN1 fragments.
4. Test of ETOH-Inducible GUS Expression
Callus cultures of Festuca rubra were co-bombarded with pUIRN-AGS (kindly provided by P. Thomsen, Syngenta, Jealott's Hill, Maidenhead, UK) and pAHC20, a selection construct with maize ubiquitin promoter fused to the BAR gene for selection with Bialaphos (kindly provided by P. Quail, Dept. of Biology, George Mason University, Fairfax, Va. 22030, USA). Plants were selected in vitro with 4 mg/l Bialaphos and transferred to soil. Plants were screend by PCR for having the GUS gene using primers 1782-56-5′ 5′-GAC TGG CAT GM CTT CGG T (SEQ ID NO: 40) and t35Srev 5′-TAT CTG GGA ACT ACT CAC ACA (SEQ ID NO: 73) and for the AlcR gene AlcR-2377-5′ 5′-CM TTT CTG GGC AGG MG TC (SEQ ID NO: 41) and tNOS-63-60-3′ 3′-CAT CGC MG ACC GGC MC (SEQ ID NO: 42). Plants that were negative for one or both primer sets were discharged, plants that were positive for both were selected for GUS staining and for RT-PCR test.
For two plants of each of 22 independent transformants and two non transgenic control plants the following induction experiment was made: Non-induced: From each plant, two tillers were cut directly into a standard X-GLUC buffer supplemented with 300 mg/l cyclohexamide, vacuum infiltrated in a speed-vac for 5 min and incubated at 37° C. for 2 nights. Cleared for chlorophyll with 2 times wash in 96% ethanol. Ethanol induction: Tillers were cut and placed in  ̃2 ml dH2O. For each tube, the volume was doubled with a 4% ethanol solution giving a  ̃2% ethanol solution in the tubes. All tubes were placed in a plastic bag and a beaker with a tissue cloth soaked in 4% ethanol. The bag was sealed and placed in a growth chamber with a 16 hours light period for 2 nights. Tillers were then GUS stained as described for un-induced.
5. Plant Transformation
Lolium perenne—Biolistic Transformation
Plasmids containing transgenes of intererst (pGOI) were introduced into Lolium perenne together with pAHC20 (Christensen and Quail, Transgene Research 5 (1996), 213-218) harboring the Bar gene, which confers resistance to the herbicide BASTA®. For particle bombardment highly embryogenic callus induced from meristems or mature embryos was used. Isolated embryos and meristems were cultured on a MS-based ((Murashige and Skoog, Physiol. Plant. 15 (1962), 473-497) callus induction medium (CM) containing 3% sucrose, 4 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D), 100 mg/l casein hydrolysate and 0.3% (w/v) gelrite (Kelco) for 12-26 weeks in the dark at 23° C. Calli were maintained by subculturing every third week on fresh CM-medium. Prior to bombardment, a osmotic pre-treatment for 4 hours were given by transferring small calli (2-4 mm) to a solid MS-based medium supplemented with 3% sucrose, 3 mg/l 2,4-D, 0.25 M sorbitol, 0.25 M mannitol and 0.3% w/v Gelrite. Bombardment was performed with a particle inflow gun (Finer et al., Plant Cell Rep. 11 (1992), 323-328) according to the optimized protocol described by Spangenberg et al. (J. Plant Physiol. 145 (1995), 693-701) with a few modifications: bombardment pressure was 8 bar and 300 :g gold particles 0.6 :m (Biorad) was coated with 0.6 :g plasmid DNA (pGOI and pAHC20 at a molar ratio of 2:1) according to Vain et al. (Plant Cell Tissue and Organ Culture 33 (1993), 237-246). The following day, calli were transferred to CM-medium supplemented with 4 mg/l bialaphos (Meiji Seika Kaisha, LTD, Tokyo) and grown at 23° C. under 16 hrs light. Selection at three weeks interval was performed until vigorously growing callus was obtained. Putative transgenic plants were regenerated by transferring calli to hormon free medium RM (MS-medium containing 3% sucrose and 4 mg/l bialaphos). Rooted plantlets were transferred to screening for stable transformation, putative transgenic plants were sprayed twice (two successive days) with a 0.5% solution of BASTA (Hoechst Schering AgrEvo A/S, Germany) supplemented with 0.1% Tween 20. The number of herbicide tolerant plants was scored after one week. Leaf material from BASTA-resistant plants was subsequently screened for the presence of pGOI by PCR. soil and grown to maturity under greenhouse conditions.
Brachypodium Distachyon—Agrobacterium Mediated Transformation
The embryos are placed on callus inducing media to initiate cell proliferation prior to transformation. After one day they were transformed with AGL1 harbouring the respective constructs and co-cultivated with Agrobacteria on callus medium for 5 days in the light. Embryos were washed in water supplemented with 250 mg/l Augmentin and drained on sterile filter paper. Selection was carried out on callus medium containing 5 mg/l bialaphos and 250 mg/l augmentin for two periods of  ̃3 weeks followed by one or two periods of  ̃2-3 weeks on selective regeneration medium inducing shoots. Green shoots were transferred to rooting medium for  ̃3 weeks and plants were potted and grown to maturity. Leaves were stained for GUS-expression as described elsewhere.
Callus medium: 4.4 g/l LS salts, 30 g/l maltose, 2.5 mg/l 2,4-D, 8 mg/l agar, pH 5.9, regeneration medium: 4.4 g/l LS salts, 30 g/l maltose, 0.2 mg/l BAP, 8 g/l agar, pH 5.9, rooting medium: 2.2 g/l LS salts, 30 g/l maltose, 8 g/l agar, pH 5.9.
6. LpTFL1 PTGS-Mediated Restoration of Flowering in Late-Flowering UBI::LpTFL1 Arabidopsis 
Plant Transformation
A 143 bp fragment of LpTFL1 (sequence XX) was amplified from a plasmid pLPTFL1 (Jensen et al., Mol. Breeding 13 (2004), 37-48) containing the LpTFL1 coding region by PCR using recombinant pfu DNA polymerase in combination with the primers LpTF1rnai5′ (5′-CACCGTGGAGCCTCTTATTGTTGGT-3′ (SEQ ID NO: 43)) and LpTFL1rnai3′ (5′-TAGATACMCTGCTGATGGGTA-3′ (SEQ ID NO: 44)). The fragment was cloned into pENTRTM/SD/D-TOPO® (Invitrogen, Carlsbad, Calif., USA) to give pENTR-LpTFL1-PTGS, which was subsequently used in a LR-recombination (Invitrogen, Carlsbad, Calif., USA) to recombine the LpTFL1-PTGS fragment into the destination vector pK7GWIWG2(I) (Karimi et al., Trends in Plant Science 7 (2002), 193-195). The resulting plasmid, pK7-LpTFL1-PTGS possesses a streptomycin and/or spectinomycin resistance gene for plasmid selection and harbors the nptII gene for plant Kanr selection. Transgenic Arabidopsis plants expressing LpTFL1 from the ubiquitin promoter (line 3 and 6, T2 generation, Jensen et al., Plant Physiol. 125 (2001), 1517-1528) were transformed with the Agrobacterium tumefaciens, strain PGV3101 (Koncz and Schell, Mol. Gen. Genet. 204 (1986), 386-396) harboring the pK7-LpTFL1-PTGS using the floral dip method described by Clough and Bent (Plant J. 16 (1998), 735-743).
7. Growth Conditions
T1 transformants were selected on MS-pates (Murashige and Skoog, 1962, loc. cit.) supplemented with 50 mg/l Kanr (pK7-LpTFL1-PTGS) and 2 mg/l Bialaphos (Shinyo Sangyo Ltd, Japan) and grown in long day (LD conditions, 16 hrs. light) at 22° C. Early flowering lines were selected and selfed for T2 flowering time analysis. Kanr, Bialaphos resistant T2 plants were stratified at 4° C. for four days and then grown in soil in short day (SD; 8 hrs light) conditions at 22° C. After three weeks half of the plants were moved to LD conditions. All lines were grown alongside the UBI::LpTFL1 line 3 and 6 and the wildtype (Col. 0) for control. Flowering time was measured as the number of days or leaves to bolting and to the opening of the first flower.
8. DNA Gel Blot Analysis
Genomic DNA for the gel blot analysis was isolated from the T2 LpTFL1-PTGS plants (hereafter referred to as PTGS-lines) and the UBI::LpTFL1 line 3 and 6 (hereafter referred to as LpTFL1-3 and LpTFL1-6, respectively) and the wild-type by the Phytopure® Genomic DNA isolation system (Nucleon). DNA (5 μg) were digested overnight with restriction endonucleases EcoRI, EcoRI in combination with HindIII (plasmid pLpTFL1) and BamHI, BamHI in combination with NcoI (plasmid pK7-LpTFL1-PTGS). It was fractionated on a 0.8% agarose gel and blotted onto Amersham Hybond N membrane in 20% SSC according to the manufacturer's recommendations. A 950 bp fragment containing the RNAi intron, LpTFL1-PTGS, and part of the 35S terminator was amplified by PCR using the primers INT#185 (5′-TAGGGGTTTAGATGCMCTGT-3′ (SEQ ID NO: 45)) in combination with T35Srev (5′-TATCTGGGAACTACTCACACA-3′ (SEQ ID NO: 46)) on plasmid DNA and used as probe for the detection of transgenes corresponding to pK7-LpTFL1-PTGS. A probe for the detection of transgenes corresponding to pLpTFL1 was generated in a similar way using the primers ACT#56 (5′-TATTTATTTGCTTGGTACTG-3′ (SEQ ID NO: 47)) together with LpTFL1ins3′ (CTCCCCCCCAAATGMGC-3′ (SEQ ID NO: 48)). Both probes were radiolabeled with β-32P-labeled dCTP (3,000 Ci/mmol) through the random primer method (Megaprime, Amersham) and hybridized to the blots containing the respective DNA digestions.
9. RNA Gel Blot Analysis
Seventy five micrograms of total RNA were isolated from the T2 PTGS plants and the LpTFL1 lines and the wild-type using the Trizol® reagent (Invitrogen, Carlsbad, Calif., USA) according to the manufactors instructions. Purified poly-A+ mRNA (Dynabeads, DYNAL, Norway) from one individual of each line was fractionated under denaturing conditions and transferred onto Hybond N membranes in 20% SSC. The membranes were hybridized to a 250 bp LpTFL1 cDNA fragment, Which was amplified by PCR using the primer LpTFL1ins5′ (5′-GACCTTATTCACATTGGTTATG-3′ (SEQ ID NO: 49)) in combination with LpTFL1ins3′ (outside the PTGS sequence), and a 330 bp AtGAPDH cDNA fragment for standardization. Relative LpTFL1 expression levels in the transgenic lines were estimated on the basis of the results from a density scan (Quantity One software, Biorad) of the autoradiograph and the highest detected value was set to 100.
EXAMPLE 1
Screening of cDNA Clones
An apex cDNA library of Lolium perenne L. (variety Green Gold) was constructed from extracted mRNA isolated from shoot apices at different growth stages after floral induction, using the ZAP-cDNA/Gigapackill Gold Cloning Kit (Stratagen, La Jolla, Calif., USA). The cDNA library containing approximately 700,000 independent clones was screened with corresponding 32P-labeled C-terminal gene probes. The LpMADS1 probe of 149 bp was made by a RT-PCR reaction using mRNA from secondary induced meristems as template and using the degenerate primers:








(SEQ ID NO: 50)









Fwd primer = 5′-SARHTGAAGMGGATAGAGAACAAGAT-3′,



 








(SEQ ID NO: 51)









Rew primer = 5′-CTCGTAGAGCTTGCCCTTGG-3′.







The probe used to isolate LpMADS2 and LpMADS3 was made by a RT-PCR reaction using mRNA from secondary induced meristems as template and using the primers:










(SEQ ID NO: 52)











Fwd primer = 5′-TCGAGAACAAGATCAACCGCC-3′,




 










(SEQ ID NO: 53)











Rew primer = 5′-TGGTGGAGAAGTTGATGAGCC-3′.







Isolation of LpMADS10, LpMADS14 and LpMADS16 by 5′- and 3′-RACE.
Purified mRNA derived from 5 μg of DNase-free RNA was used for first-strand cDNA synthesis as described by the manufacturer (Clontech Laboratories Inc.). 5′-RACE was performed with 5′-cDNA from non-induced leaves using a primer designed to be specific for MADS box genes (5′-TTGGAGMGGT(G/C)AC(G/C/T)CGGCT-3′ (SEQ ID NO: 54)) and with a nested primer (5′-GTTCTC(A/G/T)AT(C/T)CGCTT(G/C)A-3′ (SEQ ID NO: 55)). 3′-RACE was performed with 3′-cDNA with 3′-RACE primer (5′-GCCG(A/G/C)CA(AG)GT(G/C)ACCTTCTTCC-AA-3′ (SEQ ID NO: 56)) and nested primer (5′-GC(G/C/T)CT(C/T)(A/C)TCGTC(G/T)TCTC-3′ (SEQ ID NO: 57)). To isolate full-length MADS-box genes from the 5′-RACE, primers were designed in the UTR of the fragments generated in the 5′-RACE (group1-5′ primer: 5′-ACCGCAGCCACCATCTCACCTCA-3′ (SEQ ID NO: 58); group2-5′ primer: 5′-CCTCTCGCCACCACCACCAGA-3′ (SEQ ID NO: 59); group3-5′ primer: 5′-TGCTCCTGAT-TGGTCCACAGTT C-3′ (SEQ ID NO: 60)) and the 3′-RACE primer was used as the nested primer. Primers were also designed from the 3′-RACE fragments (group1-3′ primer: 5′-GAGTTGTCGTMCCAGCAGCATCACT-3′ (SEQ ID NO: 61); group2-3′ primer: 5′-AACATCACGTCATGCAGCCACMGGAT-3′ (SEQ ID NO: 62); group3-3′ primer: 5′-ATGGGACCATTCCAGTCAGTCTAGCT-3′ (SEQ ID NO: 63)) and the 5′-RACE primer was used as the nested primer. PCR parameters were an initial denaturing at 94° C. for 60 sec, followed by 30 cycles of 94° C. for 30 sec, 68° C. for 30 sec and 72° C. for 3 min.
Isolation of LpMADS10 by Yeast Two-Hybrid Screen:
A fusion library in a GAL4-activation domain vector (Matchmaker system from Clonetech, pACT2) of cDNA isolated from Lolium perenne flowers was generated and 3.6×106 colonies were screened in a Two-Hybrid assay with a fusion of the LpMADS1 K-domain to the GAL4-DNA binding domain. The K-domain of LpMADS1 (corresponding to amino acids 91 to 162) was amplified with the following primers by PCR (primer A (SEQ ID NO: 79): gcggatccggtgtcatgaatatag; primer B (SEQ ID NO: 80): gcgtcgaccagtgacctctccttc), gel purified and BamHI/SalI cloned into the pAS1 vector (Durfee et al., Genes Dev. 7 (1993), 555-569). Analysis of this Two-Hybrid screen in yeast identified a specific interaction of MADS1 and a novel MADS-box gene (Sequence ID NOS: 3-4). The cloned LpMADS10 gene was full-length.
Isolation of the LpID1-Like cDNA Clone
LpID1 was identified essentially by a PCR-based strategy. An initial strategy using the maize full-length ID1 to screen cDNA libraries for Lolium homologs led to a high number of candidates, which by sequencing showed poor homology to the maize ID1 outside the zink finger regions and thus were unlikely to represent ID1 homologs.
An alternative strategy based on the maize ID1 polypeptide was developed, in which two consensus primers (identical regions in all obtained Lolium clones) in the two zink finger regions were designed and degenerated primers based on the very C-terminal part of the maize ID1 protein. By running the lower primer—TCCTGGAGCCACMCTTCTAG (SEQ ID NO: 21)—(last 7 aa of the maize ID1) on 1st strand cDNA made from young leaves in a first reaction using upper primer—TTCCAGCGGGACCAGMCC (SEQ ID NO: 22)—in Zink finger region 1 and nesting in a second reaction with upper primer in zink finger region 2—GGATCMGMGCACTTCT (SEQ ID NO: 23)—a 700 bp fragment representing a likely partial ID1 homolog was obtained. 5′-RACE was used to extent the fragment from zink finger region 2 to the upstream zink finger region 1 and finally isolation and sequencing of a genomic clone provided the missing 5′-part. Finally, knowing the full-length sequence a full-length ID1 open reading frame (ORF) was produced by PCR and confirmed by sequencing.
In contrast to the LpID1 gene described herein, the homologoues of maize ID1 isolated from perennial ryegrass disclosed in WO 02/38768 are only distantly related to the maize ID1 outside the Zink Finger regions. Blast search results against public sequence databases including Genbank reveal that the LpID1 of the present invention represents the closest relative to the maize ID1 in comparison to any publicly available nucleotide or polypeptide sequence.
Isolation of the LpFT-Like cDNA Clone
Purified mRNA derived from 5 μg of DNase-free RNA was used for first-strand cDNA synthesis as described by the manufacturer (Clontech Laboratories Inc.).
5′-RACE and 3′-RACE was performed using the primers:








UPM long:









(SEQ ID NO: 64)









5′-CTA ATA CGA CTC ACT ATA GGGCAA GCA GTG GTA TCA



ACG CAG AGT-3′


 


3lpFT-1









(SEQ ID NO: 65)









5′-CTA CGA GAG CCC AAR GCC AAM CAT-3′



 


3lpFT-2









(SEQ ID NO: 66)









5′-AGC AAC ACA TCC TTG TGA AGG CCC A



 


3lpFT-3









(SEQ ID NO: 67)









5′-AGC TAA GTA CCG TGT GAT GCG GCT



 


3lpFT-4









(SEQ ID NO: 68)









5′-TGG CGG CGA CGG GCT TTC CGA







In particular, a cDNA library was made from a pool of L. perenne leaves harvested throughout 24 h in long day conditions. Messenger RNA was isolated from total tissues using Dynabeads Oligo (dT)25 (Dynal). A single-strand cDNA synthesis was performed with the PowerScriptTM Reverse Transcriptase according to the SMARTM RACE cDNA Amplification kit (Clontech Laboratories Inc). A 3′-RACE PCR with primer 3IpFT-1 was performed on a cDNA library following manufacturer's instructions. A 560 bp sequence was isolated which showed high homology to the rice OsHD3a sequence. To obtain the full-length cDNA of the LPFT-like 1 gene, a 5′-Race PCR was done with primer 3IpFT-2 designed on the 3′end. In total a full-length 842 bp sequence was isolated and identified as a likely LpFT-like homolog.
Isolation of the LpCO cDNA Clone
To isolate CO-like genes from Lolium perenne, a set of degenerated primers were designed based on nucleotide sequence comparison between AtCO and OsHd1. A cDNA library (Stratagene) made from L. perenne leaves, which had been induced for flowering was used as template. PCR was performed with primers LpCO-fwd1: GGGAGCGAGTGTGTGGTAC (SEQ ID NO: 69) and LpCO-rev1:: ACCCTGGCCTCCCTGTC (SEQ ID NO: 70) with 0.5 μg of template with 2 mM MgCl2, 1×PCR buffer, 0.4 μM of each primer, 0.25 mM dNTPs and 0.25 U of Taq polymerase (Life Technology) in 50 μl reaction. PCR parameters were: initial denaturation 95° C. 10 min, 35 cycles of 95° C. for 30 sec, 60° C. for 30 sec and 72° C. for 60 sec and 72° C. for 60 sec. A 300 bp PCR fragment was labelled by random labelling (Megaprime DNA labelling system, Amersham Biosciences) and used as a probe to screen 2.0 107 clones from a ZAP-cDNA phage library (Stratagene) from L. perenne (F6) leaves. 5 clones were isolated at low stringency and a unique full-length cDNA clone was isolated representing a CO homologue named LpCO.
A full-length genomic clone of LpCO was obtained by PCR using genomic ryegrass DNA (Fast DNA kit, Q-Biogene) in combination with two primers LpCO-fwd2:: ATGGTCTGTGTGGTGCMGCCA (SEQ ID NO: 71)/LpCO-rev2:: ACCGATCTACCTGAACTGCTTG (SEQ ID NO: 72) which match the sequence the in 5′ and 3′UTR respectively. PCR reaction was performed on 0.5 μg gDNA template with 2 mM MgCl2, 1×PCR buffer, 0.4 μM of each primer, 0.25 mM dNTPs and 0.25 U of Taq polymerase (Life Technology) in 50 μl reaction. PCR parameters were: 95° C. 4 min, 30 cycles of 95° C. for 20 sec, 68° C. for 15 sec and 72° C. for 90 sec and 10 cycles of 95° C. for 30 sec, 52° C. for 15 sec and 72° C. for 90 sec.
EXAMPLE 2
Restoration of WT Flowering Phenotype in an Arabidopsis thaliana Plant Otherwise Substantially Prevented in Flowering Through the Floral Suppressive Action of the Polypeptide of LpTFL1
In order to restore wild-type flowering time in late-flowering UBI::LpTFL1 Arabidopsis plants a construct was made in which a transgene encoding two 143 bp LpTFL1 inverted repeats separated by a spliceable Arabidopsis intron was placed under the control of the viral 35S CaMV promoter. The 35S::LpTFL1-PTGS construct was introduced into two different late-flowering transgenic UBI::LpTFL1 Arabidopsis lines (line 3 and 6, flowering after 77 and 66 days in LD, respectively). Several Kanr, BASTA® resistant T1 plants were regenerated of which one line from background LpTFL1-3 and three from background LpTFL1-6 flowered simultaneously with the wild-type. These four lines (PTGS3-1, PTGS6-1, PTGS6-2, and PTGS6-3) were self-pollinated and the T2 seeds were used for a detailed flowering time phenotype analysis.
The flowering time response of the PTGS lines was determined both under SD and LD conditions and compared with that of the wild-type and the late-flowering LpTFL1-3 and LpTFL1-6 background lines. All the PTGS plants were germinated and selected on MS medium containing kanamycin (50 mg/l) for selection of the 35S::LpTFL1-PTGS construct and bialaphos (2 mg/l) for selection of the UBI::LpTFL1 construct. The LpTFL1 background lines were germinated and selected on MS medium containing bialaphos (2 mg/l) and the wild-type was germinated on MS medium without selection. Flowering time was scored both as the number of days and the number of leaves produced from germination till the opening of the first flower. In the wild-type the first flower opens immediately after bolting but in the late-flowering LpTFL1-3 and LpTFL1-6 lines flowers are first formed several weeks after bolting. Time to bolting was also scored as the number of days and the number of rosette leaves produced.
In LD conditions the wild-type started bolting (and flowering) after about 37 days (FIG. 10A). At this time several of the PTGS plants had already started to flower.
The LpTFL1-3/6 background lines bolted a week later, but remained without flowers for another month (FIG. 10B). Introduction of the LpTFL1-PTGS into the LpTFL1-3 line reduced the time to flowering with 40 days from 79 to 28.8±1.8. A similar pattern was observed for the plants growing under SD conditions although flowering for all plants was considerably delayed compared with the LD grown plants (FIG. 9). The wild-type flowered after about 79 days (FIG. 9B). At this time the PTGS3-1 line had already been flowering for almost fourteen days. The LpTFL1-3 background line however, did not flower before day 150. Thus under SD conditions the presence of the LpTFL1-PTGS construct was associated with a 96 day decrease in the time to flowering in line LpTFL1-3. Two other PTGS lines (6-2 and 6-3) flowered simultaneously with the wild-type and significantly earlier than the LpTFL1-6 background line (64 days). One PTGS line (6-1) did not flower significantly earlier as the LpTFL1-6 background neither in SD nor in LD in the T2 generation.
The LpTFL1RNAi Sequence is Sufficient for Downregulation of LpTFL1 Expression and Floral Eestoration.
RNA gel blot analysis was performed to verify a PTGS-mediated downregulation of LpTFL1transcription in the early-flowering PTGS lines. The RNA blots were probed with a LpTFL1 fragment laying outside the LpTFL1-PTGS sequence in order to avoid any cross-contamination. For standardization the blot was also probed with an AtGAPDH fragment. A significant decrease in LpTFL1 mRNA was detected in the three early-flowering PTGS lines (FIG. 11). The most prominent reduction was observed in line PTGS3-1 and PTGS6-3, where the level of LpTFL1 mRNA was reduced with 91.6% and 90.4% respectively, compared to the background lines.
The presence of the two transgenes (UBI::LpTFL1 and 35S::LpTFL1-PTGS) was tested by DNA gel blot analysis in which genomic DNA from the transgenic lines, the background lines and the wild-type were digested with restriction enzymes that cuts at both T-DNA borders thereby releasing the entire cassettes or only one time in-between the borders to reveal the presence of concatamers and allow a rough prediction of transgene copy number. Digestion of the 35S::LpTFL1-PTGS cassette with BamHI and NcoI releases a fragment of 2.4 kb, which could be detected in all the PTGS lines but not in the LpTFL1 background lines or in the wild-type (FIG. 12A). Digestion of the UBI::LpTFL1 cassette with EcoRI and HindIII releases a fragment of 2.8 kb, which could be detected in all the PTGS lines and in the LpTFL1 lines but not in the wildtype (FIG. 12B). However, the intensity of the bands were markedly reduced in line PTGS6-2 and PTGS6-3. Analysis of the blot containing DNA digested with EcoRI only, revealed that the original integration pattern of UBI::LpTFL1 in the background line LpTFL1-6 had been changed in the PTGS-lines and that line PTGS6-2 and PTGS6-3 only contained a single copy of the UBI::LpTFL1 cassette (FIG. 12B). It is not possible to determine at what stage the excision of the UBI::LpTFL1 cassette has occurred and also not if it can be related to the presence of the LpTFL1-PTGS construct. The integration patterns of UBI::LpTFL1 in PTGS3-1 and PTGS6-1 were identical to their respective background lines. Thus, the reduction in flowering time observed in line PTGS6-1 and the restoration of wild-type flowering observed in line PTGS3-1 is directly linked to a LpTFL1-PTGS mediated post-transcriptional silencing of LpTFL1. This result also shows that the LpTFL1-PTGS sequence is capable of overcoming the effect of multiple UBI::LpTFL1 transgene copies.
In conclusion, it was shown that expression of the LpTFL1-PTGS construct initiates a post-transcriptional silencing of LpTFL1, which eventually will abolish the LpTFL1 repression of flowering in Arabidopsis. This result is to the inventors' knowledge the first evidence of PTGS-mediated release of transgene-induced flowering repression. This method will have wide applications for floral restoration and will not be limited to LpTFL1 but also to other floral repressors, such as the LpFT-like gene, the Lolium perenne MADS box genes LpMADS10, the LpMADS14, the LpMADS16 or the Arabidopsis thaliana Flowering Locus C/-F (FLC/FLF) gene (accession AF537203/AF116527), which may confer floral repression activity.
EXAMPLE 3
Substantial Prevention of Flowering in Arabidopsis thaliana Through the Floral Suppressive Action of Expressing the LpFT-Like Polypeptide
Blast results of LpFT-like against the NCBI sequence database revealed a close similarity to the FT subfamily of Phosphatidyl Ethanolamine Binding Proteins (PEBS). This is illustrated in FIG. 3 showing the phylogenetic relationship of LpFT-like and other Phosphatidyl Ethanolamine Binding Proteins (PEBS) including the LpTFL1 polypeptide. The LpFT-like polypeptide groups together with the FT-subfamily, being clearly distinctive from the TFL subfamily, thus indicating a floral enhancer activity of the LpFT-like polypeptide. Unexpectedly, the opposite was found to be the case. As illustrated in FIG. 4, expression of the LpFT-like polypeptide in Arabidopsis thaliana confers a strong suppression of flowering. Arabidopsis Ler and Col ecotypes constitutively expressing the LpFT-like polypeptide (under the control of the 35S promoter) showed indeterminate growth pattern and flowered in average 2-2.5 months after sowing compared to about 3 weeks after sowing for wild type plants. Some of the LpFT-like expressing plants never flowered and died without setting any seeds. These findings are very similar to the phenotype of LpTFL1 expressing plants and thus represent the first demonstration of TFL1-like functionality of a FT-like polypeptide.
The floral suppressor activity of the LpFT-like polypeptide was further demonstrated by crossing of late-flowering LpFT-like expressing Arabidopsis plants with late-flowering LpTFL1 expressing plants, as illustrated in FIG. 5. The offspring of these crossings showed an unexpected additive late-flowering effect of the LTFL1 and the LpFT-like polypeptides, the LpTFL1/LpFT-like expressing plants being significant more late flowering than any of the late-flowering LpTFL1 or LpFT-like expressing lines.
EXAMPLE 4
Constitutive Overexpression of LpFT1 Prevents Flowering in Transgenic Plants of Lolium Perenne 
Transgenic L. perenne plants expressing LpFT1 under control of the rice actin1 promoter were produced and characterised for transgene expression by RT-PCR. Control plants (wt or Act1::GUS transgene) and transgenic plants (with detectable transgene expression, yet unrespective of expression level) were vernalized and stage progression through flowering (0=non flowering, 1=elongating stem, 2=leaf sheath, 3=flower emerged, 4=anthesis) was monitored upon shift to LD conditions. Results are shown in FIG. 8. The majority of control plants had progressed through phase 1 within 24 days in LD and had reached phase 4 no later than 42 days after shift to LD. 52 days after shift to LD, 87% of all control plants had progressed through anthesis. In contrast, the gross of plants expressing the transgene never reached phase 1. In fact, 93% of all plants expressing the transgene remained non-flowering during the whole experiment.
This result clearly demonstrates the strong potential of the LpFT1 polypeptide to prevent flowering in the homologous system.
EXAMPLE 5
Constitutive Ectopic Expression of LpFT1 in the Grass Model Species Brachypodium Distachyon 
In order to demonstrate the potential of LpFT1 to repress the process of flowering in monocots, we used B. distachyon as a model system regarded as representative for the Pooidae subfamily. The Pooidea subfamily comprises the tribes Ampelodesmeae, Aveneae, Brachyelytreae, Brachypodieae, Bromeae, Diarrheneae, Lygeeae, Meliceae, Nardeae, Poeae, Stipeae and Triticeae, and thereby the large majority of agronomically important temperate grasses and cereals. Transgenic B. distachyon plants expressing LpFT1 from the rice actin1 promoter were produced by Agrobacterium-mediated transformation. In the T1 generation, at least 15 seeds were sown for each line and heading dates were monitored as days from germination to ear emergence. Lines were characterised for presence of transgene to distinguish between null segregants and transgenic offspring. Transgene expression was determined in transgenic T1 generation plants using real-time PCR. The results depicted in FIG. 21 show the comparison between heading date and LpFT1-transgene expression in 14 transgenic offspring plants of one of the lines with highest LpFT1 transgene expression. Strong transgene expression resulted in substantial delay in heading date.
In comparison to control plants, T1 individuals showing high transgene expression exhibited a pronounced branching phenotype as depicted in FIG. 22.
EXAMPLE 6
Gene Expression Profile of LpMADS1, LpMADS2, LpMADS3 in Ryegrass During the Floral Transition
The LpMADS1, LpMADS2 and LpMADS3 genes in ryegrass show an expression pattern as predicted for an AP1-like function as enhancer of floral transition. Blast results of LpMADS1, LpMADS2 and LpMADS3 against the NCBI sequence database revealed a close similarity to the AP1 subfamily of MADS box proteins. Homology to AP1-subgroup MADS box proteins/genes from other plant species based on sequence information alone, does not allow determining possible functional conservation. Considering the high degree of redundancy in function found within the MADS box proteins from, e.g., Arabidopsis additional information on e.g. expression pattern is required. For an AP1-like floral enhancer function, the expression of LpMADS1, LpMADS2 and LpMADS3 should expectedly increase early in response to floral transitional stimuli.
To determine the expression pattern of LpMADS1, LpMADS2, LpMADS3 message in ryegrass mRNA levels were examined in different tissues by real time quantitative PCR. For all three MADS genes, expression was shown to increase rapidly in the shoot apex upon exposure to 6 weeks of vernalization (see FIG. 1). The expression of LpMADS1 was more strongly induced than were LpMADS2 and LpMADS3. During secondary induction, the expression of all three genes was found to gradually decline to a level higher than found in non-induced (vegetative) plants. In all three cases, the findings together with the sequence similarity support the premise that the three MADS genes represent functional AP1-like floral enhancers with the potential of antagonistically overcoming the suppression of flowering caused by any of the polypeptides LpMADS10, LpMADS14, LpMADS16, LpTFL1 or FLC or a functional fragment, derivative, or homologue thereof.
EXAMPLE 7
Gene Expression Profile of LpMADS10, LpMADS14 and LpMADS16 in Ryegrass During the Floral Transition
The LpMADS10, LpMADS14 and LpMADS16 genes in ryegrass show an expression pattern as predicted for an inhibitor of floral transition.
To determine the expression pattern of LpMADS10, LpMADS14 and LpMADS16 message in ryegrass mRNA levels were examined in different tissues by real time quantitative PCR. For all three MADS genes, expression was shown to decrease dramatically in the shoot apex (the tissue which subsequently develops into reproductive structures e.g. the inflorescence, stem and flowers) upon exposure to a period of 12 weeks floral inductive vernalization, whereas expression in all three cases remained unchanged or increased in leaves during the floral transition, thus supporting the premise that the three MADS box genes represent possible inhibitors of the floral transition in ryegrass (see FIG. 2).
EXAMPLE 8
Ethanol Inducible Gene Expression in Grass
An ethanol inducible expression system has been described in the fungus Aspergillus nidulans (Felenbok et al., Gene 73 (1988), 385-396). It consist of the AlcA promoter with the specific AlcA box or recognition site and Alc Regulator (AlcR) that, after exposure to ethanol, binds to the AlcA box and initiates transcription of the gene fused to the AlcA promoter. The AlcA/R system has previously been tested in the dicot model plants tobacco (Caddick et al., Nature Biotechnology 16 (1988), 177-180) and Arabidopsis (Roslan et al., Plant J. 28 (2001), 225-235) and can be induced by other compounds than ethanol (WO 00/09704). The AlcA promoter has been fused to a 35S minimal promoter and in a test construct fused to the UidA reporter gene. The construct has the AlcR under control of the maize ubiquitin promoter. Here it is described that the system is applicable to grasses, exemplified by studies in Festuca rubra. Of the 22 independent transformants, 3 (14%) tested positive in the induced treatment and negative in the non-induced treatment, thus confirming the functionality of the ethanol-inducible gene expression system in grasses. The results are shown in FIG. 7.
EXAMPLE 9
Ethanol Inducible Self-Maintaining Loop
In order to avoid the potential need of multiple treatments with ethanol to induce flowering the ethanol induction system may be combined with a self-maintaining loop system.
Ideally flowering is repressed via over-expression of a repressor of flowering. In the “ethanol inducible self-maintaining loop system”, the ethanol induction is not used directly to overcome repression by expression of a “floral restoration construct”, instead it is used to induce an artificial transcription factor (434/VP16). This transcription factor activates in a second step the “floral restoration construct” from an artificial promoter (alcA-minimal 35S promoter with 434 operator sequences). In order to establish the self-maintaining loop a second gene cassette (cassette 2) is introduced expressing the 434/VP16 transcription factor itself from the artificial promoter (alcA-minimal 35S with 434 operator sequences). One ethanol pulse will produce the first 434/VP16 transcription factor molecules, which in turn will produce itself in a self-maintaining loop from gene cassette 2 and in turn further activate the expression of the “floral restoration construct”. The self-maintaining loop will reset during meiosis and seed production so that in the next generation the loop is inactivated and flowering is repressed. In order to exclude leakiness of the self-maintaining loop in the un-induced state a third gene cassette may be introduced constitutively expressing the 434-repressor protein. The 434-repressor secures the tightness of the artificial promoter (alcA-minimal 35S with 434 operator sequences) driving the expression of the artificial activator (434/VP16) and the repressor RNAi construct. Only an ethanol-induced over-expression of the 434/VP16 activator will overcome the repression of the alcA-minimal 35S promoter with 434 operator sequences by the 434-repressor. For a better understanding the system is schematically drawn in FIG. 13.
Essential components of the ethanol inducible self-maintaining loop were confirmed in planta. There is a constitutive expression of the 434-repressor protein throughout the whole life span of the plant, which ensures non-leakiness of the artificial alcA-minimal 35S promoters with 434 operator elements. Storgaard et al. (Transgenic Research 11 (2002), 151-159) have shown a minimal promoter with 434 operator elements repressed by constitutive expression of the 434-repressor to be inducible by over-expression of the 434V16 activator in Arabidopsis thaliana. 
Expression of a construct (G10) with the minimal 35S promoter and one 434 operator element driving GUS expression in Brachypodium distachyon showed no GUS staining in transgenic Brachypodium distachyon (inactivity of the promoter alone without presence of the 434/VP16 activator) whereas transgenic plants transformed with G12 (constitutive expression of the 434/VP16 activator) showed strong GUS expression (see FIG. 14).
EXAMPLE 10
Induced Restoration of WT Flowering Phenotype in an Arabidopsis thaliana Plant Otherwise Substantially Prevented in Flowering Through the Floral Suppressive Action of the Polypeptide of LpTFL1
In order to restore wild-type flowering time in late-flowering UBI::LpTFL1 Arabidopsis plants a construct was made in which a transgene encoding two 143 bp LpTFL1 inverted repeats separated by a spliceable intron (LpTFL1-RNAi) was placed under the control of a modified version of the ethanol inducible fungal promoter alcA (Caddick et al., Nat. Biotechnol. 16 (1998), 177-180). The alcR gene, which encodes a transcriptional regulator, was also incorporated into the construct under control of the maize Ubiquitin promoter (Christensen and Quail, Trans. Res. 5 (1996), 213-218). The AlcA::LpTFL1-PTGS-UBI::AlcR construct (hereafter named P05) was introduced into a late-flowering transgenic UBI::LpTFL1 Arabidopsis line 6, which flowers after approximately 66 days in LD. Several Kanr, BASTA® resistant T1 plants were regenerated which were selfed for testing at the T2 generation.
The flowering time response of the P05 lines was determined under LD conditions and compared with that of the wild-type and the late-flowering LpTFL1-6 background line. Ten to fifteen seeds from each P05 T2 line were sown in soil together with the wild-type and the LpTFL1-6 background line. The seeds were stratified at 4° C. for three days and then moved to LD conditions at 24° C. All the P05 plants and the LpTFL1-6 background plants were sprayed with BASTA® (for presence of the UBI::LpTFL1 cassette) and the surviving plants and the wild-type plants were divided in two pots per line (4-6 plants per pot), one for the ethanol induction and the other as control.
For ethanol induction of the alcA promoter, 18 days old plants were placed in trays with two 2.0 ml eppendorf tubes containing 100% ethanol and covered by a transparent plastic bag. After 8 hrs of induction the plastic bag and the ethanol was removed. Ethanol inductions of 8 hrs duration were given five days a week for three weeks. As a control to the ethanol induction, the other half of the plants were placed in similar trays but without alcohol. Flowering time was scored as the number of days from germination till the opening of the first flower. In the wild-type the first flower opens immediately after bolting but in the late-flowering LpTFL1-6 line flowers are first formed several weeks after bolting.
In LD conditions the wildtype started bolting and flowering after about 26 days whereas the LpTFL1-6 plants did not start to flower until day 50 (FIG. 16). Some of the LpTFL1-6 plants (and the un-induced P05 plants) did not flower at the termination of experiment (after 75 days) and therefore these plants were simply scored as flowering after 75 days. Six out of 22 tested P05 lines showed ethanol-induced promotion of flowering (FIG. 17) and in these plants the number of days to flowering were reduced with 31% (line P05-32) up to 49% (line P05-06) (FIG. 1). Although the un-induced P05 plants started to bolt almost simultaneously with the ethanol induced plants, they did not produce flowers before the induced plants had produced mature siliques (FIG. 17).
Since the T1 plants were heterozygous for both the UBI::LpTFL1 and the P05 transgenes, it was expected that the transgenes would segregate at the T2 generation. It was therefore expected to see plants, which were either homozygous or heterozygous for both transgenes or homozygous or heterozygous for only one of the transgenes. The plants, which only carried the P05 construct but not the UBI::LpTFL1 cassette were eliminated by spraying with BASTA®. However, the plants were not selected for kanamycin resistance (P05), plants which only contained the UBI::LpTFL1 cassette but not the P05 construct were not removed from the experiment. In order not to include such plants in which P05 had been segregated out into our flowering time data, all plants in the experiment were tested by PCR for the presence of both UBI::LpTFL1 and P05 (FIG. 18). The result showed that the few late-flowering plants present among the ethanol induced plants all lacked the P05 construct and therefore were incapable of responding to the ethanol treatment (FIG. 16). Based on these findings the average number of days to flowering were calculated on the plants which by PCR had been shown to contain both P05 and the UBI::LpTFL1 cassette. In all lines the time to flowering was significantly reduced upon ethanol induction, whereas the time to flowering in the ethanol induced LpTFL1-6 background plants remained unchanged from the non-induced plants (FIG. 19). Thus by introducing the P05 construct into the late-flowering LpTFL1-6 plants and by inducing expression of the LpTFL1-RNAi it is possible to reduce the time to flowering on average with 40% ( ̃26 days) and in some instances even up to 50% (line P05-06).
In some P05 plants we observed floral revertance in mature flowers (FIG. 20). We assume this aberrant floral development to be directly linked to a reduction in the alcA promoter activity during the two days every week, where no ethanol was applied to the plants. It has previously been shown that continuous high activity of the alcA promoter system requires continued application of ethanol and two days after the end of ethanol application, the activity of the alcA promoter is reduced by 70% (Rosland et al., Plant J. 28 (2001), 225-235). Thus decreasing the promoter activity to about 30% may result in a LpTFL1-RNAi level, which is insufficient to overcome the floral repression mediated by UBI::LpTFL1 and therefore the cells in the flowers or siliques reverted to a meristem identity. Upon new ethanol application the alcA promoter activity increased again consequently reducing the levels of LpTFL1 and new flowers could be made in place of a developing silique. Such discontinuous induction of the LpTFL1-RNAi may increase the overall seed yield since most of the floral revertance resulted in the replacement of one silique with 3-4 new similar-sized siliques.
Line P05-06 flowered after 33 days upon ethanol induction. This is only one week later than the wild-type, and by giving the ethanol induction even earlier than in this experiment (18 days post germination) it is assumed that it is possible to reduce the time to flowering even further down to wild-type levels. Similar effects may also be obtained by adding 0.5%, 1%, 2%, 4% or 5% ethanol solution, directly to the soil.
In conclusion, it has been shown that ethanol induced expression of the LpTFL1-PTGS construct initiates a post-transcriptional silencing of LpTFL1, which eventually will abolish the LpTFL1 repression of flowering in Arabidopsis. This result is to the inventor's knowledge the first evidence of a chemically induced PTGS-mediated release of transgene-induced flowering repression. This method will have wide applications for floral restoration and will not be limited to LpTFL1 but also to other floral repressors, such as the LpFT-like gene, the Lolium perenne MADS box genes LpMADS10, the LpMADS14, the LpMADS16 or the Arabidopsis thaliana Flowering Locus C/-F (FLC/FLF) gene (accession AF537203/AF116527), which may confer floral repression activity.

## Claims

1. An isolated polynucleotide which, when expressed in sense orientation in plants leads to a prevention of flowering, selected from the group consisting of
(a) polynucleotides comprising a nucleotide sequence encoding a polypeptide with the amino acid sequence of SEQ ID NO:2;
(b) polynucleotides comprising the coding region of the nucleotide sequence shown in SEQ ID NO:1; and
(c) polynucleotides comprising a nucleotide sequence having a sequence identity of at least 95% to the coding region of SEQ ID NO: 1 and polynucleotides comprising a nucleotide sequence encoding a polypeptide having a sequence identity of at least 95% to the entire amino acid sequence of SEQ ID NO: 2, wherein said nucleotide sequence when expressed in sense orientation in plants leads to the production of the encoded polypeptide and to a prevention of flowering in comparison to wild-type plants.

2. The isolated polynucleotide of claim 1 which is DNA or RNA.

3. A recombinant nucleic acid molecule comprising the polynucleotide of claim 1.

4. The recombinant nucleic acid molecule of claim 3 further comprising expression control sequences operably linked to said polynucleotide.

5. A vector comprising the polynucleotide of claim 1 or the recombinant nucleic acid molecule of claim 4.

6. The vector of claim 5 further comprising expression control sequences operably linked to said polynucleotide.

7. A method for producing genetically engineered host cells comprising introducing the isolated polynucleotide of claim 1, or the recombinant nucleic acid molecule of claim 4 into isolated host cells.

8. An isolated host cell which is genetically engineered with the polynucleotide of claim 1.

9. The host cell of claim 8 which is a bacterial, yeast, fungus, plant or animal cell.

10. A method for producing a transgenic plant comprising the steps of
(a) introducing the polynucleotide of claim 1 into the genome of a plant cell; and
(b) regenerating the cell of (a) to a transgenic plant.

11. A transgenic plant, or plant tissue comprising the plant cell of claim 9 or obtainable by the method of claim 10.

12. The transgenic plant of claim 11 which, upon an increased amount of the protein encoded by said polynucleotide as compared to a corresponding wild-type plant, shows a prevention of flowering.

13. Propagation material or harvestable parts of the transgenic plant of claim 11 comprising said plant cell.

14. The propagation material of claim 13 which is a seed.

15. The isolated polynucleotide of claim 1, which is selected from the group consisting of polynucleotides comprising a nucleotide sequence having a sequence identity of at least 97% to the coding region of SEQ ID NO: 1 and polynucleotides comprising a nucleotide sequence encoding a polypeptide having a sequence identity of at least 97% to the entire amino acid sequence of SEQ ID NO: 2, wherein said nucleotide sequence when expressed in sense orientation in plants leads to the production of the encoded polypeptide and to a prevention of flowering in comparison to wild-type plants.

16. The isolated polynucleotide of claim 1, which is selected from the group consisting of polynucleotides comprising a nucleotide sequence having a sequence identity of at least 99% to the coding region of SEQ ID NO: 1 and polynucleotides comprising a nucleotide sequence encoding a polypeptide having a sequence identity of at least 99% to the entire amino acid sequence of SEQ ID NO: 2, wherein said nucleotide sequence when expressed in sense orientation in plants leads to the production of the encoded polypeptide and to a prevention of flowering in comparison to wild-type plants.