AU779114B2 - Control of flowering - Google Patents

Description

1 CONTROL OF FLOWERING This invention relates to the control of flowering and reproduction in plants, and in particular to agents and methods for inducing or suppressing flowering.

The invention provides isolated nucleic acid molecules which are useful for inducing flowering, particularly initiating early flowering, for delaying or suppressing flowering, or for manipulating the flowering period.

DETAILED DESCRIPTION OF THE INVENTION All references, including any patents or patent applications, cited in this specification are hereby incorporated by reference. No admission is made that any eoe 15 reference constitutes prior art. The discussion of the eeo.

references states what their authors assert, and the applicants reserve the right to challenge the accuracy and pertinency of the cited documents. It will be clearly understood that, although a number of prior art publications are referred to herein, this reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art, in Australia or in any other country.

The initiation of flowering in a plant occurs in response to internal signals, such as physiological age or levels of plant growth regulators, or may result from Sochanges in environmental conditions such as day length or low temperature. It is well known that in a variety of plant species a crucial factor is day length, also known as photoperiod. In many plant species, including several ecotypes of the widely-used model plant species Arabidopsis thaliana, flowering is promoted by long day photoperiod, or by a period of low temperature (vernalization) (Napp-Zinn, 1985) H:\WcndyS\Kccp\-%pccc.\1854I-(X) CSJRO doc 20/)21)2 1A Control of flowering in both horticultural and crop plants represents a major problem in the agricultural industry, and is also a problem in forestry. Significant losses in yield of plants may result if non-uniform flowering of plants occurs; this applies both to fieldgrown and to glasshouse-grown plants. The problem is particularly acute for field-grown plants, which are frequently exposed to abnormal or unseasonal conditions which may result in induction of flowering at an inappropriate time. Efficient plant production requires the synchronization of flowering time between pollen donor and pollen receptor plants, and is particularly important to maximize market opportunities for glasshouse-grown *H plants.

e *o H:\WcndySKccpspccs\I.850I-(X) CSIROdoc 2(V02/02 WO 00/32780 PCT/AU99/01079 2 Currently-available methods for regulation of flowering in plants are expensive and labour-intensive, and require the use of plant growth regulators, and/or controlled planting regimes and controlled-environment growth conditions. Consequently there is a need in the art for more efficient, cost-effective methods for controlling flowering time. These methods are applicable to a variety of commercially-significant plants species, including both horticultural plants, particularly those used in the cutflower industry, and vegetable, cereal and other crop plants.

A class of genes known as MADS box genes encodes proteins which comprise a distinctive conserved DNA binding domain, known as the MADS box, which in certain cases has been demonstrated to bind to CC(A/T) 6 GG DNA motifs. The MADS box genes encode a class of transcription factors, which was first identified in yeast and in mammals.

Subsequently similar transcription factors were identified in a range of plants, including Arabidopsis thaliana, Antirrhinum majus, tomato, tobacco, petunia, corn, Pinus species and Eucalyptus species. In plants, the MADS box genes have a "K domain", which resembles the coiled-coil domains of keratin proteins, which are implicated in protein/protein interactions, an intervening domain, and a carboxy terminal domain. In plants the principal role of MADS box genes is in specifying inflorescence meristem identity, and floral organ identity and development. Certain MADS box genes have also been implicated as having roles in root and vegetative development.

We have now identified nucleic acid sequences comprising a MADS box in the model plant Arabidopsis thaliana which play a role in the control of flowering time. The effect on flowering depends on the degree of expression of the nucleic acid sequences.

WO 00/32780 PCT/AU99/01079 3- SUMMARY OF THE INVENTION In a first aspect, the invention provides an isolated nucleic acid molecule comprising a MADS box, which is capable of altering the flowering time of a plant.

In one preferred embodiment, the invention provides an isolated nucleic acid molecule which is capable of delaying the flowering of a plant. Preferably expression of the nucleic acid molecule in the plant, in the sense orientation under the control of a promoter sequence, is capable of delaying the flowering of the plant.

In a second preferred embodiment, the isolated nucleic acid molecule of the invention is capable of accelerating the flowering of a plant. Preferably expression of the nucleic acid molecule in the plant in the anti-sense orientation under the control of a promoter sequence is capable of accelerating the flowering of the plant.

Preferably the nucleic acid molecule of the invention comprises a nucleotide sequence corresponding to a FLOWERING LOCUS F (FLF) gene. The nucleic acid molecule may be a genomic DNA, a cDNA, or a messenger RNA.

More preferably the nucleic acid molecule comprises the nucleotide sequence set out in any one of SEQ ID NOS. i, 2, 4, and 6 to 15, or a nucleic acid molecule capable of hybridizing thereto under at least low stringency hybridization conditions, or a nucleic acid molecule with at least 70% sequence identity to at least one of SEQ ID NOS. 1, 2, 4 and 6 to 15. Methods for assessing ability to hybridize and sequence identity are well known in the art. Even more preferably the nucleic acid molecule is capable of hybridizing thereto under high stringency conditions, or has at least 80%, most preferably at least 90% sequence identity. A nucleic acid molecule having at least 70%, preferably at least 90%, more preferably at least 95% sequence identity to one or more of these sequences is also within the scope of the invention.

WO 00/32780 PCT/AU99/01079 4 In a second aspect, the invention provides a vector comprising a nucleic acid molecule according to the invention. The vector may be a virus, bacteriophage, plasmid, or bacterium. In a particularly preferred embodiment, the vector is a T-DNA vector present in a bacterium of the genus Agrobacterium, in particular Agrobacterium tumefaciens.

In a third aspect, the invention provides a plant cell transformed with a nucleic acid of the invention.

In a fourth aspect, the invention provides a plant transformed with a nucleic acid molecule of the invention.

In a fifth aspect, the invention provides a method of isolating a nucleic acid molecule capable of altering the flowering time of a target plant, comprising the step of using a nucleic acid molecule of the invention, or a functional portion thereof, as a hybridisation probe or polymerase chain reaction (PCR) primer, and optionally detecting hybridisation. Suitable methods are very well known in the art. For example, we have demonstrated that the Arabidopsis FLF sequence described herein can be used to isolate the homologous sequence from Brassica napus.

In a sixth aspect, the invention provides an FLF polypeptide. Preferably the polypeptide is encoded by a nucleic acid molecule of the invention. More preferably the polypeptide has an amino acid sequence as set out in any one of SEQ ID NO: 3, 5, and 16 to 30, or has a sequence at least 70% idntica thereto.

The polypeptide may be produced by expression of the FLF nucleic acid molecule in a convenient host, for example in a bacterial host such as Escherichia coli.

Antibodies against the polypeptide, including monoclonal antibodies, may be produced using routine methods, and it will be clearly understood that antibodies to the FLF polypeptide are within the scope of the invention. Such antibodies are useful for screening plants for high or low levels of expression of FLF polypeptide. Suitable WO 00/32780 PCT/AU99/01079 5 screening methods including Western blotting and various forms of immunoassay, for example radioimmunoassay, ELISA, and chemiluminescent or fluorescent detection immunoassays.

Genes controlling developmental stages in plants, such as the gene associated with the nucleic acid of the invention, are highly conserved during evolution.

Consequently the nucleic acid molecules and the methods of the invention are applicable to all plant species, whether the species is monocotyledonous or dicotyledonous. Thus the invention is generally applicable to flowering plants, including but not limited to ornamental, horticultural, agricultural and tree species. Methods for introducing exogenous DNA into plants of all these types, and for in vitro culture of plant tissue and regeneration of plant cells or tissues into whole plants, are known in the art.

Methods for further generation and selection of commercially useful cultivars are also well known.

Depending on the type of plant, it may be desirable to accelerate flowering ie. to induce early flowering, to synchronise flowering, to delay flowering or to suppress flowering.

For example it is desirable to suppress or delay flowering in many vegetable plants, in pasture grasses such as rye grass, or in sugar cane. Acceleration of flowering by induction of early flowering is desirable in a number of crop species, such as cotton, and in horticultural species.

We have surprisingly found that flowering can be delayed in proportion to the degree of expression of the nucleic acid molecule of the invention, and that early flowering can be induced by reducing the expression of this nucleic acid molecule.

Thus in a sixth aspect the invention provides a method of delaying flowering in a plant, comprising the step of introducing a nucleic acid molecule of the invention into cells of the plant, optionally such that expression of the nucleic acid molecule is under the control of an inducible promoter, and over-expressing the WO 00/32780 PCT/AU99/01079 -6nucleic acid molecule. Preferably the promoter is a tissue-specific promoter.

Preferably flowering is delayed for at least five days, preferably for at least twenty days, and more preferably for at least thirty days beyond the normal flowering period. Most preferably flowering is delayed for at least forty to fifty days. In at least some species it may be possible to achieve complete suppression of flowering. It will be appreciated that this further provides a method of inducing sterility in a plant.

According to a seventh aspect, the invention provides a method of inducing early flowering in a plant, comprising the step of reducing the degree of expression in the plant of a nucleic acid molecule of the invention. The reduction may be effected by any convenient means, including but not limited to transformation of the target plant with an anti-sense nucleic acid sequence, posttranscriptional gene silencing, ribozyme cleavage, disruption of the nucleic acid sequence using a transposable element or transposon, or by a procedure such as vernalisation. The person skilled in the art will readily be able to select the most suitable procedure for the particular plant species in question. Optionally the method of the invention may be supplemented by other treatments, such as an exogenous gibberellin.

Preferably flowering is at least five days earlier than the normal flowering period, more preferably at least ten days, and most preferably at least fifteen days earlier than the normal flowering period.

We have found that the degree of expression of FLF, and hence the flowering time, can be altered by modifying the activity of genes known to affect flowering time, including but not limited to FCA, FVE, FPA, LD, FLD, and VRN2. Therefore in both the sixth and seventh aspects of the invention, a further means of modifying the degree of expression of FLF is provided by modifying the activity WO 00/32780 PCT/AU99/01079 7 of one or more additional genes which affects flowering time, or vernalisation.

According to an eighth aspect, the invention provides a method of modifying the vegetative and/or floral phenotype of a plant, comprising the step of increasing the level of expression of an FLF gene, thereby to modify the level of production or activity of a gibberellin in the plant.

Preferably the vegetative or floral phenotypic characteristic is one which is regulated by gibberellic acid production or activity. More preferably the characteristic is related to plant architecture or fertility. For example, modification of gibberellic acid production and/or activity using the method of this aspect of the invention may be used to produce dwarf or sterile plants. In one particularly preferred embodiment, the invention provides sterile plants. In a second preferred embodiment, the invention provides dwarf plants; more preferably the plant is a wheat plant.

In a number of embodiments of the invention, the nucleic acid molecule of the invention is operably linked to a promoter sequence capable of regulating the expression of the nucleic acid molecule; more preferably the promoter sequence is adapted to regulate expression in a eukaryotic cell, most preferably a plant cell. The nucleic acid molecule of the invention may also be operably linked to a transcriptional terminator sequence.

Suitable promoter sequences are well known in the art, and include but are not limited to the CaMV promoter, a NOS promoter, the octopine synthetase (OCS) promoter, a subclover stunt virus promoter and the Arabidopisis thaliana ubiquitin gene promoter. The person skilled in the art will readily be able to selected the most suitable promoter for a given purpose. In particular, for some purposes an inducible promoter may be desirable, and these are also well known in the art. Suitable transcriptional terminator sequences active in plant cells WO 00/32780 PCT/AU99/01079 8 are also well known, and may be of bacterial, fungal, viral, animal or plant origin.

Suitable transcriptional terminators particularly suitable for use in the present invention include the nopaline synthase (NOS) gene transcriptional terminator of Agrobacterium tumefaciens, the transcriptional terminator of the Cauliflower mosaic virus (CaMV) 35S gene, the zein gene transcriptional terminator from Zea mays, and the Rubisco small subunit (SSU) gene transcriptional terminator sequences or subclover stunt virus (SCSV) gene sequence transcriptional terminators.

The nucleic acid molecule of the invention may be introduced into a plant cell or tissue by any suitable means. A variety of methods for introducing exogenous DNA into plant tissue (transformation) are known. These include, but are not limited to, direct DNA uptake into protoplasts (Krens et al, 1982; Paszkowski et al, 1984), polyethyleneglycol-mediated uptake to protoplasts (Armstrong et al, 1990), electrophoresis (Fromm et al, 1985), microinjection of DNA (Crossway et al, 1986), microparticle bombardment of tissue explants or cells (Christou et al, 1988; Sanford, 1993), or T-DNA-mediated transfer from Agrobacterium to the plant tissue.

Representative T-DNA vector systems are described in the following references: An et al (1985); Herrera-Estrella et al (1983a, Herrera-Estrella et al (1985). These transformation methods are applicable to plant tissue culture, or may be employed with whole plants (in planta transformation). Again a person skilled in the art will be able to select the most suitable method for any given plant.

Any plant tissue capable of subsequent clonal propagation, whether by organogenesis or embryogenesis, may be transformed with a vector of the present invention. The particular tissue chosen will vary, depending on the clonal propagation systems which are most suitable for the species being transformed. Suitable tissue targets include whole WO 00/32780 PCT/AU99/01079 9 plant, leaf discs, pollen, embryos, cotyledons, hypocotyls, megagametophytes, callus tissue, existing meristematic tissue (eg. apical meristem, axillary buds, and root meristems), and induced meristem tissue (eg. cotyledon meristem and hypocotyl meristem).

The vector of the invention may additionally comprise a dominant selectable marker to facilitate cell selection and plant breeding. A variety of suitable markers is known in the art, including but not limited to the NPTII gene, genes encoding resistance to an antibiotic such as hygromycin or ampicillin or to a herbicide such as phosphinothricin or glyphosate; a gene encoding a polypeptide which confers stress tolerance, such as superoxide dismutase; or a visually-detectable marker, such as green fluorescent protein or 3-glucuronidase. The person skilled in the art will readily be able to select the most suitable marker for use in a specific case.

The invention is applicable to any dicotyledonous or monocotyledonous plant species, including but not limited to decorative flower, vegetable, fruit, cereal, grass, tree, and other flowering species. Preferably the plant is selected from the group consisting of chrysanthemum, rose, gerbera, carnation, tulip, legumes such as soya bean, sugar beet, lettuce, cotton, oil seed rape, coriander, Lolium, wheat, barley, maize, rice, pasture grasses, Phalaris, Canola and other Brassica species, Linola species, sugar cane, Eucalyptus species, pine and poplar. Forest species are to be understood to be within the scope of the invention.

For the purposes of this specification it will be clearly understood that the word "comprising" means "including but not limited to", and that the word "comprises" has a corresponding meaning.

The term "flowering time" as used herein means the time at which floral meristem tissue is first visually detectable in the plant, for example by light microscopy or using the naked eye. The measured flowering-time includes WO 00/32780 PCT/AU99/01079 10 the time taken for the occurrence of the cellular processes in the differentiation of a floral meristem and subsequent cell divisions which enable such visual means to be used.

The term "flowering time" also includes the time taken for the transition from a vegetative meristem to a floral meristem to occur, as measured visually, following the induction of flowering in the plant by the application thereto of a specific chemical, physical or environmental stimulus, such as a plant growth regulator, photoperiod or temperature regime, including the vernalisation of the plant. Alternatively flowering may be induced in response to an internal development signal in the plant. Those skilled in the art will be aware of the specific nature of such chemical, physical or environmental stimuli or internal developmental signals.

"Altering the flowering time" means that the time period in which floral meristem tissue is first visually detected in a plant is increased, decreased, or otherwise modified or regulated. Thus, flowering may be delayed, accelerated, inhibited, suppressed, or synchronized.

The term "meristem" refers to plant tissue in which cells are undergoing, or are capable of undergoing, rapid mitotic division followed by differentiation into cell types which are capable of forming a primordium which develops into an organ such as a leaf, root, stem, floral bud or other plant organ.

"Vegetative meristem" refers to a meristem in which the differentiation process produces a cell type which develops into a vegetative organ or non-reproductive organ, such as a leaf, petiole, bract, stem or root.

"Floral meristem" refers to a meristem in which the differentiation process produces a cell type which develops into an inflorescence meristem, a secondary inflorescence meristem, a floral organ or sexual reproductive organ, in which the meristem or organ, when developed, may comprise both reproductive and nonreproductive tissues, including, but not limited to, WO 00/32780 PCT/AU99/01079 11 anthers, stamens, stigmas, ovules, carpels, petals and sepals. "Bolt" refers to an inflorescence stem of a rosette plant, and "bolting" is the development of such a stem.

The term "derived from" means that a particular integer or group of integers has originated from a particular organism or species as specified herein, but has not necessarily been obtained directly from that source.

Representative low and high stringency conditions of hybridisation as referrred to herein are as follows: High stringency: hybridization at 42 0 C in formamide, 3 x SSC, 0.1% SDS, 20 x Denhardt's, 50 gg/ml salmon sperm DNA overnight and washed with a final wash of 0.1 x SSC, 0.1% SDS at 420C.

Low stringency: hybridization at 28°C in formamide, 3 x SSC, 0.1% SDS, 20 x Denhardt's, 50 ig/ml salmon sperm DNA overnight and washed with a final wash of 0.1 x SSC, 0.1% SDS at room temperature.

A "homologue" of a nucleotide sequence refers to an isolated nucleic acid molecule which is substantially the same as the nucleic acid molecule of the present invention or its complementary nucleotide sequence, despite the occurrence within the sequence of one or more nucleotide substitutions, insertions, deletions, or rearrangements.

An "analogue" of a nucleotide sequence means an isolated nucleic acid molecule which is substantially the same as a nucleic acid molecule of the present invention or its complementary nucleic acid, despite the occurrence of any non-nucleotide constituents not normally present in the isolated nucleic acid molecule, for example carbohydrates, radiochemicals including radionucleotides, reporter molecules including, but not limited to digoxigenin, alkaline phosphatase or horseradish peroxidase.

A "derivative" of a nucleotide sequence means any isolated nucleic acid molecule which contains significant sequence similarity to the molecule or a part thereof. The WO 00/32780 PCT/AU99/01079 12 person skilled in the art will appreciate that the nucleotide sequence of the present invention may be subjected to mutagenesis to produce one or more single or multiple nucleotide substitutions, deletions and/or insertions. Nucleotide insertional derivatives of the nucleotide sequence of the present invention include 5' and 3' terminal fusions, as well as intra-sequence insertions of single or multiple nucleotides or nucleotide analogues.

Insertional nucleotide sequence variants are those in which one or more nucleotides or nucleotide analogues are introduced into a predetermined site in the nucleotide sequence of the sequence, although random insertion is also possible; suitable screening of the resulting product is performed. Deletional variants are characterised by the removal of one or more nucleotides from the nucleotide sequence. Substitutional nucleotide variants are those in which at least one nucleotide in the sequence has been removed, and a different nucleotide analogue inserted in its place.

Reference in this specification to a "gene" is to be understood in its broadest context, and includes: a classical genomic sequence comprising transcriptional and/or translational regulatory sequences and/or a coding region and/or non-translated sequences (ie.

introns and and 3'-untranslated sequences); (ii) mRNA or cDNA corresponding to the coding regions (ie. exons), optionally additionally comprising or 3'-untranslated sequences of the gene; or (iii) an amplified DNA fragment or other recombinant nucleic acid molecule produced in vitro, and comprising all or a part of the coding region and/or 5' or 3'-untranslated sequences of the gene.

The term "gene" is also used to describe synthetic or fusion molecules encoding all or part of a functional product. A functional product is one which comprises a sequence of nucleotides or is complementary to a sequence of nucleotides which encodes a functional 15/11 2004 12:01 25 35 o FAX 61 3 92438333 GRIFFITH HACK IPAUSTRALIA I]007 13polypeptide, in particular the FLF polypeptide o the invention or a homologue, analogue or derivative thereof.

In some of the examples herein the FLF gene is referred to as gene B. These two terms are synorymous.

In the claims of this application and in the description of the invention, except where the co ntext requires otherwise due to express language or necessary implication, the words "comprise" or variations such as "comprises" or "comprising" are used in an inclusive sense, i.e. to specify the presence of the stated featurps but not to preclude the presence or addition of further eatures in various embodiments of the invention.

BRIEF DESCRIPTION OF THE FIGURES Figure 1 is a photograph showing wild-t pe C24 (left), the late flowering T-DNA tagged flf mutant (middle) at 70 days after germination, and flf mutant at 10 days, showing the domed shape caused by vegetative bolt (right).

The bar represents 5 cm.

Figure 2 shows segregation of two T-DNA inserts with the late flowering phenotype A. Genomic DNA isolated from a T 2 po lation segregating for early late and very late (VL) flowering, digested with EcoRI and probed with the NPTII gene.

B. Physical map of the two T-DNA ins rts linked to the FLF locus, showing their orientatiot. EcoRI sites are labelled RI; LB and RB represent the left and right borders, respectively, of the T-DNA. The t iangle symbol represents the site of deletion of 30 bp to the right of the T-DNA. The arrows represent the dir ction of transcription of the genes.

C. Representation of a 27 kb region df Arabidopsis mutant DNA containing the gene A and lene B loci, showing the location of the T-DNA inserts. DNA fragments from the flanking plant DNA (probes 2 a d 3) were used as probes to isolate cDNA clones. HindIII BamHI and EcoRI (Rl) restriction enzyme sites are i dicated.

The positions of gene A and gene B are shown, and their directions of transcription are indicated by arrows.

D. The 6.5kb and 6.8 kb BamHI fragme ts, isolated from a genomic library of wild-type C24 'ith probes 1 and 2 from Figure 2C, spanning the site f the T-DNA insertions. Restriction sites are as in Fi ure 2C.

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COMS ID No: SBMI-00997067 Received by IP Australia: Time 12:10 Date 2004-11-15 WO 00/32780 PCT/AU99/01079 14 Figure 3 shows levels of expression of gene A and gene B in 30 day old wild-type C24 plants (lane 1), hemizygous (lane 2) and homozygous (lane 3) flf mutant plants.

Figure 4 is a photograph showing 35S::FLF T1 transgenic plants in C24 (left) and Landsberg erecta (right) ecotypes. The C24 transgenics were either earlyflowering (back) or late-flowering (front). The Landsberg erecta transgenics were either late-flowering (left) or flowered at normal time (right).

Figure 5 shows the FLF gene structure and the expression pattern of the FLF transcript A. Genomic structure of the FLF gene, showing location and size of introns and location of the MADS box, intervening domain K domain and carboxy terminal domain The numbers below the line represent the number of base pairs in each exon.

B. Pattern of expression of FLF mRNA in C24 plants: roots and rosette leaves (RL) from in vitro grown vegetative plants, cauline leaves bolt stems floral apex and buds from soil grown plants with bolt stems between 1 and 5 cm. Mature flowers and siliques were collected from older plants. Plants were grown under 16 h photoperiod conditions. The RNA gel blots for B-F were probed with a riboprobe transcribed from the FLF (Gene B) cDNA clone linearised to remove the MADS box region. The ethidium bromide-stained ribosomal bands are shown as a loading control in B-F.

C. Expression level of FLF mRNA in whole C24 or flf mutant plants, harvested every 10 days (as indicated by the numerals) until the majority of the C24 plants were bolting (50 days under these growth conditions).

D. Expression of FLF mRNA in C24 (lane 1-6) and flf (lane 7-12) plants grown for 21 days in 8 h fluorescent photoperiod, and then at the end of the 21 s photoperiod either kept in the same conditions (SD; lane 1, 2, 7, 8) or transferred to continuous dark (CD; lanes 3, 4, WO 00/32780 PCT/AU99/01079 15 9, 10) or continuous light (CL; lanes 5, 6, 11, 12) Plants were harvested either just prior to what would have been the start of the following photoperiod (dawn; lane 1, 3, 5, 7, 9, 11), or just prior to the end of the photoperiod (dusk; lanes 2, 4, 6, 8, 10, 12). Transcript levels were a little higher at the start of the photoperiod, but this pattern was not altered in the mutant.

E. Effect of gibberellic acid (GA 3 treatment and vernalization on the FLF transcript in C24 (lanes 1-3) and flf mutant (lanes 4-6) seedlings. RNA was isolated from 12 day old seedlings that had either had no treatment lanes 1 and been grown on medium containing 10-5 M

GA

3 lanes 2 and 5) or had a pretreatment of 3 weeks at 4 0 C lanes 3 and 6).

F. FLF expression in rosette leaves of C24 and the early-flowering antisense methyltransferase line 10.5 (T3 generation) harvested soon after bolting.

Figure 6 shows A. Genomic DNA isolated from individual flf, efSL3 efSL4 (M2) and C24 plants digested with EcoRI and probed with a probe directed to the 3' region of Ac.

The DNA for the flf sample was extracted from plants which contained a third T-DNA band, hence the band at about 8 kb.

The presence of this third band had no effect on flowering time, and is therefore irrelevant.

B. As for A, except that the probe was probe 4 (see Figure 2C). The DNA for the efSL3 (M2) and efSL4 (M2) samples was extracted from bulked M2 plants which contained neo-lates as well as early-flowering mutants. Therefore there is some of the 2.7 kb band present in these DNA extracts. Other DNA isolated from individual earlyflowering plants does not contain a band at 2.7 kb.

C. Location of Ac insertion in intron I. The nucleotide positions are given below, taking the A of the ATG as nucleotide 1.

16 D. Expression level of FLF gene in 15 day old rosette leaves of C24 (lane flf (lane2), ef SL3 (lane ef SL4 (lane The M2 early flowering mutants had just started to bolt, whereas the other plants remained vegetative. The ethidium bromide-stained ribosomal bands are shown as a loading control.

Figure 7 shows gel blots from neo-late plants.

A. Genomic DNA was isolated from the 6 neo-late mutant plants and from efSL3 efSL4 flf and C24 plants, and digested with EcoRI. The DNA gel blot was probed with the 3D region of Ac.

B. Total RNA was isolated from a mixture of rosette and cauline leaves from the 6 neo-late plants, flf and C24. The RNA gel blot was probed as in Figure 5. The ethidium bromide-stained ribosomal bands are shown as a loading control.

Figure 8 shows expression of the FLF gene in ecotypes and late-flowering mutants.

Total RNA was extracted from 12 d old seedlings and RNA gel bolts were probed as in Figure 5. The ethidium bromide-stained ribosomal bands are shown as a loading control in A-C.

A. Expression in a number of different Arabidopsis ecotypes B. Expression in Landsberg erecta (L.er.)and Landsberg erecta lines which contain late alleles at either the FRI (L.er.-FRf 2 or FLC (L.er.-FLCf 2 L.er.-FLC( 1 loci.

C. Expression in late-flowering mutants in either L.er. ecotype (fca, fve, fpa, gi, co, fha, fwa, fd, fe, ft) or Ws The mutants vrnl and vrn2 were isolated in the fca background, and only vrnl has been segregated away from the fca mutant locus.

Figure 9 (SEQ ID NO. 4) shows the partial genomic sequence of an FLF-like sequence from Brassica napus, HA\WendySKccpNspgcics\ 185( CSJROdoc 20/02/02 17 showing the location of exons, and the predicted sequence of the translated product.

Figure 10 (SEQ ID NO. 5) shows a comparison of the predicted translated product of a Brassica napus FLFlike sequence (top lines), and the predicted FLF translation product from Arabidopsis thaliana, showing identical amino acids highly conserved amino acids and conserved amino acids DETAILED DESCRIPTION OF THE INVENTION The invention will now be described in detail by way of reference only to the following non-limiting examples, and to the figures.

15 GENERAL METHODS Plant Material and Growth Conditions m,*r Arabidopsis was grown either in pots containing a mixture of 50% sand and 50% loam, or aseptically in testtubes or petri dishes containing a modified Murashige and Skoog (MS) medium (Langridge, 1957). Unless otherwise stated, all plants were grown in artificially lit cabinets at 210 or 230C, under long day (16 hr light, 8 hr dark) conditions using cool white fluorescent lights at an ogo 2 -1 intensity of 200 LM m s Plants were vernalized by germinating seed in the dark for either 3, 4 or 8 weeks at 40C. Following this cold treatment, seedlings were transferred to long day photoperiods at 230C and times to flower, measured as the time until stem elongation (bolting) was observed, were determined, beginning from the first day at the higher temperature.

Arabidopsis Transformation Arabidopsis was transformed either by root transformation (Valvelkens et al., 1988) for the generation of the flf mutant and gene A transgenic plants, or by in planta transformation (Bechtold et al, 1993) for the gene B HAWcndyS\Kccp\spcics\ I 0S I-(XI CSIRO.dtw 211112J02 WO 00/32780 PCT/AU99/01079 18 transgenic plants. The late-flowering mutant (flf) arose during transformation of the early-flowering ecotype C24 with a modified binary vector pBinAAc (Finnegan et al, 1993). This vector contains the neomycin phosphotransferase II (NPTII) gene under the control of the nopaline synthase (NOS) promoter, together with a deleted maize Ac transposable element inserted within the untranslated leader of the -glucuronidase gene, in the reverse orientation to the direction of transcription.

Example 1 Identification of a Mutant Locus Associated With a Late Flowering Phenotype Following transformation of the Arabidopsis thaliana ecotype C24 with a T-DNA construct containing an Ac transposable element, individual TO plants, resistant to kanamycin, were allowed to self-pollinate, and the T1 progeny screened for families that flowered significantly later than parental C24 plants.

Some of the plants of family 14-58 flowered after 70 days compared to 30 days for the C24 control plants.

Segregation analysis of the progeny of a selfed lateflowering TI plant from family 14-58, showed 53 "late" (flowering time >70 days) compared to 15 "early" flowering plants (flowering time 30 days). The result fits a 3:1 2 segregation ratio (X2=0.313 and is consistent with the late-flowering phenotype being a consequence of a single mutation. We have designated the mutant locus FLOWERING LOCUS F (FLF).

Within the segregating progeny, the lateflowering plants could be further differentiated into two classes; "lates", flowering between 70 to 90 days and "very lates", which flowered later than 150 days. Some of the "very late" plants had not flowered after a year of growth.

The "very late" flf mutant, shown in Figure 1, produced leaves at a rate similar to non-transformed C24, and had many more leaves at flowering than C24. After several months of growth, bolts arose from the internodes between WO 00/32780 PCT/AU99/01079 19 rosette leaves. These bolts elongated approximately two to three cm and formed aerial rosette structures, giving the mutant plants a dome-like appearance, similar to that described for the fld mutant (Chou and Yang, 1998).

The late-flowering phenotype observed is more extreme than any of the previously-reported late-flowering mutants and ecotypes (Koornneef et al, 1991). Progeny tests showed that selfed "very lates" produced only "very late" progeny, whereas selfed "late" plants segregated 1:2:1 for very late, late and early flowering plants. This segregation pattern is consistent with a semi-dominant mutation, with the lateness in flowering being proportional to the level of gene product.

Example 2 Construction and Screening of Genomic Libraries A genomic library of the flf mutant was constructed by partial digestion of total plant DNA with the restriction enzyme Sau3AI and ligation into the phage vector XEMBL4. The resulting library was screened using a 32 P-dCTP labelled probe of the NPTII gene (Feinberg and Vogelstein, 1983). Four positive phage clones were purified and restriction mapped. Together these spanned 27kb of plant DNA flanking the site of insertion of T-DNA.

A 2.3kb BamHI-EcoRI and a 2.7 kb EcoRI fragment (probes 1 and 2 respectively, Figure 2C) isolated from this flanking plant DNA were subsequently used to probe a genomic library of wild-type Arabidopsis C24, made from BamHI-digested DNA and cloned into XEMBL4. Probe 1 hybridized to a genomic clone containing 6.5kb of plant DNA spanning the T-DNA insertion site, and probe 2 hybridized to a genomic clone containing 6.8 kb of adjacent sequence.

Example 3 Isolation of the FLF Gene Two T-DNAs segregate with the very late flowering phenotype The very late-flowering phenotype segregates with two T-DNA bands identifiable by Southern analysis, which WO 00/32780 PCT/AU99/01079 20 were designated bands 1 and 5. Southern blotting showed that bands 1 and 5 are inverted and adjacent: RB LB LB RB 1 (LB, left border; RB, right border) The size of the bands, combined with sequence analysis, places the smaller band (band 5) closest to the FLF gene. Recombinant inbred lines were used to map the FLF region to the top of chromosome 5, 4 cM from RFLP marker 447. This places the FLF gene near FLC, a gene known to control flowering time in ecotypes of Arabidopsis.

A 27 kb segment of genomic DNA from the mutant around the site of T-DNA insertion was mapped. C24 genomic clones covering 13 kb around the T-DNA insertion site in the mutant were sequenced. Two probes, on either side of the T-DNA insert, were used to screen cDNA libraries. A 4.6 kb EcoRI/BamHI fragment from a C24 genomic clone (containing 4.4kb of sequence "upstream" of the T-DNA insertion site and 0.2 kb "downstream" of the T-DNA insertion site) was used to isolate cDNA clones identifying "gene A" as a transcribed region. A 2.7 kb EcoRI fragment, covering the region 0.4 kb "downstream" to 3.1 kb downstream of the T-DNA inserts, was used to isolate cDNA clones identifying "gene B" as a transcribed region.

Comparison of part of the intergenic region sequence between ecotypes C24 and Ws revealed an insertion into the C24 DNA of an approximately 200 bp sequence, 420 bp to the "right" of the stop codon of gene A and 120 bp to the "left" of the T-DNA insertion. The sequence is present in both C24 wild-type and the flf mutant. The 200 bp insertion shows 100 homology to ORF167 of the Arabidopsis mitochondrial genome. By PCR analysis we have determined that this sequence is absent in Landsberg erecta and Columbia ecotypes. The significance of this inserted DNA segment is unknown.

WO 00/32780 PCT/AU99/01079 21 A map of the overall region is shown in Figure 2B.

Example 4 cDNA Libraries In order to identify expressed genes closely linked to the T-DNA, three Arabidopsis cDNA libraries (Elledge et al, 1991; Weigel et al, 1992; Newman et al, 1994) were screened, using probes to plant DNA around the T-DNA insertion site (probes 2 and 3, Figure 2C). Two classes of cDNA clones were isolated. These were respectively designated gene A and gene B. Gene B was subsequently re-designated as FLF. Two gene A cDNAs were isolated with a 4.6 kb EcoRI-BamHI fragment (probe 3, Figure 2C) from a screen of 200,000 XYes clones; however, no clones were isolated with the 2.7 Kb EcoRI fragment (probe 2, Figure 2C). The gene A cDNAs were subcloned from the phage by site-specific recombination, using the CRE protein (provided by the E. coli strain BNN132) and the lox sites within the vector (Elledge et al, 1991). The larger, almost full-length gene A cDNA was further subcloned into pBluescript (Stratagene). Full-length gene A cDNAs were subsequently isolated by screening a Landsberg erecta flower cDNA library (Weigel et al, 1992). The mutant and wild-type genomic clones corresponding to the isolated cDNA were also subcloned as smaller fragments into pBluescript As no cDNA clones were isolated with probe 2 (Figure 2C) from either cDNA library, a third library was screened. Four full-length gene B cDNA clones were isolated from a XPRL2 cDNA library derived from different tissues and developmental stages (Newman et al, 1994). All cDNAs and the mutant and wild-type genomic clones were sequenced on both strands by the dideoxy chain termination method (Sanger et al, 1977) using fluorescent primers (Brumbaugh et al, 1988). The University of Wisconsin GCG software package was employed for sequence analysis (Devereux et al, 1984). The nucleotide and predicted 22 protein sequences were used to search the GenBank database for any homologous sequences; none were found.

Example 5 Construction of 35S::gene A plasmid As the larger of the initially isolated gene A cDNA clones lacked the AT of the ATG of the start codon, oligonucleotide-directed mutagenesis was employed to generate a 200bp fragment from the 5' end of the cDNA which contained the absent nucleotides. Two oligonucleotides were synthesized on an Applied Biosystems DNA Synthesizer for this purpose:- (SEQ ID NO. 31) 5' AAGCCGCGGACAATGGAAGCTGTAAGATGC 3' (2)(SEQ ID NO. 32) 5' GAGAGGCTGGTTAACCGGAG 3'.

The nucleotides indicated in bold show the locations of SacII and HpaI restriction sites within the primers. The amplification reaction was carried out in a p1 final volume that contained 2 pM of each oligonucleotide primer, 200pg of HindIII-cleaved cDNA as a template, 0.2 units of Taq polymerase and 125 pM of each of the four deoxynucleotides. Conditions for the amplification were as follows: 95C for 2 mins, 5 cycles consisting of 15 s denaturation 950C, annealing at 400C for 30 s, and polymerization at 720C for 1 min, followed by cycles where the annealing temperature was raised to 500C for 15 sec and finally 300C for 1 min. The resulting 200 bp PCR fragment was cloned into SacII and HpaI sites in the original cDNA plasmid, and then sequenced to ensure that no mutations had been introduced during the amplification procedure. Sense binary constructs were made by digesting the full length cDNA with EcoRI and SacII, end filling the recessed termini using the Klenow fragment of DNA polymerase I, and ligating the released 1.4 kb fragment into the SmaI site of the expression vector pDH51 (Pietrzak et al, 1986). This places the expression of the FLF cDNA under the control of the CaMV 35S promoter. Recombinant Il:\WcndyS\Kccpspccic%\ I 854)-(8) CSIRO.doc 2(8)2/1)2 23 plasmids, containing the cDNA in the desired orientation, were cleaved with EcoRI and cloned between the right and left border sequences of the binary vector pBinl9 (Bevan, 1984). The binary construct was transferred to Agrobacterium tumefaciens strain AGL1 (Lazo et al, 1991) by triparental mating, employing pRK2013 as the helper plasmid. Roots of wild-type C24 plants were transformed (Valvelkens et al, 1988) using the NPTII gene as a selectable marker to identify transgenic plants.

Example 6 Construction of 35S::gene B plasmid A binary construct containing gene B under the control of the CaMV 35S promoter was generated by cloning a XhoI/SpeI digested PCR product, amplified using the gene B 15 cDNA clone as template with the primers, using methods similar to those described in Example 5' CCGCTCGAGCTTAGTATCTCCGGCG 3' (SEQ ID NO. 33) and GGACTAGTCGCCCTTATCAGCGGA (SEQ ID NO. 34) in which restriction sites are shown in bold, and the sequence hybridizing to gene B cDNA is underlined, into XhoI/SpeI digested pART7 (Gleaves, 1992) containing the CaMV 35S promoter. The 35S::gene B cassette was then subcloned using NotI into pART27 (Gleaves, 1992) and introduced into A. tumefaciens strain GV3101 (Koncz and Schell, 1986) as described above. Transgenic plants were generated by in planta transformation (Bechthold et al, 1993) Example 7 DNA Gel Blot Analysis Total genomic DNA was isolated either by following the cetyltrimethylammonium bromide (CTAB) procedure (Dean et al, 1992), or as described by McNellis et al. (1998). 2-3 Lg of DNA was digested with the appropriate restriction enzyme, electrophoresed on an 0.8% agarose gel, and blotted onto Hybond N+ membranes H:\WcndyS\Kccp\spccics\I850I-() CSIRO.doc 2010210? 24 32 (Southern, 1975). Probes were labelled with 32P-dCTP using the random primer method (Feinberg and Vogelstein, 1983).

The NPTII probe was generated as described above.

The 3'Ac probe was a SphI fragment (Lawrence et al, 1993), and probe 4 was generated by amplification of the wild-type genomic clone with primers: 5'-GTATAGGGCACATGCCC-3' (SEQ ID NO. 35) and 5'-CACTCGGAGCTGTGCC-3' (SEQ ID N0.36).

This results in a 570 bp subset of probe 2 sequence, lacking the MADS box, to eliminate crosshybridization.

15 Example 8 RNA Extraction and RNA Gel Blot Analysis o. Total RNA was extracted from approximately Ig of plant tissue, following the method of Longemann et al.

(1987). 10-20 Lg of total RNA was electrophoresed on 2.2 M formaldehyde/agarose gels, and blotted onto Hybond N nylon filters. T7 or SP6 polymerase transcription of the linearised gene A or gene B plasmid template (containing the complete cDNA for gene A, linearised to remove MADS box 32 for gene B) was used to generate antisense P-dUTP labelled riboprobes. Filters were hybridized as described by Dolferus et al (1994), washed with a final solution of 0.lxSSC, 0.1% SDS at 650C. For gene A Northern blots it was necessary to treat filters with RNase A as previously described (Dolferus et al, 1994) to avoid ribosomal trapping. The filters were exposed to phosphor screens for quantification of signal intensity using a phosphorimager (Molecular Dynamics, USA). RNA size markers were used to determine the size of the gene A and gene B transcripts.

Example 9 RFLP Mapping DNA from sixty-four recombinant inbred lines (Lister and Dean, 1993) was digested with BamHI, and Southern blots were probed with gene A. The Mapmaker I :\WcndyS\K'cp\Vcccs\1 8501-(X)CSIIRO.doc 2(V0/202 WO 00/32780 PCT/AU99/01079 25 program was employed to compare the data with RFLP data for 68 mapped markers. Fine mapping of gene A was performed using DNA from F2 plants generated from a cross between Landsberg erecta and the flf mutant. A HpaI digest of 62

F

2 DNA was probed with the chromosome 5 RFLP marker 447 (Chang et al., 1988). A restriction fragment length polymorphism (RFLP) was found between the parental lines, Landsberg erecta and Columbia, by digestion of genomic DNA with BamHI and probing with the gene A cDNA.

Example 10 Insertion Of Two Inverted and Adjacent T-DNAs Produces A Partial-Dominant Late Flowering Mutant DNA gel blot analysis of a late flowering T1 parent showed five T-DNA inserts. Figure 2A shows a DNA gel blot of 16 segregating progeny plants of a selfed, hemizygous mutant plant derived from this T 1 parent. In a total of 70 progeny, only two inserts segregated with the late-flowering phenotype (bands 1 and "late" plants were hemizygous for these two T-DNAs, and "very late" plants were homozygous for the same two inserts. The two linked T-DNAs were segregated away from the other T-DNA inserts by backcrossing to non-transformed C24. Plants containing only the two T-DNA inserts (Figure 2A, bands 1 and 5) were identified by DNA gel blot analysis, and further analysis showed that the two inserts were adjacent and in inverted orientation (Figure 2B). DNA gel blot analysis, using probes derived from segments of the T-DNA construct and Ac demonstrated that no movement of the Ac transposable elements had occurred.

A genomic DNA library from a late flowering plant containing only the two linked T-DNA inserts was screened with an NPTII probe to isolate DNA segments spanning the site of T-DNA insertion. Three overlapping clones were isolated from the left side of the T-DNA. The longest of these, together with one clone isolated from the right WO 00/32780 PCT/AU99/01079 26 side, is depicted in Figure 2C. These clones accounted for a total of 27 kb of plant DNA spanning the site of the T-DNA insertion. C24 genomic clones were isolated from a genomic DNA library, which was prepared by digestion of total Arabidopsis DNA with the restriction enzyme BamHI and ligation of the digest into the phage vector XEMBL4, using probes 1 and 2 (Figure 2C). Clones containing a BamHI fragment spanning the insertion site and a 6.8 kb BamHI fragment downstream of the insertion site were characterized (Figure 2D). The genomic DNA sequence, cDNA sequence, and predicted protein sequence are set out in SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3 respectively. The genomic DNA sequence includes about 2 kb of promoter sequence, and 6 introns.

The chromosomal location of the FLF region was determined using 64 F9 recombinant inbred lines and data for several known markers (Lister and Dean, 1993). The Mapmaker program located the FLF region to the top of chromosome 5, 4cM from the RFLP marker 447, placing the gene in the vicinity of three other genes, TFL, FLC and EMF1, which are involved in the regulation of flower initiation (Shannon and Meeks-Wagner, 1991; Lee et al, 1994b; Koornneef et al, 1994; Sung et al, 1992).

Example 11 Two Genes Flanking the T-DNA Inserts Have Increased Expression In The flf Mutant Transcriptionally active regions in the plant DNA spanning the site of T-DNA insertion were identified by screening a cDNA library with probes derived from either side of the T-DNAs (probes 2 and 3, Figure 2C). Two overlapping partial-length cDNA clones were isolated with probe 3; full-length clones containing inserts of approximately 1.5 kb were isolated by screening a second cDNA library. Four identical full-length cDNA clones containing inserts of 1.0 kb were isolated with probe 2.

Comparison of cDNA and genomic sequence revealed that the T-DNAs had inserted between the two transcribed regions, WO 00/32780 PCT/AU99/01079 27 591 bp downstream of the polyadenylation site of gene A and 2.3 kb upstream of the start codon of gene B. Comparison of the mutant and C24 genomic sequences revealed a 30 bp deletion immediately downstream of the insertion site, with no further differences identifiable.

As neither gene was disrupted by the insertion of the T-DNAs, we investigated whether the expression of the genes was altered in the mutant. Figure 3A shows an RNA gel blot performed using RNA isolated from 30 d old leaf tissue of ecotype C24, and hemi- and homozygous flf mutant plants grown under identical conditions. As shown in Figure 3 antisense riboprobes specific for either gene A or gene B revealed that both genes are more highly expressed in the flf homozygote leaf tissue than in ecotype C24, with the 1.5 kb gene A transcript being approximately 10 times more highly expressed in the mutant and the 1.0 kb gene B transcript being approximately two-fold overexpressed in the homozygous mutant. The hemizygous mutant has an intermediate level of expression of both genes. Without wishing to be limited by any proposed mechanism, we believe that insertion of the T-DNA complete with Ac elements has caused this over-expression.

Example 12 Transgenic Plants Over-Expressing Gene B Have Altered Flowering Time In order to determine which gene is responsible for the late-flowering phenotype, we transformed C24 with constructs containing either gene under the control of the CaMV 35S promoter. 49 transgenic lines were generated with the gene A construct, and flowering time was assessed in the T2 generation. The majority of the transgenic lines showed no variation from wild-type flowering time; however, a few lines were slightly late-flowering. As shown in Table 1, four of the transgenic lines were significantly later flowering than the C24 wild-type. However, there was no correlation between time to flowering and the level of gene A expression. 23 transgenic lines were generated with WO 00/32780 PCT/AU99/01079 28 the gene B construct, and differences in flowering time compared to C24 were apparent in the T1 generation, ie. in the primary transformants. 17 T1 plants flowered earlier (range 15-25 d) than non-transformed C24 (30 d) under these conditions; eight of these showed either full or partial sterility. Four flowered at around the same time as C24, and two had not flowered after 90 d. Examination of the level of gene B mRNA transcript in kanamycin-resistant progeny of two early-flowering T1 plants, and rosette leaves from two late-flowering T1 plants, revealed a high level of gene B expression in all the transgenic plants.

Table 1 Flowering time of 35S::gene A transgenic plants Flowering time Relative expression level of gene A mRNA C24 29.9 0.6 1 B2 38.3 0.9 2 A53 35.8 0.8 A54 38.4 0.5 8 A93 35.4 0.5 4 flf >>50 Transgenic and flf mutant seeds were germinated on MS plates 50 g/ml kanamycin, and C24 seeds were germinated on MS plates. At least 20 12 d seedlings were transplanted into individual soil pots and grown at 23°C under fluorescent lights (16 h light, 8 h dark). Flowering time was recorded as the number of days to stem elongation.

Total RNA was extracted from 14 d in vitro grown seedlings and used for quantitation of FLF expression levels.

WO 00/32780 PCT/AU99/01079 29 Table 2 Flowering time of 35S::gene B T1 transgenic plants Flowering time Relative expression level of gene B mRNA C24 30 d 1 B4 18 d 18 d B11 80 d B12 80 d Landsberg 20 d n.d.

erecta B36 80 d 80 d flf 80 d 2 Transformant seeds, harvested from in planta transformed plants, were selected on MS plates containing ig/ml kanamycin. Kanamycin resistant T1 seedlings were transferred to soil at 20 d. The level of gene B transcript was determined in kanamycin resistant T2 plants for B2 and B5, and from young leaves from the T1 lateflowering plants (B11, B12, B36, B45). not detectable.

It is surprising that over-expression of gene B gave two completely opposite phenotypes. In order to clarify this, we generated transgenics containing gene B under the control of the 35S promoter in ecotype Landsberg erecta. Of the 24 T1 lines generated, none flowered earlier than wild-type Landsberg erecta, 12 had not bolted after 70 days, and 3 bolted after about 40 days, compared with 25 days for Landsberg erecta. Two of the three lines which bolted after 40 days exhibited floral abnormalities and partial sterility. Total RNA was isolated from rosette 30 leaves of two non-flowering T1 plants (B36, B45; Table 1), both of which had high expression levels of the transgene.

Therefore in Landsberg erecta over-expression of gene B causes a delay in flowering time, whereas in C24 it causes either a delay in flowering, or causes the plants to flower significantly earlier. This may be mediated by a dominant negative effect or by a form of posttranscriptional gene silencing. Analysis of protein expression levels is being pursued in order to clarify this point. Presumably a difference in the genetic background of the two ecotypes is responsible for the difference observed between ecotypes.

These results demonstrate that over-expression of gene B causes late-flowering, whereas gene A has little 15 effect on flowering time, indicating that over-expression of gene B is the most likely cause of the late-flowering flf phenotype and that this gene encodes a dosage-dependent repressor of flowering. Gene B will hereafter be referred to as FLF.

Example 13 Anti-Sense Constructs Anti-sense plant constructs have been generated *0*0 using an anti-sense FLF gene construct under the control of the CaMV 35S promoter. A 35S::FLF antisense binary o 25 construct was generated by cloning the EcoRI/Spel digested 'PCR product amplified with primers CGGAATTCTCACACGAATAAGGTAC (SEQ ID NO. 37) and GGACTAGTGGTCAAGATCCTTGATC (SEQ ID NO. 38) as described for the 35S::FLF construct. This amplified the region downstream of the MADS box, so that the antisense construct lacks the MADS box region. The PCR product was cloned into pART7 and pBART 27 (which is a derivative of pART27), and transgenic plants were generated as described above, except that the Bar gene was used as the selectable marker.

T1 C24 transgenic plants were generated with a construct in which the 30 end of the FLF gene, in the H:\WcndyS\Kccp'spccics1 854)1-(X) CSIRO.doc 2(Y)2102 WO 00/32780 PCT/AU99/01079 31 antisense orientation, was under the control of a promoter (35S::FLFAS). Approximately half of the T1 plants had flowered before 20 days of growth, compared to 30 days for the non-transformed strain Transgenic plants were produced in the C24 and the Columbia ecotypes. Of the six T1 plants produced in the Columbia ecotype, three bolted earlier than wild-type Columbia. Wild-type C24 plants bolt at about 30 days, and wild-type Columbia plants bolt at about 20 days.

These results indicate that the antisense construct acts to decrease flowering time, presumably by decreasing the level of the FLF transcript.

RNA gel blot analysis of early-flowering plants from three T2 C24 antisense lines revealed that the level of FLF transcript was considerably lower than in nontransformed C24, confirming that the antisense construct was acting to decrease flowering time by decreasing the level of FLF transcript.

Example 14 The FLF Gene Is A Novel MADS Box Gene The FLF cDNA sequence has strong homology to a class of transcription factors known as MADS box genes.

The FLF sequence shows greatest similarity to the MADS gene AGL14 in the M-I-K domain, but over the entire cDNA sequence it shows greater similarity to CAL (CAULIFLOWER) and AP1 (APETALA1). The location of the MADS box, I domain, K domain and C terminal domain are indicated in Figure 5A. The K domain is typical of those of other plant MADS genes. Comparison of the genomic sequence (SEQ ID NO. 1) and cDNA sequence (SEQ ID NO. 2) of the FLF gene revealed the presence of 6 introns, with intron I being kb. The predicted protein (SEQ ID NO. 3) is 196 amino acids long, which is shorter than the proteins encoded by most MADS box genes.

One of the main roles of MADS box genes in plant development which has been described to date is in specifying floral organ identity. Other roles for MADS-box WO 00/32780 PCT/AU99/01 079 32 genes include specifying root architecture and vegetative growth. To investigate whether FLF also has other roles in addition to its role in controlling the time of flowering, we examined its expression in a range of tissues. Figure 5B confirms the high expression of the FLF gene in vegetative rosette leaves, and reveals a strong expression in roots and lower expression in floral tissues, suggesting possible further roles for the FLF gene. No root phenotype has been observed in the transgenic lines. However, a number of lines had reduced fertility, which appeared to be caused by a lack of pollen in C24 lines or by abnormal carpels in Landsberg erecta lines. However, as the early Ac plants (see later) did not show these phenotypes it is unclear whether this is caused by change in expression of the FLF gene.

The expression of the FLF gene is lower in postvegetative tissues than it is in vegetative rosette leaves.

We investigated the possibility that reduction in the level of expression of the FLF gene accompanies the transition to flowering. RNA was isolated from C24 and flf whole plants every 10 days post-sowing, until the stage where the majority of C24 plants had bolted (50 The expression of the FLF gene remained unaltered in these plants (Figure suggesting that if reduction in the level of expression of the FLF gene does accompany the transition to flowering it must occur in only a few cells.

The flf mutant demonstrates a number of similarities to the Ihy mutant described by Schaffer et al.

(1998): they are both (semi-)dominant late-flowering mutants caused by insertion of foreign DNA adjacent to the gene. In wild-type plants the LHY gene is expressed for only a few hours around dawn, whereas in the lhy mutant, the LHY gene is expressed around the clock. Because of the similarities between flf and ihy, we examined the expression of the FLF gene in C24 and flf tissue harvested at dawn and dusk of an 8 h photoperiod. Although there was some difference in the expression of the genes between the WO 00/32780 PCT/AU99/01079 33 two time points, there was no alteration of this pattern in the mutant.

Example 15 The Late-Flowering Phenotype Of The flf Mutant Is Suppressed By Vernalization Or By Gibberellic Acid Treatment In a number of late-flowering mutants and ecotypes, low temperature treatment of germinating seed (vernalization) induces early flowering (Napp-Zinn, 1985), with a 4'C treatment for 21 days saturating the vernalization requirement to produce the shortest time to flower (Bagnall, 1992). The effect of vernalization on the time to flowering of hemizygous and homozygous flf mutants is shown in Table 2.

Table 2 Flowering Time of Vernalised C24 and Mutant Plants LENGTH OF VERNALISATION 0 weeks 4 weeks 8 weeks C24 (wild-type 25.2 0.2 13.6 0.3 Hemizygous flf 71.4 1.2 39.3 3.7 20.2 0.9 Homozygous flf >150 100.8 10.7 17.6 1.3 Twenty seeds of the flf mutant and wild-type C24 were grown aseptically on MS medium in test-tubes, and exposed to either 4 or 8 weeks at 4 0 C. Non-vernalized plants were grown in soil (20 plants per 20cm pot). All plants were then grown at 23 0 C under fluorescent lights (16 h light, 8 h dark). The data are presented as the average number of days standard errors) until stem elongation, excluding the period of vernalization.

A 28 day treatment at 40C resulted in a substantial reduction in the flowering time. However, eight weeks at 4 0 C was required to saturate the WO00/32780 PCT/AU99/01079 34 vernalization response in both hemizygotes and homozygotes to give a flowering time similar to that of the C24 control. This implies that there is an interaction between FLF gene expression and a component of the vernalizationinduced pathway.

RNA was extracted from 12 d old C24 seedlings that were either vernalized or were untreated controls, and probed with FLF gene-specific probe. Figure 5E shows a dramatic decrease in FLF expression in vernalized seedlings compared with unvernalized seedlings, suggesting that a component of the vernalization signalling pathway controls FLF gene expression. Day 1 is the day on which seeds were transferred to the growth room. In flf mutant plants the level of transcript was reduced in 3 week vernalized seedlings, but not to the low levels observed in C24, consistent with its only partial earlier-flowering character.

FLF mutant plants vernalized for 8 weeks had a greater reduction of FLF transcript, consistent with the greater reduction in flowering time.

As with other late-flowering vernalizationresponsive mutants and ecotypes of Arabidopsis, flf mutant plants responded to applications of gibberellic acid (GA 3 by flowering earlier. Four week-old flf homozygotes treated with 1 ig of GA 3 every second day for a total of two weeks flowered two weeks after the final GA 3 application, compared to later than 20 weeks for untreated plants. However, a single treatment of 4 week old plants with 1 jg of GA3 was not sufficient to induce flowering of flf, although this amount of GA 3 induced early flowering of the late-flowering fca mutant (Bagnall, 1992), suggesting that flf requires a greater amount of GA 3 for floral induction. In contrast to the dramatic effect of vernalization on the expression of the FLF gene, exposure of either C24 or flf seedlings to 10 M GA 3 had no effect on the expression of the FLF gene.

WO 00/32780 PCT/AU99/01079 35 Example 16 Movement Of An Ac Element Present Within The T-DNA Causes Alteration In Flowering Time Two early flowering plants (Ml plants designated efSL3 and efSL4) were identified from one seedlot comprising bulked seed from flf mutant plants. Both plants flowered after 18 days, earlier than C24 which flowered after 30 days. PCR analysis using primers from within the T-DNA sequence and flanking genomic sequence confirmed that these early-flowering plants were derived from the flf mutant, and were not contaminants.

The tandem T-DNAs present in the flf mutant each contain an Ac element, and we considered the possibility that movement of Ac was the cause of the early-flowering phenotype. DNA was isolated from individual earlyflowering M2 progeny of efSL3 and efSL4, digested with EcoRl and probed with the 3' region of Ac. Figure 6A shows the appearance of a new 2.1 kb band in the early-flowering plants, indicating the movement of Ac. The maintenance of the two original Ac bands present within the T-DNAs indicates that the Ac elements have remained in their original positions as well.

In order to determine the new location of Ac, we probed a similar DNA gel blot with probe 4 (Figure 2C), revealing a change in size of the 2.7 kb EcoRI fragment containing the promoter, MADS domain and part of intron I of the FLF gene (Figure 6B). The size of the 3' Ac fragment (2.1 kb) indicated that the Ac element had inserted near an EcoRI site, with the 3' end of the Ac nearest the EcoRI site. PCR using a primer in the 3' end of the Ac and primers near the EcoRI sites at either end of the 2.7 kb EcoRI fragment revealed that Ac had inserted within intron I in the FLF gene of efSL3. Sequencing of the PCR product determined the precise insertion point of the Ac element (Figure 6C). RNA was isolated from rosette leaves of early-flowering M2 plants and comparable-sized rosette leaves of C24 and flf plants, and probed with an FLF gene specific riboprobe. As shown in Figure 5d, FLF WO 00/32780 PCT/AU99/01079 36 gene expression was reduced to approximately 5% of the C24 expression level. It appears that the presence of Ac reduces either the transcriptional efficiency, RNA stability or the splicing efficiency of the transcript, hence reducing the amount of the normally-spliced mRNA, and resulting in early-flowering.

Example 17 Excision of Ac From Intron I Causes Later Flowering and Increased FLF Transcript Levels Twenty progeny of the M1 efSL3 plant were grown and their flowering time recorded. 15 M2 plants flowered at 18 days, the same as their M1 parent; however, 5 plants flowered later than their parent. These later-flowering plants were termed "neo-lates" to distinguish them from the original late-flowering flf mutant plants. Their approximate days to bolting were: 50 d 50 d d 85 d 100 d In the M2 progeny of efSL4, 36 plants flowered at the same time as their M1 parent but one neo-late plant, 4.6, flowered at 38 d. DNA was isolated from individual plants and probed with 3' Ac (Figure 7A). All the neo-lates appeared to be hemizygous for the presence of the 2.1 kb band, indicating hemizygosity for the presence of Ac in intron I. In some cases 3.2, 3.3) the Ac element had relocated to a new site, as indicated by the appearance of the new band in Figure 7A.

To demonstrate that in each neo-late plant one copy of the Ac in intron I had excised, PCR was performed using primers derived from intron I sequence, flanking the site of Ac insertion. In each case a PCR product was generated, indicating that at least one copy of Ac had excised. The presence of the 2.1 kb band in Figure 7A indicates that at least one copy of Ac remains in intron I; thus each plant is hemizygous for the presence of Ac in intron I The neo-late plant 4.6 is homozygous for the loss of the Ac element within the T-DNA closest to FLF (gene B).

WO 00/32780 PCT/AU99/01079 37 We believe that the sector of the Ml early-flowering parent from which the 4.6 seed derived must have been hemizygous for the presence of this Ac element, and plant 4.6 is a homozygous segregant for the loss of this Ac element.

Presumably the loss of this Ac element was an event independent of that which resulted in the insertion of Ac in intron 1, as M2 progeny of the M1 plant efSL3 are homozygous for the presence of Ac at this location.

Total RNA was extracted from approximately 80 d old rosette leaves of the neo-lates and the gene B expression level was compared to that of 80 d rosette and cauline leaves of flf and 25 d rosette leaves of C24. In all neo-lates the transcript level was higher than that of the early-flowering parent, although lower than that of the flf mutant, consistent with them being hemizygous for the late allele.

Thus insertion of an Ac element into intron I of FLF (gene B) greatly reduces FLF (gene B) transcript levels and causes early flowering, and excision of Ac from intron I restores expression of the FLF (gene B) transcript and results in later flowering. This provides compelling evidence that over-expression of FLF (gene B) in the lateflowering flf mutant is the cause of the late-flowering phenotype.

Example 18 The Expression of the FLF Gene is Controlled by Known Flowering Time Genes In order to further understand the role of the FLF gene in the control of flowering we examined its expression in a range of Arabidopsis ecotypes and lateflowering mutants. Figure 8A shows the expression of the FLF gene in a variety of ecotypes. Interestingly the FLF gene is highly expressed only in ecotypes which have a late allele at the FRI locus (Pitztal and C24), and is particularly highly expressed in C24, but not in ecotypes with an early allele at the FRI locus (Columbia, Ws, Landsberg erecta).

WO 00/32780 PCT/AU99/01079 38 It should be noted that the Pitztal seed source used in this experiment was not the late-flowering Pitztal variety. Subsequent analysis of FLF expression in the late-flowering Pitztal variety revealed a very high expression level, approximately three times that of C24.

This correlates well with the observation that Pitztal takes three times as long to flower as C24 (approximately days compared to approximately 30 days).

To investigate this further, we looked at the expression of the FLF gene in the Landsberg erecta ecotype with late alleles of either FRI or FLC (Figure 8B). Again FLF is expressed in the line of Landsberg erecta with the late FRI12 allele; however, it is also expressed in lines which contain late FLC' S 2 and FLCCO alleles. The expression of the FLF gene in Landsberg erecta-FLCfo 1 is interesting, as this plant has the same FRI and FLC genotype (FRI ear l y FLC"ate) as the ecotype Columbia, yet there is expression in Landsberg erecta-FLC

C

fo, but not in Columbia. This suggests that Landsberg erecta and Columbia differ in a third, unknown, locus, and that this locus in conjunction with late alleles at the FLC locus is able to induce expression of the FLF gene, in the absence of a late allele of FRI.

Many of the late-flowering mutants are in the Landsberg erecta ecotype, and we looked to see whether the FLF gene is upregulated in these mutants. Figure 8C shows that the FLF gene is upregulated in the fca and fve mutants and slightly upregulated in the fpa and fd mutants in the Landsberg erecta ecotype and in Id in Ws ecotype, but not in any of the other late-flowering mutants tested. FLF is also upregulated in the fld mutant in Columbia ecotype.

These data demonstrate that the function of the wild-type alleles of the FCA, FVE, FPA, LD, FD and FLD genes is to down-regulate the FLF gene.

We have shown that the higher FLF transcript levels in at least one of these mutants, fca, is the cause of the late-flowering phenotype, by using the FLF antisense construct from Example 13 to decrease both FLF transcript WO 00/32780 PCT/AU99/01079 39 level in the fca mutant and the flowering time of the mutant. We have also shown that a 28 day vernalization period is sufficient to decrease FLF transcript in all 6 mutants, and to decrease flowering time. Thus coldtreatment is able to overcome the up-regulation of the FLC gene caused by mutations of these loci, and to overcome the delay in flowering.

We also looked at the expression of the FLF gene in the mutants vrnl and vrn2, which have reduced response to vernalization. Both mutants were isolated in the fca mutant background, and as fca and vrn2 are closely linked only the fcavrn2 double mutant is currently available. No increase in expression of the FLF gene in vrnl was detectable, however, the vrn2fca double mutant had increased in FLF gene expression over the fca mutant level, suggesting that irrespective of any role in the vernalization response, the wild-type VRN2 gene acts to repress FLC expression and thereby promote flowering. fca vrnl has a similar flowering time and a similar level of FLC transcript to fca. Both fca vrnl and fca vrn2 have a smaller reduction in flowering time in response to vernalization than does fca, and this is matched by a smaller reduction in FLC transcript level. This indicates that the wild-type VRNI and VRN2 genes are involved in mediating the vernalization-induced down-regulation of the FLC gene. In our growing conditions vrnl segregated away from the fca mutant, is late-flowering and shows little vernalization response, in terms of either flowering time or alteration to FLC transcript level. This suggests that the VRNI gene may be active in an FLC-independent pathway as well as in the FLC-dependent vernalization pathway.

The expression of the FLF gene in early-flowering plants with reduced levels of methylation (Finnegan et al, 1996) is reduced (Figure 5F), suggesting that methylation may play a role in controlling the expression of the FLF gene, or a gene which is a regulator of FLF.

WO 00/32780 PCT/AU99/01079 40 Vernalisation, or some component in the vernalisation signal transduction pathway, acts either to suppress FLF transcription or to increase FLF mRNA degradation. C24 has a strong vernalisation response, with plants vernalised for 3 weeks flowering in about half the time of unvernalised plants. A four week vernalisation period decreases flf flowering time somewhat, but an eight week period is required to bring flowering back to C24 times. This incomplete effect of short vernalisation periods on flowering time of the flf mutant correlates with the incomplete decrease in transcript levels in flf. This suggests that the higher level of the FLF transcript, and presumably of the FLF protein, titrates out the promoter of flowering produced in response to vernalisation. Longer periods of vernalisation may produce more of the promoter, which might overcome the higher FLF transcript level.

Other ecotypes such as Ws, Landsberg erecta and Columbia show little response to vernalisation; under similar conditions to those which give good response with C24, the other ecotypes flower only 1 or 2 days early. We note that these ecotypes have very low FLF transcript levels, which cannot be decreased much further by vernalisation.

Pitztal is a late-flowering ecotype which has a strong vernalisation response, and has high FLF transcript levels. Vernalisation is expected to decrease the level of FLF transcript in these plants. Vernalization decreases the level of FLF transcript in this ecotype, with longer periods of vernalization resulting in a proportionally greater decrease in FLF transcript, correlating with the decrease in flowering time.

The flf mutant requires prolonged GA treatment to cause it to flower early. This suggests that the high levels of FLF transcript (and presumably FLF protein) may act to remove GA, or to decrease GA action. Since FLF is a MADS box transcription factor, it may do so by activating genes involved in the catabolism of GA, either directly or WO 00/32780 PCT/AU99/01079 41 indirectly, or by altering the expression of genes involved in GA signal transduction.

The lack of effect of GA treatment on the level of FLF transcript suggests that GA acts either downstream of FLF, or via another pathway. ie.

VERNALISATION 4 4 decrease in FLF transcript 4 4 GA 4 4 FLOWERING or VERNALISATION 4 decrease in FLF transcript 4 4 FLOWERING ?4 4 GA 4 4 FLOWERING with FLF normally acting to block the pathway between VERN and GA Tissue-Specific Expression Levels of expression of FLF in different tissues of the C24 ecotype were examined. High expression was observed in vegetative leaves, roots, flower buds and mature flowers. There was a low level of expression in cauline leaves and bolt stem, and a very low expression level in green siliques. RNA was isolated from C24 rosette leaves and "apex", ie. the tissue remaining after as many as leaves as possible and the roots were removed; this tissue includes very small leaves and the apical meristem.

There was no difference in expression level between these two tissue types.

In the flf mutant expression of FLF was twice that of C24 in vegetative tissue, and expression in floral tissues was relatively greater (about 3 times C24 level).

In the Columbia ecotype the level expression vegetative leaves was very low compared to C24, while in floral tissue there was about the same level of expression as C24.

This suggests that there may be a separate control of vegetative and floral transcription of the FLF gene.

WO 00/32780 PCT/AU99/01079 42 Developmental Expression As FLF appears to be a repressor of flowering, one prediction about its pattern of expression is that it might decrease prior to, or accompanying, the transition to flowering. RNA was extracted from whole plants that were grown on MS medium and harvested every 10 days. Under these conditions 50% of C24 plants were bolting after 50 d.

There was no change in the level of FLF transcript in either C24 or the flf mutant. This suggests that if there is a decrease in the level of FLF transcript accompanying the transition to flowering it must occur in very few cells. This result also suggest that there is no decrease in the level of transcript in older leaves, ie. the transcript is not diluted out as the leaf grows.

Circadian Response C24 and flf plants were grown in 8 h fluorescent photoperiod for 21 d and either maintained in this condition, or transferred to either continuous light or continuous dark. RNA was extracted from plants harvested at either the start or the end of what would have been the 8 h photoperiod. In each case there was slightly higher expression at the earlier time point, suggesting a subtle circadian response. There was no difference in the pattern of expression between the mutant and C24.

Example 19 Isolation of a Brassica napus FLF homologue Low stringency screening of a Brassica napus genomic library with an FLF probe lacking the MADS box region resulted in the isolation of 18 strongly-hybridizing plaques out of a total of 72,000 screened. The low stringency conditions were: hybridization at 28 0 C in formamide, 3 x SSC, 0.1% SDS, 20 x Denhardt's, 50 pg/ml salmon sperm DNA overnight and washed with a final wash of 0.1 x SSC, 0.1% SDS at room temperature. These plaques were purified, and a selection were sequenced as described in Example 4.

43 The partial genomic sequence of the Brassica napus FLF-like gene is set out in SEQ ID NO. 4 (Figure 9), and the amino acid sequence of the predicted translation product is set out in SEQ ID NO. 5 (Figure 10). The partial genomic sequence, showing the location of exons and the sequence of the corresponding translated product, is illustrated in Figure 9, and the sequence of the predicted translated product from the Brassica napus gene is compared with the corresponding product from Arabidopsis thaliana FLF in Figure 10. There is a high degree of conservation, with 79% identity and 83% similarity in the deduced FLF protein sequence (as determined by the University of Wisconsin Genetics Computer Group software package version 9.1, using default parameters).

A cDNA library was prepared from Brassica napus, and 10 cDNA clones were isolated using hybridisation to an FLF cDNA from Arabidopsis. The starting RNA was isolated from leaves of Brassica napus, cultivar Colombus. Poly(A)+ o mRNA was isolated using a mRNA purification kit (Amersham Pharmacia Biotech). A cDNA library was constructed using a SuperScript Choice System cDNA synthesis kit (Gibco BRL) in the lambda-ziplox vector (Gibco BRL) with EcoRI arms. The primary titre of the library was approximately 500,000 pfu.

Approximately 200,000 plaques from the primary library were screened, using the Arabidopsis FLF cDNA without the first two exons of the coding region. Screening was carried out at high stringency. Thirteen plaques were picked in the *first round, and of these 10 were confirmed as positive by a second round of screening at high stringency. These plaques were purified, and a plasmid containing the cDNA was excised from each clone. The complete nucleotide sequence of each clone was determined, and from this the amino acid sequences encoded by each clone were deduced.

Multiple sequence alignments were used to determine the relationships between the clones. From both amino acid and nucleotide sequence data it was concluded that the clones represent transcripts from 5 different genes.

SlAWendyS\Kccp\.pecic\I185)I-(X) CSIRO dc 2(VO2/02 44 The cDNA sequences and predicted amino acid sequences are set out in SEQ ID NOS: 6 to 15 and SEQ ID NOS: 16 to 25 respectively. The sequences probably represent 5 genes, grouped as follows: 12.1/16.1 15.1/16.2/18.2 11.2 14.1/18.1/20.1 11.3 The partial genomic sequence and translation in Figure 9 correspond to the cDNAs 12.1/16.1.

Example 20 Expression and Immunodetection of FLF protein The FLF protein could be detected on Western blots using antibodies raised to a bacterially expressed protein.

A truncated FLF protein lacking the first N-terminal amino acid residues and possessing an in-frame 20 N-terminal histidine tag was overexpressed in E.coli strain BL21 [DE3] using a pET 22b+ expression vector (Novagen). 1 ml of an overnight bacterial culture was added to 100ml of LB broth containing 50 gg/ml ampicillin and the culture grown to an OD 600 nm f 0.6 at 37 0 C. 100 1l of 1M IPTG was 25 then added, and the culture was grown for a further 4 hours Sbefore harvesting for protein isolation. Histidine-tagged protein was purified using Talon metal affinity resin (Clontech). The purified protein was injected into a rabbit for antibody production (Harlow and Lane, 1988).

Twelve day old Arabidopsis plants were ground in liquid nitrogen and homogenised in extraction buffer (0.1M NaPO 4 pH 7.2, 1mM EDTA). Insoluble material was pelleted by centrifugation at 16,000g for 2 minutes. The supernatant was immediately boiled in a one third volume of 6x SDS sample buffer (0.5M Tris pH 6.8, 10% SDS, 0.6M DTT, 0.012% bromophenol blue) for 5 minutes. Insoluble proteins were extracted by homogenising and boiling for 5 minutes in 2x H:\WcndlyS\Kccp\,pccics\ I 8501 CSIRO duc 2(02/02 WO 00/32780 PCT/AU99/01079 45 SDS sample buffer (0.167M Tris pH 6.8, 3.3% SDS, 0.2M DTT, 0.004% bromophenol blue). Protein extracts (50 gg per lane) were separated on a denaturing 12% polyacrylamide gel before blotting onto a Protran nitrocellulose membrane (Schleicher Schuell) Blots were incubated with either preimmune serum diluted 1:1000 or with FLF polyclonal antiserum diluted 1:3000. The immunoreactive protein was visualised using the ECL western blotting analysis system (Amersham) with the secondary antibody diluted 1:2000. The blots were exposed to X-ray film (Fuji RX) for 2 to minutes.

The amount of FLF protein was dramatically decreased following 35 days of low temperature treatment in the C24 and Pitztal ecotypes and in the flf mutant. The decrease in the flf mutant was not as great as in the two ecotypes, and this finding was consistent with our results for both flowering time and FLF RNA transcription. There was very little FLF protein in either the Landsberg erecta ecotype or in the efSL4 loss-of-function mutant, also consistent with the RNA data. These results suggest that protein expression parallels RNA expression, and that differences seen in the RNA transcript levels will be reflected in protein levels.

These results also demonstrate that immunoassay using antibody directed against FLF protein can be used to identify plants having either low or high level expression of this protein, and to select strains having the desired characteristics. Such immunoassays can also be used to monitor recombinant expression of the protein in bacterial or other hosts.

Example 21 Effect of transformation with FLF on Flowering Time in Brassica napus A cassette containing the Arabidopsis FLF cDNA under the control of the Cauliflower Mosaic Virus (CaMV) promoter was inserted into the vector pWBVec8. This vector contains the HPT gene, which confers resistance to WO00/32780 PCT/AU99/01079 46 the antibiotic hygromycin, also under the control of the CaMV 35S gene. The plasmid, in the Agrobacterium tumefaciens strain AGL1, was used to transform Brassica napus cultivar BLN1239 hypocotyl explants, and plants were regenerated. Hygromycin-resistant To plants were tranferred to soil. Approximately four months after the transformation experiment was begun, plants were transferred to small soil pots on a misting bench, and after approximately four weeks the plants were transferred to large pots and put in a glasshouse.

Plants were regenerated from 21 independent calli. All the plants regenerated from each callus are referred to herein as a family, and each family represents 1 or more independent transformation events.

The morphology of the plants was monitored, and the results are summarised in Table 3.

WO 00/32780 PCT/AU99/0I079 47 Table 3 Family C-lones Date re.corded PCR test for Antibody test for floral (and leaf transgene Arabidopsis FLF no. Floral protein status recorded on Days 1, 11, 33and_] 41.

CS Ex ,1 Small plant -ye -ve 1.1 /2 Day I -ve -ve /3 Day 1 -ye -ve /4 Day 1 -ye -ve Day 11 -ye -ve CS Ex /1 Day 25. 17 leaves +ve +ve 1.2 /2 Day 33. 15 leaves +ve nd /3 Day 11 -ye -ye /4 Day 33. 17 leaves -ye (false -ye +ve Day 25 +ve +ve CS Ex /1 Day 41. 21 leaves +ve +ve 1.3 /2 Day 41. 21 leaves +ve +ve /3 Day 1 -ye -ve cS Ex /1 Day 1 -ye -ve 1.4 /2 Day 1 -ye -ve CS Ex /1 Day 1 -ye -ve CS Ex /1 Day 33. 15 leaves +ve +ve 1.6 CS Ex /1 Day 33. 20 leaves +ve +ve 1.7 /2 Day 33. 17 leaves nd +ve /3 Day 33. 19 leaves +ve +ve /4 Day 33. 16 leaves +ve +ve Day 11 +ve nd CS Ex /1 Day 41. 24 leaves +ve nd 1.8 /2 Day 41. 22 leaves +ve +ve /3 Day 41. 24 leaves +ve ±ve /4 Day 41. 23 leaves +ve nd Day 41. 24 leaves +ve nd CS Ex /1 Day 25. 15 leaves Nd +ve 1.9 1 CS Ex /1 Day 25 +ve -ve 1.10 CS Ex /1 Day 25 +ve nd 1.11 /2 Day 25 -ye -ve CS Ex /1 Day 1 -ye -ve 1.12 CS Ex /1 In TC In TC In TC 1.13 CS Ex /1 Day 25 -ye -ve 1.14 Day 2 5 /2 Day 25 -ye -ve Day 25 -ye -ye WO 00/32780 WO 0032780PCT/AU99/O 1079 48 Ex /1 Day 33. 15 leaves +vte nd /2 D~ay 33. 16 leaves +ve +v~e /3 Day 33 16 leaves *vk? nGI ES x /1 Day 33. 14 leaves +ve +ve E Ex /1 Day 1 +v(e nd C x S./1X I n TC In TC In TC 1 .18 1.S E x /1 Day 25. 15 leaves +vH ncl S. 19 C.S Ex /1 Discarded Discarded Discarded 1.20 C.S' Ex In T'PC In I n 'PC In 'PC 1 .21 In TC plants remain in tissue culture stage nd not determined The plants exhibited a range of flowering times, with some plants flowering in tissue culture or while on the misting bench, while others took more than 2 months longer. Polymerase chain reaction (PCR) analysis using primers specific for the CaMV35S: :FLF transgene was performed to determine which of the putative transgenic plants contained the transgene. An antibody test (Western analysis) was also carried out to determine which plants contain the Arabidopsis FLF protein. The antibody does not cross-react with any Brassica proteins under the conditions used. Although the PCR and antibody tests were single experiments, there is a good correlation between the two tests.

Table 4 sumnmarises these results.

WO 00/32780 PCT/AU99/01079 49 Table 4 Flowering Time in Brassica napus Plants Transfomed with FLF Number of Number of Number of Number of floral floral PCR floral families plants or plants with (those Antibody negative vegetative with buds positive for both plants (18 Days or open plants PCR and families (Taking day of flowers) Antibody total) potting as day 1) test 10/41 1/26 9/15 13 Day 1 13/41 2/26 11/15 12 Day 11 24/41 9/26 15/15 7 Day 25 32/41 19/26 2 Day 33 41/41 26/26 0 Day 41 The day of potting Floral plants are into large pots is those with buds or referred to open as day 1.

flowers.

Although no wild-type flowering time control could be included in the experiment, due to the tissue culture stage of the experiment, it is reasonable to assume that the earlier flowering plants, which tested negative for the presence of the transgene and of Arabidopsis FLF protein, flowered at around the time of the wild-type plants. Thus some of the transgenic lines flowered at least 6 weeks later than the earliest flowering lines. The delay in time to flowering was also manifested by an increase in the number of leaves which the plant developed before floral buds appeared.

This example demonstrates that transformation with FLF can be used to modify the time of flowering. The person skilled in the art will be able to apply these findings to other species of plant.

WO 00/32780 WO 0032780PCT/AU99/01079 50 Example 22 FLF-like Molecules from Arabidopsis thaliana Searching of the Arabidopsis genomic sequence database revealed five putative MADS-box encoding genes with a high degree of homology to the ELF protein. One of these genes, designated FLF-LIKE1, occurs on the chromosome 1 BAC F22K20 (AC002291), while the other four, designated FLF-LIKE 2, 3, 4 and 5 respectively, occur in a cluster on the two contiguous chromosome 5 P1 clones MXK3 and MQN23 (AB019236 and AB0133 The amino acid sequences of the ELF-LIKE proteins are shown below, with amino acids identical to corresponding amino acids of the ELF protein depicted in bold type.

FLE-LIKEl (SEQ ID NO:26):

MGRRKIEIKRIENKSSRQVTFSKRRNGLIDKARQLSILCESSVAVVVVSASGKLYDSSS

GDD I SKI IDRYE IQHADELRALDLEEKI QNYL PHKELLETVQSKLEE PNVDNVSVDSL I

SLEEQLETALSVSRARKAEIMMEYIESLKEKEKLLREENQVLASQMGKNTLLATDDERG

MFPGSSSGNKIPETLPLLN.

ELF-LIKE2 (SEQ ID NO:27):

MGRKKVEIKRIENKSSRQVTFSKRRNGLIEKARQLSILCESSIAVLVVSGSGKLYKSAS

GDNMSKI IDRYEIHHADELEALDLAEKTRkNYLPLKELLEIVQSKLEESNVDNASVDTLI

SLEEQLETALSVTRARKTELXMGEVKSLQKTENLLREENQTLASQVGKKTFLVIEGDRG

MSWENGSGNKVRETLPLLK.

FLF-LIKE3 (SEQ ID NO:28): MGRRKVEIKRIENKSSRQVTFSKRRKGLIEKARQLS ILCES SIAVVAVSGSGKLYDSAS GDNMSKI IDRYE IHHADELKALDLAEKI RNYL PHKELLE IVQSKLEESNVDNVSVDSL I SMEEQLETALSVIRAKKTEIMMEDMKSLQERZKLLI EENQILASQVGKKTFLVIEGDRG

MSRENGSGNKVPETLSLLK.

FLF-LIKE4 (SEQ ID NO:29): MGRRKVEIKRIENKSSRQVTFCKRRNGLMEKARQLSILCESSVALI IISATGRLYSFSS GDSMAIKILSRYELEQADDLIKTLDLEEKTLNYLSHKELLETI QCKI EEAKSDNVS IDCLK SLEEQLKTALSVTEARKTELMMELVKTHQEKEKLLREENQSLTNQLI KMGKMKKSVEAE

DARAMSPESSSDNKPPETLLLLK.

51 (SEQ ID

MGRRRVEIKRIENKSSRQVTFCKRRNGLMEKARQLSILCGSSVALFIVSSTGKLYNSSS

GDSMAKIISRFKIQQADDPETLDLEDKTQDYLSHKELLEIVQRKIEEAKGDNVSIESLI

SMEEQLKSALSVIRARKTELLMELVKNLQDKEKLLKEKNKVLASEVGKLKKILETGDER

AVMSPENSSGHSPPETLPLLK.

Alignments of the full-length deduced amino acid sequence of the FLF-LIKE proteins with the full length FLF protein sequence revealed a 65.3 identity (86.7 similarity) for FLF-LIKE1, 61.2 identity (84.2 similarity) for FLF-LIKE2, 60.7 identity (84.2 similarity) for FLF-LIKE3, 60.7 identity (85.2 similarity) for FLF-LIKE4 and 56.1 identity (86.2 similarity) for FLF-LIKE5. In contrast, the published Arabidopsis MADS-box proteins which are most similar to FLF show only 42.9 identity (66.3 similarity) in the case of AGL14 (Rounsley et al., 1995) 40.3 identity (75.5 similarity) for CAL (CAULIFLOWER, Kempin et al., 1995) and 38.8 identity (74.0 similarity) for AP1 (APETALA1, Mandel et al., 1992). identity and similarity determined by the Wisconsin Package Version 10.0, Genetics Computer Group (GCG), Madison, Wisc., using default parameters).

cDNA from a chromosome 1 FLF -like gene (FLF- 25 LIKE1) was isolated using a RT PCR based method. First strand cDNA was generated from 5 gg of Col-0 total RNA.

Reactions were carried out using Superscript II (GIBCO BRL) in a 20 pl volume according to the manufacturer's instructions. FLF-LIKE1 transcript was amplified by PCR 30 using 1 l of the first strand cDNA synthesis reaction as template with primers 5'-ATTGAATTCGGGCATAACCCTTATCGGAGATTTG-3' (SEQ ID NO. 39) and 5'-AACGGATCCGTTGATGATGGTGGCTAATTGAGCAG-3' (SEQ ID NO. Eco RI and Bam HI restriction sites respectively are underlined. The amplification reaction was carried out in a final volume of 40 p1, which contained 2.5 pM of each oligonucleotide primer, 1.0 units of Amplitaq Polymerase (Perkin Elmer) and 250 pM of each of the four H:\WendyS\Kccp\specics\l85)1 CSIRO.doC 20/12/02 52 deoxynucleotides. Conditions for amplification were as follows: 94 0 C for 2 min, 40 cycles consisting of 15 s denaturation at 94 0 C, annealing at 55 0 C for 15 s and polymerisation at 72 0 C for 1 min, and a final extension at 72 0 C for 4 min before the temperature was decreased to 0 C. PCR products were purified using QIAquick PCR purification kit (Qiagen), digested with restriction enzymes Eco RI/Bam HI, and ligated into the corresponding restriction sites of a pBIISK+ vector (Stratagene Positive colonies were sequenced using universal primers with the Applied Biosystems Big Dye terminator sequencing mix according to the manufacturer's instructions, and analysed using an Applied Biosystems 377 sequencing machine (Perkin Elmer). cDNA sequences obtained were compared to Arabidopsis genomic sequence (BAC F22K20; AC002291). The University of Wisconsin GCG software package was employed for sequence analysis.

A binary construct containing the FLF-LIKE1 cDNA under the control of a CaMV 35S promoter was generated by 20 cloning an Eco RI/Kpn I digested PCR product into an Eco RI/Kpn I pART7 vector (Gleave, 1992) containing a CaMV promoter. The PCR product was amplified using 200 pg of the FLF-LIKE1 cDNA clone as template with primers: 5'-ATTGAATTCGGGCATAACCCTTATCGGAGATTTG-3' (SEQ ID NO. 41) and 5'-CTAGTGGTACCGTTGATGATGGTGGCTAATTGAGC-3' (SEQ ID NO. 42); Eco RI and Kpn I restriction sites respectively are underlined. The amplification reaction was carried out as described above. The cloned PCR product was sequenced to ensure that no mutations had been introduced during the amplification procedure. The 35S::FLF-LIKE1 cassette was then subcloned into pART27 using Not I (Gleave, 1992), and introduced into Agrobacterium tumefaciens strain GV3101 by electroporation. Transgenic plants were generated by in planta transformation (Bechtold et al., 1993), using the NPT II gene as a selectable marker to identify transgenic plants.

-MX) CSIROd 241VO2/02 WO 00/32780 PCT/AU99/01079 53 The 35S::FLF-LIKE1 construct was transformed into Arabidopsis thaliana ecotypes Landsberg erecta and C24.

Twenty individual Ti lines were selected for each ecotype, and of these about half showed a late-flowering phenotype.

In the Landsberg erecta background, 12 out of 20 Ti plants bolted 3-4 weeks post-germination, consistent with the bolting time of non-transformed wild type plants. The other 8 plants did not bolt until about 7-8 weeks postgermination. Similarly for C24 transformed lines, 9 out of 20 plants bolted about 4-5 weeks post-germination, consistent with the bolting time of wild-type C24 plants, while the other 11 Ti lines did not bolt until 8-10 weeks post-germination. These data suggest that the FLF-LIKE1 gene is capable of delaying flowering when overexpressed in Arabidopsis thaliana plants, similarly to what we have shown for FLF. Furthermore, it seems likely that the overexpression of the other FLF-LIKE genes described would have a similar effect on flowering time to that found for both FLF and FLF-LIKE1 genes, especially considering that FLF-LIKE1 shows greater homology to the other FLF-LIKE genes than to FLF.

Example 23 FLF Modulates Gibberellic Acid (GA) Activity in a Number of Developmental Processes The phenotypic effects of over-expression of the FLF transcript were investigated in 35S::FLF transgenic Arabidopsis thaliana. The ecotypes used and the numbers of T1 plants examined are indicated below. Many of the phenotypic characteristics found to be modified are known to be associated with growth processes which are controlled or modulated by GA. These include pollen formation, leaf expansion, decreased petiole angle, and trichome formation.

It was particularly noteworthy that some of the plants were sterile, while others showed reduced bolt height and internode length, characteristic of a dwarf phenotype, or colour changes associated with the dwarf phenotype.

WO 00/32780 PCT/AU99/01079 54 The late-flowering flf mutant requires much more GA to induce early flowering than wild type plants, suggesting that the FLF gene product may act to remove GA or GA activity. As many of the phenotypic abnormalities observed in transgenic plants expressing high levels of the FLF transcript can be attributed to an alteration in GA level or activity, it seems likely that this function of FLF is not limited to the control of GA activity in relation to the promotion of flowering, but also in relation to other roles of GA. Hence, the FLF gene may be useful in regulating GA activity in other aspects of plant growth, including, but not limited to, control of plant architecture and/or fertility.

As well as the effects on flowering time that have been discussed in previous examples, over-expression of the FLF coding sequence also produced a number of vegetative and floral phenotypes, which are outlined below.

C24 ecotype (24 T1 plants) One early-flowering plant had the appearance of a semi-dwarf (bolt height reduced, internode length reduced), typical of the GA semi-dwarfs, caused by a reduction but not abolition of GA production in the plant.

This plant was sterile.

Several plants 9, 10, 16, 19, 22, 23) exhibited partial or complete sterility. In several of these plants it appeared that the anthers had not dehisced, and no pollen was visible.

Landsberg erecta ecotype (24 T1 plants) Two plants 63) were sterile or produced very few seeds. These plants had clear floral abnormalities, having petals of reduced size that were greenish in colour, and abnormally-shaped carpels with trichomes ("hairs"). Normally Arabidopsis carpels do not have trichomes.

WO 00/32780 PCT/AU99/01079 55 The leaves of some plants which still have not flowered were reduced in size, resulting in a smaller diameter leaf rosette than normal 42, 62). Many of the late-flowering plants 37, 38, 40, 42,62) were a darker green colour than normal. GA-deficient dwarfs are dark green. Many of the late-flowering plants exhibited regions of bulging in the leaves, suggesting that leaf expansion is not occurring equally in each direction. Many plants had a reduced petiole angle, ie. the leaves were flatter.

Landsberg erecta.-FLC& f2 (17 T1 plants) Two plants had the appearance of a semi-dwarf 28). The plants had reduced fertility. One plant produced very few seeds. This plant had clear floral abnormalities, with petals of reduced size that were greenish in colour, and abnormally-shaped carpels with trichomes.

One plant which has still not flowered has extremely small rosettes (-15 mm in diameter). Others are small, but not so extremely so (#100, 103). One of the late-flowering plants (#102) was a darker green colour than normal. Many of the late-flowering plants exhibited regions of bulging in the leaves, again suggesting that leaf expansion is not occurring equally in each direction.

Many plants had a reduced petiole angle.

Landsberg erecta-FR Sf 2 (35 T1 plants) One plant had the appearance of a semidwarf. This plant had reduced fertility. One plant (#89) produced very few seeds. This plant had clear floral abnormalities: petals of reduced size that were greenish in colour, abnormally-shaped carpels with trichomes. Some of the late-flowering plants 56) were a darker green colour than normal. Two plants 98) which have still not flowered, have extremely small rosettes (-15 mm in WO 00/32780 PCT/AU99/01079 56 diameter). Others are small, but not so extremely so (#51, 53, 78, 93, 95, 96).

Many of the late-flowering plants exhibited regions of bulging in the leaves, suggesting that leaf expansion is not occurring equally in each direction. Many plants had a reduced petiole angle.

Further phenotypic abnormalities in Ac generated earlyflowering mutants The early-flowering mutants generated by insertion of Ac into intron 1 have a reduced number of trichomes on their leaves.

It will be apparent to the person skilled in the art that while the invention has been described in some detail for the purposes of clarity and understanding, various modifications and alterations to the embodiments and methods described herein may be made without departing from the scope of the inventive concept disclosed in this specification.

References cited herein are listed on the following pages, and are incorporated herein by this reference.

WO 00/32780 PCT/AU99/01079 57

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Weigel et al Cell, 1992 69 843-859 EDITORIAL NOTE APPLICATION NUMBER 18501/00 The following Sequence Listing pages 1 to 31 are part of the description. The claims pages follow on pages "62" to "66".

1- SEQUENCE LISTING <110> Commonwealth Scientific and Industrial Research organization <120> Control of Flowering <130> P42286 <140> AU 18501/00 <141> 1999-12-02 <150> PP7469 US6O/116,928 <151> 1998-12-03 :<160> 42 <170> Patentln version <210> 1 <211> 7968 <212> DNA <213> Arabidopsis thaliana *<400> 1 **ggatccaaga aataatttca tatggggcac agttaaaaaa aaaacaataa aatgataata gtaaggcttg aacttgggtt gatgtgaggc actattaagt aaaaagccat tgtactactt 120 acattttaac tacataatgt taacttatat aatatttatt gaattcagta tagggcacat 180 gccctatcca tgactaacgt gagtccgccc tgatagcgag taagaaacga gcaaaggaat 240 gcaaattatg tgaatactat acaccaacag tgtagacatg tagctacaat gcggcaatgt 300 taaaaataaa ctcaaattgg tttggaggga acaacctaat gcttataagt acacttttgt 360 ggtaaataca atccaattga aagtctttgt aggtttggtt tggtccaatg aaattgtatg 420 cgagttgagt aaaataacct tagttcaaaa cattagatat gtaatggtct agatacgatg 480 gtagccaaag atttgggtta aatttagaat aaagatcaaa gaacaaatga ctgacttcct 540 tatgtgtgtt tgtttatgta aagtcttaac tcgtgtcttg ccaaattaat aaaaaggtgc 600 -2 attatacaat aaattttgtt aacttcttgg aataccaatc agtgttagtg ttacaaaatt acctttcaag agtaattaga ttgtgttacc agtaattaat aaaatatata taaaaggaaa aaaaagaatt tttagaaatc tctcccgaac atgacgtagg taattaactg tcgatatgtc atccgtatcg aatgcaaaag gcaacgtttt aaaaatatct cgagaaaagg ccaaacctga aaaactagaa tcgcaacggt tcttctcgtc cttttcctta cttttctgag gatctcttgt tacatyagat cttttgttca gaactagagc gattatatat cacagctccg atgaatcatc aaatgtttgt tgtaatcagt tgtctaatat ttaatcatat attcaaacgg aaaattaata ataataaaat acaagctgat agtaactttg ttatatattt attattattt cgagtggttc ccaaatttta tacatagttc taggggagga tagcaaagac tattcacata ggcccgacga aaaaaaaaaa ggatcaaatt atcaagcgaa ctcatcgaga gtctccgcct cctgggtttt aaaataaaaa tg tt t ctcgg aaccaaatt t tttctctctc ttgttctcat gatttcaata agtgttactg aatatcgtta gtggctccat ttcacccact aacacttttt gtaatctatt tataatctat taatgatagt tagataaaga acaagcattt agctattgcc ccacaatata caatactatc tttgttttta agttttgaga aaagatgatg aagatagttt gctcgtcatg tttggttttt agaaaaagta atagaaagag agggcacaaa t tgagaacaa aagctcgtca ccggcaagct catttattcc taaaaaaaca ttctgtgttt aaggaagaac tatttcttaa caaaattttt ttaaattata aaatgtttgt caaaatcagt taaaaaagtt tcttttcgtc aacaaaatat acatatagta ataattatta gatattcaga agaaattggt cacccaaaaa atatgtgtgg tttactactt tgaaaaacac ctatgtaggc agtcggaaga tagagtggag tcatttagca cggtacacgt ttgcatcact gataggcaca aaaacgctta gccctctcgg aagtagccga gctttctgtt ctacagcttc cccttttatc attaatatac gtttgtgttt aatgtcgtga aaaaaaaaat aacattaaaa ttttttgctt gtggctccaa aatcagtttc aataccaatc ttgttactcg tacaaaacaa atttgtatta gcaaaacaaa atgtggtatc aaaaaataat aaaacaagct acatttaaga tcctttatta attttttttt acgactttgg gttcaaacca gttctttctg aagaaagtga ggcaatcttg ctcgtttacc aaaaatagaa gtatctccgg agagaagcca caagtcacct ct Ctg tgacg tcctccggcg ttctgttttg cgtttggttt ttttctgcga agaagctttt tctgcatgga taagttatta atagcatcaa tagaaaagtt acccactccg accaatatcg atatagcctc gaaaaat tat gtatcgttta atatagtttt taatcatgta taatgagata gatacaagca tttcttgtat tttgtgttaa ataaaatttg taacacctac gt t ttaggt t caatagttca aaactaaggc tcttcaaaac cccaaaaaaa agaaataaag cgact tgaac tgggaagaaa tctccaaacg catccgtcgc ataagtacgc tgctctttta ttttccggcg ccatgataga tagcttctta tttcattatt 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 tccttggaaa atgttgttga agtttttgaa attaaaattc ttgctatggg taagactatt ttatccaaat ggtagcttca acataatctg acaccttttg tggatttgta aaaaggatca aagatgaatt atgctatttg atttgcctca gttcaattat ttgtgaaaat attcacacag ttcatgtaag atataagttg caaatggttg catactttgt ctgactagaa aacccttagt cacaatagga gatcttctag agatatattt gggcagaaaa gaacctcaca cggcttccta tgagttttgt atctggttac tggaaaaaac aaaattgcat gtgaagtttc gagttaatta attggatctc gttaatgctg gttaatcttc tagtgtt t tt aaaaactcag gttatcgatt ctgtacataa tatgcacgtc agaagtttgg ggtaatatat aagattgtcc tatttatgtg gactttgttc cgacaatcac ccttgtttct aaataccaac tcaaaattga tagtagtttg taggatttgt ctcctggtct tactcagtta ctgatatcta gtcaatatgt atagatatgt ctctttactt gtttctataa ttcttaggag ctaagactcg cgccacatca cttgtcctca gtccttcacg aagccatctt tatatcacaa tcggatttgt aacaatgtat tatgaattcc aattgattca cctcacaatt gcgattcttt actggtctaa cgggagattt ttttaaatgt acataatatt aaaggtttat attgtatatc ctattcgtta acaacctttg ttggtgcctc ctctttggta atctggtgta gccatgttgg tcacccctag taattatgat ctctttttgc t taacaaaac agtttagttt gtgataagtt ttattgcttg acgagtatgt tcttttgata gtccatgtat tcattatcat aggttcattg -3 atttgtttga tgattgtttc gactaatgat atgcaatgca atataccaca tatctttgct tatttttcat agtacttacc gaatcacaat ttttagacta ataaataaaa aagccacatt tttaacgaat agtttcccac aattatcgcc aaattgacaa tatcttgtgt taggaaattg cgga tc tat a gtgtctacta tcaagatcgc ttaggtccag ttaataaaga atacttaggt aaattaagtg ttatctgtct tcctactaat attaatttgg aattgaatgt t ttgcaaaaa ttggagtttg cttatgggtc aaaaatccga tacgatctga ttacctttag taatgtctct cttacgggag t tgtgcagct gtggacctat acacagtagt gcacatatgc cgtcgtgtgc attaaatttc ttagtatgag aat tgggaaa ttctctcctt tcttgcagtt cttaatctta tccacaacct cttttgtcat aaaatcccac atgaatcaat caaccctcca tggccgat tc ccttggaatt agaagccttt tgatgcaaag aagttttgtc tagtcgcttc attagttagt ggtttaaata gaaataacaa aaacatatac gcttcctcat atcaatacta aaag tt tt ct tgcgtgctcg agattcctta tttcagagtg atctatagag attgactata tacttggtga tttgaatttt tacttcagta tatatatata attgttctct gttaatggta ctatgactaa tttatgggat acacacatag tcatcgttgt caatcttttg ggaaattgtc aacacttgtc ataatcctat atataataac tcacttgatg gtcgagacac tagaacgtgg agcttaactt aaaattgttg cttctatgga gtaacttcaa taggaaattg aatggaagac aatagaaata acttatggtt gctctatcgc cgtatatgtt 2580 2640 2700 2760 2820 2880 2940 3000 3060 3120 3180 3240 3300 3360 3420 3480 3540 3600 3660 3720 3780 3840 3900 3960 4020 4080 4140 4200 4260 4320 4380 4440 4500 4gatatggtat tgaactcatg agaggtaact acatgataga t tgtaaagga ggaagctctg aattggaagc aaattaagag tacttccatg tatatgattt cattttatat tctggacagt ttaatcaaag gagggaaaat attcattgga ttgagagaga taaaactttt attattgttt tgttctctca tcctaacata atgggaaaca ccctgatgga aaaatgaggt ttaccgcttc ggttcaeact tttgctctcc tgcagcaagc gaggaacacc aatgtctact cagaccgaac ccaattttat cttaacgagc tacttacaaa aaagaggcg t aataatttga aaatagaggg tataggtgtt aatctctgat agtcttccac aaatatgact tcatcacatt ctggaaaaat tgcatcaatt agaggcttat ccagcgctat gtcattttca taactaatct aaaaaaatat ttgaaaaagt ttctgaaatg atgtttcata tttttatttt gcatgctgat gttttataag ggtggctcca ttctgtccct atgagctact acctttgatt ttgtgggatc ttgagactgc cctttctgaa tcatgttgaa tgtacatcag ttgcacacat caaagagctg ttttaaaaaa taccattgtt taaataggtt ttttgatgct agaggttgca tatttgctat ttctagactc gtggctcatc aaaaattgct atttcaacat gtttagggtt cactaaactt atctgccgaa ttgagcaatt cagatattat ggaaattcag ttacgaatac gtttccagtg ttttgtcatc gatcttaaag gcgtaaattt catgcattat ttttcatggg tgaacttgtg acaaaggaat aaatgtcaaa cctctccgtg ttttgtttgc gcttgttgag atatatcctc atttgcaact atgttaccaa ttctttttaa cg tag tcc tg ttgttcttat aaaagttgtg attagaatta tgttagggaa agtctgtgta aatatatgtg tgtttgcatt agattcacaa cttatgtacc tatctgtatg atatataata tttggtaaat taaatattgc atgtgctact tagcgtgtta gccttttcaa tctccagcct ccttggtaat actattagtt gcataccgca caggatcatc gataggttag tagttttttt aatgtgagta actagagcca tgagaacaac aatcttaaag ttctgtgttg ttcttcatat ttttgacacg aactgggata atcaaatgtt aatggttttg gtatggatca tataagttaa gtctttcagt cttggaattt tgtatataca taagattggg acataaatgc ttaactagtt cctttgtatg aatacatgtt gttttggttc ttacaaagct gcttaaacat tatatatgta gggttagcta ggtcaagatc acaaacattt tgccgagtga attttcatag agtcaaaagc tactactaac tttgtcaaac tcgatgctct agaaggtaag cgtgctgctt aaaaggtcag tctctgttac gttttggatt agat tactaa caaaaagaaa ataagggtaa ataacacgct aaaccaaaat t ttgcaaatg taatttcaga tacttcggtt ttcatgagta gctgcgttta atagaaacaa tgactttaag acttttcttt agcccacata ttttcttttc aagaacaagt gaatattaag aaaggtaagg gtagttttga cttgatcgat tgaatctttt tcctaaatat cccttgtctt tctgaactat taagactata tatgaatata tg t tcaac tg ttgatttcgt ttgtttgttg atatttgcta tttaagtctg ccaaattctg 4560 4620 4680 4740 4800 4860 4920 4980 5040 5100 5160 5220 5280 5340 5400 5460 5520 5580 5640 5700 5760 5820 5880 5940 6000 6060 6120 6180 6240 6300 6360 6420 0@**e aagttgttag agaaaatgct ctaaaaatat atggacatag catttcaatc tcttctcaag tattactgaa aagttatttt attaagaaaa t tt ttcatgt tagtctggtt tatggtctgg tgtctatatt tttttttttt caagtgtcta aactggtaaa caaattatga aaaaagatat attatcgtac cctgctggac taaatcggcg aagatatgta tgggcaagag ttaagacaaa gagtgtatgt gtttagaaac gaaagaagag atatgcataa atacacactc attcactagt attagggcat taagaacaga tttatttttt aaaaattccg taaagtgttt aagtctgggt ttcagtctag taagttaatc tttttttttg gaagtagtgc agattataga gaaaaataac tccttggata agatggagaa aaatctccga gttgaaatca attattccgc actttgtgtg aggttgatga gttcttctct ttgatcggta aaccaggttt ctaataagca ttatgcttgc tagcactttc tttggttgtt aatttgataa at cgag tat a gttgttggac aattaggttt ttggtagaga tttagttcaa accttttaac tgatggttag tacttttaca tactgcttcc cttctgtagt gaagacaaaa taatcatcat caatcttccg aaatccaaaa tgataagggc tgatacttaa actttgtacc tctgtcaaaa attgctgaac tggctagcca ctgcgtgttg agatatatat ctggtcttgt ggtagtttgg cggctggtta aattacatga ataactaggt tggttcattt ttcaataaga tattggtttc caaaaaaaaa gtttggatcc tgctatatat aaacttaaaa gttttttaat agagaaagtg gtgggagcag gtgactctcc catatataat gagcgtttgt gtagacggaa ttattcgtgt acttgtgttt attttgatct ggtaacgaaa tgtgtccaat atatatatat atagttgtat tttattaggg gagttaaggg ttgctatatc tttggttctt ggagatttag tttcttgatt cttgaaggtt agtttatgga gagtggctca aggttagatt gcttaaacat ggttgttatt aatagtgatt aagctgagat cactacttaa tatgaagaag acatcttaat ctaagtcaat gagaattgca gcttcacagt ttaaatcagg gctacatttc gtccatgtac atagtcagtg tctagacaat ttagtgagat aaatcagatg attttactaa cttcttcgtt gaacctttta ctcttcaggt gtgtaaacgt ccgattagtt gttccaactc ataaattata aaagaacaca tggtggtgtg ttgacctatg ggagatgtca ttagccacct aaaaaaaaat actctctctt actatctgtt tcgagatctt gaagaagcct 6480 6540 6600 6660 6720 6780 6840 6900 6960 7020 7080 7140 7200 7260 7320 7380 7440 7500 7560 7620 7680 7740 7800 7860 7920 7968 0* 9O acggcttatt ttgcaacagg gacgtggctc tctctctctc tctgcgcg <210> 2 <211> 943 <212> DNA <213> Arabidopsis thaliana <400> 2 cgagaaaagg aaaaaaaaaa atagaaagag aaaacgctta gtatctecgg cgacttgaac 6ccaaacctga ggatcaaatt agggcacaaa gccctctcgg agagaagcca. tgggaagaaa aaaactagaa tcgcaacggt tcttctcgtc caagatcctt gtcaaaagct tgtgggatca tgagactgcc gaatcttaaa ggagaataat ctccgacaat aaatcaaaat ccgctgataa tgtgtgatac atcaagcgaa ctcatcgaga gtctccgcct gatcgatatg ctgaactatg aatgtcaaaa ctctccgtga gaaaaggaga catcatgtgg cttccggtga.

ccaaaacata gggcgagcgt ttaagtagac ttgagaacaa aagctcgtca ccggcaagct ggaaacagca gttcacacta atgtgagtat ctagagccaa aaatgctgaa gagcagaagc ctctcccact tataattatg ttgtatatct ggaactaagt aagtagccga gctttctgtt ctacagcttc tgctgatgat tgagctactt cgatgctctt gaagaccgaa agaagagaac tgagatggag acttaattag aagaaaaaaa taatactctc caatactatc caagtcacct ctctgtgacg tcCtccggcg cttaaagcct gaacttgtgg gttcaactgg ctcatgttga caggttttgg atgtcacctg ccaccttaaa aaataagata tctttggcca tgttttaaga tctccaaacg catccgtcgc ataacctggt tggatcatca.

atagcaagct aggaacacct agcttgttga ctagccagat ctggacaaat tcggcggttg tgtaattatt agagactttg caaaaggttg atgaactttg taccttattc gtgtgagaaa aaaaaaaaaa aaa <210> 3 <211> 196 <212> PRT <213> Arabidopsis thaliana <400> 3 Met Gly Arg 1 Arg Gin Val Arg Gin Leu Ser Ala Ser Lys Lys 5 Thr Phe Leu Glu Ile Lys Ile Giu Asn Lys Ser Ser Ser Lys Arg Gly Leu Ile Val Leu Cys Ser Val Ala Leu Asp Giu Lys Ala Leu Val Val Asn Leu Val Giy Lys Leu Phe Ser Ser Lys Ile Leu Asp Arg Lys Gin His Asp Leu Lys Asp His Gin Lys Ala Leu Asn Ser His Tyr Giu Leu Asn Val Leu Glu Leu Leu Giu LeuSer Lys Leu GySeAs Gly Ser Asn Vai Lys 110 Ser Ilie Asp Ala Leu Val Gin Leu Giu 115 120 Ser Val Thr Arg Ala Lys Lys Thr Giu 130 135 Asn Leu Lys Giu Lys Giu Lys Met Leu 145 150 Ala Ser Gin Met Giu Asn Asn His His 165 Giu Met Ser Pro Ala Gly Gin Ile Ser 180 185 Pro Leu Leu Asn 195 <210> 4 <211> 691 <212> DNA <213> Brassica napus -7 Giu His Leu Met Lys Giu 155 Val Gly 170 Asp Asn Leu Giu Thr Ala Leu 125 Leu Lys Leu Val Giu 140 Giu Asn Gin Val Leu 160 Ala Giu Ala Giu Met 175 Leu Pro Val Thr Leu 190 <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> Unsure (611)..(611) Unknown Unsure (655)..(655) Unknown Unsure (657)..(657) Unknown Unsure (691) (691) Unknown 8- <400> 4 ggatcctatg taacggctct gttcacacaa ctctcctcct gcaagcttgt gtgtccttga tcttctcatt gacagaacta tgattttata tattaagttt tggtctttca aaaacttgca tgaggttata tatatccctt tgagttactt ttcattacaa ggaatcaaat aaatgccctc tcttactttg atgttgaagc gcacttcaga ctctaagtgt nggatgatta actgatgaac gatccacgtg ctccgtggac gaacttgtgg aggtattagg gtcggtggtg tctctaacta tttgttgaaa ttgttgatag tatcttctcg gctttatgag aaaatattgt atgtccttca catatcaaat agaatcttca ataggttagt gtttcttgtc taagcgtgga gagctaggaa cgattgttca cctcaaagaa tgtttgaaag ctcgcaatca tttggatacc n ttctcaaatt gtcaaaagct actacctgag aatctgtgca caccctcgtt ggtacgt tga cttatattta aaggttagat cctcaaatat aacttcttca agaatctgaa cgttgccttc ctgagctatg acttttttcc tatatatgca cagctggagg cttcatactg atttgttgca atatcatata ttatgtgttg taagtgcatc aa tangnt ta 120 180 240 300 360 420 480 540 600 660 691 <210> <211> 68 <212> PRT <213> Brassica napus <400> Asn Leu Gin Ser Lys Ala Leu Ser Tyr Gly Ser 10 Lys Leu Val Glu Ser Asn Glu Leu Val Asp Ser His Asn Glu Leu Leu Val Gly Gly Val Ser Glu Asn Ala Leu Ser Val Asp Thr Leu Leu Thr Arg Ala Leu Lys Glu Lys Val Gin Leu Glu Gly Val Leu Arg Lys Thr Glu Leu Met Leu Lys Leu Val Asp Ser 55 <210> 6 <211> 908 <212> DNA <213> Brassica napus 9- <400> 6 aaaagaaaga aaacgctcag acaaagggtt agaacaaaag ctcgtcagct gcaagctata aacaacatgc cacaccatga gcgtcgattc ctaggaagac tgaaagaaga aagctgataa tcccactgct ccgaactatt ccctgtgtgt aaaaaaaa aataaaagca tatctccggc ctcggagaca tagccgacaa ttcagttctc caacttctcc tgatgatcgt gctactagag cctcgttcag agaactat tg gaaccagggt aatggaagtg ttattagccc cccctataag aaaaactacg aaaaagagag gagagctgaa gaagccatgg gtcaccttct tgcgatgcat gccggcgatg aaagctctgg cttgtcgaaa ctggagaacc ttgaagcttg ttggctagcc tcacctggac cttaagtcca ggtgagcgtt gtttgatttg aaaataaaag ccgaaccgaa gaagaaagaa ccaaacgacg ccgtcgctct acctggtcaa atcttcagtc gtaagcttgt accttgagac ttgatagcct agatggagaa agatctctga aaacttgtga gtatatcttc agtaaaaata caaaaataag cctcaggatc actagagatc caatggtctc cctcgttgtc gatcgttgat agaagctccg ggaatcaaat tgccctctcc caaagaaaag gaataatctt catcaattgt ctaaaaacaa acactctctt tatatttaag aaagaacaaa aaattagggc aagcgaat tg atcgagaaag tcagcctctg cgatatggaa aagtatggtt tctgatgtaa gtaactagag gagaaattgc gcgggagccg c cgg taact c aaataagtta ggctgagaga aaaaaaaaaa <210> 7 <211> 847 <212> DNA <213> Brassica napus <220> <221> <222> <223> Unsure (742)..(742) Unknown <400> 7 ggcgacttta accgtacctc agaatcaaat tagggcacag agacctctcg gagacagaag ctatgggaag aaaaaaacta gaaatcaagc gaatcgagaa aaacagtagc agacaagtca ccttctgcaa acgacgcaac ggtctcatcg agaaagctcg tcagctttct gttctctgcg aggcatctgt tgggcttctc gttgtctccg cctccgacaa actctacagc ttctcctccg gggatagact ccctggatct tggaaagcaa aagatcacct agcttgttga ctagtcagat ctggacaaat tactcggcgg cttttcgcag ttaacaaata aaanaaa ggagaagatc tcagtcaaaa gcttgtggaa tgagactgcc aagtctcaaa tgagaagaaa ctccgacatc ttaatcaaaa cttaattaac acctatgtca cttgatcgat tctctgaact tcaattgatg ctctctgtaa gaaaaggaga aatcttgagg aatcttcctg taagaaacat cncctttact gtttaacccc 10 a tgggaaaaa atagttcaca atgtaagcgt c tag agc tcg atctgctgaa gagccgaagc taactctccc ataatctaaa cggcggttaa aaaaaaaaaa acatgctgat ccatgagcta ggattccctC gaaggcagaa agaagagaac tgataatata gctgcttaat gataacctat tcgaaattaa aaaaaaaaaa gatctcaatg ctagaacttg gttgagctag ctaatgttaa caggttttgg gagatgtcat taaccacctt gtaggtttta aaaca tat aa gaaaaaaaaa <210> 8 <211> 969 <212> DNA <213> Brassica napus <400> 8 ctctggatca ctagaaatca agcggtctca ctcgttgtct atccttgatc aaagctctga gaatcaaatg aatgccctct ctcaaagaaa aagaagaatc gacatcaatc caaattaaaa aagggtgcac tgttagatat ttgtagtgag aattagggca agcgaat tga ttgagaaagc cctcctccgg gatatggaaa gctatggttc tcggtggtgt ctctaactag aggagaagct ttgcgggagc ttccggtaac aatccaaaac gttgtatatc aatgagtaaa tgtgcttgtg cagagaccac gaacaaaagt tcgtcagctt caagctctac acagcatgct acacaatgag aagcgtggac agctaggaag gctgaaagaa cgaagctgat tctcccactg atacaactaa tcaatactcc ataaaaataa tgttcttttt ttggagacag agccgacaag tctgttctct agcttctccg gatgatctta ttacttgaac accctcgttc acagaactaa gagaatcagg aatatggaga cttaattagc ataaataagc cttggctgag aaagat ctt t ctcttctttg aaaccatggg tcaccttctc gcgatgcatc ccggtgataa aagccctgaa ttgtggatag agctggaggg tgttgaagct ctttggctgg tgtcacctgg caccgttaga tttgttgttt agattgtgtg gtaccttcgt cttcatggcg aagaaaaaaa caaacgacgc cgtcgcgctt cctggtcagg tcttcagtca caagcttgtg tgtccttgaa tgttgatagc ccagaaggag acaaatctcc cggggctgat ttcacccttg tttactccta cgagagagaa aagaagccta ccgtctaatt tgtaacggag acgtggccct ctctgccctt ttgtattcgt aattcctttg 11 tattaaaaa <210> 9 <211> 868 <212> DNA <213> Brassica napus <400> 9 aacgctcagt actgttggag aagtagccga gctttccgtt ctacagcttc tgatgatgat tgagctactg gggttccctg gaagacagaa agaggagaac tgataatatg actgcttaat caaacaagat ctccggctcg atgcaagaca atctccggct acagaagcca caagttacct ctctgtgacg tcctccggtg cttaaagcct gaacttgtgg gttcagctgg ctaatgttga catgttttgg gatgtctcac tagtcacctt gtgtaattat agaggcttcg ctttcaaact agtggaaacc tggggaggaa tctctaaacg catccgtcgc ataacctggt tggatcgtca aaagcaagct aggaacacct agcttgtcga ctagccagat caggacaaat taatcggcga ccccttgtaa ggtgtaaaac taaaaaaa ggacctcaag gaaacttgaa acgcaacggt tcttcttgtc caagatcctt gtcaaaagct tgaggaatca tgagaacgcc gaaccttaaa ggagaagagt ctccgacatc ataaataaaa agggtgtacg tatttcagat atcaaattag atcaagcgaa ctcatcgaga gtctccgcct gatcgatatg ttggactgtg aatgtcgata ctctccgtaa gaaaaggaga aatcttgtgc aatcttccgg tccaaaacat ttgtataatc ttatgtaaga ggcgcaaagc t tgagaacaa aagc tcg tca ccgggaaact gaaagcaaca gttcacacca atgtaagtgt caagagctag agttgctgga gagccgaagc taacgctccc ataactaaaa tatactctct tagaaaatct 09 <210> <211> <212> <213> 792

DNA

Brassica napus <220> <221> Unsure <222> (619)..(619) <223> Unknown 12 <400> caaaggcttc gaacaaaagt tcgtcagctt caagctttac acaacatgct acaccatgag cgtcgactcc taggaagaca gaaagaagag agctgataaa ccgactgctt tcgaactatt cccgtgtgta aaaaaaaaaa tcggagacag agccgacaag tcagttctct aacttctccg gatgatctta ctactagagc ctcgttcagc gaactaatgt aaccagggt t atggagatgt tattagccnc cccctataag aaactatggt aa aagccatggg tcaccttctc gcgatgcatc ccggcgataa aagctctgga ttgtcgaaag tggaggacca tgaagcttgt tggctagcca cacctggaca cttaagtcca ggtgaacgtt tagatttaag aagaaagaaa caaacgacgc cgtcgctctt cctggtcaag tcttcagtca taagcttgtg cct tgagact tgatagcctc gatggagaag aatctctgac aaacttgtga gtatatcttc taaaaatata ctagagatca aatggtctca ctcgttgtct atccttgatc aaagctccga gaatcaaatt gccctctccg aaagaaaagg aataatcttg atcaatcgtc ctaaaaacaa attctctctg tatttaagac agcgaattga tcgagaaagc cagcctccgg gatatggaaa agtatggttc ctgatgtaag taactagagc agaaattgct cgggagccga cggtaactct aaataagtta gctgagagac atactaaaaa 120 180 240 300 360 420 480 540 600 660 720 780 792 <210> 11 <211> 990 <212> DNA <213> Brassica napus <400> 11 gggcacagag attgagaaca aaagctcgtc tccggcaagc ggaaaacagc ggttcacaca ggtgtaagcg act ag agc ta aagctgctga ggagccgaag gtaactctcc accacttgga aaagtagccg agctttctgt tctacagctt atgctgatga atgagttact tggacaccct ggaagacaga aagaagagaa ctgataatat cactgcttaa gacagaaacc acaagtcacc tctctgcgat ctccgccggt tcttaaagcc tgaacttgtg cgttcagctg actaatgttg tcaggctttg ggagatgtca t tagccaccg atgggaagaa ttctccaaac gcatccgtcg gataacctgg ctgaatcttc gatagcaagc gagggtgtcc aagcttgttg gctggccaga cctggacaaa ttagacgggg aaaaactaga gacgcagcgg cgcttctcgt tcaggatcct agtcaaaagc ttgtggaatc ttgaaaatgc atagcctcaa aggagaagaa tctccgacat ctgatcaaat aatcaagcga tctcattgag tgtctcctcc tgatcgatat tctgagctat aaatgtcggt cctctctcta agaaaaggag gaatcttgcg caatcttccg taaaaaatcc 13 aaaacataca actaaataaa taagctttgt tgtttttcac ccttgaaggg tgcacgttgt atatctcaat actcccttgg ctgagagatt gtgtgtttac tcctatgtta gatataatga gtaaaataaa aataaaaaga tctttgtacc ttcgtcgaga gagaattgta gtgagtgtgc ttgtgtgttc tttttctctt ctttgcttca tggcgaagaa gcctaccgtc taatttgtaa cggagacgtg gccctctctg cccttttgta ttcgtaattc ctttgtattt atccacaacg catagaggtt gtcatggttt aaaaaaaaaa <210> <211> <212> <213> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> 12 780

DNA

Brassica napus Unsure (748) (748) Unknown Unsure (769) (769) Unknown Unsure (779)..(779) Unknown <400> 12 ttagggcaca aaggcttctc cgaattgaga acaaaagtag gagaaagctc gtcagctttc gcctccggca agctttacaa tatggaaaac aacatgctga tatggttcac accatgagct ggagacagaa gccatgggaa gaaagaaact agagatcaag ccgacaagtc accttctcca aacgacgcaa tggtctcatc agttctctgc gatgcatccg tcgctcttct cgttgtctca cttctccgcc ggcgataacc tggtcaagat ccttgatcga tgatcttaaa gctctggatc ttcagtcaaa agctccgaag actagagctt gtcgaaagta agcttgtgga atcaaattct 14 gatgtaagcg actagagcta aaattgctga ggagccgaag gtaactctcc ataagttatc tgagagaccc tcgactccct ggaagacaga aagaagagaa ctgataaaat gactgcttta gaactattcc ccgtgtgtaa cgttcagctg actaatgttg ccagggtttg ggagatgtca ttagccacct cctataaggg actatggnta gaggaccacc aagcttgttg gctagccaga cctggacaaa taagtccaaa tgaacgttgt gatttaagta t tgagactgc atagcctcaa tggagaagaa tctctgacat acttgtgact atatcttcat aaatatatnt cctctccgta agaaaaggag taatcttgcg caatcgtccg aaaaacaaaa tctctctggc ttaagacana <210> 13 <211> 845 <212> DNA <213> Brassica napus

S

S S 0 0* S S S S

S.

S

S..

S. S

S*

5505

S

5.55 SS S 0* .5 5055 55

S

S.

0* S

S.

55 <400> 13 ctccggctag agaagccatg agttaccttc ctgtgacgca ctccggtgat taaagccttg acttgtggaa tcagctggag aatgttgaag tgttttggct tgtctcacca gtcaccttta gtaattatcc aggcttcggg t tcaa tggaaaccgg gggaggaaga tctaaacgac tccgtcgctc aacctggtca gatcgtcagt agcaagcttg gaacaccttg cttgtcgaga agccagatgg ggacaaatct atcggcgaat ccttgtaaag tgtaaaacta acctcaagat aacttgaaat gcaacggtct ttcttgtcgt agatccttga caaaagcttt aggaatcaaa agaacgccct accttaaaga agaagagtaa ccgacatcaa aaataaaatc ggtgtacgtt tttcagattt caaattaggg caagcgaatt catcgagaaa ctccgcctcc tcgatatgga ggactgtggt tgtcgataat ctccgtaaca aaaggagaag tcttgtgcga tcttccggta caaaacatat gtataatcta atgtaagata cgcaaagcac gagaacaaaa gctcgtcagc gggaaactct aagcaacatg tcacaccatg gtaagtgtgg agagctagga ttgctggaag gccgaagctg acqctcccac aactaaaaca tactctctct gaaaatctat tgttggagac gtagccgaca tttccgttct acagcttctc atgatgatct agctactgga gttccctggt agacagaact aggagaacca ataatatgga tgcttaatta aacaagatgt ccggctcgag gcaagacact <210> <211> <212> 15 <213> Brassica napus <400> 14 cggcgagagt gaagccatgg gtcaccttct tgcgatgcat gccggcgata aaagctctgg cttgtcgaaa c tggaggacc ttgaagcttg ttggctagcc tcacctggac ccttaagtcc gggtgaacgt ttagatttaa tgaaaccgaa gaagaaagaa ccaaacgacg CCgtcgctct acctggtcaa atcttcagtc gtaagcttgt accttgagac ttgatagcct agatggagaa aaatctctga aaaacttgtg tgtatatctt gtaaaaatat tctcaggatc aaattagggc acaaaggctt ctcggagaca act ag agat c caatggtctc tctcgttgtc gatccttgat aaaagctccg ggaatcaaat tgccctctcc caaagaaaag gaataatctt catcaatcgt actaaaaaca cattctctct atatttaaga aagcgaattg atcgagaaag tcagcctccg cgatatggaa aagtatggtt tctgatgtaa gtaactagag gagaaat tgc gcgggagccg ccggtaactc aaaataagtt ggctgagaga catactaaaa agaacaaaag ctcgtcagct gcaagcttta aacaacatgc cacaccatga gcgtcgactc ctaggaagac tgaaagaaga aagctgataa tccgactgct atcgaactat ccccgtgtgt aaaaa tagccgacaa ttcagttctc caacttctcc tgatgatctt gctactagag cctcgttcag agaactaatg gaaccagggt aatggagatg ttattagcca tcccctataa aaaactatgg <210> <211> 891 <212> DNA <213> Brassica napus <400> tccggctagt gaagccatgg gttaccttct tgtgacgcat tccggtgata aaagcct tgg cttgtggaaa cagctggagg atgt tgaagc gttttggcta ggaaaccgga ggaggaagaa ctaaacgacg ccgtcgctct acctggtcaa atcgtcagtc gcaagcttga aacaccttga ttgtcgagaa gccagatgga cctcaagatc acttgaaatc caacggtctc tcttgtcgtc gatccttgat aaaagctttg ggaatcaaat gaacgccctc ccttaaagaa gaagagtaat aaat tagggc aagcgaattg atcgagaaag tccgcctccg cgatatggaa gactgtggtt gtcgataatg tccgtaacaa aaggagaagt cttgtgcgag gcaaagcact agaacaaaag ctcgtcagct ggaaactcta agcaacatga cacaccatga taagtgtggg gagctaggaa tgctggaaga ccgaagctga gt tggagaca tagccgacaa ttccgttctc cagcttctcc tgatgatctt gctactggaa ttccctggtt gacagaacta ggagaaccat taatatggat 16 gtctcaccag gacaaatctc cgacatcaat cttccggtaa cgctcccact gcttaattag 660 tcacctttaa tcggcgaata aataaaatcc aaaacatata actaaaacaa acaagatgtg 720 taattatccc cttgtaaagg gtgtacgttg tataatctat actctctctc cggctcgaga 780 ggcttcgggt gtaaaactat ttcagattta tgtaagatag aaaatctatg caagacactt 840 tcaaactttg taccttgctt tgtcgacaga gaattacttc gagctaaaaa a 891 <210> 16 <211> 196 <212> PRT <213> Brassica napus <400> 16 Met Gly Arg Lys Lys Leu Glu Ile Lys Arg Ile Glu Asn Lys Ser Ser .1 5 10 Arg Gin Val Thr Phe Ser Lys Arg Arg Asn Gly Leu Ile Giu Lys Ala *20 25 *Arg Gin Leu Ser Val Leu Cys Asp Ala Ser Val Ala Leu Leu Val Val *35 40 *Ser Ala Ser Gly Lys Leu Tyr Asn Phe Ser Ala Gly Asp Asp Leu Val 55 Lys Ile Val Asp Arg Tyr Gly Lys Gin His Ala Asp Asp Arg Lys Ala *:65 70 75 Leu Asp Leu Gin Ser Giu Ala Pro Lys Tyr Giy Ser His His Glu Leu 90 *Leu Giu Leu Val Giu Ser Lys Leu Val Giu Ser Asn Ser Asp Val Ser *100 105 110 *Val Asp Ser Leu Val Gin Leu Glu Asn His Leu Glu Thr Ala Leu Ser 115 120 125 Val Thr Arg Ala Arg Lys Thr Glu Leu Leu Leu Lys Leu Val Asp Ser 130 135 140 Leu Lys Giu Lys Giu Lys Leu Leu Lys Giu Glu Asn Gln Gly Leu Aia 145 150 155 160 Ser Gin Met Giu Lys Asn Asn Leu Aia Gly Ala Glu Ala Asp Lys Met 165 170 175 Glu Val Ser Pro Gly Gln Ile Ser Asp Ilie Asn Cys Pro Val Thr Leu 180 185 190 Pro Leu Leu Tyr 195 <210> 17 17 <211> 196 <212> PRT <213> Brassica napus <400> 17 Met Gly Arg Lys Lys Leu Giu Ile Lys Arg Ile Giu Lys Asn Ser Ser 1 5 10 Arg Gin Val Thr Phe Cys Lys Arg Arg Asn Gly Leu Ile Glu Lys Ala 25 Arg Gin Leu Ser Val Leu Cys Giu Ala Ser Val Gly Leu Leu Val Vai 40 Ser Ala Ser Asp Lys Leu Tyr Ser Phe Ser Ser Gly Asp Arg Leu Giu 55 Lys Ile Leu Asp Arg Tyr Gly Lys Lys His Ala Asp Asp Leu Asn Ala 70 75 *Leu Asp Leu Gin Ser Lys Ser Leu Asn Tyr Ser Ser His His Giu Leu **85 90 .*Leu Giu Leu Val Giu Ser Lys Leu Val Glu Ser Ile Asp Asp Val Ser ::100 105 110 Val Asp Ser Leu Val Giu Leu Giu Asp His Leu Giu Thr Ala Leu Ser *115 120 125 Val Thr Arg Ala Arg Lys Ala Giu Leu Met Leu Lys Leu Val Giu Ser 130 135 140 Leu Lys Giu Lys Glu Asn Leu Leu Lys Glu Glu Asn Gin Val Leu Ala 145 150 155 160 :Ser Gin Ile Giu Lys Lys Asn Leu Giu Gly Ala Giu Ala Asp Asn Ile *165 170 175 Giu Met Ser Ser Gly Gin Ile Ser Asp Ile Asn Leu Pro Val Thr Leu 180 185 190 *Pro Leu Leu Asn 195 <210> 18 <211> 197 <212> PRT <2i3> Brassica napus <400> i8 Met Gly Arg Lys Lys Leu Giu Ile Lys Arg Ile Glu Asn Lys Ser Ser 1 5 10 Arg Gin Val Thr Phe Ser Lys Arg Arg Ser Gly Leu Ile Giu Lys Ala 18 25 Arg Gin Leu Ser Vai Leu Cys Asp Ala Ser Vai Ala Leu Leu Val Val 40 Ser Ser Ser Gly Lys Leu Tyr Ser Phe Ser Ala Gly Asp Asn Leu Val 55 Arg Ilie Leu Asp Arg Tyr Giy Lys Gin His Ala Asp Asp Leu Lys Ala 70 75 Leu Asn Leu Gin Ser Lys Ala Leu Ser Tyr Gly Ser His Asn Giu Leu 90 Leu Giu Leu Val Asp Ser Lys Leu Val Glu Ser Asn Val Gly Gly Val 100 105 110 Ser Vai Asp Thr Leu Val Gin Leu Giu Gly Val Leu Giu Asn Ala Leu 115 120 125 Ser Leu Thr Arg Ala Arg Lys Thr Glu Leu Met Leu Lys Leu Val Asp 130 135 140 Ser Leu Lys Giu Lys Glu Lys Leu Leu Lys Glu Giu Asn Gin Ala Leu *145 150 155 160 Ala Gly Gin Lys Giu Lys Lys Asn Leu Ala Gly Ala Giu Ala Asp Asn *165 170 175 Met Giu Met Ser Pro Gly Gin Ile Ser Asp Ile Asn Leu Pro Val Thr 180 185 190 *Leu Pro Leu Leu Asn 195 <210> 19 <211> 197 <212> PRT <213> Brassica napus *<400> 19 Met Gly Arg Lys Lys Leu Giu Ile Lys Arg Ilie Glu Asn Lys Ser Ser 1 5 10 Arg Gin Val Thr Phe Ser Lys Arg Arg Asn Gly Leu Ile Giu Lys Ala 25 Arg Gin Leu Ser Val Leu Cys Asp Ala Ser Val Ala Leu Leu Val Val 40 Ser Ala Ser Gly Lys Leu Tyr Ser Phe Ser Ser Gly Asp Asn Leu Val 55 Lys Ile Leu Asp Arg Tyr Gly Lys Gin His Asp Asp Asp Leu Lys Ala 70 75 Leu Asp Arg Gin Ser Lys Ala Leu Asp Cys Gly Ser His His Glu Leu 90 Leu Glu Leu Val Giu S 100 Ser Val Gly Ser Leu V 115 Ser Val Thr Arg Ala A 130 Asn Leu Lys Glu Lys G 145 1 Ala Ser Gin Met Glu L 165 Met Asp Val Ser Pro G 180 Leu Pro Leu Leu Asn 195 <210> <211> 196 <212> PRT <213> Brassica napus 19 Glu Ser Glu His Leu Met Glu Glu 155 Val Arg 170 Asp Ile <400> Met Gly Arg Lys Lys Leu Giu Ile Lys Arg Ile Glu Asn Lys Ser Ser 1 5 10 Arg Arg Ser Lys 65 Leu Leu Val1 Val1 Leu 145 Thr Phe Ser 20 Ser Val Leu Gly Lys Leu Asp Arg Tyr 70 Gin Ser Lys Val Glu Ser 100 Leu Val Gin Ala Arg Lys Lys Glu Lys 150 Gly Leu Ile Glu Lys Ala Val Ala Leu Leu Val Val Ala Gly Asp Asn Leu Val Ala Asp Asp Leu Lys Ala 75 Gly Ser His His Glu Leu Ser Asn Ser Asp Val Ser 110 Leu Glu Thr Ala Leu Ser 125 Leu Lys Leu Val Asp Ser 140 Glu Asn Gin Gly Leu Ala 155 160 Ala Glu Ala Asp Lys Met 175 Ser Gin Met Glu Lys Asn Asn Leu Ala Gly 165 170 20 Giu Met Ser Pro Gly Gin Ile Ser Asp Ile Asn Arg Pro Val Thr Leu 180 185 190 Arg Leu Leu Tyr 195 <210> 21 <211> 197 <212> PRT <213> Brassica napus <400> 21 Met Gly Arg Lys Lys Leu Giu Ile Lys Arg Ile Giu Asn Lys Ser Ser 1 5 10 Arg Gin Val Thr Phe Ser Lys Arg Arg Ser Gly Leu Ile Giu Lys Ala 25 *Arg Gin Leu Ser Val Leu Cys Asp Ala Ser Val Ala Leu Leu Vai Val 40 .*Ser Ser Ser Gly Lys Leu Tyr Ser Phe Ser Ala Gly Asp Asn Leu Vai 55 Arg Ile Leu Asp Arg Tyr Gly Lys Gin H-is Ala Asp Asp Leu Lys Ala 70 75 Leu Asn Leu Gin Ser Lys Ala Leu Ser Tyr Gly Ser His Asn Giu Leu 90 Leu Giu Leu Val Asp Ser Lys Leu Vai Giu Ser Asn Vai Gly Giy Val 100 105 110 ***Ser Val Asp Thr Leu Val Gin Leu Giu Gly Val Leu Giu Asn Ala Leu 115 120 125 Ser Leu Thr Arg Ala Arg Lys Thr Giu Leu Met Leu Lys Leu Val Asp 130 135 140 Ser Leu Lys Giu Lys Giu Lys Leu Leu Lys Giu Giu Asn Gin Ala Leu 145 150 155 160 Ala Giy Gin Lys Giu Lys Lys Asn Leu Ala Gly Ala Giu Aia Asp Asn 165 170 175 Met Giu Met Ser Pro Gly Gin Ilie Ser Asp Ilie Asn Leu Pro Vai Thr 180 185 190 Leu Pro Leu Leu Asn 195 <210> 22 <211> 196 <212> PRT 21 <213> Brassica napus 4 <400> 22 Met Gly Arg Lys 1 Arg Gin Val Thr Arg Gin Leu Ser Ser Ala Ser Gly Lys Ile Leu Asp Leu Asp Leu Gin Leu Glu Leu Val 100 Val Asp Ser Leu 115 Val Thr Arg Ala 130 Leu Lys Giu Lys 145 Ser Gin Met Giu Giu Met Ser Pro 180 Arg Leu Leu Tyr 195 <210> 23 <211> 197 Lys Arg Al a Phe Gin Lys Val 105 Asp Leu Lys Ala Asp 185 Ser Lys Val Leu Lys Glu Val Leu Asp Leu Lys 175 0e Gly Gin Ile Ser Ile Asn Arg Pro Val Thr Leu 190 <212> PRT <213> Brassica napus <400> 23 Met Gly Arg Lys Lys Leu Glu Ile Lys Arg Ile Glu Asn Lys Ser Ser 1 5 10 Arg Gin Val Thr Phe Ser Lys Arg Arg Asn Gly Leu Ile Glu Lys Ala 25 Arg Gin Leu Ser Val Leu Cys Asp Ala Ser Val Ala Leu Leu Val Val 40 22 Ser Ala Ser Gly Lys Leu Tyr Ser Phe Ser Ser Gly Asp Asn Leu Val 55 Lys Ilie Leu Asp Arg Tyr Gly Lys Gin His Asp Asp Asp Leu Lys Ala 70 75 Leu Asp Arg Gin Ser Lys Ala Leu Asp Cys Gly Ser His His Giu Leu 90 Leu Giu Leu Val Giu Ser Lys Leu Giu Giu Ser Asn Val Asp Asn Val 100 105 110 Ser Val Giy Ser Leu Val Gin Leu Giu Giu His Leu Giu Asn Aia Leu 115 120 125 Ser Val Thr Arg Ala Arg Lys Thr Giu Leu Met Leu Lys Leu Val Giu 130 135 140 Asn Leu Lys Giu Lys Giu Lys Leu Leu Giu Giu Giu Asn His Val Leu 145 150 155 160 Ala Ser Gin Met Giu Lys Ser Asn Leu Val Arg Ala Giu Ala Asp Asn 165 170 175 *Met Asp Val Ser Pro Gly Gin Ile Ser Asp Ile Asn Leu Pro Val Thr 180 185 190 *Leu Pro Leu Leu Asn 195 <210> 24 <211> 196 <212> PRT <213> Brassica napus <400> 24 Met Gly Arg Lys Lys Leu Giu Ile Lys Arg Ilie Glu Asn Lys Ser Ser 00*1 5 10 sO.Arg Gin Val Thr Phe Ser Lys Arg Arg Asn Gly Leu Ile Glu Lys Ala 25 Arg Gin Leu Ser Val Leu Cys Asp Ala Ser Val Ala Leu Leu Val Val 40 Ser Ala Ser Gly Lys Leu Tyr Asn Phe Ser Ala Gly Asp Asn Leu Val 55 Lys Ilie Leu Asp Arg Tyr Gly Lys Gin His Ala Asp Asp Leu Lys Ala 70 75 Leu Asp Leu Gin Ser Lys Ala Pro Lys Tyr Gly Ser His His Giu Leu 90 Leu Giu Leu Val Giu Ser Lys Leu Val Giu Ser Asn Ser Asp Val Ser 100 105 110 Val Asp Ser Leu Val Gin Leu Giu Asp His Leu Glu Thr Ala Leu Ser 23 115 120 125 Val Thr Arg Ala Arg Lys Thr Glu Leu Met Leu Lys Leu Val Asp Ser 130 135 140 Leu Lys Glu Lys Glu Lys Leu Leu Lys Giu Giu Asn Gin Gly Leu Ala 145 150 155 160 Ser Gin Met Giu Lys Asn Asn Leu Ala Gly Ala Giu Ala Asp Lys Met 165 170 175 Giu Met Ser Pro Gly Gin Ilie Ser Asp Ile Asn Arg Pro Val Thr Leu 180 185 190 Arg Leu Leu Tyr 195 <210> <211> 197 <212> PRT <213> Brassica napus <400> Met Gly Ai 1 Arg Gin Vi Arg Gin L( Ser Ala S( Lys Ilie LE 65 Leu Asp Ai Leu Glu LE Ser Val GI Ser Val T1 130 Asn Leu L 145 Ala Ser G3 Ile Gly Val Ser Asp 75 Gly Ser His Met Giu 155 Arg Asn Ile Leu Asp Asp His Val1 Giu 125 Lys Asn Glu Ser Ser Lys Ala Val Val Leu Val Lys Ala Giu Leu Asn Val Ala Leu Val Glu Val Leu 160 Asp Asn 175 met Asp Val Ser Pro Gly Gin Ile Ser Asp Ilie Asn Leu Pro Val Thr 180 185 190 24 Leu Pro Leu Leu Asn 195 <210> 26 <211> 196 <212> PRT <213> Arabidopsis thaliana <400> 26 Met Gly Arg Arg Lys Ile Giu Ile Lys Arg Ile Giu Asn Lys Ser Ser 1 5 10 Arg Gin Val Thr Phe Ser Lys Arg Arg Asn Gly Leu Ile Asp Lys Ala 25 Arg Gin Leu Ser Ilie Leu Cys Giu Ser Ser Val Ala Val Val Val Val 40 Ser Ala Ser Gly Lys Leu Tyr Asp Ser Ser Ser Gly Asp Asp Ile Ser 55 Lys Ile Ile Asp Arg Tyr Giu Ile Gin His Ala Asp Giu Leu Arg Aia 70 75 Leu Asp Leu Glu Giu Lys Ile Gin Asn Tyr Leu Pro His Lys Giu Leu 90 Leu Giu Thr Val Gin Ser Lys Leu Giu Giu Pro Asn Val Asp Asn Val 100 105 110 Ser Val Asp Ser Leu Ilie Ser Leu Giu Giu Gin Leu Glu Thr Ala Leu 115 u 120 125 Ser Val Ser Arg Ala Arg Lys Ala Giu Leu Met Met Giu Tyr Ile Giu 130 135 140 Ser Leu Lys Giu Lys Giu Lys Leu Leu Arg Glu Giu Asn Gin Val Leu 145 150 155 160 Ala Ser Gin Met Gly Lys Asn Thr Leu Leu Ala Thr Asp Asp Giu Arg *165 170 175 Gly Met Phe Pro Gly Ser Ser Ser Gly Asn Lys Ilie Pro Giu Thr Leu 180 185 190 Pro Leu Leu Asn 195 <210> 27 <211> 196 <212> PRT <213> Arabidopsis thaliana <400> 27 25 Met Gly Arg 1 Arg Gin Val Arg Gin Leu Ser Gly Ser Lys Ilie Ile Leu Asp Leu Leu Giu Ile Ser Vai Asp 115 Ser Val Thr 130 Ser Leu Gin 145 Aia Ser Gin Giy Met Ser Pro Leu Leu 195 <210> 28 <211> 196 Lys Val 5 Phe Ser Ile Leu Lys Leu Arg Tyr 70 Giu Lys Gin Ser Leu Ile Ala Arg Thr Giu 150 Gly Lys 165 Giu Asn Lys Arg Ser Ser His Asn Giu 105 Giu Giu Leu Phe Giy 185 Ile Giu Asn Gly Leu Ile Ile Ala Val Ser Giy Asp Ala Asp Giu 75 Leu Pro Leu Ser Asn Val Gin Leu Giu 125 Met Met Gly 140 Giu Giu Asn 155 Vai Ile Giu Lys Val Arg Ser Ser Lys Ala Val Vai Met Ser Giu Ala Giu Leu Asn Ala Ala Leu Vai Lys Thr Leu 160 Asp Arg 175 Thr Leu 0 .0* o I.: <212> PRT <213> Arabidopsis thaliana <400> 28 Met Gly Arg Arg Lys Val Giu Ilie Lys Arg Ile Glu Asn Lys Ser Ser 1 5 10 Arg Gin Val Thr Phe Ser Lys Arg Arg Lys Gly Leu Ile Giu Lys Ala 25 Arg Gin Leu Ser Ile Leu Cys Giu Ser Ser Ile Ala Val Val Ala Val 40 Ser Gly Ser Gly Lys Leu Tyr Asp Ser Ala Ser Gly Asp Asn Met Ser 55 Lys Ilie Ile Asp Arg Tyr Glu Ilie His His Ala Asp Glu Leu Lys Ala 26 70 75 Leu Asp Leu Ala Giu Lys Ile Arg Asn Tyr Leu Pro His Lys Giu Leu 90 Leu Giu Ile Val Gin Ser Lys Leu Giu Giu Ser Asn Val Asp Asn Val 100 105 110 Ser Val Asp Ser Leu Ile Ser Met Giu Giu Gin Leu Glu Thr Ala Leu 115 120 125 Ser Vai Ile Arg Ala Lys Lys Thr Glu Leu Met Met Giu Asp Met Lys 130 135 140 Ser Leu Gin Giu Arg Giu Lys Leu Leu Ilie Giu Giu Asn Gin Ile Leu 145 150 155 160 Ala Ser Gin Val Giy Lys Lys Thr Phe Leu Val Ile Glu Gly Asp Arg 165 170 175 Gly Met Ser Arg Giu Asn Gly Ser Gly Asn Lys Val Pro Glu Thr Leu 180 185 190 Ser Leu Leu Lys 195 <210> 29 *<211> 200 <212> PRT <213> Arabidopsis thaliana <400> 29 Met Gly Arg Arg Lys Val Glu Ilie Lys Arg Ile Giu Asn Lys Ser Ser 1 5 10 .Arg Gin Val Thr Phe Cys Lys Arg Arg Asn Gly Leu Met Giu Lys Ala 25 *Arg Gin Leu Ser Ile Leu Cys Giu Ser Ser Val Ala Leu Ile Ile Ilie 40 *Ser Ala Thr Gly Arg Leu Tyr Ser Phe Ser Ser Giy Asp Ser Met Ala 55 Lys Ilie Leu Ser Arg Tyr Giu Leu Glu Gin Ala Asp Asp Leu Lys Thr 70 75 Leu Asp Leu Giu Giu Lys Thr Leu Asn Tyr Leu Ser His Lys Giu Leu 90 Leu Giu Thr Ile Gin Cys Lys Ile Giu Giu Ala Lys Ser Asp Asn Val 100 105 110 Ser Ilie Asp Cys Leu Lys Ser Leu Giu Glu Gin Leu Lys Thr Ala Leu 115 120 125 Ser Val Thr Arg Ala Arg Lys Thr Giu Leu Met Met Giu Leu Val Lys 130 135 140 27 Thr His Gin Giu Lys Giu Lys Leu Leu Arg Giu Giu Asn Gin Ser Leu 145 150 155 160 Thr Asn Gin Leu Ile Lys Met Gly Lys Met Lys Lys Ser Val Giu Ala 165 170 175 Giu Asp Ala Arg Ala Met Ser Pro Giu Ser Ser Ser Asp Asn Lys Pro 180 185 190 Pro Giu Thr Leu Leu Leu Leu Lys 195 200 <210> <211> 198 <212> PRT <213> Arabidopsis thaliana <400> Met Gly Arg Arg Arg Val Giu Ile Lys Arg Ilie Giu Asn Lys Ser Ser 1 5 10 Arg Gin Val Arg Gin Leu 35 Ser Ser Thr Lys Ilie Ilie 65 Leu Asp Leu Leu Giu Ile Ser Ile Giu 115 Ser Val Ile 130 Asn Leu Gin 145 Ala Ser Giu Arg Aia Val Thr Leu Pro 195 <210> 31 Thr Phe Cys 20 Ser Ile Leu Gly Lys Leu Ser Arg Phe Giu Asp Lys Val Gin Arg 100 Ser Leu Ile Arg Ala Arg Asp Lys Giu 150 Val Gly Lys 165 Met Ser Pro 180 Leu Leu Lys Arg Arg Gly Ser Asn Ser Ile Gin Gin Asp Ile Giu 105 Met Giu 120 Thr Giu Leu Leu Lys Lys Giu Asn Ser Ser Gly His Ser Pro Pro Giu 28 <211> <212> DNA <213> Artificial <220> <223> synthesised DNA primer <400> 31 aagccgcgga caatggaagc tgtaagatgc <210> 32 <211> <212> DNA <213> Artificial <220> <223> synthesised DNA primer <400> 32 *gagaggctgg ttaaccggag <210> 33 <211>

DNA

<213> Artificial <220> :<223> synthesised DNA primer <400> 33 ccgctcgagc ttagtatctc cggcg <210> 34 <211> 24 <212> DNA <213> Artificial <220> <223> synthesised DNA primer 29 <400> 34 ggactagtcg cccttatcag cgga 24 <210> <211> 17 <212> DNA <213> Artificial <220> <223> synthesised DNA primer <400> gtatagggca catgccc 17 <210> 36 *<211> 16

DNA

Artificial <220> <223> synthesised DNA primer <400> 36 cactcggagc tgtgcc 16 <210> 37 <211> <212> DNA <213> Artificial <220> <223> synthesised DNA antisense primer <400> 37 cggaattctc acacgaataa ggtac <210> 38 <211> <212> DNA 30 <213> Artificial <220> <223> synthesised DNA antisense primer <400> 38 ggactagtgg tcaagatcct tgatc <210> 39 <211> 34 <212> DNA <213> Artificial <220> 0.0*0:<223> synthesised DNA primer *~e <400> 39 attgaattcg ggcataaccc ttatcggaga tttg 34 0. <210> <211> <212> DNA o. :<213> Artificial .00. <220> synthesised DNA primer 0* <400> :00 aacggatccg ttgatgatgg tggctaattg agcag <210> 41 <211> 34 <212> DNA <213> Artificial <220> <223> synthesised DNA primer <400> 41 attgaattcg ggcataaccc ttatcggaga tttg 34 31 <210> 42 <211> <212> DNA <213> Artificial <220> <223> synthesised DNA primer <400> 42 ctagtggtac cgttgatgat ggtggctaat tgagc 0 0000 00 0 0 0 0 000 0

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0 0 0 0 00 555555

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0050 S 05 S S 0 S SO S. S S 0*

Claims (4)

1. An isolated nucleic acid molecule comprising box, which is capable of altering the flowering plant, and which comprises the nucleotide sequence set out in of SEQ ID NOS. 1, 2, 4, and 6 to a nucleic acid molecule capable of! hybridizing to a sequence set out in (a! 0 than to the MADS box region thereof, un following hybridization conditions: hyb overnight at 280 C in 50% formamide, 3 x 0.1% SDS, 20 x Denhardt's, 50 pg/ml sal DNA and washing with a final wash of 0. 0.1% SDS at room temperature, or under hybridization conditions of greater str or a nucleic acid molecule which has 70% sequence identity, outside the MADSi 0 region, with a sequence set out in

2. An isolated nucleic acid molecule according t comprising a MADS ime of a any one other ler the :idization SSC, ion sperm x SSC, .ngency; it least box SClaim 1, S@ S S a nucleic acid molecule capable ofi hybridizing to a nucleotide sequence as set out 25 in any one of SEQ ID NOS: 1, 2, 4, and to under the following hybridization conditions: hybridization overnight at 42 0 C in 50% formamide, 3 x SSC, 0.1% SDS, 20 x Denhardt's, 50 g/ml salmon sperm DNA and washing with a fi al wash of 0.1 x SSC, 0.1% SDS at 42 0 C; or a nucleic acid molecule having at least sequence identity with a sequence set oit in Claim l(a).

3. An isolated nucleic acid molecule according to Claim 1 or Claim 2, which, when expressed in a plant in tLe sense orientation under the control of a promoter seque ce, is capable of delaying the flowering of the plant. H;\cKulacUKCccpiiSS01.00doc 1511IMD COMS ID No: SBMI-00997067 Received by IP Australia: Time 12:10 Date 2004-11-15 15/11 2004 12:02 FAX 61 3 92438333 GRIFFITH HACK IPAUSTRALIA ~009

63- 4. An isolated nucleic acid molecule according to Claim 1 or Claim 2, which is capable of accelerating the flowering of a plant. An isolated nucleic acid molecule according to Claim 4, which, when expressed in a plant in the anti-sense orientation under the control of a promoter sequence is capable of accelerating the flowering of the plant. 6. An isolated nucleic acid molecule according tD Claim 1 or Claim 2, which encodes a polypeptide having an amino acid sequence as set out in any one of SEQ ID Nos: 3, 5 or 16 to 30, or a sequence at least 70% identical threto. 7. A vector comprising an isolated nucleic acid nolecule according to any one of Claims 1 to 6. 8. A plant cell transformed with an isolated nucleic acid according to any one of Claims 1 to 6. 9. A plant transformed with an isolated nucleic 'cid molecule according to any one of Claims 1 to 6. A method of isolating a nucleic acid molecule capable of altering the flowering time of a target plant, i 20 comprising the step of using an isolated nucleic hcid Smolecule according to any one of Claims 1 to 6, or a functional portion thereof, as a hybridisation prbbe or polymerase chain reaction (PCR) primer, and optionally detecting hybridisation. 11. A method according to Claim 10, in which the nucleic o*!o acid molecule is capable of hybridizing to a nuci otide sequence as set out in any one of SEQ ID NOS: 1, 4, and 6 to 15 under the following hybridization conditi ns: hybridization overnight at 28 0 C in 50% formamide, 3 x SSC, 30 0.1% SDS, 20 x Denhardt's, 50 .g/ml salmon sperm iNA and washing with a final wash of 0.1 x SSC, 0.1% SDS at room temperature, or under hybridization conditions of greater stringency, and the nucleic acid molecule does no include a MADS box region. 12. A method of delaying flowering in a plant, co]prising the step of introducing an isolated nucleic acid molecule IClr.uoUj lA1 1501.00.doc 1511 L4 COMS ID No: SBMI-00997067 Received by IP Australia: Time 12:10 Date 2004-11-15 15/11 2004 12:03 FAX 61 3 92438333 GRIFFITH HACK -IPAUSTRALIA R010 -64 according to any one of Claims 1 to 6 into cells of the plant, and over-expressing the nucleic acid mole ule. 13. A method according to claim 12, wherein the isolated nucleic acid molecule is expressed under the control of an inducible promoter. 14. A method of inducing early flowering in a pl t, comprising the step of reducing the degree of ex ession of a nucleic acid molecule in the plant, wherein the nucleic acid molecule comprises a) the nucleotide sequence set out in any one of SEQ ID NOS. 1, 2, 4, and 6 to b) a nucleic acid molecule capable of hybridizing to a sequence set out in (a other than to the MADS box region thereof, unier the following hybridization conditions: hybridization overnight at 28 0 C in 50% formamide, 3 xJSSC, 0.1% SDS, 20 x Denhardt's, 50 gg/ml salmon s erm DNA, and washing with a final wash of 0.1 x SSC, 0.1% SDS at room temperature, or under hybricization 20 conditions of greater stringency; or c) a nucleic acid molecule which has at least 70% sequence identity, outside the MADS box region, with a sequence set out in A method of modifying the vegetative and/or floral phenotype of a plant, comprising the step of increasing the level of expression of a nucleic acid molecule in the plant, wherein the nucleic acid molecule comprises the nucleotide sequence set out in any one of SEQ ID NOS. 1, 2, 4, and 6 to 30 a nucleic acid molecule capable of hybridizing to a sequence set out in (a other than to the MADS box region thereof, under the following hybridization conditions: hyblidization overnight at 28 0 C in 50% formamide, 3 x 2SC, 0.1% SDS, 20 x Denhardt's, 50 gg/ml salmon sperm DNA, and washing with a final wash of 0.1 x SSC, 0.1% Hclau&KccpAc\l 1 501.00.doc 15/I1/4 COMS ID No: SBMI-00997067 Received by IP Australia: Time 12:10 Date 2004-11-15 15/11 2004 12:03 FAX 61 3 92438333 GRIFFITH BACK 4IPAUSTRALIA Q011 4 65 SDS at room temperature, or under hybri conditions of greater stringency; or a nucleic acid molecule which has sequence identity, outside the MADS region, with a sequence set out in thereby modifying the level of production or acti gibberellin in the plant. 16. A method of modifying the response of a pla vernalisation, comprising the step of increasing decreasing the level of expression of a nucleic a molecule in the plant, wherein the nucleic acid m comprises the nucleotide sequence set out in any ID NOS. 1, 2, 4, and 6 to a nucleic acid molecule capable of hybr a sequence set out in other than t< box region thereof, under the following hybridization conditions: hybridization at 28 0 C in 50% formamide, 3 x SSC, 0.1% 20 Denhardt's, 50 ug/ml salmon sperm DNA, washing with a final wash of 0.1 x SSC, at room temperature, or under hybridizat conditions of greater stringency; or a nucleic acid molecule which has at lea 25 sequence identity, outside the MADS box with a sequence set out in 17. An isolated polypeptide encoded by a nucleic molecule according to any one of Claim 1 to 6. 18. A polypeptide according to Claim 17, compriE amino acid sequence set out in any one of SEQ ID I and 16 to 30, or having at least 70% sequence ider thereto. 19. An isolated antibody directed against a pol according to Claim 17 or Claim 18. 20. A method of assaying the level of expressio polypeptide according to Claim 17 or Claim 18, coI the step of using an antibody according to Claim 2 lization t least box Lity of a t to Br id jlecule one of SEQ .dizing to the MADS overnight SDS, 20 x Lnd 0.1% SDS .ion .st region, acid ing the [OS: 3, .tity peptide of a prising 0. H1~cin ~4 KeD 'A8501.00.doc 1511/04 COMS ID No: SBMI-00997067 Received by IP Australia: Time 12:10 Date 2004-11-15 15/11 2004 12:03 FAX 61 3 92438333 GRIFFITH HACK IPAUSTRALIA 0012 66 21. A method of selecting plants with low or higi h levels of expression of a nucleic acid molecule, comprising the step of determining the level in the plant of an mRNA capable of hybridizing to a nucleotide sequence set out in any one of SEQ ID NDS. 1, 2, 4, and 6 to 15, other than to the MADS pox region thereof, under the following hybridizatLon conditions: hybridization overnight at 28 0 C in formamide, 3 x SSC, 0.1% SDS, 20 x Denhardt's, 50 tg/ml salmon sperm DNA, ind washing with a final wash of 0.1 x SSC, 0.1% SDS at room temperature, or under hybridization conditions of greater stringency; or a nucleic acid molecule which has at least 70% se uence identity, outside the MADS box region, ith a sequence set out in any one of SEQ ID N 1S. 1, 2, 4, and 6 to 15, or a polypeptide according to claim 16 or Tlaim 17 22. A method according to claim 21, in which the plants oe 20 are members of a naturally-occurring population. 23. An isolated nucleic acid molecule according to claim 1, substantially as herein described with referen.e to any S. one of the examples and drawings. 24. A method according to any one of claims 14, 15, 16 or 21, substantially as herein described with refere ce to any one of the examples and drawings. Dated this 15th day of November 2004 COMMONWEALTH SCIENTIFIC AND INDUSTRIAL RESEARCH 30 ORGANISATION and PASCUAL PEREZ By their Patent Attorneys GRIFFITH HACK Fellows Institute of Patent and Trade Mark Attorneys of Australia Itcmln!Kecp c iflOLOfcdoc IS/11/04 COMS ID No: SBMI-00997067 Received by IP Australia: Time 12:10 Date 2004-11-15