CA2353080A1 - Control of flowering - Google Patents

Abstract

The invention relates to 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. 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.
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. 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.

Description

CONTROL OF FLOTnIERTNG
This invention relates to the control of flowering and reproduction in plants, and in particular to S 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.
is DETAILED DESCRIPTION OF THE INVENTION
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 15 changes 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 20 thal.zana, flowering is promoted by long day photoperiod, or by a period of low temperature (vernalization} (Napp-Zinn, 1985} .
Control of flowering in both horticultural and crop plants represents a major problem in the agricultural 25 industry, and is also a problem in forestry. Significant losses in yield of plants may result if non-uniform flowering of plants accurs; this applies both to field--grown and to glasshouse-grown plants. The problem is particularly acute for field-grown plants, which axe 30 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 3S to maximize market opportunities for glasshouse-grown plants.

WO 00/32780 PCT'/AU99101079 Currently-available methods far 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 cut-flower 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)~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, Ant.irrh.inum maws, tomato, tobacco, petunia, corn, Pinus species and Eucalyptus species. In plants, the MARS box genes have a "K domain", which resembles the coiled-coil domains of keratin proteins, which are implicated in protein/protein interactions, an intervening (I) domain, and a carboxy terminal (C) 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 00/327$0 PCT/AU99/01079 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.
_5 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. 1, 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 o 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 800, most preferably at least 90o sequence identity. A nucleic acid molecule having at least 70%, preferably at least 900, more preferably at least 95a sequence identity to one or more of these sequences is also within the scope of the invention.

WO 00!32780 PCT/AU99/01079 Tn 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 l6 to 30, or has a sequence at least 70o identical 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 00132780 PCTIAU99/01079 screening methods including Western blotting and various forms of immunoassay, fox 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 nucleic 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, post-transcriptional gene silencing, ribozyme cleavage, disruption of the nucleic acid sequence using a transposable element or transposon, or by a procedure such as vernali.sation. 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 VI~N2. Therefore in both the sixth and seventh aspects 3S of the invention, a further means of modifying the degree of expression of FLF is provided by modifying the activity WO 00/32780 PCTlAU99101079 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 _5 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 35S
promoter, a NOS promoter, the octopine synthetase (OCS) promoter, a subclover stunt virus promoter and the Arabidopisis thalzana 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 g 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 _S nopaline synthase (N4S) gene transcriptional terminator of Agrobacterium tumefaciens, the transcriptional terminator of the Cauliflower mosaic virus (CaMV) 35S gene, the zero gene transcrip-tional 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 a1, 1984), polyethyleneglycol-mediated uptake to protoplasts (Armstrong e~ a1, 1990), electrophoresis (Fromm et a1, 1985), microinjection of DNA (Crossway et a1, 1986), microparticle bombardment of tissue explants or cells (Christou et a1, 1988; Sanford, 1993), or T-DNA-mediated transfer from Agrobac~erium to the plant tissue.
Representative T-DNA vector systems are described in the following references: An et al (1985); Herrera-Estrella et a1 (1983a, b); Herrera-Estrella et a1 (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 _ g _ 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 far 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, Lo.lium, wheat, barley, maize, rice, pasture grasses, Phalaris, Canala and other Brassica species, .L.inola 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 - ~.a -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 S 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 i0 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 15 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, 20 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 25 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 30 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 35 reproductive organ, in which the meristem or organ, when developed, may comprise both reproductive and non-reproductive tissues, including, but not limited to, 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°C in 50%
formamide, 3 x SSC, 0.1o SDS, 20 x Denhardt's, 50 ~tg/ml salmon sperm DNA overnight and washed with a final wash of 0.1 x SSC, 0.1% SDS at 42°C.
Low stringency: hybridization at 28°C in 50%
formamide, 3 x SSC, 0.1% SDS, 20 x Denhardt's, 50 ~.g/ml salmon sperm DNA overnight and washed with a final wash of 0.1 x SSC, 0.1o 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 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 intro-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:
(i) a classical genomic sequence comprising transcriptional and/or translational regulatory sequences and/or a coding region and/or non-translated sequences (ie.
introns and 5'- and 3'-untranslated sequences);
(ii) mRNA or cDNA corresponding to the coding regions (ie. exons), optionally additionally comprising 5'-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 polypeptide, in particular the FLF polypeptide of the invention or a homologue, analogue or derivative thereof.
In some of the examples herein the FLF gene is referred to ws gene B. These two terms are synonymous.
S
BRIEF DESCRIPTION OF THE FIGURES
Figure 1 is a photograph showing wild-type C24 (left), the late flowering T-DNA tagged flf mutant (middle) at 70 days after germination, and f1f mutant at 250 days, showing the domed shape caused by vegetative bolts (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 T2 population segregating for early (E), late (L) and very late (VL) flowering, digested with EcoRI and probed with the NPTII
gene.
B. Physical map of the two T-DNA inserts linked to the FLF locus, showing their orientation. EcoRI
sites are labelled RT; LB and RB represent the left and right borders, respectively, of the T-DNA. The triangle symbol represents the site of deletion of 30 by to the right of the T-DNA. The arrows represent the direction of transcription of the genes.
C. Representation of a 27 kb region of Arabidopsis mutant DNA containing the gene A and gene B
loci, showing the location of the T-DNA inserts. DNA
fragments from the flanking plant DNA (probes 2 and 3) were used as probes to isolate cDNA clones. HindIII (H), BamHI
(B) and EcoRT (R1) restriction enzyme sites are indicated.
The positions of gene A and gene B are shown, and their directions of transcription are indicated by arrows.
n. The 6.5kb and 6.8 kb BamHI fragments, isolated from a genomic library of wild-type C24 with probes 1 and 2 from Figure 2C, spanning the site of the T-DNA insertions. Restriction sites are as in Figure 2C.

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) f1f 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 early-flowering (back) or late-flowering (front). The Landsberg erecta transgenics were either late-flowering Cleft) 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 (I), K domain (K) and carboxy terminal domain (C). 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 (R) and rosette leaves (RL) from .in vitro grown vegetative plants, cauline leaves (CL), bolt stems (BS), floral apex and buds (B) from soil grown plants with bolt stems between 1 and 5 cm. Mature flowers (F) and siliques (S) 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 f1f 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 2l~' photoperiod either kept in the same conditions (SD; lane 1, 2, 7, 8) or transferred to continuous dark (CD; lanes 3, 4, 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, 5, 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 (GA3) treatment and vernalization on the FLF transcript in C24 (lanes 1-3) and f1f mutant (lanes 4-6) seedlings. RNA was isolated from 12 day old seedlings that had either had no treatment (C; lanes 1 and 4), been grown on medium containing 10-5 M
GA3 (G; lanes 2 and 5) or had a pretreatment of 3 weeks at IS 4°C (V; 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 (M2), efSL4 (M2) and C24 plants digested with EcoRI
and probed with a probe directed to the 3' region of Ac.
The DNA for the fIf 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.
8. 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-later 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 early-flowering 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.

D. Expression level of FLF gene in 15 day old rosette leaves of C24 (lane 1), fIf (lane2), ef SL3 (lane 3), ef SL4 (lane 4). 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 (M2), efSL4 (M2), flf and C24 plants, and digested with EcoRI. The DNA gel blot was probed with the 3' region of Ac.
8. Total RNA was isolated from a mixture of rosette and cauline leaves from the 6 neo-late plants, f1f 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 8. Expression in Landsberg erecta (L.er.)and Landsberg erecta lines which contain late alleles at either the FR.I ( L . er. -FRIsFZ ) or FLC ( L . er. -FLC'sfa , 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 VJs ( 1d) . The mutants vrnZ and vrn2 were isolated in the fca background, and only vrn2 has been segregated away from the fca mutant locus.
Figure 9 shows the partial genomic sequence Ofan FLF-lzke sequence from Brassica napus, showing the location - 17 _ of exons, and the predicted sequence of the translated product.
Figure 10 shows a comparison of the predicted translated product of a Brassica napus FLF-like sequence (top lines), and the predicted FLF translation product from Arabidopsis thaliana, showing identical amino acids (), highly conserved amino acids (:) and conserved amino acids (.) a 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.
GENERAL METHODS
Plant Material arid Growth Conditions Arabidopsis was grown either in pots containing a mixture of 50% sand and 50a loam, or aseptically in test-tubes 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 21° or 23°C, under long day (16 hr light, 8 hr dark) conditions using cool white fluorescent lights at an intensity of 200 /.t.M m 2s 1.
Plants were vernalized by germinating seed in the dark for either 3, 4 or 8 weeks at 4°C. Following this cold treatment, seedlings were transferred to long day photoperiods at 23°C 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.
Arab,idopsis 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 a1, 1993) for the gene B

transgenic plants. The late-flowering mutant (fIf) arose during transformation of the early-flowering ecotype C24 with a modified binary vector pBinOAc (Finnegan et a1, 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 (3-glucuronidase gene, in the reverse orientation to the direction of transcription.
i0 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 late-flowering T1 plant from family l4-58, showed 53 "late"
(flowering time >70 days) compared to 15 "early" flowering plants (flowering time 30 days). The result fits a 3:1 2S segregation ratio (x2=0.313 P>0.5), 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 late-flowering plants could be further differentiated into two classes; "laces", flowering between 70 to 90 days and "very laces", which flowered later than 150 days. Some of the "very late" plants had not flowered after a year of growth.
The "very late" f1f 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 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 f1d 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 a1, 1991). Progeny tests showed that selfed "very later" 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 T.; hrnr; o~
A genomic library of the f1f mutant was constructed by partial digestion of total plant DNA with the restriction enzyme Sau3AI and ligation into the phage vector ~,EMBL4. The resulting library was screened using a 32P-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 a,EMBL4. 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 vexy late flowering phenotype The very late-flowering phenotype segregates with two T-DNA bands identifiable by Southern analysis, which were designated bands 1 and 5. Southern blotting showed that bands 1 and 5 are inverted and adjacent:
RB < LB LB ~-> RB

(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 by sequence, 420 by to the "right" of the stop codon of gene A and 120 by to the "left" of the T-DNA insertion. The sequence is present in both C24 wild-type and the f1f mutant. The 200 by insertion shows 100 % homology to ORF167 of the Arabidops.is mitochondrial gename. By PCR analysis we have 3S determined that this sequence is absent in Landsberg erecta and Columbia ecotypes. The significance of this inserted DNA segment is unknown.

_ WO 00/327$0 PCT/AU99101079 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 a1, 1992; Newman et a1, 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 .Yes clones; however, 1S 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 SK(-) (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 SK(-). 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 ~,PRL2 cDNA library derived from different tissues and developmental stages (Newman et a1, 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 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:-(1) 5' AAGCCGCGGACAATGGAAGCTGTAAGATGC 3' (2) 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 10 ~,l final volume that contained 2 ~,M of each oligonucleotide primer, 200pg of HindIII-cleaved cDNA as a template, 0.2 units of Taq polymerase and 125 ja,M of each of the four deoxynucleotides. Conditions for the amplification were as follows: 95°C for 2 mins, 5 cycles consisting of 15 s denaturation 95°C, annealing at 40°C for 30 s, and polymerization at 72°C for 1 min, followed by 25 cycles where the annealing temperature was raised to SO°C
for 15 sec and finally 30°C for 1 min. The resulting 200 by 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 usirxg the Klenow fragment of DNA polymerase I, and ligating the released 2.4 kb fragment into the SmaI site of the expression vector pDH51 (Pietrzak et a1, 1985). This places the expression of the FLF cDNA
under the control of the CaMV 35S promoter. Recombinant 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 tBevan, 1984). The binary construct was transferred to Agrobacte.r.ium tumefaciens strain AGL1 (Lazo et aI, 1991) by triparental mating, employing pRK2013 as the helper plasmid. Roots of wild-type C24 plants were transformed (Valvelkens et a1, 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
cDNA clone as template with the primers, using methods similar to those described in Example 5:
5' CCGCTCGAGCTTAGTATCTCCGGCG 3' and 5' GGACTAGTCGCCCTTATCAGCGGA 3', in which restriction sites are shown in bold, and the sequence hybridizing to gene B cDNA is underlined, into XhoI/SpeI digested pART7 (cleaves, 1992) containing the CaMV 35S promoter. The 35S::gene B cassette was then subcloned using NotI into pART27 (cleaves, 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 ~g of DNA was digested with the appropriate restriction enzyme, electrophoresed on an 0.8%
agarose gel, and blotted onto Hybond N+ membranes (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' and 5'-CACTCGGAGCTGTGCC-3'.
This results in a 570 by subset of probe 2 sequence, lacking the MARS box, to eliminate cross-hybridization.
Example 8 RNA Extraction and RNA Gel Blot Analysis Total RNA was extracted from approximately 1g of plant tissue, following the method of Longemann et al.
(1987). 10-20 ~.g of total RNA was electrophoresed on 2.2 M
formaldehyde/agarose gels, and blotted onto Hybond N nylon filters. T7 or SP6 polymerise transcription of the linearised gene A or gene B plasmid template (containing the complete cDNA for gene A, linearised to remove MARS box for gene B) was used to generate antisense 32P-dUTP
labelled riboprobes. Filters were hybridized as described by Dolferus et a1 (1994), washed with a final solution of 0.lxSSC, 0.1% SDS at 65°C. For gene A Northern blots it was necessary to treat filters with RNase A as previously described (Dolferus et a1; 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 WO 00/32780 PCTlAU99/01079 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 f1f mutant. A HpaI digest of 62 F2 DNA was probed with the chromosome 5 RFLP marker 447 (Chang et aZ., 1988). A restriction fragment length polymorphism (RFLP) was found between the parental lines, Landsberg erecta and Columbia, by digestion of genomic DNA
with Ba.mHI and probing with the gene A cDNA.
lU
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 T1 parent. In a total of 70 progeny, only two inserts segregated with the late-flowering phenotype (bands 1 and 5); "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 2S 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
3U 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 35 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 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 S total Arabidopsis DNA with the restriction enzyme BamHI and ligation of the digest into the phage vector ~,EMBL4, using probes 1 and 2 (Figure 2C). Clones containing a 6.5kb BamHI fragment spanning the insertion site and a 5.8 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 E'MF1, which are involved in the regulation of flower initiation (Shannon and Meeks-Wagner, 1991; Lee et al, 1994b; Koornneef et a1, 1994; Sung et a1, 1992).
Example 11 Two Genes Flanking the T-DNA Inserts Have Increased Expression In The f1f 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, 591 by 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 by 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 lU 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 f1f 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 _ 2g _ 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 expressiori level of gene A mRNA

C24 29.9 0.6 1 B2 38.3 0.9 2 A53 35.8 0.8 20 A54 38.4 0.5 8 A93 35.4 0.5 4 f1f 50 10 Transgenic and fIf mutant seeds were germinated on MS plates + 50 ~.g/ml kanamycin, and C24 seeds were germinated on MS plates. At least 20 22 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.

- as -Table 2 Flowering time of 35S::gene B T1 transgenic plants Flowering time Relative expression level of gene 8 mRNA

C24 30 d 1 B4 18 d > 10 B5 18 d > 10 B11 > 80 d > 10 B12 > 80 d > 10 Landsberg 20 d n.d.

erecta ~

IB36 > 80 d > 10 B45 > $0 d > 10 f3f > 80 d 2 Transformant seeds, harvested from in planta transformed plants, were selected on MS plates containing 50 ~,g/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 BS, and from young leaves from the T1 late-flowering plants (B11, B12, B36, B45). n.d.: 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 355 promoter in ecotype Landsberg erecta. Of the 24 T1 lines generated, none flowered earlier than wild-type Landsberg erector, 12 had not bolted after 70 days, and 3 bolted after about 40 days, compared with 25 days for Landsberg erector. Two of the three lines which bolted after 40 days exhibited floral abnormalities and partial sterility. Total RNA was isolated from rosette 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 post-transcriptional gene silencing. Analysis of protein expression levels is being pursued in order to clarify this IO 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 effect on flowering time, indicating that over-expression of gene B is the most likely cause of the late-flowering f1f 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 using an anti-sense FLF gene construct under the control of the CaMV 35S promoter. A 35S::FLF antisense binary construct was generated by cloning the EcoRI/SpeI digested PCR product amplified with primers CGGAATTCTCACACGAATAAGGTAC and GGACTAGTGGTCAAGATCCTTGATC
as described for the 35S::FLF construct. This amplified the region downstream of the MARS box, so that the antisense construct lacks the MARS 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.
25 T1 C24 transgenic plants were generated with a construct in which the 3' end of the FLF gene, in the WO 00/327$0 PCT/AU99/01079 antisense orientation, was under the control of a 35S
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 non transformed 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 MARS Box Gene The FLF cDNA sequence has strong homology to a class of transcription factors knoiun as MADS box genes.
The FLF sequence shows greatest similarity to the MARS gene AGL14 in the M-I-K domain, but over the entire cDNA
sequence it shows greater similarity to CAL (CAULIFLOWER) and APl (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 MARS 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 3.5 kb. The predicted protein (SEQ ID NO. 3) is 196 amino acids long, which is shorter than the proteins encoded by mast MARS 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 MARS-box WO 00/32780 PCT/AU99l01079 genes include specifying root architecture and vegetative growth. To investigate whether FL.F 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 1S the FLF gene.
The expression of the FLF gene is lower in post-vegetative 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 f.~f whole plants every 10 days post-sowing, until the stage where the majority of C24 plants had bolted (50 d). The expression of the FLF gene remained unaltered in these plants (Figure 5C), 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 fIf mutant demonstrates a number of similarities to the 1hy 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 Ihy mutant;
the LHY gene is expressed around the clock. Because of the similarities between flf and lhy, 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 two time points, there was no alteration of this pattern in the mutant.
Example 15 The Late-Flowering Phenotype Of The fif 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 f1f mutants is shown in Table 2.
IS
Tabla 2 Flowering Time of Vernalised C24 and Mutant Plants hENGTH 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 f1f >150 100.8 10.? 17.6 1.3 Twenty seeds of the f1f mutant and wild-type C24 were grown aseptically on MS medium in test-tubes, and exposed to either 4 or 8 weeks at 4°C. Non-vernalized plants were grown in soil (20 plants per 20cm pot). All plants were then grown at 23°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 4°C resulted in a substantial reduction in the flowering time. However, eight Weeks at 4°C was required to saturate the WO 00!32780 PCT/AU99101079 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 vernalization-S induced 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 SE 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 f1f 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 vernalization-responsive mutants and ecotypes of Arabidopsis, f1f mutant plants responded to applications of gibberellic acid (GA3) by flowering earlier. Four week-old fIf homozygotes treated with 1 ~,g of GA3 every second day for a total of two weeks flowered two weeks after the final GA3 application, compared to later than 20 weeks for untreated plants. However, a single treatment of 4 week old plants with l ~Lg of GA3 was not sufficient to induce flowering of flf, although this amount of GA3 induced early flowering of the late-flowering fca mutant (Bagnall, 1992), suggesting that f1f requires a greater amount of GA3 for floral induction. In contrast to the dramatic effect of vernalization on the expression of the FLF gene, exposure of either C24 or f.If seedlings to 10-~' M GA3 had no effect on the expression of the FLF gene.

Example 1& Movement Of An Ac Element Present Within The T-DNA Causes Alteration In Flowering Time Two early flowering plants (M1 plants designated efSL3 and efSL4) were identified from one seedlot S comprising bulked seed from f1f 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 fIf lU mutant, and were not contaminants.
The tandem T-DNAs present in the f1f 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 early-15 flowering M2 progeny of efSL3 and efSL4, digested with EcoR1 and probed with the 3' region of Ac. Figure 5A 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 2U 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 25 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 3U 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 35 leaves of early-flowering M2 plants and comparable-sized rosette leaves of C24 and f1f plants, and probed with an FLF gene specific riboprobe. As shown in Figure 5d, FLF

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 ar the splicing efficiency of the transcript, S 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-fates" to distinguish them from the original late-flowering f1f mutant plants. Their approximate days to bolting were: 50 d (3.2), 50 d (3.4), 60 d (3.5), 85 d (3.5), 100 d (3.1). 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-fates 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 (3.1, 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 PCTlAU99l01079 We believe that the sector of the M1 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 l, 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-laces and the gene B
expression level was compared to that of 80 d rosette and cauline leaves of f1f and 25 d rosette leaves of C24. In all neo-laces the transcript level was higher than that of the early-flowering parent, although lower than that of the f1f 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 late-flowering f1f 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 late-flowering 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 3S particularly highly expressed in C24, but not in ecotypes with an early allele at the FRI locus (Columbia, Ws, Landsberg erector).

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 S 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 90 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 FRI'f2 allele; however, it is also expressed in lines which contain late FLC'f2 and FLC~~l alleles . The expression 1S of the FLF gene in Landsberg erecta-FLf~°1 is interesting, as this plant has the same FRS and FLC genotype (FRIearly~
F,L~aate) as the ecotype Columbia, yet there is expression 'in Landsberg erecta-FLf~°1, 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 1d in Ws ecotype, but not in any of the other late-flowering mutants tested. FLF is also upregulated in the f1d 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 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 cold-s treatment 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. Bath 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 vrn2 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 vrn.1 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 VRN1 and VRN2 genes are involved in mediating the vernalization-induced down-regulation of. the FLC gene. In our growing conditions vrn.Z 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 VRN1 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 a1, 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.

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 S plants vernalised for 3 weeks flowering in about half the time of unvernalised plants. A four week vernalisation period decreases f1f 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 flawering time of the fIf 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 fIf 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 MARS box transcription factor, it may do so by activating genes involved in the catabolism of GA, either directly or WO 00/327$0 PCTIAU99101079 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 S of FLF, or via another pathway. ie.
VERNALISATION -~ -~ decrease in FLF transcript ~ ~ GA -~ -~ FL04JERING
or VERNALISATION ~ ~ decrease in FLF transcript -j -~ FLOWERING
I0 ?-~ ~ GA -~ -~ FLOWERING
with FLF normally acting to block the pathway between VERN
and GA
IS 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 20 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.
2S There was no difference in expression level between these two tissue types.
In the fIf 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?.
30 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 35 control of vegetative and floral transcription of the FLF
gene.

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 500 of C24 plants were bolting after 50 d.
There was no change in the level of FLF transcript in either C24 or the f1f 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 f1f 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 2S of expression between the mutant and C24.
Example 19 Isolation of a Brassica napus FLF homologue Low stringency screening of a Brassica napus genoinic 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°C in 50%
formamide, 3 x SSC, 0.1% SDS, 20 x Denhardt's, 50 ~.g/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.

The partial genomic sequence of the Brassica napes FLF-like gene is set out in SEQ ID NO. 4, and the amino acid sequence of the predicted translation product is set out in SEQ ID N0. 5. The partial genomic sequence, showing the location of exons and the sequence of the correspanding translated product, is illustrated in Figure 9, and the sequence of the predicted translated product from the Brassica napes gene is compared with the corresponding product from Arabidopsis thaliana FLF in IO 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 Gdisconsin Genetics Computer Group software package version 9.1, using default parameters).
A cDNA library was prepared from Brassica napes, and 10 cDNA clones were isolated using hybridisation to an FLF cDNA from Arabidopsis. The starting RNA was isolated from leaves of Brassica napes, cultivar Colombus. Poly(A)+
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 3S relationships between the clones. From both amino acid and nucleotide sequence data it was concluded that the clones represent transcripts from 5 different genes.

The cDNA sequences and predicted amino acid sequences are set out in SEQ ID NOS: 6 to 15 and SEQ ID
NOS: 16 to 23 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 80 N-terminal amino acid residues and possessing an in-frame 2U N-terminal histidine tag was overexpressed in E.co.t.i strain BL21 [DE3] using a pET 22b+ expression vector (Novagen). 1 ml of an overnight bacterial culture was added to 100m1 of LB broth containing 50 ~Lg/ml ampicillin and the culture grown to an OD~oonm of 0.6 ~at 37°C. 100 ~l of 1M IPTG was then added, and the culture was grown for a further 4 hours before 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 Arabi.dopsis plants were ground in liquid nitrogen and homogenised in extraction buffer (0.1M
NaP04 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
3S sample buffer (0.5M Tris pH 6.8, 10% SDS, 0.6M DTT, 0.0120 bromophenol blue) for 5 minutes. Insoluble proteins were extracted by homogenising and boiling for 5 minutes in 2x _ 4g _ SDS sample buffer (0.167M Tris pH 6.8, 3.3% SDS, 0.2M DTT, 0.0040 bromophenol blue). Protein extracts (50 ~Lg per lane) were separated on a denaturing 12% polyacrylamide gel before blotting onto a Protran nitrocellulose membrane S (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 10 mznutes.
The amount of FLF protein was dramatically decreased following 35 days of low temperature treatment in the C24 and Pitztal ecotypes and in the fZf mutant. The decrease in the f1f 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 2S 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) 35S promoter was inserted into the vector pWBVec8. This vector contains the HPT gene, which confers resistance to 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 lU 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.

_ 47 _ Table 3 Family clones Date recorded PCR test for Antibody test floral (anr.3 transgene for leaf Arabidopsis no.). Floral FLF
status recorded protein on Days 1, 11, 25, 33 and 41.

s:':~ J1 small plant -ve -ve Ex 1.1 /2 Day 1 -ve -ve /3 Day 1 -ve -ve /4 Day 1 -ve -ve --/5 Day 11 - _ve -v~, CS Ex /1 Day 25. i7 leaves+ve +ve 1.2 /2 Day 33. 15 leaves+ve nil /3 Day 11 -ve -ve /4 Day 33. 17 leaves-ve (false -ve +ve ?) /5 Day 25 +ve +ve Ex /1 Day 41. 21 leaves+ve +ve 1.3 /2 Day 41. 21 leave.>+ve +ve /3 Day 1 -ve -ve C; Ex / 1 Day 1 -ve -ve 1.4 /2 Day 1 -ve -ve Cs Ex /1 Day 1 -ve -ve 1.5 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 leavesnd +ve /3 Day 33. 19 leaves+ve +ve /4 Day 33. 15 leave:+ve +ve /5 Day 11 +ve nd C:~ 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 /5 Day 41. 24 leaves+ve nd CS Ex /1 Day 25. 15 leavesNd +ve 1.9 Cs Ex /1 Day 25 +ve -ve 1.10 CS Ex /1 Day 25 +ve nd 1.11 /2 Day 25 -ve -ve C:~ Ex /1 Bay 1 -ve -ve 1.12 CS Ex /1 In TC In TC In TC
1.13 CS Ex /1 Day 25 -ve -ve 1.14 /2 Day 25 -ve -ve /3 Day 25 -ve ._..: /1 Day 3s . 15 +ve nd Ex leaves 1.15 /2 Day .33. 16 +ve +ve leaves i3 Day 33. 16 leaves+ve nd Ex /1 Day 33. 14 leave.~-.+ve +ve 1.16 c: ~ :' 1 Isay 1 +ve nd Ex 1.17 Ex /1 In Tr:' In TC In Tc' 1.18 ._..; /1 Lsay 25. 15 +ve nd Ex leave:

1.19 C:: Ex /1 Isisc~.ar<:ieciDiscarded Discarded 1.20 Ex In T.' In TC' In TC In TC

2.21 In TC = plants remain in tissue culture stage nd - not determined The plants exhibited a range of flowering times, with same 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 far 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 ~rassica proteins under the conditions 1S used. Although the PCR and antibody tests were single experiments, there is a good correlation between the two tests:
Table 4 summarises these results.

Table 4 Flowering Time in Brassica napus Plants Transfomed with FLF
Number Number Number Number of of of of floral floral floral families PCR

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 Day 1 Day 11 Day 25 Day 33 Day 41 The day of potting into large pots is referred to as day 1. Floral plants are those with buds or open 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.

_ 5p _ Example 22 FLF-like Molecules from Arabidopsis t-~~ ~;a"~
Searching of the Arab,idopsis genomic sequence database revealed five putative MARS-box encoding genes with a high degree of homology to the FLF protein. One of these genes, designated FLF-LIKE2, 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 AB013395).
The amino acid sequences of the FLF-LIKE proteins are shown below, with amino acids identical to corresponding amino acids of the FLF protein depicted in bold type.
FLF-LIKE1 (SEQ ID N0:26):
MGRRKIEIKRIENKSSRQVTFSKRRNGLIDKARQLSILGESSVAVVW'SASGKLYDSSS
GDDISKIIDRYEIQHADELRALDLEEKIQNYLPHKELLETVQSKLEEPNVDNVSVDSLT
SLEEQLETALSVSRARKAELMMEYIESLKEKEKLLREENQVLASQMGKNTLLATDDERG
MFPGSSSGNKIPETLPLLN.
FLF-LIKE2 (SEQ ID N0:27):
MGRKKVEIKRIENKSSRQVTFSKRRNGLIEKARQLSILCESSIAVLVVSGSGRLYKSAS
GDNMSKIIDRYEIHHADEhEALDLAEKTRNYLPLKELLEIVQSKLEESNVDNASVDTLI
SLEEQLETALSVTRARKTELMMGEVKSLQKTENLLREENQTLASQVGKKTFLVIEGDRG
MSWENGSGNKVRETLPLLK.
FLF-LIKE3 (SEQ ID N0:28):
MGRRKVEIKRIENKSSRQVTFSRRRKGLIEKARQLSILCESSIAVVAVSGSGKLYDSAS
GDNMSKIIDRYEIHHADELKALDLAEKIRNYLPHKELLEIVQSKLEESNVDNVSVDSLI
SMEEQLETALSVIRAKKTELMMEDMKSLQEREKLLIEENQILASQVGKKTFLVIEGDRG
MSRENGSGNKVPETLSLLK.
FLF-LIKES (SEQ ID N0:29):
MGRRKVEIKRIENKSSRQVTFCKRRNGLMEKARQLSILCESSVALIIISATGRLYSFSS
GDSMAKILSRYELEQADDLKTLDLEEKTLNYLSHKELLETIQCKIEEAKSDNVSIDCLK
SLEEQLKTALSVTRARKTELMMELVKTHQEKERLLREENQSLTNQLIKMGKMKKSVEAE
DARAMSPESSSDNKPPETLLLLK.

FLF-LIKE5 (SEQ ID N0:30):
MGRRRVEIKRIENKSSRQVTFCKRRNGLMEKARQLSILCGSSVALFIVSSTGKLYNSSS
GDSMAKIISRFKIQQADDPETLDLEDRTQDYLSHKELLEIVQRKIEEAKGDNVSIESLI
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, 62.2 % identity ($4.2 similarity) for FLF-LTKE2, 60.7 % identity (84.2 %
similarity) for FLF-LIKES, 60.7 % identity (85.2 similarity) for FLF-LIKE4 and 56.1 % identity (86.2 %
similarity) for FLF-LIKE5. In contrast, the published Arabidops.is 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 arid 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-LIKES) was isolated using a RT PCR based method. First strand cDNA was generated from 5 ~.Lg of Col-0 total RNA.
Reactions were carried out using Superscript II (GIBCO BRL) in a 20 ~L1 volume according to the manufacturer°s instructions. FLF-LIKE2 transcript was amplified by PCR
using 1~.1 of the first strand cDNA synthesis reaction as template with primers .
5'-ATTGAATTCGGGCATAACCCTTATCGGAGATTTG-3' and 5'-AACGGATCCGTTGATGATGGTGGCTAATTGAGCAG-3';
Eco RI and Bam HI restriction sites respectively are underlined. The amplification reaction was carried out in a final volume of 40 ~L1, which contained 2.5 N,M of each oligonucleotide primer, 1.0 units of Amplitaq Polymerase (Perkin Elmer) and 250 ~,M of each of the four deoxynucleotides. Conditions for amplification were as follows: 94"C for 2 min, 40 cycles consisting of 15 s denaturation at 94°C, annealing at 55°C for 15 s and polymerisation at 72"C for 1 min, and a final extension at 72"C for 4 min before the temperature was decreased to 25"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 I5 l~rabidopsis genomic sequence (BAC F22K20; AC002291). The University of Wisconsin GCG software package was employed for sequence analysis.
A binary construct containing the FLF-LIKE.1 cDNA
under the control of a CaMV 35S promoter was generated by cloning an Eco RI/Kpn I digested PCR product into an Eco RI/Kpn I pART7 vector (cleave, 1992) containing a CaMV 35S
promoter. The PCR product.was amplified using 200 pg of the FLF-LIKE2 cDNA clone as template with primers:
5'-ATTGAATTCGGGCATAACCCTTATCGGAGATTTG-3' and 5'-CTAGTGGTACCGTTGATGATGGTGGCTAATTGAGC-3';
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 (cleave, 1992), and introduced into Agrobacter.zum tumefaciens strain GV3101 by electroporation. Transgenic plants were generated by in planta transformation (Bechtold e~ al., 1993), using the NPT II gene as a selectable marker to identify transgenic plants.

The 35S::FLF-LIKEZ construct was transformed into Arabidopsis thaliana ecotypes Landsberg erecta and C24.
Twenty individual Tl 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 T1 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 post-germination. 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-hIKE~
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-LIKEI 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.

The late-flowering f1f 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 wn 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 Tl plants) One early-flowering plant (#6) 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 plant s (#6, 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 erects ecotype (24 T1 plants) Two plants (#59, 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 3S trichomes ("hairs"). Normally Arabidopsis carpels do not have trichomes.

The leaves of some plants which still have not flowered were reduced in size, resulting in a smaller diameter leaf rosette than normal (#40, 42, 62). Many of the late-flowering plants (#36, 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, 3e. the leaves were flatter.
Landsberg erecta.-FLCsf2 (17 T1 plants) Two plants had the appearance of a semi-dwarf (#26, 28). The plants had reduced fertility. One plant 1S (#25) produced very few seeds. This plant had clear floral abnormalities, with petals of reduced size that were greenish in colour, and abnormally-shaped carpets with trichomes.
One plant (#32) which has stitl 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-FRIsf2 (35 Tl plants) One plant (#47) had the appearance of a semi-dwarf. 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 carpets with trichomes. Some of the late-flowering plants (#48, 56) were a darker green colour than normal. Two plants (#97, 98) which have still not ftowered, have extremely small rosettes (~15 mm in 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 early-flowering 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 .

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Weigel et a1 Cell, 1992 69 843--859 SEQUENCE LISTING
( 1 ) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: COMMONWEALTH SCIENTIFIC AND INDUSTRIAL
RESEARCH ORGANISATION
(B) STREET: LIMESTONE AVENUE
{C) CTTY: CAMPBELL
(D) STATE: ACT
(E) COUNTRY: AUSTRALIA
(F~ POSTAL CODE (ZIP): 2612 (ii) TITLE OF INVENTION: CONTROL OF FLOWERING
(iii) NUMBER OF SEQUENCES: 3 (iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: Patentln Release #1.0, Version #1.30 (EPO) (2) INFORMATION FOR SEQ ID NO: I:
(i) SEQUENCE CHARACTERISTICS:
{A) LENGTH: 7968 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Arabidopsis thaliana (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
GGATCCAAGA AATAATTTCA TATGGGGCAC AGTTAAAAAA AAAACAATAA

GTAAGGCTTG AACTTGGGTT GATGTGAGGC ACTATTAAGT AAAAAGCCAT TGTACTACIT

ACATTTTAAC TACATAATGT TAAC'TTATAT AATATTTATT GAATTCAGTA TAGGGCACAT

GCCCTATCCA TGACTAACGT GAGTCCGCCC TGATAGCGAG TAAGAAACGA

SS
GCAAATTATG TGAATACTAT ACACCAACAG TGTAGACATG TAGCfACAAT GCGGCAATGT

TAAAA,ATAAA CTCAAATTGG TTTGGAGGGA ACAACCTAAT GCTTATAAGT ACACTTTTGT

GGTAAATACA ATCCAATTGA AAGTCTTTGT AGGTTTGGTT TGGTCCAATG AAATTGTATG

CGAGTTGAGT AAAATAACCT TAGTTCAAAA CATTAGATAT GTAATGGTCT AGATACGATG

GTAGCCAAAG ATTTGGGTTA AATTTAGAAT AAAGATCAAA GAACAAATGA CTGACTTCCT

TATGTGTGTT TGTTTATGTA AAGTCTTAAC TCGTGTCTTG CCAAATTAAT AAAAAGGTGC

ATTATACAAT GAACTAGAGC TTGTTCTCAT CAAAATT'I'IT AACATTAAAA TAAGTTATTA

AAATTTTGTT GATTATATAT GATTTCAATA TTAAATTATA TTTTTTGCTT ATAGCATCAA

AACTTCTTGG CACAGCTCCG AGTGTTACTG AAATGTTTGT GTGGCTCCAA TAGAAAAGTT
7$0 AATACCAATC ATGAATCATC AATATCGTTA CAAAATCAGT AATCAGTTTC ACCCACTCCG

AGTGTTAGTG AAATGTTTGT GTGGCTCCAT TAAAAAAGTT AATACCAATC ACCAATATCG

TTACAAAATT TGTAATCAGT TTCACCCACT TCTITTCGTC TTGTTACTCG ATATAGCCTC

ACCTTTCAAG TGTCTAATAT AACACTITI'T AACAAAATAT TACAAAACAA GAAAAATTAT

AGTAATTAGA TTAATCATAT GTAATCTATT ACATATAGTA ATTTGTATTA GTATCGTTTA

TTGTGTTACC ATTCAAACGG TATAATCTAT ATAATTATTA GCAAAACAAA ATATAGTTTT

AGTAATTAAT AAAATTAATA TAATGATAGT GATATTCAGA ATGTGGTATC TAATCATGTA

AAAATATATA ATAATAAAAT TAGATAAAGA AGAAATTGGT AAAAAATAAT

TAAAAGGAAA ACAAGCTGAT ACAAGCATTT CACCCAAAAA AAAACAAGGT

AAAAAGAATT AGTAACTTTG AGCTATTGCC ATATGTGTGG ACATTTAAGA TTTCTTGTAT

TTTAGAAATC TTATATATTT CCACAATATA TTTACTACTT TCCTTTATTA TTTGTGTTAA

WO 00!32780 - 3 - PCT/AU99101079 TCTCCCGAAC ATTATTATTT CAATACTATC TGAAAAACAC ATTTTTTTTT ATAAAATTTG

ATGACGTAGG CGAGTGGTTC TTTG'TTITTA CTATGTAGGC ACGACTTTGG TAACACCTAC

TAATTAACTG CCAAATTTTA AGTTTTGAGA AGTCGGAAGA GTTCAAACCA GTTTTAGGTT

TCGATATGTC TACATAGTTC AAAGATGATG TAGAGTGGAG GTTCTTTCTG CAATAGTTCA

ATCCGTATCG TAGGGGAGGA AAGATAGTTT TCATTTAGCA AAGAAAGTGA

AATGCAAAAG TAGCAAAGAC GCTCGTCATG CGGTACACGT GGCAATCTTG TCTTCAAAAC

GCAACGTTTT TATTCACATA TTTGG'ITI"IT TTGCATCACT CTCGTTTACC CCCA~~AA.AAA

AAAAATATCT GGCCCGACGA AGAAAAAGTA GATAGGCACA AAAAATAGAA

CGAGAAAAGG AAAAAAAAAA ATAGAAAGAG AAAACGCTTA GTATCTCCGG

CCAAACCTGA GGATCAAATT AGGGCACAAA GCCCTCTCGG AGAGAAGCCA

AAAACTAGAA ATCAAGCGAA TTGAGAACAA AAGTAGCCGA CAAGTCACCT

TCGCAACGGT CTCATCGAGA AAGCTCGTCA GCTTTCTGTT CTCTGTGACG CATCCGTCGC

TCTTCTCGTC GTCTCCGCCT CCGGCAAGCT CTACAGCTTC TCCTCCGGCG ATAAGTACGC

CTTTTCCTTA CCTGGGTTTT CATTTATTCC CCCTTTTATC TTCTGTTTTG TGCTCTTTTA

CTZTTCTGAG AAAA.TAA,AAA TAAAAAAACA ATTAATATAC CGTTTGGTTT TTTTCCGGCG

GATCTCTTGT TGTTTCTCGG TTCTGTGTTT GTTTGTGTTT TTTTCTGCGA CCATGATAGA

TACATGAGAT AACCAAATTT AAGGAAGAAC AATGTCGTGA AGAAGCTTTT TAGCTTCTTA

CTTTTGTTCA TTTCTCTCTC TATTTCTTAA AAAA.AAAAAT TCTGCATGGA TTTCATTATT

TCCTTGGAAA AAAATTGCAT GTCCTTCACG ATTTGTTTGA TACGATCTGA TGCGTGCTCG

ATGTTGTTGA GTGAAGTTTC AAGCCATCTT TGATTGTTTC TTACCTTTAG AGATTCCTTA

AGT1'TITGAA GAGTTAATTA TATATCACAA GACTAATGAT TAATGTCTCT TTTCAGAGTG

ATTAAAATTC ATTGGATCTC TCGGATTTGT ATGCAATGCA CTTACGGGAG ATCTATAGAG

TTGCTATGGG GTTAATGCTG AACAATGTAT ATATACCACA TTGTGCAGCT ATTGACTATA

TAAGACTATT GTTAATCTTC TATGAATTCC TATCTTTGCT GTGGACCTAT TACTTGGTGA

TTATCCAAAT TAGTGTITfT AATTGATTCA TATTITI'CAT ACACAGTAGT TTTGAATTTT

GGTAGCTTCA AAAAACTCAG CCTCACAATT AGTACTTACC GCACATATGC TACTTCAGTA

ACATAATCTG GTTATCGATT GCGATTCTTT GAATCACAAT CGTCGTGTGC TATATATATA

ACACCTTTTG CTGTACATAA ACTGGTCTAA TTTTAGACTA ATTAAATTTC ATTGTTCTCT

TGGATTTGTA TATGCACGTC CGGGAGATTT ATAAATAAAA TTAGTATGAG GTTAATGGTA

AAAAGGATCA AGAAGTTTGG TTTTAAATGT AAGCCACATT AATTGGGAAA CTATGACTAA

AAGATGAATT GGTAATATAT ACATAATATT TTTAACGAAT TTCTCTCCTT TTTATGGGAT

ATGCTATTTG AAGATTGTCC AAAGGTTTAT AGTTTCCCAC TCITGCAGTT ACACACATAG

ATTTGCCTCA TATTTATGTG ATTGTATATC AATTATCGCC CTTAATCTTA TCATCGTTGT

GTTCAATTAT GACTTTGTTC CTATTCGTTA AAATTGACAA TCCACAACCT CAATCTTTTG

ATTCACACAG CCTTGTTTCT TTGGTGCCTC TAGGAAATTG AAAATCCCAC AACACTTGTC

TTCATGTAAG AAATACCAAC CTCTTTGGTA CGGATCTATA ATGAATCAAT ATAATCCTAT

ATATAAGTTG TCAAAATTGA ATCTGGTGTA GTGTCTACTA CAACCCTCCA ATATAATAAC

CAAATGGTTG TAGTAGTTTG GCCATGTTGG TCAAGATCGC TGGCCGATTC TCACTTGATG

CATACTTTGT TAGGATTTGT TCACCCCTAG TTAGGTCCAG CCTTGGAATT GTCGAGACAC

CTGACTAGAA CTCCTGGTCT TAATTATGAT TTAATAAAGA AGAAGCCTTT TAGAACGTGG

AACCCTTAGT TACTCAGTTA CTCI°iTI'TGC ATACTTAGGT TGATGCAAAG
AGCTTAACTT

CACAATAGGA CTGATATCTA TTAACAAAAC AAATTAAGTG AAGTTTTGTC AAAATTGTTG

GATCTTCTAG GTCAATATGT AGTTTAGTTT TTATCTGTCT TAGTCGCTTC CTTCTATGGA

AGATATATTT ATAGATATGT GTGATAAGTT TCCTACTAAT ATTAGTTAGT GTAACTTCAA

GGGCAGAAAA CTCTTTACTT TTATTGCTTG ATTAATTTGG GGTTTAAATA TAGGAAATTG

GAACCTCACA GTTTCTATAA ACGAGTATGT AATTGAATGT GAAATAACAA AATGGAAGAC

CGGCTTCCTA TTCTTAGGAG TCTTTTGATA TTTGCAAAAA AAACATATAC AATAGAAATA

TGAGTTTTGT CTAAGACTCG GTCCATGTAT TTGGAGTTTG GCTTCCTCAT ACTTATGGTT

ATCTGGTTAC CGCCACATCA TCATTATCAT CTTATGGGTC ATCAATACTA GCTCTATCGC

TGGAAAAAAC CTTGTCCTCA AGGTTCATTG AAAAATCCGA AAAGTTTTCT CGTATATGTT

GATATGGTAT TAC'TTACAAA CAAAGAGCTG ATGTTACCAA TTTTGACACG AGATTACTAA

TGAACTCATG AAAGAGGCGT TTTTAAAAAA TTCTTTTTAA AACTGGGATA CAAAAAGAAA

AGAGGTAACT AATAATTTGA TACCATTGTT CGTAGTCCTG ATCAAATGTT ATAAGGGTAA

ACATGATAGA AAATAGAGGG TAAATAGGTT TTGTTCTTAT AATGGTTTTG ATAACACGCT

TTGTAAAGGA TATAGGTGTT TTTTGATGCT AAAAGTTGTG GTATGGATCA AAACCA.A,AAT

GGAAGCTCTG AATCTCTGAT AGAGGTTGCA ATTAGAATTA TATAAGTTAA TTTGCAAATG

AATTGGAAGC AGTCTTCCAC TATTTGCTAT TGTTAGGGAA GTCTTTCAGT TAATTTCAGA

AAATTAAGAG AAATATGACT TTCTAGACTC AGTCTGTGTA CTTGGAATTT TACTTCGGTT

TACTTCCATG TCATCACATT GTGGCTCATC AATATATGTG TGTATATACA TTCATGAGTA

lU TATATGATTT CTGGAAAAAT AAAAATTGCT TGTTTGCATT TAAGATTGGG GCTGCGTTTA

CATTTTATAT TGCATCAATT ATTTCAACAT AGATTCACAA ACATAAATGC ATAGAAACAA

TCTGGACAGT AGAGGCTTAT GTTTAGGGTT CTTATGTACC TTAACTAGTT TGAC°ITTAAG

TTAATCAAAG CCAGCGCTAT CACTAAACTT TATCTGTATG CC"I"ITGTATG ACTTTTCTTT

GAGGGAAAAT GTCATTTTCA ATCTGCCGAA ATATATAATA AATACATGTT AGCCCACATA

ATTCATTGGA TAACTAATCT TTGAGCAATT TTTGGTAAAT GTIZTGGTTC TTTTCITI'~'C

TTGAGAGAGA AAAAAAATAT CAGATATTAT TAAATATTGC TTACAAAGCT AAGAACAAGT

TAAAACTTTT TTGAAAAAGT GGAAATTCAG ATGTGCTACT GCTTAAACAT GAATATTAAG

ATTATTGTTT TTCTGAAATG TTACGAATAC TAGCGTGTTA TATATATGTA AAAGGTAAGG

TGTTCTCTCA ATGTTTCATA GTTTCCAGTG GCCTTTTCAA GGGTTAGCTA GTAGTTTTGA

TCCTAACATA TTTTTATTTT TTTTGTCATC TCTCCAGCCT GGTCAAGATC CTTGATCGAT

ATGGGAAACA GCATGCTGAT GATCTTAAAG CCTTGGTAAT ACAAACATTT TGAATCTTTT

CCCTGATGGA GTTTTATAAG GCGTAAATTT ACTATTAGTT TGCCGAGTGA TCCTAAATAT

AAAATGAGGT GGTGGCTCCA CATGCATTAT GCATACCGCA ATTTTCATAG CCCTTGTCTT

TTACCGCTTC TTCTGTCCCT TTTTCATGGG CAGGATCATC AGTCAAAAGC TCTGAACTAT

GGTTCACACT ATGAGCTACT TGAACTTGTG GATAGGTTAG TACTACTAAC TAAGACTATA

WO 00/32780 ! ~ - PCT/AU99/01079 TTTGCTCTCC ACCTTTGATT ACAAAGGAAT TAGTTTTTTT TTTGTCAAAC TATGAATATA

TGCAGCAAGC TTGTGGGATC AAATGTCAAA AATGTGAGTA TCGATGCTCT TGTTCAACTG

GAGGAACACC TTGAGACTGC CCTCTCCGTG ACTAGAGCCA AGAAGGTAAG TTGATTTCGT

AATGTCTACT CCTTTCTGAA TTTTGTTTGC TGAGAACAAC CGTGCTGCTT TTGTTTGTTG

CAGACCGAAC TCATGTTGAA GCTTGTTGAG AATCTTAAAG AAAAGGTCAG ATATTTGCTA

CCAATTTTAT TGTACATCAG ATATATCCTC TTCTGTGTTG TCTCTGTTAC TTTAAGTCTG
636t?
CTTAACGAGC TTGCACACAT ATTTGCAACT TTCTTCATAT GTT'ITGGATT CCAAATTCTG

AAGTTGTTAG GTTTAGAAAC TTGATCGGTA ATTGCTGAAC ATTTTGATCT TTAAATCAGG

AGAAAATGCT GAAAGAAGAG AACCAGGTTT TGGCTAGCCA GGTAACGAAA

CTAAAAATAT ATATGCATAA CTAATAAGCA CTGCGTGTTG TGTGTCCAAT GTCCATGTAC

ATGGACATAG ATACACACTC TTATGCTTGC AGATATATAT ATATATATAT ATAGTCAGTG

CATTTCAATC ATTCACTAGT TAGCACTTTC CTGGTCTTGT ATAGTTGTAT TCTAGACAAT

TCTTC1'CAAG ATTAGGGCAT TTTGGTTGTT GGTAGTTTGG TTTATTAGGG TTAGTGAGAT

TATTACTGAA TAAGAACAGA AATTTGATAA CGGCTGGTTA GAGTTAAGGG AAATCAGATG

AAGTTATTTT TTTATTTTTT ATCGAGTATA AATTACATGA TTGCTATATC ATTTTACTAA

ATTAAGAAAA AAAAATTCCG GTTGTTGGAC ATAACTAGGT TTTGGTTCTT CTTCTTCGTT

TTTTTCATGT TAAAGTGTTT AATTAGGTTT TGGTTCATTT GGAGATTTAG GAACCTTrTA

TAGTCTGGTT AAGTCTGGGT TTGGTAGAGA TTCAATAAGA TTTCTTGATT CTCTTCAGGT

TATGGTCTGG TTCAGTCTAG TI"TAGTTCAA TATTGGTTTC CTTGAAGGTT GTGTAAACGT

WO 00/32780 ' 8 ' PCT/AU99/O10'I9 TGTGTATATT TAAGTTAATC ACCTTTTAAC CAAAAAAAAA AGTTTATGGA CCGATTAGTT

TTZT1'TI"1'IT TTTTTTTTTG TGATGGTTAG GTTTGGATCC GAGTGGCTCA GTTCCAACTC

CAAGTGTCTA GAAGTAGTGC TACTTTTACA TGCTATATAT AGGTTAGATT ATAAATTATA

AACTGGTAAA AGATTATAGA TACTGCTTCC AAACTTAAAA GCTTAAACAT AAAGAACACA

CAAATTATGA GAAAAATAAC CTTCTGTAGT GTTI"I"ITAAT GGTTGTTATT TGGTGGTGTG

IS
AAAAAGATAT TCCTTGGATA GAAGACAAAA AGAGAAAGTG AATAGTGATT

ATTATCGTAC AGATGGAGAA TAATCATCAT GTGGGAGCAG AAGCTGAGAT

CCTGCTGGAC AAATCTCCGA CAATCTTCCG GTGACTC'TCC CACTACTTAA TTAGCCACCT

TAAATCGGCG GTTGAAATCA AAATCCAAAA CATATATAAT TATGAAGAAG

3a AAGATATGTA ATTATTCCGC TGATAAGGGC GAGCGTTTGT ACATCTTAAT ACTCTCTCTT

TGGGCAAGAG ACTTTGTGTG TGATACTTAA GTAGACGGAA CTAAGTCAAT ACTATCTGTT

TTAAGACAAA AGGTTGATGA ACTTTGTACC TTATTCGTGT GAGAATTGCA TCGAGATCTT

GAGTGTATGT GTTCTTCTCT TCTGTCAAAA ACTTGTGTTT GCTTCACAGT GAAGAAGCCT

(2) INFORMATION FOR SEQ ID NO: 2:
{i) SEQUENCE CHARACTERISTICS:
{A) LENGTH: 943 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii} HYPOTI~TICAL: NO
SS (iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A} ORGANISM: Arabidopsis thaliana WO 00132780 _ 9 _ PCT/AU99/01079 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
CGAGAAAAGG AAAAAAAAAA ATAGAAAGAG AAAACGCTTA GTATCTCCGG

CCAAACCTGA GGATCAAATT AGGGCACAAA GCCCTCTCGG AGAGAAGCCA

AAAACTAGAA ATCAAGCGAA TTGAGAACAA AAGTAGCCGA CAAGTCACCT

TCGCAACGGT CTCATCGAGA AAGCTCGTCA GCTTTCTGTT CTCTGTGACG CATCCGTCGC
TCTTCTCGTC GTCTCCGCCT CCGGCAAGCT CTACAGCTTC TCCTCCGGCG ATAACCTGGT

CAAGATCCTT GATCGATATG GGAAACAGCA TGCTGATGAT CTTAAAGCCT TGGATCATCA

GTCAAAAGCT CTGAACTATG GTTCACACTA TGAGCTACTT GAACTTGTGG ATAGCAAGCT

TGTGGGATCA AATGTCAAAA ATGTGAGTAT CGATGCTCTT GTTCAACTGG AGGAACACCT

TGAGACTGCC CTCTCCGTGA CTAGAGCCAA GAAGACCGAA CTCATGTTGA AGCTTGTTGA

GAATCTTAAA GAAAAGGAGA AAATGCTGAA AGAAGAGAAC CAGGTTTTGG

GGAGAATAAT CATCATGTGG GAGCAGAAGC TGAGATGGAG ATGTCACCTG

CTCCGACAAT CITCCGGTGA CTCTCCCACT ACTTAATTAG CCACCTTAAA TCGGCGGTTG

AAATCAAAAT CCAAAACATA TATAATTATG AAGAAAAAAA AAATAAGATA

CCGCTGATAA GGGCGAGCGT TTGTATATCT TAATACTCTC TCTTTGGCCA AGAGACTTTG
$40 TGTGTGATAC TTAAGTAGAC GGAACTAAGT CAATACTATC TGTTTTAAGA CAAAAGGTTG

(2) INFORMATION FOR SEQ ID NO: 3:
SS (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 196 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Arabidopsis thaliana (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
Met Gly Arg Lys Lys Leu Glu Ile Lys Arg Ile Glu Asn Lys Ser Ser Arg Gln VaI Thr Phe Ser Lys Arg Arg Asn Gly Leu Ile Glu 20 Lys Ala Arg Gln Leu Ser Val Leu Cys Asp Ala Ser Val Ala Leu Leu Val Val Ser Ala Ser Gly Lys Leu Tyr Ser Phe Ser Ser Gly Asp Asn Leu Vat Lys Ile Leu Asp Arg Tyr Gly Lys Gin His Ala b5 70 75 Asp Asp Leu Lys Ala Leu Asp His Gln Ser Lys Ala Leu Asn Tyr Gly Ser His Tyr Glu Leu Leu Glu Leu Val Asp Ser Lys Leu Val Gly Ser Asn Val Lys Asn Val Ser Ile Asp Ala Leu VaI Gln Leu Glu Glu His Leu Glu Thr Ala Leu Ser Val Thr Arg Ala Lys Lys Thr Glu Leu Met Leu Lys Leu Val Glu Asn Leu Lys Glu Lys Glu Lys Met Leu Lys Glu Glu Asn Gln Val Leu Ala Ser Gln Met Glu Asn Asn His His Val Gly Ala Glu Ala Glu Met Glu Met Ser Pro Aia Gly Gln Ile Ser Asp Asn Leu Pro Val Thr Leu Pro Leu Leu Asn 6 to 25 SEQUENCE LISTTNG
<110> CSIRO
<120> CONTROL OF FLOWERING
<130> FP10512 <140> PP7469 <141> 1998-12-03 <160> 27 <170> PatentIn Ver. 2.1 <210> 1 <221> 908 <212> DNA
<213> Brassica napus <400> 1 aaaagaaaga aataaaagca aaaaagagag aaaataaaag caaaaataag aaagaacaaa 60 aaacgctcag tatctccggc gagagctgaa ccgaaccgaa cctcaggatc aaattagggc 120 acaaagggtt ctcggagaca gaagccatgg gaagaaagaa actagagatc aagcgaattg 180 agaacaaaag tagccgacaa gtcaccttct ccaaacgacg caatggtctc atcgagaaag 240 ctcgtcagct ttcagttctc tgcgatgcat ccgtcgctct cctcgttgtc tcagcctctg 300 gcaagctata caacttctcc gccggcgatg acctggtcaa gatcgttgat cgatatggaa 360 aacaacatgc tgatgatcgt aaagctctgg atcttcagtc agaagctccg aagtatggtt 420 cacaccatga gctactagag cttgtcgaaa gtaagcttgt ggaatcaaat tctgatgtaa 480 gcgtcgattc cctcgttcag ctggagaacc accttgagac tgccctctcc gtaactagag 540 ctaggaagac agaactattg ttgaagcttg ttgatagcct caaagaaaag gagaaattgc 600 tgaaagaaga gaaccagggt ttggctagcc agatggagaa gaataatctt gcgggagccg 660 aagctgataa aatggaagtg tcacctggac agatctctga catcaattgt ccggtaactc 720 tcccactgct ttattagccc cttaagtcca aaacttgtga ctaaaaacaa aaataagtta 780 ccgaactatt cccctataag ggtgagcgtt gtatatcttc acactctctt ggctgagaga 840 ccctgtgtgt aaaaactacg gtttgatttg agtaaaaata tatatttaag aaaaaaaaaa 900 aaaaaaaa 908 <210> 2 <211> 847 <212> DNA
<213> Brassica napus <400> 2 ggcgacttta accgtacctc agaatcaaat tagggcacag agacctctcg gagacagaag 60 ctatgggaag aaaaaaacta gaaatcaagc gaatcgagaa aaacagtagc agacaagtca 120 ccttctgcaa acgacgcaac ggtctcatcg agaaagctcg tcagctttct gttctctgcg 280 7$0 - 12 - PCT/AU99/01079 aggcatctgt tgggcttctc gttgtctccg cctccgacaa actctacagc ttctcctccg 240 gggatagact ggagaagatc cttgatcgat atgggaaaaa acatgctgat gatctcaatg 300 ccctggatct tcagtcaaaa tctctgaact atagttcaca ccatgagcta ctagaacttg 360 tggaaagcaa gcttgtggaa tcaattgatg atgtaagcgt ggattccctc gttgagctag 420 aagatcacct tgagactgcc ctctctgtaa ctagagctcg gaaggcagaa ctaatgttaa 480 agcttgttga aagtctcaaa gaaaaggaga atctgctgaa agaagagaac caggttttgg 540 ctagtcagat tgagaagaaa aatcttgagg gagccgaagc tgataatata gagatgtcat 6004.
ctggacaaat ctccgacatc aatcttcctg taactctccc gctgcttaat taaccacctt 660 tactcggcgg ttaatcaaaa taagaaacat ataatctaaa gataacctat gtaggtttta 720 cttttcgcag cttaattaac cncctttact cggcggttaa tcgaaattaa aaacatataa 780 ttaacaaata acctatgtca gtttaacccc aaaaaaaaaa aaaaaaaaaa gaaaaaaaaa 840 aaanaaa 847 <210> 3 <211> 969 <212> DNA
<213> Brassica napus <400> 3 ctctggatca aattagggca cagagaccac ttggagacag aaaccatggg aagaaaaaaa 60 ctagaaatca agcgaattga gaacaaaagt agccgacaag tcaccttctc caaacgacgc 120 agcggtctca ttgagaaagc tcgtcagctt tctgttctct gcgatgcatc cgtcgcgctt 180 CtCgttgtCt CCtCCtCCgg caagctctac agcttctccg ccggtgataa cctggtcagg 240 atccttgatc gatatggaaa acagcatgct gatgatctta aagccctgaa tcttcagtca 300 aaagctctga gctatggttc acacaatgag ttacttgaac ttgtggatag caagcttgtg 360 gaatcaaatg tcggtggtgt aagcgtggac accctcgttc agctggaggg tgtccttgaa 420 aatgccctct ctctaactag agctaggaag acagaactaa tgttgaagct tgttgatagc 480 ctcaaagaaa aggagaagct gctgaaagaa gagaatcagg ctttggctgg ccagaaggag 540 aagaagaatc ttgcgggagc cgaagctgat aatatggaga tgtcacctgg acaaatctcc 600 gacatcaatc ttccggtaac tctcccactg cttaattagc caccgttaga cggggctgat 660 caaattaaaa aatccaaaac atacaactaa ataaataagc tttgttgttt ttcacccttg 720 aagggtgcac gttgtatatc tcaatactcc cttggctgag agattgtgtg tttactccta 780 tgttagatat aatgagtaaa ataaaaataa aaagatcttt gtaccttcgt cgagagagaa 840 ttgtagtgag tgtgcttgtg tgttcttttt ctcttctttg cttcatggcg aagaagccta 900 ccgtctaatt tgtaacggag acgtggccct ctctgccctt ttgtattcgt aattcctttg 960 tattaaaaa 969 <210> 4 <211> 868 <212> DNA
<213> Brassica napus <400> 4 aacgctcagt atctccggct agtggaaacc ggacctcaag atcaaattag ggcgcaaagc 60 actgttggag acagaagcca tggggaggaa gaaacttgaa atcaagcgaa ttgagaacaa 120 aagtagccga caagttacct tctctaaacg acgcaacggt ctcatcgaga aagctcgtca 180 WO 00/32780 _ 13 ~ PCTIAU99/01079 gctttccgtt ctctgtgacg catccgtcgc tcttcttgtc gtctccgcct ccgggaaact 240 ctacagcttc tcctccggtg ataacctggt caagatcctt gatcgatatg gaaagcaaca 300 tgatgatgat cttaaagcct tggatcgtca gtcaaaagct ttggactgtg gttcacacca 360 tgagctactg gaacttgtgg aaagcaagct tgaggaatca aatgtcgata atgtaagtgt 420 gggttccctg gttcagctgg aggaacacct tgagaacgcc ctctccgtaa caagagctag 480 gaagacagaa ctaatgttga agcttgtcga gaaccttaaa gaaaaggaga agttgctgga 540 agaggagaac catgttttgg ctagccagat ggagaagagt aatcttgtgc gagccgaagc 600 tgataatatg gatgtctcac caggacaaat ctccgacatc aatcttccgg taacgctccc 660 actgcttaat tagtcacctt taatcggcga ataaataaaa tccaaaacat ataactaaaa 720 caaacaagat gtgtaattat ccccttgtaa agggtgtacg ttgtataatc tatactctct 780 ctccggctcg agaggcttcg ggtgtaaaac tatttcagat ttatgtaaga tagaaaatct 840 atgcaagaca ctttcaaact taaaaaaa 868 <210> 5 <211> 792 <212> DNA
<213> Brassica napus <400> 5 caaaggcttc tcggagacag aagccatggg aagaaagaaa ctagagatca agcgaattga 60 gaacaaaagt agccgacaag tcaccttctc caaacgacgc aatggtctca tcgagaaagc 120 tcgtcagctt tcagttctct gcgatgcatc cgtcgctctt ctcgttgtct cagcctccgg 180 caagctttac aacttctccg ccggcgataa cctggtcaag atccttgatc gatatggaaa 240 acaacatgct gatgatctta aagctctgga tcttcagtca aaagctccga agtatggttc 300 acaccatgag ctactagagc ttgtcgaaag taagcttgtg gaatcaaatt ctgatgtaag 360 cgtcgactcc ctcgttcagc tggaggacca ccttgagact gccctctccg taactagagc 420 taggaagaca gaactaatgt tgaagcttgt tgatagcctc aaagaaaagg agaaattgct 480 gaaagaagag aaccagggtt tggctagcca gatggagaag aataatcttg cgggagccga 540 agctgataaa atggagatgt cacctggaca aatctctgac atcaatcgtc cggtaactct 600 ccgactgctt tattagccnc cttaagtcca=a~aacttgtga ctaaaaacaa aaataagtta 660 tcgaactatt cccctataag ggtgaacgtt gtatatcttc attctctctg gctgagagac 720 cccgtgtgta aaactatggt tagatttaag taaaaatata tatttaagac atactaaaaa 780 aaaaaaaaaa as 792 <210> 6 <211> 990 <212> DNA
<213> Brassica napus <400> 6 gggcacagag accacttgga gacagaaacc atgggaagaa aaaaactaga aatcaagcga 60 attgagaaca aaagtagccg acaagtcacc ttctccaaac gacgcagcgg tctcattgag 120 aaagctcgtc agctttctgt tctctgcgat gcatccgtcg cgcttctcgt tgtctcctcc 180 tccggcaagc tctacagctt ctccgccggt gataacctgg tcaggatcct tgatcgatat 240 ggaaaacagc atgctgatga tcttaaagcc ctgaatcttc agtcaaaagc tctgagctat 300 ggttcacaca atgagttact tgaacttgtg gatagcaagc ttgtggaatc aaatgtcggt 360 ggtgtaagcg tggacaccct cgttcagctg gagggtgtcc ttgaaaatgc cctctctcta 420 actagagcta ggaagacaga actaatgttg aagcttgttg atagcctcaa agaaaaggag 480 aagctgctga aagaagagaa tcaggctttg gctggccaga aggagaagaa gaatcttgcg 540 ggagccgaag ctgataatat ggagatgtca cctggacaaa tctccgacat caatcttccg 600 gtaactctcc cactgcttaa ttagccaccg ttagacgggg ctgatcaaat taaaaaatcc 660 aaaacataca actaaataaa taagctttgt tgtttttcac ccttgaaggg tgcacgttgt 720 atatctcaat actcccttgg ctgagagatt gtgtgtttac tcctatgtta gatataatga 780 gtaaaataaa aataaaaaga tctttgtacc ttcgtcgaga gagaattgta gtgagtgtgc 840 ttgtgtgttc tttttctctt ctttgcttca tggcgaagaa gcctaccgtc taatttgtaa 900 cggagacgtg gccctctctg cccttttgta ttcgtaattc ctttgtattt atccacaacg 960 catagaggtt gtcatggttt aaaaaaaaaa 990 <210> 7 <211> 780 <212> DNA
<213> Brassica napus <400> 7 ttagggcaca aaggcttctc ggagacagaa gccatgggaa gaaagaaact agagatcaag cgaattgaga acaaaagtag ccgacaagtc accttctcca aacgacgcaa tggtctcatc 120 gagaaagctc gtcagctttc agttctctgc gatgcatccg tcgctcttct cgttgtctca 180 gCCtCCggca agctttacdd CttCtCCCJCC ggcgataacc tggtcaagat ccttgatcga 240 tatggaaaac aacatgctga tgatcttaaa gctctggatc ttcagtcaaa agctccgaag 300 tatggttcac accatgagct actagagctt gtcgaaagta agcttgtgga atcaaattct 360 gatgtaagcg tcgactccct cgttcagctg gaggaccacc ttgagactgc cctctccgta 420 actagagcta ggaagacaga actaatgttg aagcttgttg atagcctcaa agaaaaggag 480 aaattgctga aagaagagaa ccagggtttg gctagccaga tggagaagaa taatcttgcg 540 ggagccgaag ctgataaaat ggagatgtca cctggacaaa tctctgacat caatcgtccg 600 gtaactctcc gactgcttta ttagccacct taagtccaaa acttgtgact aaaaacaaaa 660 ataagttatc gaactattcc cctataaggg tgaacgttgt atatcttcat tctctctggc 720 tgagagaccc ccgtgtgtaa actatggnta gatttaagta aaatatatnt ttaagacana 780 <210> 8 <211> 845 <212> DNA
<213> Brassica napus <400> 8 ctccggctag tggaaaccgg acctcaagat caaattaggg cgcaaagcac tgttggagac 60 agaagccatg gggaggaaga aacttgaaat caagcgaatt gagaacaaaa gtagccgaca 120 agttaccttc tctaaacgac gcaacggtct catcgagaaa gctcgtcagc tttccgttct 180 ctgtgacgca tccgtcgctc ttcttgtcgt CtCCgCC.tCC gggaaactct acagcttctc 240 ctccggtgat aacctggtca agatccttga tcgatatgga aagcaacatg atgatgatct 300 taaagccttg gatcgtcagt caaaagcttt ggactgtggt tcacaccatg agctactgga 360 acttgtggaa agcaagcttg aggaatcaaa tgtcgataat gtaagtgtgg gttccctggt 420 tcagctggag gaacaccttg agaacgccct ctccgtaaca agagctagga agacagaact 480 WO 00/32780 _ i 5 _ PCT/AU99/01079 aatgttgaag cttgtcgaga accttaaaga aaaggagaag ttgctggaag aggagaacca 540 tgttttggct agccagatgg agaagagtaa tcttgtgcga gccgaagctg ataatatgga 600 tgtctcacca ggacaaatct ccgacatcaa tcttccggta acgctcccac tgcttaatta 660 gtcaccttta atcggcgaat aaataaaatc caaaacatat aactaaaaca aacaagatgt 720 gtaattatcc ccttgtaaag ggtgtacgtt gtataatcta tactctctct ccggctcgag 780 aggcttcggg tgtaaaacta tttcagattt atgtaagata gaaaatctat gcaagacact 840 ttcaa 845 <210> 9 <211> 825 <212> DNA
<213> Brassica napus <400> 9 cggcgagagt tgaaaccgaa tctcaggatc aaattagggc acaaaggctt ctcggagaca 60 gaagccatgg gaagaaagaa actagagatc aagcgaattg agaacaaaag tagccgacaa 120 gtcaccttct ccaaacgacg caatggtctc atcgagaaag ctcgtcagct ttcagttctc 180 tgcgatgcat ccgtcgctct tctcgttgtc tcagcctccg gcaagcttta caacttctcc 240 gccggcgata acctggtcaa gatccttgat cgatatggaa aacaacatgc tgatgatctt 300 aaagctctgg atcttcagtc aaaagctccg aagtatggtt cacaccatga gctactagag 360 cttgtcgaaa gtaagcttgt ggaatcaaat tctgatgtaa gcgtcgactc cctcgttcag 420 ctggaggacc accttgagac tgccctctcc gtaactagag ctaggaagac agaactaatg 480 ttgaagcttg ttgatagcct caaagaaaag gagaaattgc tgaaagaaga gaaccagggt 540 ttggctagcc agatggagaa gaataatctt gcgggagccg aagctgataa aatggagatg 600 tcacctggac aaatctctga catcaatcgt ccggtaactc tccgactgct ttattagcca 660 ccttaagtcc aaaacttgtg actaaaaaca aaaataagtt atcgaactat tcccctataa 720 gggtgaacgt tgtatatctt cattctctct ggctgagaga ccccgtgtgt aaaactatgg 780 ttagatttaa gtaaaaatat atatttaaga catactaaaa aaaaa 825 <210> 10 <211> 891 <212> DNA
<213> Brassica napus a <400> 10 tccggctagt ggaaaccgga cctcaagatc aaattagggc gcaaagcact gttggagaca 60 gaagccatgg ggaggaagaa acttgaaatc aagcgaattg agaacaaaag tagccgacaa 120 gttaccttct ctaaacgacg caacggtctc atcgagaaag ctcgtcagct ttccgttctc 180 tgtgacgcat ccgtcgctct tcttgtcgtc tccgcctccg ggaaactcta cagcttctcc 240 tccggtgata acctggtcaa gatccttgat cgatatggaa agcaacatga tgatgatctt 300 aaagccttgg atcgtcagtc aaaagctttg gactgtggtt cacaccatga gctactggaa 360 cttgtggaaa gcaagcttga ggaatcaaat gtcgataatg taagtgtggg ttccctggtt 420 cagctggagg aacaccttga gaacgccctc tccgtaacaa gagctaggaa gacagaacta 480 atgttgaagc ttgtcgagaa ccttaaagaa aaggagaagt tgctggaaga ggagaaccat 540 gttttggcta gccagatgga gaagagtaat cttgtgcgag ccgaagctga taatatggat 600 gtctcaccag gacaaatctc cgacatcaat cttccggtaa cgctcccact gcttaattag 660 acttgtggaa agcaagcttg aggaatcaaa 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> 11 <211> 196 <212> PRT
<213> Brassica napus <400> 11 Met Gly Arg Lys Lys Leu Glu Ile Lys Arg Ile Glu Asn Lys Ser Ser Arg Gln Val Thr Phe Ser Lys Arg Arg Asn Gly Leu Ile Glu Lys Ala Arg Gln Leu Ser VaI Leu Cys Asp Ala Ser Val Ala Leu Leu Val Val Ser Ala Ser Gly Lys Leu Tyr Asn Phe Ser Ala Gly Asp Asp Leu Val Lys Ile Val Asp Arg Tyr Gly Lys Gln His Ala Asp Asp Arg Lys Ala Leu Asp Leu Gln Ser Glu Ala Pro Lys Tyr Gly Ser His His Glu Leu Leu Glu Leu Val Glu Ser Lys Leu Val Glu Ser Asn Ser Asp Val Ser Val Asp Ser Leu Val Gln Leu Glu Asn His Leu Glu Thr Ala Leu Ser Val Thr Arg Ala Arg Lys Thr Glu Leu Leu Leu Lys Leu Val Asp Ser Leu Lys Glu Lys Glu Lys Leu Leu Lys Glu Glu Asn Gln Gly Leu Ala 245 3.50 155 160 Ser Gln Met Glu Lys Asn Asn Leu Ala Gly Ala Glu Ala Asp Lys Met Glu Val Ser Pro Gly Gln Ile Ser Asp Ile Asn Cys Pro Val Thr Leu Pro Leu Leu Tyr <210> 12 <211> 196 <212> PRT
<213> Brassica napus <400> 12 Met Gly Arg Lys Lys Leu Glu ile Lys Arg Ile Glu Lys Asn Ser Ser Arg Gln Val Thr Phe Cys Lys Arg Arg Asn Gly Leu I1e Glu Lys Ala Arg Gln Leu Ser Val Leu Cys Glu Ala Ser Val Gly Leu Leu Val Val Ser Ala Ser Asp Lys Leu Tyr Ser Phe Ser Ser Gly Asp Arg Leu Glu Lys Ile Leu Asp Arg Tyr Gly Lys Lys His Ala Asp Asp Leu Asn Ala Leu Asp Leu Gln Ser Lys Ser Leu Asn Tyr Ser Ser His His Glu Leu Leu Glu Leu Val Glu Ser Lys Leu Val Glu Ser Ile Asp Asp Val Ser Val Asp Ser Leu Val Glu Leu Glu Asp His Leu Glu Thr Ala Leu Ser Val Thr Arg Ala Arg Lys Ala Glu Leu Met Leu Lys Leu Val Glu Ser Leu Lys Glu Lys Glu Asn Leu Leu Lys Glu G1u Asn Gln Val Leu Ala Ser Gin Ile Glu Lys Lys Asn Leu Glu Gly Ala Glu Ala Asp Asn Ile Glu Met Ser Ser Gly Gln Ile Ser Asp Ile Asn Leu Pro Val Thr Leu Pro Leu Leu Asn WO 00132780 - 1$ - PCTIAU99/01079 <210> 13 <211> 197 <212> PRT
<213> Brassica napus <400> 13 Met Gly Arg Lys Lys Leu Glu Ile Lys Arg Ile Glu Asn Lys Ser Ser Arg Gln Val Thr Phe Ser Lys Arg Arg Ser Gly Leu Ile Glu Lys Ala Arg Gln Leu Ser Val Leu Cys Asp Ala Ser Val Ala Leu Leu Val Val Ser Ser Ser Gly Lys Leu Tyr Ser Phe Ser Ala Gly Asp Asn Leu Val Arg Ile Leu Asp Arg Tyr Gly Lys Gln His AIa Asp Asp Leu Lys Ala Leu Asn Leu Gln Ser Lys Ala Leu Ser Tyr Gly Ser His Asn Glu Leu Leu Glu Leu Val Asp Ser Lys Leu Val Glu Ser Asn Val Gly Gly Val Ser Val Asp Thr Leu Val Gln Leu Glu Gly Val Leu Glu Asn Ala Leu Ser Leu Thr Arg Ala Arg Lys Thr. Glu Leu Met Leu Lys Leu Val Asp Sex Leu Lys Glu Lys Glu Lys Leu Leu Lys Glu Glu Asn Gln Ala Leu Ala Gly Gln Lys Glu Lys Lys Asn Leu Ala Gly Ala Glu Ala Asp Asn Met Glu Met Ser Pro Gly Gln Ile Ser Asp Ile Asn Leu Pro Val Thr Leu Pro Leu Leu Asn WO 00/327$0 ~ 1 ~ ~ PCTIAU99/01079 <210> 24 <211> 197 <212> PRT
<213> Brassica napus <400> 14 Met Gly Arg Lys Lys Leu Glu Ile Lys Arg Ile Glu Asn Lys Ser Ser Arg Gln Val Thr Phe Ser Lys Arg Arg Asn Gly Leu Ile Glu Lys Ala Arg Gln Leu Ser Val Leu Cys Asp Ala Ser Val Ala Leu Leu Val Val Ser Ala Ser Gly Lys Leu Tyr Ser Phe Ser Ser Gly Asp Asn Leu Val Lys Ile Leu Asp Arg Tyr Gly Lys Gln His Asp Asp Asp Leu Lys Ala Leu Asp Arg Gln Ser Lys Ala Leu Asp Cys Gly Ser His His Glu Leu Leu Glu Leu Val Glu Ser Lys Leu Glu GIu Ser Asn Val Asp Asn Val Ser VaI Gly Ser Leu Val Gln Leu GIu Glu His Leu Glu Asn Ala Leu Ser Val Thr Arg Ala Arg Lys Thr Glu Leu Met Leu Lys Leu Val Glu Asn Leu Lys Glu Lys Glu Lys Leu Leu Glu Glu G1u Asn His Val Leu Ala Ser Gln Met Glu Lys Ser Asn Leu Val Arg Ala Glu AIa Asp Asn Met Asp Val Ser Pro Gly Gln Ile Ser Asp Ile Asn Leu Pro VaI Thr Leu Pro Leu Leu Asn <210> 15 <211> 196 WO 00!32780 - 2 ~ _ PCT/AU99101079 <212> PRT
<213> Brassica napus <400> 15 Met Gly Arg Lys Lys Leu Glu Ile Lys Arg Ile Glu Asn Lys Ser Ser Arg Gln Val Thr Phe Ser Lys Arg Arg Asn Gly Leu Ile Glu Lys Ala Arg Gln Leu Ser Val Leu Cys Asp Ala Ser Val Ala Leu Leu Val Val Ser Ala Ser Gly Lys Leu Tyr Asn Phe Ser Ala Gly Asp Asn Leu Val Lys Ile Leu Asp Arg Tyr Gly Lys Gln His Ala Asp Asp Leu Lys Ala Leu Asp Leu Gln Ser Lys Ala Pro Lys Tyr Gly Ser His His Glu Leu Leu Glu Leu Val Glu Ser Lys Leu Val Glu Ser Asn Ser Asp Val Ser Val Asp Ser Leu Val Gln Leu Glu Asp His Leu Glu Thr Ala Leu Ser Val Thr Arg Ala Arg Lys Thr Glu Leu Met Leu Lys Leu Val Asp Ser Leu Lys Glu Lys Glu Lys Leu Leu Lys Glu Glu Asn Gln Gly Leu Ala Ser Gln Met Glu Lys Asn Asn Leu Ala Gly Ala Glu Ala Asp Lys Met Glu Met Ser Pro Gly Gln Ile Ser Asp Ile Asn Arg Pro Val Thr Leu Arg Leu Leu Tyr <210> 16 <211> 197 <212> PRT
<213> Brassica napus WO 00/32780 _ 2 Z _ PCT/AU99/01079 <400> 16 Met Gly Arg Lys Lys Leu Glu Ile Lys Arg Ile Glu Asn Lys Ser Ser Arg Gln Val Thr Phe Ser Lys Arg Arg Ser Gly Leu Ile Glu Lys Ala Arg Gln Leu Ser Val Leu Cys Asp Ala Ser Val Ala Leu Leu Val Val Ser Ser Ser Gly Lys Leu Tyr Ser Phe Ser Ala Gly Asp Asn Leu Val Arg Ile Leu Asp Arg Tyr Gly Lys Gln His Ala Asp Asp Leu Lys Ala &5 70 75 80 Leu Asn Leu Gln Ser Lys Ala Leu Ser Tyr Gly Ser His Asn Glu Leu Leu Glu Leu Val Asp Ser Lys Leu Val Glu Ser Asn Val Gly Gly Val Ser Val Asp Thr Leu Val Gln Leu Glu Gly Val Leu Glu Asn Ala Leu Ser Leu Thr Arg Ala Arg Lys Thr Glu Leu Met Leu Lys Leu Val Asp Sex Leu Lys Glu Lys Glu Lys Leu Leu Lys Glu Glu Asn Gln Ala Leu Ala Gly Gln Lys Glu Lys Lys Asn, Leu Ala Gly Ala Glu Ala Asp Asn 1&5 170 175 Met Glu Met Ser Pro Gly Gln Ile Ser Asp Ile Asn Leu Pro Val Thr Leu Pro Leu Leu Asn <210> 17 <211> 196 <212> PRT
<213> Brassica napus <400> 17 WO 00/32780 _ 22 _ PCTlAU99/01079 Met Gly Arg Lys Lys Leu Glu Ile Lys Arg Ile Glu Asn Lys Ser Ser Arg Gln Val Thr Phe Ser Lys Arg Arg Asn Gly Leu Ile Glu Lys Ala Arg Gln Leu Ser Val Leu Cys Asp Ala Ser Val Ala Leu Leu Val Val Ser Ala Ser Gly Lys Leu Tyr Asn Phe Ser Ala Gly Asp Asn Leu Val Lys Ile Leu Asp Arg Tyr Gly Lys Gln His Ala Asp Asp Leu Lys Aia Leu Asp Leu Gln Ser Lys Ala Pro Lys Tyr Gly Ser His His Glu Leu Leu Glu Leu Val Glu Ser Lys Leu Val Glu Ser Asn Ser Asp Val Ser Val Asp Ser Leu Val Gln Leu Glu Asp His Leu Glu Thr Ala Leu Ser Val Thr Arg Ala Arg Lys Thr Glu Leu Met Leu Lys Leu Val Asp Ser Leu Lys Glu Lys Glu Lys Leu Leu Lys Glu Glu Asn Gln Gly Leu Ala Ser Gln Met Glu Lys Asn Asn Leu Ala Gly Ala Glu Ala Asp Lys Met Glu Met Ser Pro Gly Gln Ile Ser Asp Ile Asn Arg Pro Val Thr Leu Arg Leu Leu Tyr <210> 18 <211> 197 <212> PRT
<213> Brassica napus <400> 18 Met Gly Arg Lys Lys Leu Glu Ile Lys Arg Ile Glu Asn Lys Ser Ser WO 00132780 _ 2 3 _ PCT/AU99/Ot 079 Arg Gln Val Thr Phe Ser Lys Arg Arg Asn Gly Leu Ile Glu Lys Ala Arg Gln Leu Ser Val Leu Cys Asp Ala Ser Val Ala Leu Leu Val Val Ser Ala Ser Gly Lys Leu Tyr Ser Phe Ser Ser Gly Asp Asn Leu Val Lys Ile Leu Asp Arg Tyr Gly Lys Gln His Asp Asp Asp Leu Lys Ala Leu Asp Arg Gln Ser Lys Aia Leu Asp Cys Gly Ser His His Glu Leu Leu Glu Leu Val Glu Ser Lys Leu Glu Glu Ser Asn Vai Asp Asn Val Ser Val Gly Ser Leu Val Gln Leu Glu Glu His Leu Glu Asn Ala Leu Ser Val Thr Arg Ala Arg Lys Thr Glu Leu Met Leu Lys Leu Val Glu Asn Leu Lys Glu Lys Glu Lys Leu Leu Glu Glu Glu Asn His Va1 Leu Ala Ser Gln Met Glu Lys Ser Asn Leu Val Arg Ala Glu Ala Asp Asn Met Asp Val Ser Pro Gly Gln Ile Ser Asp Ile Asn Leu Pro Val Thr Leu Pro Leu Leu Asn <210> 19 <211> 196 <212> PRT
<213> Brassica napus <400> 19 Met Gly Arg Lys Lys Leu Glu Ile Lys Arg Ile Glu Asn Lys Ser Sex Arg Gln Val Thr Phe Ser Lys Arg Arg Asn Gly Leu Ile Glu Lys Ala Arg Gln Leu Ser Val Leu Cys Asp Ala Ser Va1 Ala Leu Leu Val Val Ser A3a Ser Gly Lys Leu Tyr Asn Phe Ser Ala Gly Asp Asn Leu Val Lys Ile Leu Asp Arg Tyr Gly Lys Gln His Ala Asp Asp Leu Lys Ala Leu Asp Leu Gln Ser Lys Ala Pro Lys Tyr Gly Ser His His Glu Leu Leu Glu Leu Val Glu Ser Lys Leu Val Glu Ser Asn Ser Asp Val Ser Val Asp Ser Leu Val Gln Leu Glu Asp His Leu Glu Thr Ala Leu Ser Val Thr Arg Ala Arg Lys Thr Glu Leu Met Leu Lys Leu Val Asp Ser Leu Lys Glu Lys G1u Lys Leu Leu Lys Glu Glu Asn Gln Gly Leu Ala Ser Gln Met Glu Lys Asn Asn Leu Ala Gly Ala Glu Ala Asp Lys Met Glu Met Ser Pro Gly Gln Ile Ser Asp Ile Asn Arg Pro Val Thr Leu Arg Leu Leu Tyr <210> 20 <211> 197 <212> PFtT
<213> Brassica napus <400> 20 Met Gly Arg Lys Lys Leu Glu Ile Lys Arg Ile Glu Asn Lys Ser Ser Arg Gln Val Thr Phe Ser Lys Arg Arg Asn Gly Leu Ile Glu Lys Ala WO 00/32780 ~ 2 ~ ~ PCT/AU99101079 Arg Gln Leu Ser Val Leu Cys Asp Ala Ser Val Ala Leu Leu Val Val Ser A1a Ser Gly Lys Leu Tyr Ser Phe Ser Ser Gly Asp Asn Leu Val Lys Ile Leu Asp Arg Tyr Gly Lys Gln His Asp Asp Asp Leu Lys Ala Leu Asp Arg Gln Ser Lys Ala Leu Asp Cys Gly Ser His His Glu Leu Leu Glu Leu Val Glu Ser Lys Leu Glu Glu Ser Asn Val Asp Asn Val Ser Val Gly Ser Leu Val Gln Leu Glu Glu His Leu Glu Asn Ala Leu Ser Val Thr Arg Ala Arg Lys Thr Glu Leu Met Leu Lys Leu Va1 Glu Asn Leu Lys Glu Lys Glu Lys Leu Leu Glu Glu Glu Asn His Val Leu Ala Ser Gln Met Glu Lys Ser Asn Leu Val Arg Ala Glu Ala Asp Asn Met Asp Val Ser Pro Gly Gln Ile Ser Asp Ile Asn Leu Pro Val Thr Leu Pro Leu Leu Asn -<210> 21 <211> 196 <212> PRT
<213> Brassica napus <400> 22 Met Gly Arg Arg Lys Ile Glu Ile Lys Arg Ile Glu Asn Lys Ser Ser Arg G1n Val Thr Phe Ser Lys Arg Arg Asn Gly Leu Ile Asp Lys Ala Arg Gln Leu Ser Ile Leu Cys Glu Ser Ser Val Ala Val Val Val Val WO 00/327$0 _ 2 6 ~ PCT/AU99/01079 Ser Ala Ser Gly Lys Leu Tyr Asp Ser Ser Ser Gly Asp Asp Ile Ser Lys Ile I1e Asp Arg Tyr Glu Ile Gln His Ala Asp Glu Leu Arg Ala Leu Asp Leu Glu Glu Lys Ile Gln Asn.Tyr Leu Pro His Lys Glu Leu Leu Glu Thr Val Gln Ser Lys Leu Glu Glu Pro Asn Val Asp Asn Val Ser VaI Asp Ser Leu Ile Ser Leu Glu Glu Gln Leu G1u Thr Ala Leu Ser Val Ser Arg Ala Arg Lys Ala Glu Leu Met Met Glu Tyr Ile Glu Ser Leu Lys Glu Lys Glu Lys Leu Leu Arg Glu Glu Asn Gln Val Leu Ala Ser Gln Met Gly Lys Asn Thr Leu Leu Ala Thr Asp Asp Glu Arg Gly Met Phe Pro Gly Ser Ser Ser Gly Asn Lys Ile Pro Glu Thr Leu Pro Leu Leu Asn <210> 22 <211> 296 <212> PRT
<213> Brassica napus <400> 22 Met Gly Arg Arg Lys Val Glu Ile Lys Arg Ile Glu Asn Lys Ser Ser Arg Gln Val Thr Phe Ser Lys Arg Arg Lys Gly Leu Ile Glu Lys Aia Arg Gln Leu Ser Iie Leu Cys Glu Ser Ser Ile Ala Val Val Ala Val Ser Gly Ser Gly Lys Leu Tyr Asp Ser Ala Ser Gly Asp Asn Met Ser WO OOI32780 _ 2 ~ _ PCT/AU99/01079 Lys Ile Ile Asp Arg Tyr Glu Ile His His Ala Asp Glu Leu Lys Ala Leu Asp Leu Ala Glu Lys Ile Arg Asn Tyr Leu Pro His Lys Glu Leu Leu Glu Ile Val Gln Ser Lys Leu Glu Glu Ser Asn Val Asp Asn Val Ser Val Asp Ser Leu Ile Sex Met Glu Glu Gln Leu Glu Thr Aia Leu Ser Val Ile Arg Ala Lys Lys Thr Glu Leu Met Met Glu Asp Met Lys Ser Leu G1n Glu Arg Glu Lys Leu Leu Ile Glu Glu Asn Gln Ile Leu Ala Ser Gln Val Gly Lys Lys Thr Phe Leu Val Ile Glu Gly Asp Arg Gly Met Ser Arg Glu Asn Gly Ser Gly Asn Lys Val Pro Glu Thr Leu Ser Leu Leu Lys <210>23 <211>200 <212>PRT

<213>Brassica napus <400> 23 Met Gly Arg Arg Lys Val Glu Ile Lys Arg Ile Glu Asn Lys Ser Ser Arg Gln Vai Thr Phe Cys Lys Arg Arg Asn Gly Leu Met Glu Lys A1a Arg Gln Leu Ser Ile Leu Cys Glu Ser Ser Val Ala Leu Ile Ile Ile Ser Ala Thr Gly Arg Leu Tyr Ser Phe Ser Ser Gly Asp Ser Met Ala WO OOI32780 _ 2 8 _ PCT/AU99/01079 Lys Ile Leu Ser Arg Tyr Glu Leu Glu Gln Ala Asp Asp Leu Lys Thr Leu Asp Leu Glu Glu Lys Thr Leu Asn Tyr Leu Ser His Lys Glu Leu Leu Glu Thr Ile Gln Cys Lys Ile Glu Glu Ala Lys Ser Asp Asn Val Ser Ile Asp Cys Leu Lys Ser Leu Glu Glu Gln Leu Lys Thr Ala Leu Ser Val Thr Arg Ala Arg Lys Thr Glu Leu Met Met Glu Leu Val Lys Thr His Gln Glu Lys Glu Lys Leu Leu Arg Glu Glu Asn Gln Ser Leu Thr Asn Gln Leu Ile Lys Met Gly Lys Met Lys Lys Sex Val Glu A1a 165 1?0 175 Glu Asp Ala Arg Ala Met Ser Pro Glu Ser Ser Ser Asp Asn Lys Pro Pro Glu Thr Leu Leu Leu Leu Lys <210> 24 <211> 198 <212> PRT
<213> Brassica napus <400> 24 Met Gly Arg Arg Arg Val Glu Ile Lys Arg Ile Glu Asn Lys Ser Ser Arg Gln Val Thr Phe Cys Lys Arg Arg Asn Gly Leu Met Glu Lys Ala Arg Gln Leu Ser Ile Leu Cys Gly Ser Ser Val Ala Leu Phe Ile Val Ser Ser Thr Gly Lys Leu Tyr Asn Ser Ser Ser Gly Asp Ser Met Ala Lys Ile Ile Ser Arg Phe Lys Ile Gln Gln Ala Asp Asp Pro Glu Thr WO00/32780 _ 29 _ PCTlAU99/0I079 Leu Asp Leu Glu Asp Lys Thr Gln Asp Tyr Leu Ser His Lys Glu Leu Leu Glu Ile Val Gln Arg Lys Ile Glu Glu Ala Lys Gly Asp Asn Val Ser Ile Glu Ser Leu Ile Ser Met Glu Glu Gln Leu Lys Ser Ala Leu Ser Val Ile Arg Ala Arg Lys Thr Glu Leu Leu Met Glu Leu Val Lys Asn Leu Gln Asp Lys Glu Lys Leu Leu Lys Glu Lys Asn Lys Val Leu Ala Ser Glu Val Gly Lys Leu Lys Lys Ile Leu Glu Thr Gly Asp Glu Arg Ala Val Met Ser Pro Glu Asn Ser Ser Gly His Ser Pro Pro Glu Thr Leu Pro Leu Leu Lys <210> 25 <211> 196 <212> PRT
<213> Brassica napus <400> 25 Met Gly Arg Lys Lys Val Glu Ile Lys Arg Ile Glu Asn Lys Ser Ser Arg G1n Va1 Thr Phe Ser Lys Arg Arg Asn Gly Leu Ile Glu Lys A7.a Arg Gln Leu Ser Ile Leu Cys Glu Ser Ser Lle Ala Val Leu Val Val Ser Gly Ser Gly Lys Leu Tyr Lys Ser Ala Ser Gly Asp Asn Met Ser Lys Ile Ile Asp Arg Tyr Glu Ile His His Ala Asp Glu Leu Glu Ala Leu Asp Leu Ala Glu Lys Thr Arg Asn Tyr Leu Pro Leu Lys Glu Leu WO 00/327$0 ~ 3 0 _ PCT/AU99/01079 Leu Glu Ile Val Gln Ser Lys Leu Glu Glu Ser Asn Val Asp Asn Ala Ser Val Asp Thr Leu Ile Ser Leu Glu Glu Gln Leu Glu Thr Ala Leu Ser Val Thr Arg Ala Arg Lys Thr Glu Leu Met Met Gly Glu Val Lys Ser Leu Gln Lys Thr Glu Asn Leu Leu Arg Glu Glu Asn Gln Thr Leu Ala Ser G1n Val Gly Lys Lys Thr Phe Leu Val Ile Glu Gly Asp Arg Gly Met Ser Trp Glu Asn Gly Ser Gly Asn Lys Val Arg Glu Thr Leu Pro Leu Leu Lys <210> 26 <211> 691 <212> DNA
<213> Brassica napus <400> 26 ggatcctatg tgaggttata gatccacgtg catatcaaat ttctcaaatt cgttgccttc 60 taacggctct tatatccctt ctccgtggac agaatcttca gtcaaaagct ctgagctatg 120 gttcacacaa tgagttactt gaacttgtgg ataggttagt actacctgag acttttttcc 180 ctctcctcct ttcattacaa aggtattagg gtttcttgtc aatctgtgca tatatatgca 240 gcaagcttgt ggaatcaaat gtcggtggtg taagcgtgga caccctcgtt cagctggagg 300 gtgtccttga aaatgccctc tctctaacta gagctaggaa ggtacgttga cttcatactg 360 tcttctcatt tcttactttg tttgttgaaa cgattgttca cttatattta atttgttgca 420 gacagaacta atgttgaagc ttgttgatag cctcaaagaa aaggttagat atatcatata 480 tgattttata gcacttcaga tatcttctcg tgtttgaaag cctcaaatat ttatgtgttg 540 tattaagttt ctctaagtgt gctttatgag ctcgcaatca aacttcttca taagtgcatc 600 tggtctttca nggatgatta aaaatattgt tttggatacc agaatctgaa aatangntta 660 aaaacttgca actgatgaac atgtccttca n 691 <210> 27 <211> 68 <212> PRT
<213> Brassica napus <400> 27 Asn Leu Gln Ser Lys A1a Leu Ser Tyr Gly Ser His Asn Glu Leu Leu Glu Leu Val Asp Ser Lys Leu Val Glu Ser Asn Val Gly Gly Val Ser Val Asp Thr Leu Va1 Gln Leu Glu Gly Val Leu GIu Asn Ala Leu Ser Leu Thr Arg Ala Arg Lys Thr Glu Leu Met Leu Lys Leu Val Asp Ser Leu Lys GIu Lys

Claims (22)

CLAIMS:

1. An isolated nucleic acid molecule comprising a MARS
box, which is capable of altering the flowering time of a plant, and which comprises (a) the nucleotide sequence set out in any one of SEQ ID NOS. 1, 2, 4, and 6 to 15;
(b) a nucleic acid molecule capable of hybridizing to a sequence set out in (a), other than to the MADS box region thereof, under at least low stringency hybridization conditions; 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).

2. A nucleic acid molecule according to Claim 1, in which the nucleic acid molecule is (a) capable of hybridizing to a nucleotide sequence as set out in any one of SEQ ID
NOS: 1, 2, 4, and 6 to 15 under high stringency hybridization conditions; or (b) has at least 80% sequence identity with a sequence set out in Claim 1(a).

3. An isolated nucleic acid molecule according to Claim 1 or Claim 2, in which 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.

4. An isolated nucleic acid molecule according to Claim 1 or Claim 2, which is capable of accelerating the flowering of a plant.

5. An isolated nucleic acid molecule according to Claim 4, in which 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.

6. An isolated nucleic acid molecule according to Claim 1 or Claim 2, which comprises a nucleotide sequence corresponding to a FLOWERING LOCUS F (FLF) gene, or a PCR primer or a biologically active fragment derived therefrom.

7. A vector comprising a nucleic acid molecule according to any one of Claims 1 to 6.

8. A plant cell transformed with a nucleic acid according to any one of Claims 1 to 6.

9. A plant transformed with a nucleic acid molecule according to any one of Claims 1 to 6.

10. 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 according to any one of Claims 1 to 6, or a functional portion thereof, as a hybridisation probe or polymerase chain reaction (PCR) primer, and optionally detecting hybridisation.

11. A method according to Claim 10, in which the nucleic acid molecule is capable of hybridizing to a nucleotide sequence as set out in any one of SEQ ID NOS: 1, 2, 4, and 6 to 15 under at least low stringency hybridization conditions, and the nucleic acid molecule does not include a MARS box region.

12. A method of delaying flowering in a plant, comprising the step of introducing a nucleic acid molecule according to any one of Claims 1 to 6 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 nucleic acid molecule.

13. A method of inducing early flowering in a plant, comprising the step of reducing the degree of expression of a nucleic acid molecule according to any one of Claims 1 to 6 in the plant.

14. 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.

15. A method of modifying the response of a plant to vernalisation, comprising the step of increasing or decreasing the level of expression of an FLF gene.

16. A method according to Claim 14 or Claim 15, in which the FLF gene comprises a nucleic acid molecule according to any one of Claims 1 to 6.

17. A polypeptide encoded by a nucleic acid molecule according to any one of Claim 1 to 6.

18. An FLF polypeptide, comprising the amino acid sequence set out in any one of SEQ ID NOS: 3,5, and 16 to 30, or having at least 70% sequence identity thereto.

19. An antibody directed against a polypeptide according to Claim 17 or Claim 18.

20. A method of assaying the level of expression of FLF
polypeptide, comprising the step of using an antibody according to Claim 19.

21. A method of selecting plants with low or high levels of expression of FLF, comprising the step of determining the level of FLF mRNA or FLF polypeptide in the plant.

22. A method according to Claim 20, in which the plants are members of a naturally-occurring population.