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  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
  • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
  • C12N15/8262—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield involving plant development
  • C12N15/827—Flower development or morphology, e.g. flowering promoting factor [FPF]
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
  • C12N15/8218—Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]

Definitions

• This invention relates to the control of the time of flowering in plants by genetic engineering. Specifically, this invention relates to the control of the timing of flowering by manipulation of the activity of the FLOWERING LOCUS C (FLC) family of genes.
• FLC FLOWERING LOCUS C
• day-neutral species of tobacco flower after producing a specific number of nodes and thus could be classified as flowering entirely through an autonomous pathway, but grafting studies indicate that day-neutral and photoperiod-responsive tobacco species respond to similar translocatable flowering signals (Lang et al., Proc. Natl. Acad. Sci., USA, 74, 2412-2416, 1977; McDaniel et al., Plant J., 9, 55-61, 1996). Thus aspects of the underlying biochemistry of these pathways appear to be conserved. Genetic analyses in several species has identified genes that affect the timing of flowering. The most extensive genetic analysis of flowering-time genes has been performed in Arabidopsis thaliana.
• flowering-time genes have been identified by two approaches.
• One approach has been to induce mutations that affect flowering time in early-flowering varieties. Such mutations can cause either late- flowering or even earlier flowering.
• Late-flowering mutations identify genes whose wild-type role is to promote flowering and early-flowering mutations identify inhibitory ones.
• Studies in Arabidopsis have identified over 20 loci for which mutations specifically affect flowering time and several other loci that affect flowering time as well as other aspects of development (e.g., det2, copl, gal and phyB) (Koornneef et al., Ann. Rev. Plant Physiol, Plant Mol Biol. , 49, 345-370, 1998; Weigel, Ann. Rev.
• Another group of late- flowering mutants exhibit a slight or no difference in flowering time when grown in short days compared to long days. Furthermore, this group shows little or no response to vernalization. Double mutants within a group do not flower significantly later than either single-mutant parent, whereas double mutants containing a mutation in each group flower later than the single-mutant parents (Koornneef et al., Genetics, 148, 885-92, 1998). Thus, there appears to be parallel flowering pathways that mediate the flowering response to environmental and developmental cues. A photoperiod pathway promotes flowering in long days.
• a pathway referred to in the literature as autonomous appears to control the age, or more specifically the developmental stage, at which plants are competent to flower. Recent support of the developmental role of this pathway is the demonstration that autonomous pathway mutants exhibit changes such as alterations of trichome patterns that indicate such mutant plants are delayed in the juvenile to adult transition (Telfer et al., Development, 124, 645-654, 1997).
• VFR2 vernalization-responsive flowering time in rapa 2
• VFR2 appears to correspond to FLC from Arabidopsis: VFR2 was mapped at high resolution using hybridization probes that permit a comparison of Arabidopsis and Brassicas after introgression of the late allele into the early-flowering annual variety, and only a probe corresponding to FLC detected no recombination events with VFR2 ( ⁇ 0.44cm) indicating that VFR2 is an FLC homolog.
• the timing of flowering is of great importance in agriculture and horticulture.
• the product In horticultural crops the product is often the flowers.
• food, feed crops, or fiber crops such as the cereals rice, wheat, maize, barley, and oats, and dicots such as soybeans, canola, and cotton, sunflower, tomato, broccoli, and other members of the legume family, the product is often flowers or the result of flowering — fruits, seeds, or seedpods. Understanding the molecular basics of flowering-time control will lead to strategies to optimize flower, fruit, and seed production by genetic manipulations that modify the timing of flowering.
• the present invention encompasses a gene family for the FLOWERING LOCUS C (FLC) genes that are one of the significant controlling factors in the repression of flowering.
• the invention includes the DNA sequences for these genes as well as the expressed polypeptides and proteins from these genes.
• the present invention is also directed at transgenic plants which have altered flowering characteristics from non-transgenic plants of the same species due to the presence of a transgene which affects the level or timing of FLC protein activity in the transgenic plants.
• the timing of flowering can be made earlier or later, depending on the desires of the plant breeder, by changes to the levels of the FLC genes in the plants. This permits plants to be modified in a very useful way. Since flowering is an important physiological stage for a flowering plant, the ability to manipulate flowering time in a plant species makes it possible either to increase vegetative growth or flower creation by a plant, whichever is more desired in the instance.
• FIGURE 1 is a phylogenic diagram of the relative degree of relatedness among members of the MADS box class of plant genes.
• This disclosure is directed at the nucleotide and protein sequences for the genes of the FLOWERING LOCUS C (FLC) family of plant genes.
• FLC FLOWERING LOCUS C
• the FLC genes are, however, similar and homologous. It is disclosed here that these genes can be used to make transgenic plants that have altered flowering characteristics.
• FLC genes described here it becomes possible to both advance flowering time in a transgenic plant, by repressing FLC activity, or to retard flowering time in a plant, by increasing FLC activity in a plant. This thus gives plant breeders and creators a unique tool so as to sculpt the flowering time characteristics of a crop plant to more closely follow the desires of the breeder.
• the FLC genes are part of a category of genes referred to as MADS box genes.
• the MADS box is a highly conserve motif shared by a group of evolutionarily related transcription factors.
• the name MADS is an acronym for the original genes first identified as sharing the common MADS box domain.
• MADS box genes There are a large number of MADS box genes which have been identified so far, and there is ongoing work to further organize these genes into sub-groups. In plants, the MADS box genes are known to affect many aspects of plant development, including structural development.
• Two of the FLC genes identified here, (Arabidopsis FLC2 and FLC3) have been previously sequenced as a part of the Arabidopsis genome sequencing effort, although their function was not previously known.
• Alvarez-Buylla UC San Diego , is a phylogenic tree illustration of the relative degree of relationship among MADS-box proteins so far identified from Arabidopsis and maize. Note that the three identified FLC genes are all more closely related to each other than they are related to any other MADS box genes.
• sequence listing is the cDNA sequence, and the deduced amino acid sequence, for each of FLC 1, FLC2 and FLC3 from Arabidopsis thaliana, as well as BrFLCl A and BrFLClB from Brassica rapa. Also presented below is some sequence comparison data.
• FLC2 to the Brassica genes is slightly less than 50% outside of the MADS box, so among the variants in the FLC gene family, identity at the amino acid levels is believed to generally be above 40%. Thus, amino acid identity of over 40% outside the MADS box regions is one indication of a member of the FLC gene family.
• Fig. 1 Note again the phylogenic chart of Fig. 1. It is significant that the three identified FLC genes from Arabidopsis are all more closely related to each other than to any other Arabidopsis MADS box genes. Since it is believed that the full spectrum of MADS box genes used by the plant is known, (at least in Arabidopsis from the nearly complete genome sequencing effort), one can chart out, using commonly available sequence analysis and matching software, the relatedness of any newly sequenced gene to the members of the MADS box family of genes as shown on Fig. 1. A member of the FLC gene family is a gene which maps by phylogenic analysis to be closer to Arabidopsis FLC1, FLC2 or FLC3, by phylogenic sequence analysis, than to any other MADS box gene from Arabidopsis.
• a way of confirming that a gene is a member of the FLC gene family is by testing the gene for effect on the timing of plant flowering. Described in the examples below are tests to demonstrate that the FLC genes do, in fact, act to delay flowering in transgenic plants. By testing a possible FLC gene for similar effect in transgenic plants, using Arabidopsis as a model, the activity of a putative FLC gene from another plant species can be confirmed.
• extra copies of the gene can be introduced into the plant, preferably by germ line transformation of the plant genome, and by properly choosing the strength and characteristics of the plant promoter chosen, the level of activity of the protein produced by the native gene can be increased in the cells and tissues of the plant.
• transgenic plant As is known to those of skill in the art, one needs to make a genetic construction capable of expressing an inserted protein coding sequence, whether foreign or endogenous, in a plant. One also needs a method to insert the genetic construction into the plant.
• Any genetic construction intended to cause the synthesis in the cells of the plant of a polypeptide or protein must include a sequence of DNA known as a protein coding sequence, which specifies the sequence of the polypeptide or protein to be produced in the resultant plant.
• a protein coding sequence to be expressed in a plant to produce a polypeptide or protein it must be placed under the control of a plant expressible promoter and be followed by a plant transcriptional terminator sequence, also known as a polyadenlyation sequence.
• the plant expressible promoter is a promoter which will work in plants, usually either of plant origin or from a plant pathogen like a virus (e.g.
• Cauliflower mosaic virus or a bacteria
• Plant promoters from pathogens tend to be constitutive promoters, meaning that they actually express the protein coding sequence in all of the tissues of the plant at all times.
• Other plant promoters are known to be tissue specific (e.g. to fruit or to flower) or developmentally specific (e.g. to stage of plant life such as emergent specific or senescent specific), while others are intended to be inducible (e.g. heat shock or metal ion induced promoters). Any of these types of promoters may by used in the practice of this invention depending on the intended affect on the transgenic plant to be produced.
• the genetic construction can be one intended to lower endogenous levels of protein activity without producing a protein in the plant.
• One well-known method to accomplish this is through the use of antisense technology, in which a genetic construct is created which causes the synthesis in the cells of the plant of an mRNA strand complementary to some portion of the mRNA created during the expression of the target gene. The antisense RNA interferes with the translation of the target mRNA and less protein is produced in the affected plant cells.
• Agrobacterium-mediated transformation or accelerated particle mediated transformation.
• the various techniques of Agrobacterium-mediated plant transformation make use of the natural ability of the plant pathogens of the Agrobacterium genus to transfer DNA from a plasmid in the bacteria into the genome of a plant cell.
• Particle-mediated plant transformation techniques utilize DNA-coated small carrier particles accelerated from a device, often referred to as a gene gun, into the cells of a plant. The full implementation of either approach requires techniques to recover a fully mature, morphologically normal plant from the transformed cells.
• transgenic plants have been worked out for many plant species and many, and perhaps all, of the economically important plant species.
• Other techniques, such as electroporation have also been used to make transgenic plants.
• the particular technique of plant transformation does not matter. Once the plant has been genetically engineered, and a transgenic plant has been created, the method of transformation of the original plant becomes irrelevant. A transgene inserted into the genome of one plant is then fully inheritable by progeny plants of the original genetically engineered plant by normal rules of classical plant breeding.
• transgene is here used to apply to an inserted genetic construction carried in the cells of a target plant.
• transgenic plant refers to a plant that carries such a transgene.
• FLC genes Disclosed here is information about a set of plant genes, the FLC genes. While these genes have existed previously in their native, or altered, state in plants, this disclosure is believed to be the first disclosure of these genes in isolated form. By isolated form, it is meant that the genes have been isolated from their host plants. Now the information from those genes becomes available for use in in vitro manipulations of the genes and their components to create genetic constructions for several uses.
• One contemplated use is the creation of transgenic plants.
• Another contemplated use is the diagnosis and analysis of plants, both transgenic and non-transgenic, to analyze and determine their pattern of FLC gene activity as an aid to breeding or creating plants having desired flowering time characteristics.
• plants that lack FLC activity flower much earlier than those containing an active FLC allele.
• Plants with wild type FLC activity had about 6 times the number of leaves as the plants with decreased FLC activity.
• plants genetically engineered to express FLC at higher than normal levels exhibit substantially delayed flowering. While the examples set forth below are executed in Arabidopsis, due to the simplicity in the genetic manipulation of that plant, the same techniques will work in other plants species. In fact, the high degree of sequence identity among the FLC genes indicates that the members of the FLC gene family from one plant species will function, as a general rule, even in other plants species.
• the flower time regulation FLC gene is a repressor of flower initiation in plants.
• the flower inititation polynucleotide fragment of FLC plays a central role in flower initiation control because other genes that regulate flowering act by modulating the activity of this gene.
• This gene is, however, not the sole factor in determing flowering time in a plant.
• FRIGIDA FRI
• the locus denominated FRIGIDA FRI
• the gene LUMINIDEPENDENS which acts to promote flowering, does so by decreasing FLC levels.
• mutants that lack LUMINIDEPENDENS activity which are delayed in flowering, FLC levels are substantially increased.
• modulating a flower initiation regulator is a reliable means to control the time of flower initiation.
• FRI acts to increase FLC levels.
• the two genes FLC and FRI are two dominant alleles which interact to cause late flowering.
• lower case e.g. fri, flc
• upper case e.g. FRI, FLC
• Both dominant alleles are normally required to achieve late flowering.
• plants which will be early flowering include the genetic combinations: fri/fri and flc 1 /flc 1 ; fri/fri and FLCl/flcl; fri/fri and FLC1/FLC1; FRI/fri and flcl/flcl; and FRI/FRI and flcl/flcl.
• Genetic combinations which will be late flowering will be: FRI/fri/ and FLCl/flcl : FRI/FRI and FLCl/flcl; FRI/fri and FLC1FLC1; and FIR FRI and FLCl/FLCl.
• the lifetime of a plant can be divided into at least two phases, the vegetative phase and the reproductive phase.
• the vegetative phase the plant continues growth, which includes increasing in size and in the number of leaves present on the plant.
• the reproductive phase begins with flower initiation. At that point much of the plant's further growth is the growth (or development) of flowers, fruits, and seeds.
• Commercially important crop plants have been bred for desirable characteristics, including uniformity in the time the plants are ready for harvesting. This has resulted in a high degree of uniformity in the number of leaves present on each plant in a population of plants grown under the same conditions.
• alterations in the time of flower initiation can often be measured as a function of the number of leaves on a plant. For instance, if flower initiation is activated early in a plant, that plant will have fewer leaves relative to the same plant grown under the same conditions that do not activate flower initiation early. Moreover, a plant that activates flower initiation early can also be said to have a shortened vegetative phase relative to the same plant grown under the same conditions that do not activate flower initiation early. Likewise, if flower initiation is repressed such that the plant undergoes flower initiation later, that plant will have more leaves relative to the same plant grown under the same conditions that do not repress flower initiation until later.
• a plant that represses flower initiation may also be said to have a prolonged vegetative phase relative to the same plant grown under the same conditions that do not repress flower initiation. Alterations in the time of flower initiation can also be measured as a function of time.
• Plants in which a copy of an FLC gene is introduced may also contain a wild- type (i.e., endogenous) flower time regulation coding region which acts to repress flower initiation.
• the FLC gene can act to augment the activity of an endogenous flower time regulation coding region to make flower initiation occur later.
• a second copy of a flower time regulation coding region can be introduced into a plant to increase the amount of flower time regulation FLC protein present in the plant. Expression of a portion of an FLC protein encoded by a portion of the flower time regulation coding region can also lead to activation of flowering in a plant.
• a portion of a polypeptide which leads to activation of flowering in a plant can be referred to as a dominant negative mutant, and is further described herein.
• the present invention also provides a genetically modified plant, characterized as having the phenotypic trait of altered time of flower initiation. By this it is meant that the modified plants of the present invention, whether modified by incorporating an FLC gene expressing a new or additional FLC protein in the plant, or by inhibiting activity of an endogenous FLC gene in the plant, demonstrate a different length of time to the onset of flower initiation relative to the same plant without the transgene inserted.
• flowering initiation (on average) in the transgenic plant occurs at least about 3 days, more preferably at least about 7 days, most preferably at least about 12 days after initiation of flowering in the same plant without the transgene.
• flowering initiation (on average) in the transgenic plant occurs at least about 3 days, more preferably at least about 7 days, most preferably at least about 12 days before initiation of flowering in the same plant without the transgene.
• the genetically modified plant and the same plant without the transgene are grown under the same conditions.
• the different length of time to the onset of the flowering stage of the plant relative to the same plant without the transgene can also be measured by determining the difference in the number of leaves on the genetically modified plant at the time of flower initiation and the number of leaves on the same plant without the transgene at the time of flower initiation.
• the transgenic plant exhibits at least about 10% more, more preferably at least about 50% more, most preferably at least about 80% more leaves at flower initiation than the same plant without the transgene.
• the transgenic plant exhibits at least about 10% fewer, more preferably at least about 50% fewer, most preferably at least about 80% fewer leaves at flower initiation than the same plant without the transgene.
• the genetically modified plant and the same plant without the transgene are grown under the same conditions.
• a nucleic acid molecule in one embodiment, includes a polynucleotide having a nucleotide sequence that represents the coding region of the gene, FLOWERING LOCUS C (FLC), from Arabidopsis thaliana, or a portion thereof, as well as allelic variants in sequence of the FLC gene and homologs of the coding region of the FLC gene derived from other species.
• Homology is a relatedness that can be determined by, but not limited to, nucleic acid hybridization techniques, computer searches of databases, computer or manual comparisons of amino acid and nucleotide sequences, and protein detection with the use of FLC-specific antibodies.
• Two nucleotide sequences are "similar” if they can be aligned so that a percentage of corresponding residues are identical.
• two nucleotide sequences have greater than about 31%, more preferably at least about 50%, even more preferably at least about 70%, and most preferably at least about 80% identity.
• the invention includes genes and proteins which are members of the FLC family of plant genes and share a significant level of primary structure with the sequence of FLC 1 presented below.
• the two amino acid sequences i.e., the amino acid sequence of the homo log and the sequence of FLC 1 are aligned such that the residues that make up the MADS domain, i.e., amino acids 1-60, are aligned in that region and then the entire length of the two amino acid sequences are further aligned to maximize the number of amino acids that they have in common along the lengths of their sequences.
• Gaps in either or both sequences are permitted in making the alignment in order to place the residues of the MADS domain in register and to maximize the number of shared amino acids, although the amino acids in each sequence must nonetheless remain in their proper order.
• the percentage amino acid identity is the higher of the following two numbers: (a) the number of amino acids that the two sequences have in common within the alignment, divided by the number of amino acids in FLC 1 , multiplied by 100; or (b) the number of amino acids that the two sequences have in common within the alignment, divided by the number of amino acids in the candidate polypeptide, multiplied by 100.
• a flower time regulation polypeptide has greater than 40% identity, more preferably at least about 50% identity, most preferably at least about 70% identity, over the entire length of the FLCl protein.
• Sequence homology or identity is less important at the nucleotide level. As is well understood by those in the art, the degeneracy of the genetic code permits many DNA protein coding sequences to code for the same amino acid sequence. It is even possible, and common, to alter the coding sequences of native protein coding sequences as a part of making plant expression cassettes, without altering the amino acids sequence of the resultant proteins, for various cloning convenience reasons or for reasons of codon usage or preference in the target plant.
• Isolated nucleic acid molecules of the invention can be obtained by several methods. For example, they can be isolated using procedures which are well known in the art. These include, but are not limited to: 1) hybridization of detectably labeled probes representing all or part of any one of the FLC genes to genomic or cDNA libraries to detect similar nucleic acid sequences; 2) antibody screening of expression libraries to detect similar structural features; 3) synthesis by the polymerase chain reaction (PCR); and 4) chemical synthesis of a nucleic acid molecule. Sequences for specific coding regions of genes can also be found in GenBank, the National Institutes of Health computer database. The coding region can then be isolated and ligated into a vector as described below.
• a polynucleotide e.g., a probe
• SDS sodium dodecyl sulfate
• 10 mM EDTA 10 mM EDTA
• a polynucleotide will hybridize to the nucleotide sequence set forth in SEQ ID NO: 1 under standard stringency hybridizing conditions.
• the polynucleotide e.g., probe
• the polynucleotide does not have to be complementary to all the nucleotides of the polynucleotide fragment as long as there is hybridization under the above-stated conditions.
• higher stringency conditions can be used, for example by increasing the temperature of the hybridization and wash steps to 65°C , 60°C , 55°C , or 50°C .
• the length of time required for hybridization can vary from 12 hours stated above.
• lower stringency hybridization conditions permit hybridization of related but not identical FLC genes, and thereby allow identification of FLC genes in other species.
• hybridization and washing temperatures are, in increasing order of preference, at 68 °C hybridization and 65 °C wash, 60° C hybridization and wash, 55 °C hybridization and wash, 50°C hybridization and wash, most preferably 45 °C hybridization and wash.
• Plants included in the invention are any flowering plants amenable to transformation techniques, including both monocotyledonous and dicotyledonous plants.
• monocotyledonous plants include, but are not limited to, vegetables such as asparagus, onions and garlic; cereals such as maize, barley, wheat, rice, sorghum, pearl millet, rye and oats; and grasses such as forage grasses and turfgrasses.
• dicotyledonous plants include, but are not limited to, vegetables, feed, and oil crops such as tomato, beans, soybeans, peppers, lettuce, peas, alfalfa, clover, Brassica species (e.g., cabbage, broccoli, cauliflower, brussel sprouts, rapeseed, and radish), carrot, beets, eggplant, spinach, cucumber, squash, melons, cantaloupe, sunflowers; fiber crops such as cotton; and various ornamentals such as flowers and shrubs.
• vegetables, feed, and oil crops such as tomato, beans, soybeans, peppers, lettuce, peas, alfalfa, clover, Brassica species (e.g., cabbage, broccoli, cauliflower, brussel sprouts, rapeseed, and radish), carrot, beets, eggplant, spinach, cucumber, squash, melons, cantaloupe, sunflowers; fiber crops such as cotton; and various ornamentals such as flowers and shrubs.
• Naturally occurring late flowering in Arabidopsis is caused primarily by the interaction of two dominant genes, FLCl (Lee, et al. Plant Journal 6, 903-909 1994; Koornneef, et al. Plant Journal 6, 911-919 1994) and FRIGIDA (FRI) (Lee, et al. Mol Gen Genet 237, 171-176 1993; Clarke and Dean. Mol Gen Genet 242, 81-89 1994).
• FLCl Lee, et al. Plant Journal 6, 903-909 1994; Koornneef, et al. Plant Journal 6, 911-919 1994
• FRIGIDA FRIGIDA
• Short days-FRI -containing wild type >100 Short days-flcl-3 44 (2.7)
• the lines described here were obtained from the Arabidopsis Biological Resource Center, Columbus, OH. Unless otherwise noted, the growth conditions were those generally known and used by a person of skill in the art. Due to the effect of vernalization on flower initiation, growth conditions did not include prolonged exposure of plants to cold temperatures (e.g., 0°C to 8°C ).
• the techniques used to genetically analyze Arabidopsis thaliana are know to a person of skill in the art (see, e.g., Koornneef et al., Genetic Analysis, In: "Arabidopsis Protocols," Methods in Molecular Biology Series, vol. 8, Martinez-Zapater et al., (eds.), Humana Press, Totowa, New Jersey, pp. 105-227, 1998).
• the F] plants generated from a cross of Eer (fri/fri ;flcl /flcl) to Col (fri/fri; FLCl /FLCl) were crossed to the tester line containing the FRI in Ler(FRI/FRI;flcl/flcl).
• This tester line contained a late-flowering allele of FRI, but also contains the flcl- Ler allele and is, therefore, early flowering.
• Plants containing recombination events between ngal58 and 151 were then tested with a third microsatellite marker nga249, which revealed that FLCl resided in the interval between nga249 and ngal 51.
• the region between nga249 and ngal 51 was contained within four yeast artificial chromosome (YAC) clones.
• YAC yeast artificial chromosome
• dCAPS derived cleaved amplified polymorphic sequence
• Sau3Al were used to create a library in the binary vector pPZP211 (Hajdukiewicz et al., 1994), and individual clones from this library were used to transform the FRI in Ler line.
• the library was constructed with DNA from the Col background, which contains a late-flowering allele of FLCl.
• FRI in Ler plants transformed with a construct containing the Col allele of FLCl will be late flowering.
• One of the clones from this library, 211-31 produced T, plants that were very late-flowering. Over one third of the plants underwent senescence without flowering after 8 months of growth.
• flcl-1 contains a 104 bp deletion that removes the start codon and flc 1-4 contains a 7 bp deletion that results in a frame shift after the first 20 amino acids
• flcl-1 contains a single-base transition at the first exon/intron junction that changes the conserved GT donor site to AT and presumably disrupts splicing.
• FLCl cDNA was isolated by RT- PCR from the Col background and the sequence is presented below in the sequence listing.
• transgenic Arabidopsis were created containing two different FLCl constructs.
• the first construct, 211-31 was made from genomic DNA containing FLCl and its native promoter.
• the second construct, pSM7 contained the genomic coding region of FLCl under control of the constitutive 35S promoter from cauliflower mosaic virus (Odell, et al. Nature 313, 810-2 1985.).
• 211-31 was transformed into FRI in Ler by Agrobacterium mediated transformation (Bechtold, et al., C.R. Acad. Sci. Paris, 316: 1194, 1993). Untransformed FRIin Ler flowers after forming approximately 14 primary rosette leaves(Lee, et al. Plant Journal 6, 903-909 1994.). FRI in Ler plants transformed with 211-31 showed a dramatic delay in flowering due to the synergistic interaction of FRI and FLCl to delay flowering (Table 3). Greater than 90% of transformants formed 50 or more leaves before flowering and 38% never flowered, even when grown under far-red-enriched light - conditions that strongly promote flowering in Arabidopsis.
• FLCl over- expression can prevent flowering altogether for the life of the plant. Due to the increased duration of the vegetative phase of development in plants transformed with 211-31, biomass was increased by 10 fold. This demonstrates that over-expression of an FLC gene is capable of turning an early flowering plant into a late flowering plant.
• pSM7 was transformed into wild-type Ler by Agrobacterium mediated transformation (Bechtold, et al., C.R. Acad. Sci. Paris, 316:1194, 1993) to determine the effect of constitutive expression of FLCl in a normally early-flowering line. The results are summarized in Table 5.
• transgenic plants obtained 30% were not significantly later than the Ler parent, 35% showed a moderate delay in flowering time, and 35% were quite late flowering.
• Part of the variation in flowering time of the transgenic plants may be due to differences in expression levels stemming from their insertion at different locations in the genome.
• FLCl expression is sufficient to substantially delay flowering.
• the delay in flowering caused by constitutive FLCl expression is insensitive to vernalization (vernalization is effective in promoting flowering in naturally-occurring late-flowering lines containing FRI and FLCl).
• vernalization is effective in promoting flowering in naturally-occurring late-flowering lines containing FRI and FLCl.
• FLCl homologs were isolated from Brassica rapa mRNA by RT-PCR using primers designed to the Arabidopsis FLCl sequence.
• the nucleotide sequences of the B. rapa homologs (BrFLCl A and BrFLClB) are presented in the sequence listing below.
• the amino acid identity between FLCl and BrFLCl A and BrFLClB is shown in Table 6.
• Overexpression constructs were created by placing BrFLCl A and BrFLClB under control of the constitutive 35S promoter. These constructs were transformed into an early-flowering strain of Arabidopsis and the flowering time of the Tl plants was determined.
• amino acids 1-60 are considered to be the MADS domain. Because of the high degree of conservation within the MADS domain between family members, similarity inside and outside of the MADS domain is presented.
• FLC2 and FLC3 Two other MADS domain genes, FLC2 and FLC3, with significant homology to FLCl. These genes were previously identifed by the Arabidopsis genome sequencing effort, although their function was unknown, and they are here designated FLC 2 and FLC 3. cDNA clones were obtained for FLC2 and FLC3 by RT-PCR. The nucleotide sequences of FLC2 and FLC3 are presented in the sequence listing below. The amino acid identity between FLCl and FLC2 and FLC3 is shown in Table 7.
• Loss-of-function alleles were identified in FLC2 by PCR based screening of a T-

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