WO2006020122A2 - Methods and compositions for modulating flowering time in plants - Google Patents

Abstract

The subject invention provides methods and compositions for modulating time to flowering in plants. The subject methods and compositions generally relate to modulating expression of certain gene products and variants thereof in plants, e.g., dicot or monocot plants. Transgenic plants in which expression of those gene products is reduced or increased are provided, as well as methods for modulating flowering time by reducing or increasing expression of those gene products. The invention finds use in a variety of agricultural and horticultural applications.

Description

METHODS AND COiViPOSlTIONS FOR MODULATING FLOWERING TIME IN

PLANTS

FIELD OF THE INVENTION [01] The field of this invention is modulation of flowering time in plants.

BACKGROUND OF THE INVENTION

[02] The transition from vegetative growth to flowering is a major developmental switch in a plant life cycle. The timing of this transition is critical for the plant's reproductive success. Accordingly, most plant species have evolved systems to precisely regulate flowering time. These systems monitor both environmental cues and the developmental state of the plant.

[03] Photoperiod and temperature are two environmental cues commonly monitored by plants. In plants responsive to photoperiod cues so examined, flowering is promoted by flowering signals which are translocated from leaves to meristems as leaves detect day length changes (e.g., Zeevaart, Light and the Flowering Process, 137-142 (Eds., D. Vince-Prue, B. Thomas and K. E. Cockshull, Academic Press, Orlando, 1984)). In temperature-responsive plants, exposure to cold temperatures promotes flowering by a process known as vernalization. Vernalization affects meristems directly, perhaps by causing them to become competent to perceive flowering signals (Lang, Encyclopedia of Plant Physiology, 15(Part 1): 1371 -1536, (ed., W. Ruhland, Springer-Verlag, Berlin, 1965)). Other environmental cues that can affect flowering include light quality and nutritional status.

[04] The developmental state of the plant can also influence flowering time. Most species go through a juvenile phase during which flowering is suppressed and then undergo a transition to an adult phase in which the plant becomes competent to flower (Poethig, Science, 250:923-930 (1990)). This "phase change" permits the plant to reach a proper size for productive flowering.

[05] Physiological analyses of the flowering timing mutants and the naturally occurring flowering timing variants indicate that flowering is controlled in by multiple pathways (e.g., Koomneef et al., Ann. Rev. Plant Physiol., Plant MoI. Biol., 49:345-370 (1998)). For example, plants containing one group of late-flowering mutants (fca, fpa, fve, fy, Id) and plants containing the late-flowering FLC and FRI alleles are delayed in flowering during inductive (long-day) conditions and more severely delayed during short-day conditions. Studies have shown that vernalization of these late-flowering lines can suppress the late-flowering phenotype. Another group of late-flowering mutants (co, fd, fe, fha, ft, fwa, gi) exhibit minimal or no difference in flowering time when grown in short days compared to long days. This group also shows little or no response to vernalization. Moreover, double mutants within a group do not flower 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)). A separate autonomous pathway appears to control the age or, more specifically, the developmental stage at which plants are competent to flower. This pathway is referred to as autonomous because mutations in this pathway do not affect the plant's photoperiod response. Recent studies of these mutations have shown changes in these mutants, such as alterations of trichome patterns, which indicate that such mutant plants are delayed in transitioning from the juvenile to adult states (Telfer et al., Development, 124:645-654 (1997)). Accordingly, parallel flowering pathways which mediate flowering time in response to environmental and developmental cues may exist.

Flowering time may be manipulated to increase plant output. For example, accelerating the onset of flowering in certain crops may permit those crops to be grown in regions where the growing season is otherwise too short, or permit multiple crops to be grown in regions where only one crop is currently possible. In addition, preventing or substantially delaying flowering will increase the yield of the useful parts of certain crops. For example, delaying or preventing flowering in forage crops (e.g., alfalfa and clover) and vegetables crops (e.g., cabbage and related Brassicas, spinach, and lettuce) should increase crop yields. Likewise, the yields of crops in which underground parts are used (e.g., sugar beets or potatoes), may also be increased by delaying or preventing flowering. In sugar beets, the prevention of flowering will also permit more energy to be devoted to sugar production. Likewise the yield of wood and biomass crops may also be increased by delaying flowering.

[07] Accordingly, there is a great need for new means for manipulating flowering time in plants. The invention described herein meets these needs, and others.

[08] Literature of interest includes: Genbank accession number AAF04909 and Lim et al.,

Plant Cell. 2004 16:731-40.

SUMMARY OF THE INVENTION

[09] The subject invention provides methods and compositions for modulating time to flowering in plants. The subject methods and compositions generally relate to modulating expression of certain gene products herein termed flowering time modulators and variants thereof in plants, e.g., dicot or monocot plants. Transgenic plants in which expression of those gene products is reduced or increased are provided, as well as methods for modulating flowering time by reducing or increasing expression of those gene products. The invention finds use in a variety of agricultural and horticultural applications.

BRIEF DESCRIPTION OF THE DRAWINGS

[10] Figs. 1A and 1B. flk is a late-flowering mutant. (A) 35-day-old plants of the wild-type

(WT) and three flk mutant alleles (flk-1, flk-2, flk-3) grown in long-day photoperiods (LD:18L/6D). (B) A comparison of the flowering-time of the wild type and the flk mutants. Results of two separate experiments are shown. In one experiment that includes flk1 and flk∑, the flk mutants failed to flower in SD when the experiment was terminated (more than 100 days after germination). In the other experiment, only flkS was included which flowered eventually in SD.

[11] Figs 2A-2D. The FLK gene encodes a KH-domain protein. (A) The FLK gene structure and the location of the T-DNA insertions in four different flk mutant alleles. Filled boxes represent exons of FLK, the numbers on the top or the bottom of the gene indicate nucleotide positions relative to the start codon ATG, relative positions of three KH domains are shown in the diagram underneath depicting the FLK protein. (B) FLK mRNA in the wild type and flk mutant alleles was analyzed by RT-PCR, the top and bottom panels show the RT-PCR results using primers specific to FLK or UBQ, respectively. (C) Transgenic plants expressing the 35S::GFP-FLK gene flowered earlier than the wild type. (D) GFP fluorescence images showing cellular localization of the GFP-FLK fusion protein.

[12] Figs. 3A-3B. The flk mutation affects FLC expression. (A). A representative RT-PCR result showing the expression of FLC, FT, and SOC1 in the wild type (WT) and the flk-1 mutant. Plant tissue was harvested shortly after light-on from 12-, 13-, 14- 15- or 22-day-old seedlings grown in LD photoperiods. (B) The lack of apparent effect of flk mutation on the mRNA stability of FLC. Seedlings of the wild type (WT) or flk mutant (flk) were treated with transcription inhibitors for up to 4 h (see Supplemental Materials and Methods). The FLC mRNA level was examined using RT-PCR at different time points (h) after incubation in transcription inhibitors.

[13] Figs. 4A and 4B. Q-PCR showing mis-expression of genes in the flk mutant. (A)

Real-time Q-PCR results showing relative expression levels of selected genes in wild type (white bar) and flk mutant plants (black bar) grown in continuous cool white fluorescent light for 16 days. Data represent the mean and standard deviation of three replicates. For each gene, the relative amount of calculated message was normalized to the level of the control gene ubiquitin (At5g15400). (B). Q-PCR results showing relative expression levels of six circadian clock-regulated genes in wild type (solid circles with solid line) and in flk mutant plants (solid triangles with dashed-line). Wild-type (CoI-O) and flk1 plants were entrained in 12:12 (Light: Dark) photoperiods for 7 days and then transferred to continuous light. Plant tissue was harvested in 4-hour increments from plants that had been in continuous light at the time indicated (see Supplemental Materials and Methods). Data represent the mean of four independent biological replicates. The relative expression of the indicated genes was normalized to the level of the control gene ubiquitin (At5g15400).

[14] Figs 5A - 5C Expression profiles of selected genes analyzed in three DNA microarray experiments. The flklWT score represents the relative expression levels of a gene in the wild type and the flk mutant. A flkNΨT score >1 indicates an higher expression of the respective gene in the flk mutant than in the wild type. A flk/WT score <1 indicates an lower expression in the flk mutant. A score with the false-discovery rate (FDR) < 20%, P-value < 0.05, and fold- change^ 1.3 represents one putative mis-expression event in the flk mutant and denoted by an asterisk. Genes showing the FLK-dependent expression change in all three microarray experiments (i.e. with three putative mis-expression events) are listed in (A). Representative genes associated with floral development or autonomous/vernalization control of flowering- time are listed in (B), regardless of their expression change. Representative genes associated with photoperiod regulation of flowering are listed in (C), regardless of their expression change. Samples used in three microarray experiments are: 16-day-old wild type and flk mutant seedlings grown in long days and harvested 16h after light-on (Exp. 1), 16-day- old seedlings grown in continuous light (Exp. 2), and 7-day-old seedlings grown in long days (18hL/6hD) and harvested 1h after light-on (Exp. 3). The entire array dataset can be found online at the gene expression omnibus, accession, GSE1512).

[15] Fig. 6. A comparison of the flowering time of the wild type and the flk mutants in response to vernalization. For the vernalization treatment, seeds were imbibed in 0.1% agarose at 4⁰C in a walk-in cooler for about 2 months. Most seeds germinated by the end of the cold treatment, and they were transplanted to grow in soil in LD. The flowering time was measured as described in the Materials and Methods.

[16] Fig. 7. Oligonucleotide sequence of primers used in the experiments described.

[17] Figs. 8A-8C Sequence comparison of KH-domain genes. (A) Amino acid sequence alignment of Arabidopsis FLK and other KH-domain proteins. (B) An amino acid sequence alignment of FLK and At3g32940. (C) A phylogenetic analysis of Arabidopsis KH-domain genes using the neighbor-joining Cluster W method. ^* indicates the two genes for which the expression is affected by the flk mutation. Conserved amino acid sequences are highlighted.

[18] Fig. 9. shows the sequences of an exemplary FLK nucleic acid and polypeptide.

[19] Fig. 10 shows the GenBank accession numbers and polypeptide sequences of flowering time modulators, along with a description of the traits that can be modulated by increasing or decreasing the expression of those sequences. The genes defined by these sequences may also be termed SCC (suppressor of cry1cry2 double mutant) genes. These genes were identified by transforming cry1cry2 double mutant plants (which grow tall and flower later) with an enhancer vector to produce a library. The library was screened for those that grow short and/or flower earlier. The genes identified in Fig. 10 are dominant, and there overexpression of these genes or an ortholog thereof confers a short status and/or early- flowering phenotype in a plant. DEFINITIONS

[20] The terms "nucleic acid molecule" and "polynucleotide" are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three-dimensional structure, and may perform any function, known or unknown. Non-limiting examples of polynucleotides include a gene, a gene fragment, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymβs, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. The nucleic acid molecule may be linear or circular.

[21] By "transformed" it is meant an alteration in a cell resulting from the uptake of foreign nucleic acid, usually DNA, as described in great detail herein. Use of the term "transformation" is not intended to limit introduction of the foreign nucleic acid to any particular method. Suitable methods include viral infection, transfection, conjugation, protoplast fusion, electroporation, particle gun technology, calcium phosphate precipitation, direct microinjection, and the like. The choice of method is generally dependent on the type of cell being transformed and the circumstances under which the transformation is taking place (i.e. in vitro, ex vivo, or in vivo). A general discussion of some of these methods can be found in Ausubel, et al, Short Protocols in Molecular Biology, 3rd ed., Wiley & Sons, 1995. Transgenic plants contain transformed cells, and, in most embodiments, are regenerated from transformed cells.

[22] A "coding sequence" or a sequence which "encodes" a selected polypeptide, is a nucleic acid molecule which is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide, for example, in vivo when placed under the control of appropriate regulatory sequences (or "control elements"). The boundaries of the coding sequence are typically determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3¹ (carboxy) terminus. A coding sequence can include, but is not limited to, cDNA from viral, prokaryotic or eukaryotic mRNA, genomic DNA sequences from viral or procaryotic DNA, and even synthetic DNA sequences. A transcription termination sequence may be located 3' to the coding sequence. Other "control elements" may also be associated with a coding sequence. A DNA sequence encoding a polypeptide can be optimized for expression in a selected cell by using the codons preferred by the selected cell to represent the DNA copy of the desired polypeptide coding sequence. A "polypeptide-coding sequence" may be a nucleic acid sequence of the chloroplast or nuclear genome, or may be a nucleic acid sequence of a vector.

[23] "Encoded by" refers to a nucleic acid sequence which codes for a polypeptide sequence, wherein the polypeptide sequence or a portion thereof contains an amino acid sequence of at least 3 to 5 amino acids, more preferably at least 8 to 10 amino acids, and even more preferably at least 15 to 20 amino acids from a polypeptide encoded by the nucleic acid sequence.

[24] "Operably linked" refers to an arrangement of elements wherein the components so described are configured so as to perform their usual function. Thus, a given promoter that is operably linked to a coding sequence (e.g., a reporter expression cassette) is capable of effecting the expression of the coding sequence when the proper enzymes are present. The promoter or other control elements need not be contiguous with the coding sequence, so long as they function to direct the expression thereof. For example, intervening untranslated yet transcribed sequences can be present between the promoter sequence and the coding sequence and the promoter sequence can still be considered "operably linked" to the coding sequence.

[25] By "nucleic acid construct" it is meant a nucleic acid sequence that has been constructed to comprise one or more functional units not found together in nature. Examples include circular, linear, double-stranded, extrachromosomal DNA molecules (plasmids), cosmids (plasmids containing COS sequences from lambda phage), viral genomes comprising non-native nucleic acid sequences, and the like.

[26] A "vector" is capable of transferring gene sequences to target cells. Typically, "vector construct," "expression vector," and "gene transfer vector," mean any nucleic acid construct capable of directing the expression of a gene of interest and which can transfer gene sequences to target cells. Thus, the term includes cloning, and expression vehicles, as well as integrating vectors.

[27] An "expression cassette" comprises any nucleic acid construct capable of directing the expression of a gene/coding sequence of interest. Such cassettes can be constructed into a "vector," "vector construct," "expression vector," or "gene transfer vector," in order to transfer the expression cassette into target cells. Thus, the term includes cloning and expression vehicles, as well as viral vectors.

[28] Techniques for determining nucleic acid and amino acid "sequence identity" also are known in the art. Typically, such techniques include determining the nucleotide sequence of the mRNA for a gene and/or determining the amino acid sequence encoded thereby, and comparing these sequences to a second nucleotide or amino acid sequence. In general, "identity" refers to an exact nucleotide-to-nucleotide or amino acid-to-amino acid correspondence of two polynucleotides or polypeptide sequences, respectively. Two or more sequences (polynucleotide or amino acid) can be compared by determining their "percent identity." The percent identity of two sequences, whether nucleic acid or amino acid sequences, is the number of exact matches between two aligned sequences divided by the length of the shorter sequences and multiplied by 100. An approximate alignment for nucleic acid sequences is provided by the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981). This algorithm can be applied to amino acid sequences by using the scoring matrix developed by Dayhoff, Atlas of Protein Sequences and Structure, M. O. Dayhoff ed, 5 suppl. 3:353-358, National Biomedical Research Foundation, Washington, D.C., USA, and normalized by Gribskov, Nucl. Acids Res. 14(6):6745-6763 (1986). An exemplary implementation of this algorithm to determine percent identity of a sequence is provided by the Genetics Computer Group (Madison, Wl) in the "BestFit" utility application. The default parameters for this method are described in the Wisconsin Sequence Analysis Package Program Manual, Version 8 (1995) (available from Genetics Computer Group, Madison, Wl). A preferred method of establishing percent identity in the context of the present invention is to use the MPSRCH package of programs copyrighted by the University of Edinburgh, developed by John F. Collins and Shane S. Sturrok, and distributed by IntelliGenetics, Inc. (Mountain View, CA). From this suite of packages the Smith-Waterman algorithm can be employed where default parameters are used for the scoring table (for example, gap open penalty of 12, gap extension penalty of one, and a gap of six). From the data generated the "Match" value reflects "sequence identity." Other suitable programs for calculating the percent identity or similarity between sequences are generally known in the art, for example, another alignment program is BLAST, used with default parameters. For example, BLASTN and BLASTP can be used using the following default parameters: genetic code = standard; filter = none; strand = both; cutoff = 60; expect = 10; Matrix = BLOSUM62; Descriptions = 50 sequences; sort by = HIGH SCORE; Databases = non-redundant, GenBank + EMBL + DDBJ + PDB + GenBank CDS translations + Swiss protein + Spupdate + PIR.

[29] The term "stringent hybridization conditions" as used herein refers to conditions that are compatible to produce binding pairs of nucleic acids, e.g., surface bound and solution phase nucleic acids, of sufficient complementarity to provide for the desired level of specificity in the assay while being less compatible to the formation of binding pairs between binding members of insufficient complementarity to provide for the desired specificity. Stringent assay conditions are the summation or combination (totality) of both hybridization and wash conditions.

[30] A "stringent hybridization" and "stringent hybridization wash conditions" in the context of nucleic acid hybridization (e.g., as in array, Southern or Northern hybridizations) are sequence dependent, and are different under different experimental parameters. Stringent hybridization conditions that can be used to identify nucleic acids within the scope of the invention can include, e.g., 0.2χSSC and 0.1% SDS at 65°C.

[31] A first polynucleotide is "derived from" a second polynucleotide if it has the same or substantially the same nucleotide sequence as a region of the second polynucleotide, its cDNA, complements thereof, or if it displays sequence identity as described above. [32] A first polypeptide is "derived from" a second polypeptide if it is (i) encoded by a first polynucleotide derived from a second polynucleotide, or (ii) displays sequence identity to the second polypeptides as described above.

[33] "Substantially purified" general refers to isolation of a substance (compound, polynucleotide, protein, polypeptide, polypeptide composition) such that the substance comprises the majority percent of the sample in which it resides. Typically in a sample a substantially purified component comprises 50%, preferably 80%-85%, more preferably 90- 95% of the sample. Techniques for purifying polynucleotides and polypeptides of interest are well-known in the art and include, for example, ion-exchange chromatography, affinity chromatography and sedimentation according to density.

[34] The term "exogenous" is defined herein as DNA which is introduced into a cell by the method of the present invention, such as with the DNA constructs defined herein. Exogenous DNA can possess sequences identical to or different from the endogenous DNA present in the cell prior to transfection.

[35] By "transgene" or "transgenic element" is meant an artificially introduced, chromosomally integrated nucleic acid sequence present in the genome of a host organism.

DETAILED DESCRIPTION OF THE INVENTION

[36] The subject invention provides methods and compositions for modulating time to flowering in plants. The subject methods and compositions generally relate to modulating expression of certain gene products and variants thereof in plants, e.g., dicot or monocot plants such as soybean, wheat and canola. Transgenic plants in which expression of those gene products is reduced or increased are provided, as well as methods for modulating flowering time by reducing or increasing expression of those gene products. The invention finds use in a variety of agricultural and horticultural applications.

[37] Before the present subject invention is described further, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[38] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[3S] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.

[40] It must be noted that as used herein and in the appended claims, the singular forms

"a", "and", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a vector" includes a plurality of such vectors and reference to "the target cell" includes reference to one or more target cells and equivalents thereof known to those skilled in the art, and so forth.

[41] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

[42] As discussed briefly above, the invention provides methods of modulating flowering time in plants by altering (i.e., increasing or decreasing) the activity of certain gene products, herein termed flowering time modulators.

[43] Flowering time is the time taken, usually calculated in days, from emergence of a seedling from a seed to initiation of flowering (i.e., floral transition). In many embodiments of the subject invention, the subject methods result in a plant in which flowering time is increased or decreased by at least 10%, at least 20%, at least 30%, at least 40%, at least 60%, at least 80% or at least 90% or 95%, or more. In certain embodiments, flowering time may be increased by at least 100%, by at least 200%, by at least 500% or more and in some embodiments, the subject plants do not flower in their lifetime. As discussed in the background section, methods for measuring flowering time are well known in the art, and, as such, need not be described in any more detail.

[44] Flowering time modulators on interest include the following Arabidopsis genes and orthologs thereof:

[45] Variants of interest include orthologs of these sequences from non-Arabidopsis species (i.e., the phylogenetically most similar sequence to one of the above sequences from a non-Arabidopsis species), and variants in which any of the sequences (including the Arabidopsis or orthologous sequences) has been modified. In general, a variant of a subject gene product possesses at least 70%, at least 80%, at least 85%, at least 90% or at least 95% sequence identity to a sequence discussed above. In certain embodiments, a variant may be encoded by a nucleic acid that hybridizes under stringent hybridization conditions to a polynucleotide encoding any of the polypeptide sequences discussed above. The amino acid and nucleic acid sequences and descriptions thereof (e.g., any annotation of the sequences) set forth in the NCBI GenBank database entries discussed above are explicitly incorporated by reference in their entirety.

[46] In certain embodiments, the subject methods involve modulating the activity of FLK

(Flowering Locus KH-domain) protein in plants. The sequence of an exemplary FLK polypeptide and polynucleotide from Arabidopsis is set forth in Fig. 9 (SEQ ID NOS: 1 and 2), and non-Arabidopsis orthologs of these sequences are also known (see Fig. 8A). In other embodiments, the subject methods involve modulating the activity of a protein selected from the group consisting of SEQ ID NO:3; SEQ ID NO:4; SEQ ID NO:5; SEQ ID NO:6; SEQ ID NO:7; or SEQ ID NO:8; or a variant or ortholog thereof.

[47] The subject methods typically involve increasing or decreasing expression of a gene encoding a subject gene product in a plant. In general, these methods involve reducing the expression of a product of gene that is endogenous to a plant (i.e., found in the genome of a plant in its natural form), or by increasing the expression of the gene product by introducing a recombinant vector for expression of that gene product in the plant.

[48] Plants of interest include monocot and dicot plant species, for example crops such as soybean, wheat, corn, potato, cotton, rice, oilseed rape (including canola), sunflower, alfalfa, sugarcane, castor and turf; or fruits and vegetables, such as banana, blackberry, blueberry, strawberry, and raspberry, cantaloupe, carrot, cauliflower, coffee, cucumber, eggplant, grapes, honeydew, lettuce, mango, melon, onion, papaya, peas, peppers, pineapple, spinach, squash, sweet corn, tobacco, tomato, watermelon, rosaceous fruits (such as apple, peach, pear, cherry and plum) and vegetable brassicas (such as broccoli, cabbage, cauliflower, brussβl sprouts and kohlrabi). Other crops, fruits and vegetables whose ceils may be targetted include barley, rye, millet, sorghum, currant, avocado, citrus fruits such as oranges, lemons, grapefruit and tangerines, artichoke, cherries, nuts such as the walnut and peanut, endive, leek, roots, such as arrowroot, beet, cassava, turnip, radish, yarn, and sweet potato, Arabidopsis, beans, mint and other labiates. Woody species, such as pine, poplar, yew, rubber, palm, eucalyptus etc. and lower plants such as mosses, ferns, and algae are also of interest. Plants of interest may belong to the following plant families: Brassicaceae, Compositae, Euphorbiaceae, Leguminosae, Linaceae, Malvaceae, Umbilliferae and Graminae. Of particular interest are oilseed crops, such as canola, soybean and corn. Orthologs of the above-described sequences are present in these plants. Modulation of endogenous gene expression

[4S] As discussed above, it is often desirable to increase or reduce the activity of an endogenous polypeptide in a plant to reduce or increase flowering time. Methods for reducing endogenous gene expression are generally well known in the art and include gene "silencing" and "knock-out" methods.

[50] Methods for gene silencing, including antisense, RNAi, ribozyme and cosuppression technologies are based in hybridization of an expressed exogenously supplied nucleic acid with an RNA transcribed from an endogenous gene of interest in a plant cell.

[51] In certain embodiments, gene expression can be modified by gene silencing using double-strand RNA (Sharp (1999) Genes and Development 13: 139-141). RNAi, otherwise known as double-stranded RNA interference (dsRNAi), has been extensively documented in the nematode C. elegans (Fire, A., et al, Nature, 391 , 806-811 , 1998) and an identical phenomenon occurs in plants, in which it is usually referred to as post-transcriptional gene silencing (PTGS) (Van Blokland, R., et al., Plant J., 6: 861-877, 1994; deCarvalho-Niebel, F., et al., Plant Cell, 7: 347-358, 1995; Jacobs, J. J. M. R. et al., Plant J., 12: 885-893, 1997; reviewed in Vaucheret, H., et al., Plant J., 16: 651-659, 1998). The phenomenon also occurs in fungi (Romano, N. and Masino, G., MoI. Microbiol., 6: 3343-3353, 1992, Cogoni, C, et al., EMBO J., 15: 3153-3163; Cogoni, C. and Masino, G., Nature, 399: 166-169, 1999), in which it is often referred to as "quelling". RNAi silencing can be induced many ways in plants, where a nucleic acid encoding an RNA that forms a "hairpin" structure is employed in most embodiments. Alternative strategies include expressing RNA from each end of the encoding nucleic acid, making two RNA molecules that will hybridize. Current strategies for RNAi induced silencing in plants are reviewed by Carthew et al (Curr Opin Cell Biol. 2001 13:244-8).

[52] In other embodiments, gene expression may be down-regulated by antisense or cosupression technology (see, e.g., Lichtenstein and Nellen (1997) Antisense Technology: A Practical Approach IRL Press at Oxford University, Oxford, England). In general, sense or antisense sequences are introduced into a cell, where they may be amplified, e.g., by transcription. A reduction or elimination of expression (i.e., a "knock-out") of a gene in a transgenic plant can be obtained by introducing a sense or antisense construct containing a cDNA corresponding to that gene. For sense suppression, the cDNA is arranged in forward orientation (with respect to the coding sequence) relative to the promoter sequence in the expression vector, whereas for antisense suppression, the cDNA is arranged in antisense (i.e., reverse) orientation. The introduced sequence need not be the full length cDNA or gene, and need not be identical to the cDNA or gene found in the plant type to be transformed. Typically, the antisense sequence need only be capable of hybridizing to the target gene or RNA of interest. Thus, where the introduced sequence is of shorter length, a higher degree of homology to the endogenous sequence may be needed for effective suppression. While sequences of various lengths can be utilized, preferably, the introduced sequence in the vector should be at least 30 nucleotides in length, and improved suppression may be observed as the length of the sequence increases. Many embodiments, the length of the antisense sequence in the vector will be greater that 100 nucleotides. Transcription of an antisense construct as described results in the production of RNA molecules that are the reverse complement of mRNA molecules transcribed from the endogenous transcription factor gene in the plant cell.

[53] Vectors in which RNA encoded by a cDNA is over-expressed can also be used to obtain co-suppression of a corresponding endogenous gene, e.g., in the manner described in U.S. Patent No. 5,231 ,020 to Jorgensen. Such co-suppression (also termed sense suppression) does not require that the entire cDNA be introduced into the plant cells, nor does it require that the introduced sequence be exactly identical to the endogenous gene. However, as with antisense suppression, the suppressive efficiency will be enhanced as specificity of hybridization is increased, e.g., as the introduced sequence is lengthened, and/or as the sequence similarity between the introduced sequence and the endogenous transcription factor gene is increased. Vectors expressing an untranslatable form of an mRNA, e.g., sequences comprising one or more stop codon, or nonsense mutation) can also be used to suppress expression of an endogenous gene, thereby reducing or eliminating it's activity and modifying one or more traits. Methods for producing such constructs are described in U.S. Patent No. 5,583,021. Preferably, such constructs are made by introducing a premature stop codon into a cDNA.

[54] In other embodiments, gene expression may be abolished is by insertion mutagenesis using the T- DNA of Agrobacterium tumefaciens. After generating a library of insertion mutants, the mutants can be screened to identify those containing an insertion in a gene of interest. Plants containing a single transgene insertion event at the desired gene can be crossed to generate homozygous plants for the mutation (Koncz et al. (1992) Methods in Arabidopsis Research, World Scientific). [55] Alternatively, gene expression may be modulated by altering or eliminating an endogenous gene, e.g., by homologous recombination (Kempin et al. (1997) Nature 389:802). A gene can also be modified by using the cre-lox system (for example, as described in US Pat. No. 5,658,772). A plant genome can be modified to include first and second lox sites that are then contacted with a Cre recombinase. If the lox sites are in the same orientation, the intervening DNA sequence between the two sites is excised. If the lox sites are in the opposite orientation, the intervening sequence is inverted.

[56] Because the methods are based on hybridization, the methods are particularly applicable to silencing of gene families that share a level of sequence identity, for example for families of genes that contain 60% or more, 70% or more, 80% or more, 90% or more or 95% or more sequence identity over 100, 200, or 500 or more nucleotides. However, by choosing sequences that are found only in certain genes, the expression of only those genes can be reduced. RNA-induced silencing strategies for plants are reviewed in Matzke et al (Curr Opin Genet Dev. 2001 11:221-7).

[57] A subject polynucleotide may be incorporated into recombinant nucleic acid, e.g. DNA or RNA, that directs expression of polypeptides of the invention in appropriate plant host cells, transgenic plants, or the like. Due to the inherent degeneracy of the genetic code, nucleic acid sequences which encode substantially the same or a functionally equivalent amino acid sequence can be substituted for any listed sequence to provide for cloning and expressing the relevant homologue.

[58] The present invention includes recombinant constructs comprising one or more of the nucleic acid sequences herein. The constructs typically comprise a vector, such as a plasmid, a cosmid, a phage, a virus (e.g., a plant virus), a bacterial artificial chromosome (BAC), a yeast artificial chromosome (YAC), or the like, into which a nucleic acid sequence of the invention has been inserted, in a forward or reverse orientation. In a preferred aspect of this embodiment, the construct further comprises regulatory sequences, including, for example, a promoter, operably linked to the sequence. Large numbers of suitable vectors and promoters are known to those of skill in the art, and are commercially available.

[59] In general, expression plasmids will contain a selectable marker and subject nucleic acid sequences. The selectable marker provides resistance to toxic chemicals and allows selection of cells containing the marker over cells not containing the marker. Conveniently, the marker encodes resistance to a herbicide, e.g. phosphinothricin, glyphosate etc, or an antibiotic, such as kanamycin, G418, bleomycin, hygromycin, chloramphenicol, or the like. The particular marker employed is one that allows for selection of transformed cells over cells lacking the introduced recombinant DNA. Antibiotic or herbicide resistance markers including cat (chloramphenicol acetyl transferase), npt Il (neomycin phosphotransferase II), PAT (phosphinothricin acetyltransferase), ALS (acetolactate synthetase), EPSPS (5-enolpyruvyl- shikirnate-3-phosphate synthase), and bxn (bromoxynil-specific nitrilase) may be used. A preferred marker sequence is a DNA sequence encoding a selective marker for herbicide resistance and most particularly a protein having enzymatic activity capable of inactivating or neutralizing herbicidal inhibitors of glutamine synthetase. The non-selective herbicide known as glufosinate (BASTA™ or LIBERTY ™) is an inhibitor of the enzyme glutamine synthetase. It has been found that naturally occurring genes or synthetic genes can encode the enzyme phosphinothricin acetyl transferase (PAT) responsible for the inactivation of the herbicide. Such genes have been isolated from Streptomyces. Specific species include Streptomyces hygroscopicus (Thompson C. J. et al., EMBO J., vol. 6:2519-2523 (1987)), Streptomyces coelicolor (Bedford et al, Gene 104: 39-45 (1991)) and Streptomyces viridochromogenes (Wohlleben et al. Gene 80:25-57 (1988)). These genes including those that have been isolated or synthesized are also frequently referred to as bar genes. These genes have been cloned and modified for transformation and expression in plants (EPA 469 273 and U.S. Pat. No. 5,561 ,236). Through the incorporation of the pat gene, corn plants and their offspring can become resistant against phosphinothricin (or glufosinate).

General texts that describe molecular biological techniques useful herein, including the use and production of vectors, promoters and many other relevant topics, include Berger, Sambrook and Ausubel, supra. Any of the identified sequences can be incorporated into a cassette or vector, e.g., for expression in plants. A number of expression vectors suitable for stable transformation of plant cells or for the establishment of transgenic plants have been described including those described in Weissbach and Weissbach, (1989) Methods for Plant Molecular Biology, Academic Press, and Gelvin et al., (1990) Plant Molecular Biology Manual, Kluwer Academic Publishers. Specific examples include those derived from a Ti plasmid of Agrobacterium tumefaciens, as well as those disclosed by Herrera-Estrella et al. (1983) Nature 303:209, Bevan (1984) Nucl Acid Res. 12: 8711-8721 , Klee (1985) Bio/Technology 3: 637-642, for dicotyledonous plants.

[61] Alternatively, non-Ti vectors can be used to transfer the DNA into monocotyledonous plants and cells by using free DNA delivery techniques. Such methods can involve, for example, the use of liposomes, electroporation, microprojectile bombardment, silicon carbide whiskers, and viruses. By using these methods transgenic plants such as wheat, rice (Christou(1991) Bio/Technology 9: 957-962) and corn (Gordon-Kamm (1990) Plant Cell 2: 603-618) can be produced. An immature embryo can also be a good target tissue for monocots for direct DNA delivery techniques by using the particle gun (Weeks et al. (1993) Plant Physiol 102: 1077-1084; Vasil (1993) Bio/Technology 10: 667-674; Wan and Lemeaux (1994) Plant Physiol 104: 37-48, and for Agrobacterium-mediated DNA transfer (Ishida et al.

(1996) Nature Biotech 14: 745-750). [62] Typically, plant transformation vectors include one or more cloned plant coding sequence (genomic or cDNA) under the transcriptional control of 5' and 3' regulatory sequences and a dominant selectable marker. Such plant transformation vectors typically also contain a promoter (e.g., a regulatory region controlling inducible or constitutive, environmentally-or developmentally-regulated, or cell-or tissue-specific expression), a transcription initiation start site, an RNA processing signal (such as intron splice sites), a transcription termination site, and/or a polyadenylation signal.

[63] Examples of constitutive plant promoters which can be useful for expressing a sequence include: the cauliflower mosaic virus (CaMV) 35S promoter, which confers constitutive, high-level expression in most plant tissues (see, e.g., Odel et al. (1985) Nature 313:810); the nopaline synthase promoter (An et al. (1988) Plant Physiol 88:547); and the octopine synthase promoter (Fromm et al. (1989) Plant cell 1 :977).

[64] A variety of plant gene promoters that regulate gene expression in response to environmental, hormonal, chemical, developmental signals, and in a tissue-active manner can be used for expression of sequence in plants. Choice of a promoter is based largely on the phenotype of interest and is determined by such factors as inducibility (e.g., in response to wounding, heat, cold, drought, light, pathogens, etc.), timing, developmental state, and the like. Constitutive promoters (e.g., the CaMV 35S promoter), inducible promoters and meristem specific promoters are of particular interest.

[65] Plant expression vectors can also include RNA processing signals that can be positioned within, upstream or downstream of the coding sequence. In addition, the expression vectors can include additional regulatory sequences from the 3'-untranslated region of plant genes, e.g., a 3'terminator region to increase mRNA stability of the mRNA, such as the Pl-Il terminator region of potato or the octopine or nopaline synthase 3' terminator regions.

[66] Specific initiation signals can aid in efficient translation of coding sequences. These signals can include, e.g., the ATG initiation codon and adjacent sequences. In cases where a coding sequence, its initiation codon and upstream sequences are inserted into the appropriate expression vector, no additional translational control signals may be needed. However, in cases where only coding sequence (e.g., a mature protein coding sequence), or a portion thereof, is inserted, exogenous transcriptional control signals including the ATG initiation codon can be separately provided. The initiation codon is provided in the correct reading frame to facilitate transcription. Exogenous transcriptional elements and inititation codons can be of various origins, both natural and synthetic. The efficiency of expression can be enhanced by the inclusion of enhancers appropriate to the cell system in use. Plant transformation

[67j Transgenic plants (or plant cells, or plant explants, or plant tissues) incorporating the polynucleotides described above can be produced by a variety of well established techniques. Following construction of a vector, most typically an expression cassette, including a polynucleotide standard techniques can be used to introduce the polynucleotide into a plant, a plant cell, a plant explant or a plant tissue of interest. Optionally, the plant cell, explant or tissue can be regenerated to produce a transgenic plant.

[68] The plant can be any higher plant, including gymnosperms, monocotyledonous and dicotyledenous plants. Suitable protocols are available for Leguminosae (alfalfa, soybean, clover, etc.), Umbelliferae (carrot, celery, parsnip), Cruciferae (cabbage, radish, rapeseed, broccoli, etc.), Curcurbitaceae (melons and cucumber), Gramineae (wheat, corn, rice, barley, millet, etc.), Solanaceae (potato, tomato, tobacco, peppers, etc.), and various other crops. See protocols described in Ammirato et al. (1984) Handbook of Plant Cell Culture-Crop Species. Macmillan Publ. Co. Shimamoto et al. (1989) Nature 338:274-276; Fromm et al. (1990) Bio/Technology 8:833-839; and Vasil et al. (1990) Bio/Technology 8:429-434.

[69] Transformation and regeneration of both monocotyledonous ad dicotyledonous plant cells is now routine, and the selection of the most appropriate transformation technique will be determined by the practitioner. The choice of method will vary with the type of plant to be transformed; those skilled in the art will recognize the suitability of particular methods for given plant types. Suitable methods can include, but are not limited to: electroporation of plant protoplast; liposome-mediated transformation; polyethylene glycol (PEG) mediated transformation; transformation using viruses: micro-injection of plant cells; micro-projectile bombardment of plant cells; vacuum infiltration; whiskers technology, and Agrobacterium tumeficiens mediated transformation. Transformation means introducing a nucleotide sequence into a plant in a manner to cause stable or transient expression of the sequence.

[70] Successful examples of the modification of plant characteristics by transformation with cloned sequences which serve to illustrate the current knowledge in this field of technology, and which are herein incorporated by reference, include: U.S. Patent Nos. 5,571 ,706; 5,677,175; 5,510,471; 5,750,386; 5,597,945; 5,589,615; 5,750,871 ; 5,268,526; 5,780,708; 5,538,880; 5,773,269; 5,736,369 and 5,610,042.

[71] Following transformation, plants are preferably selected using a dominant selectable marker incorporated into the transformation vector. Typically, such a marker will confer antibiotic or herbicide resistance on the transformed plants, and selection of transformants can be accomplished by exposing the plants to appropriate concentrations of the antibiotic or herbicide.

[72] After transformed plants are selected and grown to maturity, those plants showing modified flowering time are identified. Additionally, to confirm that the modified flowering time is due to changes in expression levels or activity of the polypeptide or polynucleotide of the invention can be determined by analyzing rriRNA expression using Northern blots, RT-PCR or microarrays, or protein expression using immunoblots or Western blots or gel shift assays, for example.

EXAMPLES

[73] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.

MATERIALS AND METHODS

[74] Plant and DNA Materials: Approximately 1100 T-DNA insertion lines (in the CoI-O background) affecting 326 putative RNA-binding proteins were selected (http://signal.salk.edu/cgi-bin/tdnaexpress) and the seeds of these lines were obtained from the Arabidopsis Biological Resource Center or the SaIk Institute Genomic Analysis Laboratory. These lines were grown in a green house, flowering time was scored as the rosette leaf number at bolting and days from the end of imbibition to bolting (Mockler et al., (2003) Proc Natl Acad Sci U S A 100, 2140-5), and plants that showed apparent flowering- time variations from the wild type were selected for further analysis. Among the putative mutants studied, Salk_007750 (flk-1), Salk_001523 (flk-2), SaIkJ 39230 (flk-3) and SaIkJ 12850 (flk-4) affected the same locus (Locus: At3g04610, Accession: AY070475), which was referred to as FLK by this and a recently published reports.

[75] Genomic DNA of four allelic flk mutants was extracted using the CTAB method. The genomic flanking sequences of T-DNA insertions were PCR-amplified using FLK-specific primers and T-DNA-specific primers (see Fig. 7). The amplified DNA was gel purified and sequenced. To examine flowering time, plants were grown in compound soil in long days (LD:18hr L/6hr D, 21⁰C), or short days (SD: 9hr L/15hr D, 21⁰C), or continuous cool-white fluorescence light (~23°C) as described previously. Flowering time was scored as the rosette leaf number at bolting and days from the end of imbibition to bolting. The FLK coding sequence was amplified from total RNA using the primers described (see Fig. 7). The FLK cDNA fragment was fused at ATG to the N-terminus of GFP (Green Fluorescent Protein) in the vector pEGAD (Cutler et al, )2000) Proc Natl Acad Sci U S A 97, 3718-23), and transformed into Arabidopsis (Col-4 and CoI-O). The GFP-FLK transgenic plants were selected using Basta resistance (10mg/l Ammonium-DL-homoalanine-4-yl-(methyl) phosphinate) on soil and the GFP-FLK expression was verified using RT-PCR analysis and fluorescence imaging of GFP. Homozygous Basta-resistant transgenic plants were scored for flowering time at the T3 generation. The cellular localization of GFP-FLK was examined using a fluorescence microscope (Dialux 20, Leitz Co, Germany).

[763 RMA Isolation and Semi-quantitative Reverse Trsnscription-PGR (RT-PCR):

Seeds of Arabidopsis wild type (CoI-O or Col-4) or flk mutants were surface-sterilized (in a sealed jar vaporized with 100 ml bleach and 3 ml concentrated HCL for 6-12 hr), and sown on IViS medium. The plates were stratified at 4⁰C for 4 days, exposed to continuous white light for 6 hours, and placed in continuous white light or in photoperiodic conditions. The aerial parts of the seedlings were harvested, and the total RNA was isolated using a Qiagen RNeasy kit (Qiagen, Valencia, CA), and DNA-free RNA was obtained by RQ1 DNase I treatment (1 unit DNase per 5.0 μg in 20 μl reaction) according to the manufacturer's instructions (Promega, Madison, Wisconsin). cDNA was prepared from 5.0 μg total RNA using the M- WlLV reverse transcriptase or Superscript First-strand cDNA Synthesis System according to the manufacturer's instructions (Invitrogen, Carlsbad, CA). The cDNA was diluted 10 fold, and 1 μl of diluted cDNA was used in a 20 μl PCR reaction with the primers described (see Fig. 7).

[77] DNA microarray analysis: Three separate microarray experiments were conducted comparing WT and flk mutant plants of different developmental stages and grown under different growth conditions. In the first experiment, three independent biological replicates of sixteen-day-old WT (CoI-O) and flk-1 seedlings were grown in long days (18L:6D) and seedlings were harvested for total RNA preparation 16 hrs after lights-on. In the second experiment, three independent biological replicates of sixteen-day old continuous light-grown WT (CoI-O) and flk-2 seedlings were harvested for total RNA preparation. In the third experiment, three independent biological replicates of seven-day-old WT (CoI-O) and flk-1 seedlings were grown in long days (18L:6D) and seedlings were harvested for total RNA preparation 1 hr after lights-on. Labeled cRNA targets were prepared and hybridized to Affymetrix ATH 1 genechip arrays according to the manufacturer's protocols (Affymetrix; Santa Clara, CA). Background-corrected, quantile-normalized gene expression summaries for the replicates were generated by the Robust Multichip Average (RMA) method using RMAExpress software. The triplicate datasets were further subjected to permutation analysis using a script to identify genes whose expression differences in the two genotypes corresponded to acceptable false-discovery rates.

[78] Quantitative Real-Time Polymerase Chain Reaction (Q-PCR). The quantitative real¬ time RT-PCR (Q-PCR) was carried out using standard procedures with minor modifications.

Primer Express v2.0 software (Applied Biosystems, Foster City, CA) was used to design oligonucleotide primers (see Fig. 7). Total RNA was isolated from three independent replicates of seedlings grown in conditions indicated. cDNAs were prepared as described above, diluted twenty-fold, and 1 μl of diluted cDNA was used in a 25μl Q-PCR reaction with SYBR Green Master Mix (Applied Biosystems). All reactions were performed in triplicate using an ABI Prism 7700 Sequence Detection System (Applied Biosystems, Foster City, California). Data analysis was performed according to the protocol provided by Applied Biosystems using Sequence Detector Systems v1.7 software. A standard curve was constructed for each primer pair using an equal mixture of all cDNAs according to the manufacturer's instructions (Applied- Biosystems). The expression level of each gene was calculated based on the standard curve for a given primer set. The relative amount of calculated message was normalized to the level of a control ubiquitin gene (At5g15400). All sequences of primers used in Q-PCR are as described (see Fig. 7).

[79] RNA stability assay: 10-day-old seedlings were excised and placed in tubes containing the transcription inhibitor solution (ImIVI PIPES buffer pH 6.3, I mM KCI, 1mM Na- citrate, 15mM sucrose, 100μg/ml cyclohexamide (A.G. Scientific, San Diego, CA), 100μg/ml actinomycin D and 100μg/ml cordycepin (Sigma, St. Louis, MO). Samples from the wild type and flk-1 were collected at the time points indicated up to 4hr. RNAs isolation and RT-PCR are as described. For each sample 2μg of total RNA was reverse transcribed and diluted to 100μl, from which 2μl was used for RT-PCR with gene specific primers (see Fig. 7). RESULTS

[80] To understand how Arabidopsis photoreceptors regulate photoperiodic flowering, we investigated how RNA-binding proteins contribute to the photoperiodic regulation of gene expression. We screened T-DNA insertion mutations that affect genes encoding putative RNA-binding proteins for variations in flowering time. Four independent insertion mutations of the same gene (exemplified by GenBank Accession number At3g04610) on Arabidopsis chromosome 3 were found to have a similar late-flowering phenotype (Fig. 1). All four mutants flowered later than the wild type in both long days and short days. The mutant plants did not flower until more than 40 days (long-day) or 120 days (short-day) after germination, in contrast to wild type plants that flowered within 30 days (long-day) or 60 days (short-day) after germination (Fig. 1). The flk mutant remains responsive to vernalization, because the mutant plants flowered significantly earlier after a vernalization treatment (Fig. 6). These results show that the corresponding gene plays an important role in the regulation of floral initiation.

[81] The gene corresponding to the mutations was identified based the analysis of DNA sequences flanking the T-DNA inserts. It encodes a 577-residue protein that contains three K-homology (KH) motifs (Fig. 2A, Fig. 8A). Because none of the 4 mutant alleles showed readily discernable morphological changes other than delayed flowering, we reasoned that the major function of the corresponding gene is to regulate flowering time and referred to it as FLK (for Flowering Locus KH-domain). The T-DNAs of the two flk alleles (flk-1 and flk-2) were inserted in the first intron, the T-DNAs of other two alleles (flk-3, flk-4) were inserted in the second intron (Fig. 2A). It appears that all of the flk alleles identified are loss-of-function or null mutations because none of the flk alleles tested expressed detectable amounts of FLK mRNA (Fig. 2B). In the wild type plant, FLK is expressed in various tissues including flowers, leaves, roots and siliques, and it is relatively abundant in the young infloresence.

[82] The KH domain is an evolutionarily conserved RNA-binding motif found in proteins of diverse organisms including eubacteria, archaea, and eukaryotes. The core consensus sequence V/l IG)(XG)(XIA/ in the middle of the KH domain (~50-70 residues) is perfectly conserved in all three KH domains of FLK (Fig. 8A). This core consensus sequence has been found to be important for RNA-binding activity of KH-domain proteins. For example, a single amino acid substitution in the KH domain core sequence of the human FMR1 protein abolishes its RNA-binding activity and causes fragile X mental-retardation syndrome. The first two KH domains of FLK are grouped near the N-terminus and the third KH domain is located at the C-terminus (Fig. 8A). Such an overall architecture of KH domain arrangement is conserved in the PCBP (polyC-binding protein) type of KH-domain RNA-binding proteins, including the founding member of the KH-domain protein family, hnRNP K. The N-terminus of FLK preceding the first KH domain is rich in glutamine (33/150 residues or 22%). This region also contains three perfect 8-residue repeats (LEPQQYEV), although these repeats are not strictly conserved in the putative rice ortholog of FLK (Fig. 8A).

[83] To study the function and cellular localization of the FLK protein, we prepared transgenic plants overexpressing the GFP-FLK fusion protein under the constitutive 35S promoter. The 35S::GFP-FLK transgenic plants flowered earlier than the wild type (Fig. 2C). Because the loss-of-function flk mutant flowered later whereas 35S::GFP-FLK transgenic plants flowered earlier, we concluded that FLK is a positive regulator of floral initiation. The GFP-FLK fusion protein was enriched in the nucleus, although it was also found in the cytosol (Fig. 2D). The nuclear localization of GFP-FLK is consistent with a proposition that FLK may be involved in the regulation of mRNA trafficking or metabolism associated with expression of flowering-time genes.

[84] We investigated whether the flk mutation affects gene expression by analyzing genome-wide expression profiles of three different samples: 16-day-old seedlings grown in long days and harvested 16h after light-on (Exp. 1), 16-day-old seedlings grown in continuous light (Exp. 2), and 7-day-old seedlings grown in long days (18hL/6hD) but harvested 1h after light-on (Exp. 3). Affymetrix ATH1 genechip arrays representing approximately 24,000 Arabidopsis genes were used in all three experiments. The complete DNA microarray results of the three experiments can be found online at the world wide website of the NCBI, accession, GSE1512). We also used Q-PCR and RT-PCR to re-examine the gene expression changes detected by the DNA microarray studies. Results of the three microarray experiments are summarized in Fig. 5, for which three statistical criteria (see Fig. 5) were used to determine a putative mis-expression event in the flk mutant. Although a number of genes showed altered expression in at least one microarray experiment (referred to as one mis-expression event and marked by asterisks in Fig. 5), only 8 genes showed altered mRNA expression in the flk mutant in all three microarray experiments (Fig. 5A). The two genes for which the expression showed the most profound change in the flk mutant are FLC and FLK. The level of FLK expression is close to the background level in the flk mutant as demonstrated by RT-PCR (Fig. 5B) and Q-PCR (Fig. 4A). The mRNA level of FLC was approximately 6 to 10 fold higher in the flk mutant than in the wild type in all three microarray experiments. Increased FLC expression in the flk mutant was confirmed by both RT-PCR (Fig. 3A) and Q- PCR (Fig. 4A). Because KH-domain proteins are often associated with RNA metabolism, we examined whether the elevated FLC mRNA level in the flk mutant was due to a defect in FLC mRNA turnover (Fig. 3B). In this experiment, tissues were excised and incubated with transcription inhibitors (cordycepin, cycloheximide, and actinomycin D), and FLC mRNA level was examined at different time points following the inhibitor treatment. Fig. 3B shows that FLC mRNA level decreased gradually in tissues treated with transcription inhibitors in both the wild type and flk mutant, but there was no obvious difference of the rate of FLC mRNA decay between the two genotypes. This result suggests that FLK may regulate FLC mRNA expression via a mechanism other than RNA turnover. The FLK protein expressed and purified from the in vitro translation system or from E. coli also failed to bind FLC mRNA or polyribonucleotides in various conditions tested.

[85] In addition to FLK, two other KH-domain genes also showed altered expression profile in the flk mutation in all three microarray experiments (Fig 5A). The mRNA level of one KH- domain gene (At5g06770) was about 100% higher in the flk mutant than in the wild type, whereas the other KH-domain gene (At3g32940) showed an approximately 50% decreased expression in the flk mutant in all three microarray experiments (Fig. 5A). In contrast, the expression of other KH-domain genes, including HEN4 that regulates floral organ development, showed normal expression in the flk mutant in all microarray experiments (Fig. .. 5B). The modest mis-expression of At5g06770 was confirmed by RT-PCR, in which this KH- domain gene showed modestly elevated expression in three different flk mutant alleles tested (Fig. 6). A phylogenetic analysis indicates that at least one of the two KH-domain genes affected by the flk mutation is closely related to FLK, although the sequence similarity between these KH-domain proteins are not particularly strong (Figs. 8B and 8C). Because the expression of these two KH-domain genes is affected by the flk mutation, their functions are likely associated with FLK. F86] Results of the expression comparison for genes that play roles in the autonomous pathway controlling flowering time or genes that regulate floral development, such as floral organ identity, are summarized in Fig. 5B. The expression profiles of genes known or likely to be involved in photoperiodic control of flowering time are listed in the Fig. 5C. Fig. 5B shows that, with few exceptions, genes associated with autonomous pathway or floral development generally were expressed normally in the different samples analyzed in all three microarray experiments. For example, two autonomous pathway genes involved in RNA metabolism and regulation of FLC expression, FCA, and FY, showed no change of expression in the flk mutant in all three microarray experiments (FPA is not represented on the ATH1 array). The expression of other genes that are known to regulate FLC expression, such as FR/, FLD, FVE, VRN1, VRN2, were also not apparently affected by the flk mutation. Genes known for their roles in the regulation of floral organ development, such as AG, AP1, AP2, CLV1, SUP, also demonstrated normal expression in the flk mutant in all three microarray experiments (Fig. 5B).

[87] In contrast to the genes associated with autonomous pathway or floral development

(Fig. 5B), the expression profiles of genes associated with photoperiodic regulation of flowering time showed more complicated pattern in the flk mutant (Fig. 5C). For example, among the 26 genes listed in Fig. 5B, only two putative mis-expression events (marked by asterisks in Fig. 5) were detected according to the selection criteria (see Fig. 5). In contrast, a total of 15 putative mis-expression events were detected among 13 genes listed in Fig. 5C. At least one putative mis-expression event was detected for almost every gene listed in Fig. 5C (and 4 out of 13 genes showed mis-expression in two microarray experiments). Among the genes listed in Fig. 5C, FT and SOC1 are known to be positively regulated by CO and negatively regulated by FLC in response to different signals. One or two putative mis- expression events were detected for the FT or SOC1 genes, respectively, both showing decreased expression in the flk mutant (Fig. 5C). The expression of FT and SOC1 were re- examined using RT-PCR or Q-PCR analyses. Fig. 3A showed that FT and SOC1 both were expressed at lower levels in the flk mutant in samples collected from 12 to 22 days after germination. A modestly decreased mRNA accumulation of FT and SOC1 was also demonstrated using a Q-PCR assay (Fig. 4A). Because FLC expression was significantly . increased in the flk mutant, our results are consistent with FLC being a negative regulator of the mRNA expression of FT and SOC1, resulting in delayed flowering. We concluded that FLK is a negative regulator of FLC expression that suppresses FLC expression to de-repress floral initiation.

[88] It is noted that most of the putative mis-expression events were detected in 16-day-old samples grown in continuous light (Fig. 5C, Exp. 2), but not in 7-day-old samples grown in long days and collected 1h after light-on (Fig. 5C, Exp. 3). Most of the gene expression changes detected in the 16-day-old flk mutant seedlings were confirmed by Q-PCR experiment (Fig. 4). For example, AGL24, APRRS₁ APRR5, APRR7, and CO genes each registered at least one decreased expression in the flk mutant (Fig. 5C), and similarly decreased expression of these genes were detected in the Q-PCR experiment (Fig. 4A). In contrast, CCA1 and LHY showed slightly increased expression in the 16-day-old flk mutant in both microarray and Q-PCR experiments (Table 1C, Fig. 4A). This discrepancy does not seem to be due to the developmental difference between the wild type and the flk mutant that flower later, because few of the 26 genes, which are associated with autonomous/vernalization control of flowering time or floral development and are also known to increase expression at later developmental stages, showed similar bias (Fig. 5B, Exp. 2). Moreover, the genes that showed decreased expression in the flk mutant, such as CO, FT, SOC1, APRR5, and APRR7 are known positive regulators of floral initiation, whereas the genes that showed increased expression in the flk mutant, such as CCA1 and LHY are known negative regulators of flowering. In both cases, the function of those genes for which the expression was modestly altered by the flk mutation correlated with the delayed flowering phenotype of flk. This again suggests that the mis-expression of these genes in the flk mutant may not be due to random experimental variations. The function of RVE2 is not known, but it is a MYB proteins related to CCA1 and LHY. Moreover, a recently reported protein called EPR1 that shares ~57% amino acid similarity to RVE2 was found to suppress flowering. [89] Most of the photoperiodic pathway genes mentioned above are regulated by the circadian clock, which may explain the different results derived from samples grown in different photoperiods and collected at different time. So we investigated whether the expression of the photoperiodic pathway genes in young seedlings may be detected under a free-running condition. In this experiment, 7-day-old wild-type and flk mutant seedlings were entrained in photoperiods (12hL/12hD) and transferred to continuous light for two days of "free running"; samples were collected every 4h for 24 hours and analyzed using real-time Q-PCR. Among the 6 genes tested, four {APRR7, CO, FT, TOC1) showed lower amplitude of the circadian rhythm, whereas the other two (CCA1 and REV2) showed increased expression at some points of free running. Both results are consistent with their registered mis-expression events found in the microarray (Fig. 5) or Q-PCR studies using adult (16-day-old) plants, which indicate that the modest changes in the expression of photoperiodic pathway genes can also be detected in young (7-day-old) seedlings of the flk mutation using a more sensitive method. We conclude that FLK mainly regulates the expression of FLC, but it may also play a minor role, directly or indirectly, in the expression of genes associated with photoperiodic pathway. In this regard, it is interesting to note that flc mutations have been recently found to cause shortened period of the circadian rhythm; and the increased FLC expression or activity may suppresses the expression of the photoreceptor gene CRY2. . It is evident from the above results and discussion that the subject invention provides an important new means for modulating flowering time in plants. Specifically, the subject invention provides plants in which expression of certain genes and gene products are modulated to alter flowering time, and methods for modulating the flowering time of plants using those polynucleotides and polypeptides. As such, the subject methods and systems find use in a variety of different applications, including research, trait improvement and other applications. Accordingly, the present invention represents a significant contribution to the art. [91] While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto.

Claims

We Claim:

1 „ A transgenic plant in which activity of a flowering time modulating protein is modulated as compared to an equivalent non-transgenic plant, wherein modulation of said activity causes an alteration in the time flowering in said transgenic plant as compared to said equivalent non-transgenic plant.

2. The transgenic plant of claim 1 , wherein said activity is increased, and said transgenic plant flowers earlier than said equivalent non-transgenic plant.

3. The transgenic plant of claim 2, wherein said activity is increased using a recombinant construct comprising a promoter active in said transgenic plant, operably linked to a nucleic acid encoding an protein.

4. The transgenic plant of claim 1 , wherein said activity is decreased, and said transgenic plant flowers later than said equivalent non-transgenic plant.

5. The transgenic plant of claim 4, wherein said activity is decreased by reducing expression of an FLK gene that is endogenous to said plant.

6. The transgenic plant of claim 5, wherein said activity is decreased by antisense, co-suppression or RNA inhibition.

7. The transgenic plant of claim 5, wherein said activity is decreased by altering the sequence of an FLK gene.

8. The transgenic plant of claim 1 , wherein said activity is provided by a polypeptide having an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 2.

9. The transgenic plant of claim 1 , wherein said activity is encoded by a polynucleotide having an nucleotide sequence set forth as SEQ ID NO:1 or a polynucleotide that hybridizes to SEQ ID NO:1.

10. A seed of a transgenic plant according to Claim 1.

11. A transgenic plant according to Claim 1 , wherein said flowering time modulating protein is selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4; SEQ ID NO:5, SEQ ID NQ:6, SEQ ID NO:7, and SEQ ID NQ:8 or an ortholog thereof.

12. A method of altering flowering time in a transgenic plant, comprising modulating activity of a flowering time modulating protein in said plant.

13. The method of claim 12, wherein said activity is increased as compared to an equivalent non-transgenic plant, and said transgenic plant flowers earlier than said equivalent non-transgenic plant.

14. The method of claim 13, wherein said activity is increased using a recombinant construct comprising a promoter active in said transgenic plant, operably linked to a nucleic acid encoding a flowering time modulating protein.

15. The method of claim 12, wherein said activity is decreased as compared to an equivalent non-transgenic plant, and said transgenic plant flowers later than said equivalent non-transgenic plant.

16. The method of claim 15, wherein said activity is decreased by reducing expression of a gene that is endogenous to said plant.

17. The method of claim 16, wherein said activity is decreased by antisense, co- suppression or RNA inhibition.

18. The method of claim 16, wherein said activity is decreased by altering the sequence of a flowering time modulating gene.

19. The method of Claim 12, wherein said flowering time modulating protein is selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4; SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8 or an ortholog thereof.