WO2002070661A2 - Identification and characterization of a waxy curled leaf phenotype (wcl1) in arabidopsis - Google Patents

Abstract

The present invention is directed to a novel plant phnenotype, designated Waxy Curled Leaf (WCL1), a nucleic acid sequence expressed in plants demonstrating the WCL1 phenotype and the corresponding amino acid sequence. Also provided are plant cells and plants that exhibit modified WCL1 expression.

Description

IDENTIFICATION AND CHARACTERIZATION OF A

Waxy Curled Leaf PHENOTYPE (WCLl) IN ARABIDOPSIS

BACKGROUND OF THE INVENTION The traditional methods for gene discovery, including chemical mutagenesis, irradiation and T-DNA insertion, used to screen loss of function mutants have limitations. Mutagenic methods such as these rarely identify genes that are redundant in the genome, and gene characterization is time-consuming and laborious.

Activation tagging is a method by which genes are randomly and strongly up- regulated on a genome-wide scale, after which specific phenotypes are screened for and selected. Isolation of mutants by activation tagging has been reported (Hayashi et al,

1992). An activation T-DNA tagging construct was used to activate genes in tobacco cell t culture allowing the cells to grow in the absence of plant growth hormones (Walden et al,

1994). Genes have been isolated from plant genomic sequences flanking the T-DNA tag and putatively assigned to plant growth hormone responses. (See, e.g., Miklashevichs et al. 1997, Harling et al, 1997; Walden et. al., 1994; and Schell et al, 1998, which discusses related studies.)

The first gene characterized in Arabidopsis using activation tagging was a gene encoding the histone kinase involved in the cytokinin signal transduction pathway. The gene sequence was isolated from plant genomic DNA by plasmid rescue and the role of the gene, CK71, in cytokinin responses in plants was confirmed by re-introduction into Arabidopsis (Kakimoto, 1996). This was followed by reports of several dominant mutants such as TINY, LFfY and SFfl using a similar approach along with the Ds transposable element (Wilson et al, 1996, Schaffer et ah, 1998, Fridborg et ah, 1999). In a more recent report, activation T-DNA tagging and screening plants for an early flowering phenotype led to the isolation of the FT gene (Kardailsky et ah, 1999).

The potential application of activation tagging as a high through put technology for gene discovery has been demonstrated based on screening of several dominant mutant genes involved in photoreceptor, brassinosteroid, gibberellin and flowering signal pathways, as well as disease resistance. (See, e.g., Weigel et ah, 2000, Christensen et ah, 1998; Kardailsky et ah, 1999).

SUMMARY OF THE INVENTION

The invention provides nucleic acid and amino acid sequences associated with the Waxy Curled Leaf ("WCLl ") phenotype in plants, identified for its altered leaf, flowering, and stature phenotypes relative to wild-type Arabidopsis plants.

In one aspect, the invention provides one or more isolated WCLl nucleic acid sequences comprising a nucleic acid sequence that encodes or is complementary to a sequence that encodes a WCLl polypeptide having at least 70%, 80%, 90% or more sequence identity to the amino acid sequence presented as SEQ ID NO: 2.

In another aspect, the polynucleotide comprises a nucleic acid sequence that hybridizes, under high, medium, or low stringency conditions to the nucleic acid sequence, or fragment thereof, presented as SEQ ED NO:l, or the complement thereof. In a related aspect, expression of one or more of such WCLl polynucleotides in a plant is associated with the WCLl phenotype.

The invention further provides plant transformation vectors, plant cells, plant parts and plants comprising a WCLl nucleic acid sequence.

Expression of such a WCLl nucleic acid sequence in a plant is associated with the WCLl phenotype, presented as an altered leaf morphology, late flowering, and compact stature phenotype.

The expression of a WCLl nucleic acid sequence may be modified in ornamental plants, fruit and vegetable-producing plants, grain-producing plants, oil-producing plants and nut-producing plants, as well as other crop plants, resulting in the WCLl phenotype. In a further aspect the invention provides a method of modifying the WCLl phenotype in a plant by introducing a WCLl nucleic acid sequence into plant progenitor cells and growing the cells to produce a transgenic plant.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions.

Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as they would to one skilled in the art of the present invention. Practitioners are particularly directed to Sambrook et ah, 1989, and Ausubel FM et ah, 1993, for definitions and terms of the art. It is to be understood that this invention is not limited to the particular methodology, protocols, and reagents described, as these may vary.

All publications cited herein, and listed below immediately after the examples, are expressly incorporated herein by reference for the purpose of describing and disclosing compositions and methodologies that might be used in connection with the invention. All cited patents, patent publications, and sequence information in referenced websites are also incorporated by reference.

As used herein, the term "vector" refers to a nucleic acid construct designed for transfer between different host cells. An "expression vector" refers to a vector that has the ability to incorporate and express heterologous DNA fragments in a foreign cell. Many prokaryotic and eukaryotic expression vectors are commercially available. Selection of appropriate expression vectors is within the knowledge of those having skill in the art.

A "heterologous" nucleic acid construct or sequence has a portion of the sequence which is not native to the plant cell in which it is expressed. Heterologous, with respect to a control sequence refers to a control sequence (i.e. promoter or enhancer) that does not function in nature to regulate the same gene the expression of which it is currently regulating. Generally, heterologous nucleic acid sequences are not endogenous to the cell or part of the genome in which they are present, and have been added to the cell, by infection, transfection, microinjection, electroporation, or the like. A "heterologous" nucleic acid construct may contain a control sequence/DNA coding sequence combination that is the same as, or different from a control sequence/DNA coding sequence combination found in the native plant.

As used herein, the term "gene" means the segment of DNA involved in producing a polypeptide chain, which may or may not include regions preceding and following the coding region, e.g. 5' untranslated (5' UTR) or "leader" sequences and 3' UTR or "trailer" sequences, as well as intervening sequences (introns) between individual coding segments (exons).

The term "% homology" is used interchangeably herein with the term "% identity."

As used herein, "recombinant" includes reference to a cell or vector, that has been modified by the introduction of a heterologous nucleic acid sequence or that the cell is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found in identical form within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all as a result of deliberate human intervention. As used herein, the terms "transformed", "stably transformed" or "transgenic" with reference to a plant cell means the plant cell has a non-native (heterologous) nucleic acid sequence integrated into its genome which is maintained through two or more generations.

As used herein, the term "gene expression" refers to the process by which a polypeptide is produced based on the nucleic acid sequence of a gene. The process includes both transcription and translation; accordingly, "expression" may refer to either a polynucleotide or polypeptide sequence, or both. Sometimes, expression of a polynucleotide sequence will not lead to protein translation. "Over-expression" refers to increased expression of a polynucleotide and/or polypeptide sequence relative to its expression in a wild-type plant and may relate to a naturally-occurring or non-naturally occurring sequence. "Under-expression" refers to decreased expression of a polynucleotide and/or polypeptide sequence, generally of an endogenous gene, relative to its expression in a wild-type plant. The term "mis-expression" encompasses both over- expression and under-expression. The term "introduced" in the context of inserting a nucleic acid sequence into a cell, means "transfection", or "transformation" or "transduction" and includes reference to the incorporation of a nucleic acid sequence into a eukaryotic or prokaryotic cell where the nucleic acid sequence may be incorporated into the genome of the cell (for example, chromosome, plasmid, plastid, or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (for example, transfected mRNA).

As used herein, a "plant cell" refers to any cell derived from a plant, including cells from undifferentiated tissue (e.g., callus) as well as plant seeds, pollen, progagules and embryos.

As used herein, the terms "native" and "wild-type" relative to a given plant trait or phenotype refers to the form in which that trait or phenotype is found in the same variety of plant in nature.

As used herein, the term "modified" regarding a plant trait, refers to a change in the phenotype of a transgenic plant relative to a non-transgenic plant, as it is found in nature. As used herein, the term "Ti" refers to the generation of plants from the seed of T₀ plants. The Ti generation is the first set of transformed plants that can be selected by application of a selection agent, e.g., an antibiotic or herbicide, for which the transgenic plant contains the corresponding resistance gene.

As used herein, the term "T₂" refers to the generation of plants by self-fertilization of the flowers of Ti plants, previously selected as being transgenic. As used herein, the term "plant part" includes any plant organ or tissue including, without limitation, seeds, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen, and microspores. Plant cells can be obtained from any plant organ or tissue and cultures prepared therefrom. The class of plants which can be used in the methods of the present invention is generally as broad as the class of higher plants amenable to transformation techniques, including both monocotyledenous and dicotyledenous plants.

As used herein, "transgenic plant" includes reference to a plant that comprises within its genome a heterologous polynucleotide. Generally, the heterologous polynucleotide is stably integrated within the genome such that the polynucleotide is passed on to successive generations. The heterologous polynucleotide may be integrated into the genome alone or as part of a recombinant expression cassette. "Transgenic" is used herein to include any cell, cell line, callus, tissue, plant part or plant, the genotype of which has been altered by the presence of heterologous nucleic acid including those transgenics initially so altered as well as those created by sexual crosses or asexual propagation from the initial transgenic.

Thus a plant having within its cells a heterologous polynucleotide is referred to herein as a "transgenic plant". The heterologous polynucleotide can be either stably integrated into the genome, or can be extra-chromosomal. Preferably, the polynucleotide of the present invention is stably integrated into the genome such that the polynucleotide is passed on to successive generations. The polynucleotide is integrated into the genome alone or as part of a recombinant expression cassette. "Transgenic" is used herein to include any cell, cell line, callus, tissue, plant part or plant, the genotype of which has been altered by the presence of heterologous nucleic acids including those transgenics initially so altered as well as those created by sexual crosses or asexual reproduction of the initial transgenics.

A plant cell, tissue, organ, or plant into which the recombinant DNA constructs containing the expression constructs have been introduced is considered "transformed", "transfected", or "transgenic". A transgenic or transformed cell or plant also includes progeny of the cell or plant and progeny produced from a breeding program employing such a transgenic plant as a parent in a cross and exhibiting an altered phenotype resulting from the presence of a recombinant nucleic acid sequence. Hence, a plant of the invention will include any plant which has a cell containing a construct with introduced nucleic acid sequences, regardless of whether the sequence was introduced into the directly through transformation means or introduced by generational transfer from a progenitor cell which originally received the construct by direct transformation.

The terms "Waxy Curled Leaf " and "WCLl", as used herein encompass native Waxy Curled Leaf (WCLl) nucleic acid and amino acid sequences, homologues, variants and fragments thereof. An "isolated" WCLl nucleic acid molecule is a WCLl nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the natural source of the WCLl nucleic acid. An isolated WCLl nucleic acid molecule is other than in the form or setting in which it is found in nature. However, an isolated WCLl nucleic acid molecule includes WCLl nucleic acid molecules contained in cells that ordinarily express WCLl where, for example, the nucleic acid molecule is in a chromosomal location different from that of natural cells.

As used herein, the term "mutant" with reference to a polynucleotide sequence or gene differs from the corresponding wild type polynucleotide sequence or gene either in terms of sequence or expression, where the difference contributes to a modified plant phenotype or trait. Relative to a plant or plant line, the term "mutant" refers to a plant or plant line which has a modified plant phenotype or trait, where the modified phenotype or trait is associated with the modified expression of a wild type polynucleotide sequence or gene. Generally, a "variant" polynucleotide sequence encodes a "variant" amino acid sequence which is altered by one or more amino acids from the reference polypeptide sequence. The variant polynucleotide sequence may encode a variant amino acid sequence having "conservative" or "non-conservative" substitutions. Variant polynucleotides may also encode variant amino acid sequences having amino acid insertions or deletions, or both.

As used herein, the term "phenotype" may be used interchangeably with the term "trait". The terms refer to a plant characteristic which is readily observable or measurable and results from the interaction of the genetic make-up of the plant with the environment in which it develops. Such a phenotype includes chemical changes in the plant make-up resulting from enhanced gene expression which may or may not result in morphological changes in the plant, but which are measurable using analytical techniques known to those of skill in the art.

As used herein, the term "interesting phenotype" with reference to a plant produced by the methods described herein refers to a readily observable or measurable phenotype demonstrated by a Ti and/or subsequent generation plant, which is not displayed by a plant that has not been so transformed (and/or is not the progeny of a plant that has been so transformed) and represents an improvement in the plant. An "improvement" is a feature that may enhance the utility of a plant species or variety by providing the plant with a unique quality. By unique quality is meant a novel feature or a change to an existing feature of the plant species which is a quantitative change (increase or decrease) or a qualitative change in a given feature or trait.

II. The Identified WCLl Phenotype and Gene. The gene and phenotype of this invention were identified in a large-scale screen using activation tagging. Activation tagging is a process by which a heterologous nucleic acid construct comprising a nucleic acid control sequence, e.g. an enhancer, is inserted into a plant genome. The enhancer sequences act to enhance transcription of a one or more native plant genes (See, e.g., Walden R, et ah, 1994; Weigel D et al. 2000). Briefly, a large number of Arabidopsis plants were transformed with the activation tagging vector pSKI015 (Weigel et al, 2000), which comprises a T-DNA (i.e., the sequence derived from the Ti plasmid of Agrobacterium tumifaciens that are transferred to a plant cell host during Agrobacterium infection), an enhancer element and a selectable marker gene. Following random insertion of pSKI015 into the genome of transformed plants, the enhancer element can result in up-regulation genes in the vicinity of the T-DNA insertion, generally within 5-10 kilobase (kb) of the insertion. In the Ti generation, plants were exposed to the selective agent in order to specifically recover those plants that expressed the selectable marker and therefore harbored insertions of the activation-tagging vector. Transformed plants were observed for interesting phenotypes, which are generally identified at the Ti, T₂ and or T₃ generations. Interesting phenotypes may be identified based on morphology, a biochemical screen, herbicide tolerance testing, herbicide target identification, fungal or bacterial resistance testing, insect or nematode resistance testing, screening for stress tolerance, such as drought, salt or antibiotic tolerance, and output traits, such as oil, starch, pigment, or vitamin composition. Genomic sequence surrounding the T-DNA insertion is analyzed in order to identify genes responsible for the interesting phenotypes. Genes responsible for causing such phenotypes are identified as attractive targets for manipulation for agriculture, food, ornamental plant, and/or pharmaceutical industries.

It will be appreciated that in most cases when a modified phenotype results from the enhanced expression of a tagged gene, the phenotype is dominant. In some cases, the enhanced expression of a given native plant gene or a fragment thereof may result in decreased expression or inactivation of its homologue or another native plant gene, which results in the interesting phenotype. The T-DNA insertion may also result in disruption ("loss-of-function") of a native plant gene, in which case the phenotype is generally recessive.

The present invention provides an altered leaf morphology, late flowering, and compact stature phenotype (the "WCLl phenotype"), identified in an ACTTAG Arabidopsis line, where Ti plants were observed as having waxy leaf epidermis, high chlorophyll content, no leaf petiole, reduced stem height, and late flowering. T₂ plants were observed as having waxy leaf epidermis, curled leaf margins, no leaf serration, no petiole, reduced stem height, compact stature and late flowering. The phenotype and associated gene have been designated Waxy Curled Leaf ("WCLl"). The invention also presents a novel correlation between the WCLl phenotype and the nucleic acid (cDNA) sequence provided in SEQ ID NO:l, which was identified by analysis of the genomic DNA sequence surrounding the T-DNA insertion correlating with the WCLl phenotype. The characterization of the correlation between the WCLl gene and phenotype is described in detail in the Examples.

III. Compositions of the Invention

A. WCLl Nucleic acids and Polypeptides

Arabidopsis WCLl nucleic acid (cDNA) sequence is provided in SEQ ED NO:l and in Genbank entry GI 9502365, complement of nucleotides 76139-75219. The corresponding protein sequence is provided in SEQ ID NO:2 and in GI 9743351.

The WCLl gene is predicted to encode a protein with an AP2, DNA-binding domain. As used herein, the term "WCLl polypeptide" refers to a full-length WCLl protein or a fragment, derivative (variant), or ortholog thereof that is "functionally active," meaning that the protein fragment, derivative, or ortholog exhibits one or more or the functional activities associated with the polypeptide of SEQ ID NO:2. In one preferred embodiment, a functionally active WCLl polypeptide causes a WCLl phenotype when mis-expressed in a plant. In another embodiment, a functionally active WCLl polypeptide is capable of rescuing defective (including deficient) endogenous WCLl activity when expressed in a plant or in plant cells; the rescuing polypeptide may be from the same or from a different species as that with defective activity. In another embodiment, a functionally active fragment of a full length WCLl polypeptide (i.e., a native polypeptide having the sequence of SEQ ID NO:2 or a naturally occurring ortholog thereof) retains one of more of the biological properties associated with the full-length WCLl polypeptide, such as signaling activity, binding activity, catalytic activity, or cellular or extra-cellular localizing activity. Preferred WCLl polypeptides display DNA binding activity. A WCLl fragment preferably comprises a WCLl domain, such as a C- or N-terminal or catalytic domain, among others, and preferably comprises at least 10, preferably at least 20, more preferably at least 25, and most preferably at least 50 contiguous amino acids of a WCLl protein. A preferred WCLl fragment comprises an AP2 domain. Functional domains can be identified using the PFAM program (Bateman A et al., 1999 Nucleic Acids Res 27:260- 262; website at pfam.wustl.edu). Functionally active variants of full-length WCLl polypeptides or fragments thereof include polypeptides with amino acid insertions, deletions, or substitutions that retain one of more of the biological properties associated with the full-length WCLl polypeptide. In some cases, variants are generated that change the post-translational processing of a WCLl polypeptide. For instance, variants may have altered protein transport or protein localization characteristics or altered protein half-life compared to the native polypeptide.

As used herein, the term "WCLl nucleic acid" encompasses nucleic acids with the sequence provided in or complementary to the sequence provided in SEQ ID NO: 1, as well as functionally active fragments, derivatives, or orthologs thereof. A WCLl nucleic acid of this invention may be DNA, derived from genomic DNA or cDNA, or RNA.

In one embodiment, a functionally active WCLl nucleic acid encodes or is complementary to a nucleic acid that encodes a functionally active WCLl polypeptide. Included within this definition is genomic DNA that serves as a template for a primary RNA transcript (i.e., an mRNA precursor) that requires processing, such as splicing, before encoding the functionally active WCLl polypeptide. A WCLl nucleic acid can include other non-coding sequences, which may or may not be transcribed; such sequences include 5' and 3' UTRs, polyadenylation signals and regulatory sequences that control gene expression, among others, as are known in the art. Some polypeptides require processing events, such as proteolytic cleavage, covalent modification, etc., in order to become fully active. Accordingly, functionally active nucleic acids may encode the mature or the pre-processed WCLl polypeptide, or an intermediate form. A WCLl polynucleotide can also include heterologous coding sequences, for example, sequences that encode a marker included to facilitate the purification of the fused polypeptide, or a transformation marker.

In another embodiment, a functionally active WCLl nucleic acid is capable of being used in the generation of loss-of -function WCLl phenotypes, for instance, via antisense suppression, co-suppression, etc. In one preferred embodiment, a WCLl nucleic acid used in the methods of this invention comprises a nucleic acid sequence that encodes or is complementary to a sequence that encodes a WCLl polypeptide having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or more sequence identity to the polypeptide sequence presented in SEQ ID NO:2.

In another embodiment a WCLl polypeptide of the invention comprises a polypeptide sequence with at least 50% or 60% identity to the WCLl polypeptide sequence of SEQ ID NO:2, and may have at least 70%, 80%, 85%, 90% or 95% or more sequence identity to the WCLl polypeptide sequence of SEQ ID NO: 2. In another embodiment, a WCLl polypeptide comprises a polypeptide sequence with at least 50%, 60%, 70%, 80%, 85%, 90% or 95% or more sequence identity to a functionally active fragment of the polypeptide presented in SEQ ID NO:2, such as an AP2 domain. In yet another embodiment, a WCLl polypeptide comprises a polypeptide sequence with at least 50%, 60 %, 70%, 80%, or 90% identity to the polypeptide sequence of SEQ ID NO:2 over its entire length and comprises an AP2 domain.

In another aspect, a WCLl polynucleotide sequence is at least 50% to 60% identical over its entire length to the WCLl nucleic acid sequence presented as SEQ ED NO:l, or nucleic acid sequences that are complementary to such a WCLl sequence, and may comprise at least 70%, 80%, 85%, 90% or 95% or more sequence identity to the WCLl sequence presented as SEQ ID NO: 1 or a functionally active fragment thereof, or complementary sequences.

As used herein, "percent (%) sequence identity" with respect to a specified subject sequence, or a specified portion thereof, is defined as the percentage of nucleotides or amino acids in the candidate derivative sequence identical with the nucleotides or amino acids in the subject sequence (or specified portion thereof), after aligning the sequences and introducing gaps, if necessary to achieve the maximum percent sequence identity, as generated by the program WU-BLAST-2.0al9 (Altschul et ah, J. Mol. Biol. (1997) 215:403-410; website atblast.wustl.edu/blast/README.html) with search parameters set to default values. The HSP S and HSP S2 parameters are dynamic values and are established by the program itself depending upon the composition of the particular sequence and composition of the particular database against which the sequence of interest is being searched. A "% identity value" is determined by the number of matching identical nucleotides or amino acids divided by the sequence length for which the percent identity is being reported. "Percent (%) amino acid sequence similarity" is determined by doing the same calculation as for determining % amino acid sequence identity, but including conservative amino acid substitutions in addition to identical amino acids in the computation. A conservative amino acid substitution is one in which an amino acid is substituted for another amino acid having similar properties such that the folding or activity of the protein is not significantly affected. Aromatic amino acids that can be substituted for each other are phenylalanine, tryptophan, and tyrosine; interchangeable hydrophobic amino acids are leucine, isoleucine, methionine, and valine; interchangeable polar amino acids are glutamine and asparagine; interchangeable basic amino acids are arginine, lysine and histidine; interchangeable acidic amino acids are aspartic acid and glutamic acid; and interchangeable small amino acids are alanine, serine, threonine, cysteine and glycine.

Derivative nucleic acid molecules of the subject nucleic acid molecules include sequences that hybridize to the nucleic acid sequence of SEQ ED NO: 1. The stringency of hybridization can be controlled by temperature, ionic strength, pH, and the presence of denaturing agents such as formamide during hybridization and washing. Conditions routinely used are well known (see, e.g., Current Protocol in Molecular Biology, Vol. 1, Chap. 2.10, John Wiley & Sons, Publishers (1994); Sambrook et ah, Molecular Cloning, Cold Spring Harbor (1989)). In some embodiments, a nucleic acid molecule of the invention is capable of hybridizing to a nucleic acid molecule containing the nucleotide sequence of SEQ ID NO: 1 under stringent hybridization conditions that comprise: prehybridization of filters containing nucleic acid for 8 hours to overnight at 65° C in a solution comprising 6X single strength citrate (SSC) (IX SSC is 0.15 M NaCl, 0.015 M Na citrate; pH 7.0), 5X Denhardt's solution, 0.05% sodium pyrophosphate and 100 μg/ml herring sperm DNA; hybridization for 18-20 hours at 65° C in a solution containing 6X SSC, IX Denhardt's solution, 100 μg/ml yeast tRNA and 0.05% sodium pyrophosphate; and washing of filters at 65° C for 1 h in a solution containing 0.2X SSC and 0.1% SDS (sodium dodecyl sulfate). In other embodiments, moderately stringent hybridization conditions are used that comprise: pretreatment of filters containing nucleic acid for 6 h at 40° C in a solution containing 35% formamide, 5X SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.1%) PVP, 0.1% Ficoll, 1 % B SA, and 500 μg/ml denatured salmon sperm DNA; hybridization for 18-20 h at 40° C in a solution containing 35% formamide, 5X SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 μg/ml salmon sperm DNA, and 10% (wt/vol) dextran sulfate; followed by washing twice for 1 hour at 55° C in a solution containing 2X SSC and 0.1% SDS. Alternatively, low stringency conditions can be used that comprise: incubation for 8 hours to overnight at 37° C in a solution comprising 20% formamide, 5 x SSC, 50 mM sodium phosphate (pH 7.6), 5X Denhardt's solution, 10% dextran sulfate, and 20 μg/ml denatured sheared salmon sperm DNA; hybridization in the same buffer for 18 to 20 hours; and washing of filters in 1 x SSC at about 37° C for 1 hour.

As a result of the degeneracy of the genetic code, a number of polynucleotide sequences encoding a WCLl polypeptide can be produced. For example, codons may be selected to increase the rate at which expression of the polypeptide occurs in a particular host species, in accordance with the optimum codon usage dictated by the particular host organism (see, e.g., Nakamura et al, 1999). Such sequence variants may be used in the methods of this invention.

The methods of the invention may use orthologs of the Arabidopsis WCLl. Methods of identifying the orthologs in other plant species are known in the art. Normally, orthologs in different species retain the same function, due to presence of one or more protein motifs and/or 3-dimensional structures. In evolution, when a gene duplication event follows speciation, a single gene in one species, such as Arabidopsis, may correspond to multiple genes (paralogs) in another. As used herein, the term "orthologs" encompasses paralogs. When sequence data is available for a particular plant species, orthologs are generally identified by sequence homology analysis, such as BLAST analysis, usually using protein bait sequences. Sequences are assigned as a potential ortholog if the best hit sequence from the forward BLAST result retrieves the original query sequence in the reverse BLAST (Huynen MA and Bork P, Proc Natl Acad Sci (1998) 95:5849-5856; Huynen MA et ah, Genome Research (2000) 10:1204-1210). Programs for multiple sequence alignment, such as CLUSTAL (Thompson JD et al, 1994, Nucleic Acids Res 22:4673-4680) may be used to highlight conserved regions and/or residues of orthologous proteins and to generate phylogenetic trees. In a phylogenetic tree representing multiple homologous sequences from diverse species (e.g., retrieved through BLAST analysis), orthologous sequences from two species generally appear closest on the tree with respect to all other sequences from these two species. Structural threading or other analysis of protein folding (e.g., using software by ProCeryon, Biosciences,

Salzburg, Austria) may also identify potential orthologs. Nucleic acid hybridization methods may also be used to find orthologous genes and are preferred when sequence data are not available. Degenerate PCR and screening of cDNA or genomic DNA libraries are common methods for finding related gene sequences and are well known in the art (see, e.g., Sambrook, 1989; Dieffenbach and Dveksler, 1995). For instance, methods for generating a cDNA library from the plant species of interest and probing the library with partially homologous gene probes are described in Sambrook et al. A highly conserved portion of the Arabidopsis WCLl coding sequence may be used as a probe. WCLl ortholog nucleic acids may hybridize to the nucleic acid of SEQ ID NO: 1 under high, moderate, or low stringency conditions. After amplification or isolation of a segment of a putative ortholog, that segment may be cloned and sequenced by standard techniques and utilized as a probe to isolate a complete cDNA or genomic clone. Alternatively, it is possible to initiate an EST project to generate a database of sequence information for the plant species of interest. In another approach, antibodies that specifically bind known WCLl polypeptides are used for ortholog isolation (see, e.g., Harlow and Lane, 1988, 1999). Western blot analysis can determine that a WCLl ortholog (i.e., an orthologous protein) is present in a crude extract of a particular plant species. When reactivity is observed, the sequence encoding the candidate ortholog may be isolated by screening expression libraries representing the particular plant species. Expression libraries can be constructed in a variety of commercially available vectors, including lambda gtl 1, as described in Sambrook, et ah, 1989. Once the candidate ortholog(s) are identified by any of these means, candidate orthologous sequence are used as bait (the "query") for the reverse BLAST against sequences from Arabidopsis or other species in which WCLl nucleic acid and/or polypeptide sequences have been identified.

WCLl nucleic acids and polypeptides may be obtained using any available method. For instance, techniques for isolating cDNA or genomic DNA sequences of interest by screening DNA libraries or by using polymerase chain reaction (PCR), as previously described, are well known in the art. Alternatively, nucleic acid sequence may be synthesized. Any known method, such as site directed mutagenesis (Kunkel TA et ah, 1991), may be used to introduce desired changes into a cloned nucleic acid.

In general, the methods of the invention involve incorporating the desired form of the WCLl nucleic acid into a plant expression vector for transformation of in plant cells, and the WCLl polypeptide is expressed in the host plant.

B. Antibodies.

The present invention further provides anti- WCLl polypeptide antibodies. The antibodies may be polyclonal, monoclonal, humanized, bispecific or heteroconjugate antibodies. Methods of preparing polyclonal antibodies are known to the skilled artisan. Such polyclonal antibodies can be produced in a mammal, for example, following one or more injections of an immunizing agent, and preferably, an adjuvant. Typically, the immunizing agent and/or adjuvant will be injected into the mammal by a series of subcutaneous or intraperitoneal injections. The immunizing agent may include a WCLl polypeptide or a fusion protein thereof. It may be useful to conjugate the antigen to a protein known to be immunogenic in the mammal being immunized. The immunization protocol may be determined by one skilled in the art based on standard protocols or by routine experimentation. Alternatively, the anti- WCLl polypeptide antibodies may be monoclonal antibodies.

Monoclonal antibodies may be produced by hybridomas, wherein a mouse, hamster, or other appropriate host animal, is immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent [Kohler et ah, 1975]. Monoclonal antibodies may also be made by recombinant DNA methods, such as those described in U.S. Patent No. 4,816,567.

The anti- WCLl polypeptide antibodies of the invention may further comprise humanized antibodies or human antibodies. The term "humanized antibody" refers to humanized forms of non-human (e.g., murine) antibodies that are chimeric antibodies, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')₂ or other antigen- binding partial sequences of antibodies) which contain some portion of the sequence derived from non-human antibody. Methods for humanizing non-human antibodies are well known in the art, as further detailed in Jones et ah, 1986; Riechmann et ah, 1988; and Verhoeyen et ah, 1988. Methods for producing human antibodies are also known in the art. See, e.g., Jakobovits, A, et ah, 1995; Jakobovits, A, 1995. In one exemplary approach, anti- WCLl polyclonal antibodies are used for gene isolation. Western blot analysis may be conducted to determine that WCLl or a related protein is present in a crude extract of a particular plant species. When reactivity is observed, genes encoding the related protein may be isolated by screening expression libraries representing the particular plant species. Expression libraries can be constructed in a variety of commercially available vectors, including lambda gtl 1, as described in Sambrook, et al. , 1989. IV. Utility Of the WCLl Phenotype and Gene

From the foregoing, it can be appreciated that the WCLl nucleotide sequence, protein sequence and phenotype find utility in modulated expression of the WCLl protein and the development of non-native phenotypes associated with such modulated expression.

The WCLl altered leaf morphology, late flowering, and compact stature phenotype has features that distinguish the mutant from wild type Arabidopsis.

The compact stature is an attractive trait to incorporate into turf grass for reducing mowing/grooming requirements and for producing thicker lawns. Reducing plant height would also be beneficial in fruit trees that are several meters high. Reducing height in such plants would lead to more efficient fruit harvest with less wastage. The generally compact phenotype also offers potential for higher planting density with some crops - shortened petioles would allow closer spacing, and reduced height would allow more tiers of planting, especially in hydroponic systems. Higher fruit yield per unit resource (such as water and fertilizer) is another potential advantage when the plant size is reduced since the total consumption of resources will be reduced for plants of smaller stature. The waxy cuticle may confer increased drought tolerance, which could have utility in a variety of arid land agricultural crops. The waxy cuticle may confer increased pathogen resistance, and the unusual curled leaf phenotype could be of value in ornamental plants. The late flowering trait has potential utility for extending a crop's growing season, as well as for extending the functional range of a fruit producing plant (later fruiting would protect against the frosts that occur later in the growing season at more extreme latitudes). The observed morphology is one version of the phenotype.

In practicing the invention, the WCLl phenotype and modified WCLl expression is generally applicable to any type of plant.

The methods described herein are generally applicable to all plants. Although activation tagging and gene identification is carried out in Arabidopsis, following identification of a nucleic acid sequence and associated phenotype, the selected gene, a homologue, variant or fragment thereof, may be expressed in any type of plant. In one aspect, the invention is directed to fruit- and vegetable-bearing plants. In a related aspect, the invention is directed to the cut flower industry, grain-producing plants, oil-producing plants and nut-producing plants, as well as other crops including, but not limited to, cotton (Gossypium), alfalfa (Medicago sativa), flax (Linum usitatissimum), tobacco (Nicotiana), turfgrass (Poaceae family), and other forage crops. The skilled artisan will recognize that a wide variety of transformation techniques exist in the art, and new techniques are continually becoming available. Any technique that is suitable for the target host plant can be employed within the scope of the present invention. For example, the constructs can be introduced in a variety of forms including, but not limited to as a strand of DNA, in a plasmid, or in an artificial chromosome. The introduction of the constructs into the target plant cells can be accomplished by a variety of techniques, including, but not limited to Agrobacterium-mediated transformation, electroporation, microinjection, microprojectile bombardment calcium-phosphate-DNA co-precipitation or liposome-mediated transformation of a heterologous nucleic acid construct comprising the WCLl coding sequence. The transformation of the plant is preferably permanent, i.e. by integration of the introduced expression constructs into the host plant genome, so that the introduced constructs are passed onto successive plant generations.

In one embodiment, binary Ti-based vector systems may be used to transfer and confirm the association between mis-expression (e.g. , over-expression or under- expression) of an identified gene with a particular plant trait or phenotype. Standard Agrobacterium binary vectors are known to those of skill in the art and many are commercially available, such as pBI121 (Clontech Laboratories, Palo Alto, CA).

The optimal procedure for transformation of plants with Agrobacterium vectors will vary with the type of plant being transformed. Exemplary methods for

Agrobacterium-msdiat d transformation include transformation of explants of hypocotyl, shoot tip, stem or leaf tissue, derived from sterile seedlings and or plantlets. Such transformed plants may be reproduced sexually, or by cell or tissue culture. Agrobacterium transformation has been previously described for a large number of different types of plants and methods for such transformation may be found in the scientific literature.

Depending upon the intended use, a heterologous nucleic acid construct may be made which comprises a nucleic acid sequence associated with the WCLl phenotype, and which encodes the entire protein, or a biologically active portion thereof for transformation of plant cells and generation of transgenic plants.

The expression of a WCLl nucleic acid sequence or a homologue, variant or fragment thereof may be carried out under the control of a constitutive, inducible or regulatable promoter. In some cases expression of the WCLl nucleic acid sequence or homologue, variant or fragment thereof may regulated in a developmental stage or tissue- associated or tissue-specific manner. Accordingly, expression of the nucleic acid coding sequences described herein may be regulated with respect to the level of expression, the tissue type(s) where expression takes place and/or developmental stage of expression leading to a wide spectrum of applications wherein the expression of a WCLl coding sequence is modulated in a plant.

Strong promoters with enhancers may result in a high level of expression. When a low level of basal activity is desired, a weak promoter may be a better choice. Expression of WCLl nucleic acid sequence or homologue, variant or fragment thereof may be controlled at the level of transcription, by the use of cell type specific promoters or promoter elements in the plant expression vector.

Numerous promoters useful for heterologous gene expression are available. Exemplary constitutive promoters include the raspberry E4 promoter (U.S. Patent Nos. 5,783,393 and 5,783,394), the 35S CaMV (Jones JD et al, 1992), the CsVMV promoter (Verdaguer B et ah, 1998) and the melon actin promoter (PCT application WO0056863). Exemplary tissue-specific promoters include the tomato E4 and E8 promoters (U.S. Patent No. 5,859,330) and the tomato 2AII gene promoter (Van Haaren MJJ et ah, 1993).

When WCLl sequences are intended for use as probes, a particular portion of a WCLl encoding sequence, for example a highly conserved portion of a coding sequence may be used. In yet another aspect, in some cases it may be desirable to inhibit the expression of endogenous WCLl sequences in a host cell. Exemplary methods for practicing this aspect of the invention include, but are not limited to antisense suppression (Smith, et αZ., 1988); co-suppression (Napoli, et αZ.,1989); ribozymes (PCT Publication WO 97/10328); and combinations of sense and antisense (Waterhouse, et ah, 1998). Methods for the suppression of endogenous sequences in a host cell typically employ the transcription or transcription and translation of at least a portion of the sequence to be suppressed. Such sequences may be homologous to coding as well as non-coding regions of the endogenous sequence. In some cases, it may be desirable to inhibit expression of the WCLl nucleotide sequence. This may be accomplished using procedures generally employed by those of skill in the art together with the WCLl nucleotide sequence provided herein.

Standard molecular and genetic tests may be performed to analyze the association between a cloned gene and an observed phenotype. A number of other techniques that are useful for determining (predicting or confirming) the function of a gene or gene product in plants are described below. 1. DNA/RNA analysis

DNA taken form a mutant plant may be sequenced to identify the mutation at the nucleotide level. The mutant phenotype may be rescued by overexpressing the wild type (WT) gene. The stage- and tissue-specific gene expression patterns in mutant vs. WT lines, for instance, by in situ hybridization, may be determined. Analysis of the methylation status of the gene, especially flanking regulatory regions, may be performed. Other suitable techniques include overexpression, ectopic expression, expression in other plant species and gene knock-out (reverse genetics, targeted knock-out, viral induced gene silencing (VIGS, see Baulcombe D, 1999). In a preferred application, microarray analysis, also known as expression profiling or transcript profiling, is used to simultaneously measure differences or induced changes in the expression of many different genes. Techniques for microarray analysis are well known in the art (Schena M et ah, Science (1995) 270:467-470; Baldwin D et ah, 1999; Dangond F, Physiol Genomics (2000) 2:53-58; van Hal NL et ah, J Biotechnol (2000) 78:271-280; Richmond T and Somerville S, Curr Opin Plant Biol (2000) 3:108-116).

Microarray analysis of individual tagged lines may be carried out, especially those from which genes have been isolated. Such analysis can identify other genes that are coordinately regulated as a consequence of the overexpression of the gene of interest, which may help to place an unknown gene in a particular pathway. 2. Gene Product Analysis

Analysis of gene products may include recombinant protein expression, antisera production, immunolocalization, biochemical assays for catalytic or other activity, analysis of phosphorylation status, and analysis of interaction with other proteins via yeast two-hybrid assays. 3. Pathway Analysis

Pathway analysis may include placing a gene or gene product within a particular biochemical or signaling pathway based on its overexpression phenotype or by sequence homology with related genes. Alternatively, analysis may comprise genetic crosses with WT lines and other mutant lines (creating double mutants) to order the gene in a pathway, or determining the effect of a mutation on expression of downstream "reporter" genes in a pathway. 4. Other Analyses

Other analyses may be performed to determine or confirm the participation of the isolated gene and its product in a particular metabolic or signaling pathway, and to help determine gene function. All publications, patents and patent applications are herein expressly incorporated by reference in their entirety.

While the invention has been described with reference to specific methods and embodiments, it will be appreciated that various modifications and changes may be made without departing from the invention.

EXAMPLE 1 Generation of Plants with a WCLl Phenotype by Transformation with an Activation Tagging Construct

A. Agrobacterium vector preparation. Mutants were generated using the activation tagging "ACTTAG" vector, pSKI015

(GenBank Identifier [GI] 6537289; Weigel D etah, 2000).).

Transformed E. coli colonies and cultures containing the pSKI015 activation tagging construct was confirmed by selection on media containing 100 μg/ml ampicillin. Agrobacterium colonies and cultures were grown in selective media containing 100 μg/ml carbenicillin. The presence of the pSKI015 construct was verified in colonies by

PCRprimers that span the ocs terminator in the BAR selection cassette under the following PCR conditions: 30 cycles of 94°C 30 seconds; 63°C 40 seconds; 72°C 120 seconds. For long-term storage, PCR-positive colonies were grown in selective media, glycerol added to a final concentration of 30% and cultures quick frozen then stored at - 80°C. For the initiation of dense Agrobacterium cultures for plant transformation, stock cultures were grown in selective media, glycerol added to a final concentration of 30%, and a number of 20 μl aliquots quick frozen in liquid nitrogen and stored at -80°C. pSKI015 was maintained in Agrobacterium GV3101 without the helper plasmid and in Agrobacterium strain EHA 105. An Agrobacterium culture was prepared by starting a 50 ml culture 4-5 days prior to plant transformation (e.g., by "dunking"). Liquid cultures were grown at 28°C, on an orbital shaker at 200 rpm, in LBB with Carbenicillin (Cb) at lOOmg/1 to select for the plasmid, with 50mg/l Kanamycin (Kan) added to select for the helper plasmid. After 2 days, this small culture was used to inoculate 6-8 liters (L) of LBB with Cb lOOmg/1 and Kan 50mg/l, IL each in 2000ml Erienmeyer flasks. Cultures are placed on a shaker for 2-3 days, checked for cell concentration by evaluating the ODβoQ (visible light at 600nm) using a spectrophotometer with an OD₆oo reading for between 1.5- 2.5 preferred. The cultures were then centrifuged at 4,500 RCF for 15 minutes at room temperature (18-22° C), the bacteria resuspended to approximately OD₆oo=0-8 with about 500 ml per dunking vessel. Approximately 15-20 liters were prepared for 200 pots, and 20-30 plants dunked at a time.

B. Growth and Selection of Arabidopsis thaliana Plants

Arabidopsis plants were grown in Premier HP soil which contains peat moss and perlite, using a minimal amount of N-P-K (171-2-133) fertilizer diluted to 1/10 the strength, with sub-irrigation, as needed and a n 18 hr day length using natural light supplemented by high pressure sodium lamps at a temperature of 20-25° C. Seeds were sown under humidity domes for the first 4-7 days, then transferred to a greenhouse having approximately 70% humidity. Healthy Arabidopsis plants were grown from wild type Arabidopsis seed, Ecotype:

Col-0, under long days (16 hrs) in pots in soil covered with bridal veil or window screen, until they flowered.

Plants began flowering after about 3-4 weeks, with watering and fertilizing continued as needed until a majority of the siliques turned yellow/brown. Then plants were then left to dry out and seed collected by breaking open siliques to release the seed.

Seed was stored at room temperature for a few days, then stored at 4°C in an airtight container with desiccant.

Plants are monitored for pests and pathogens, particularly, fungus gnats, white flies, and aphids, with pest control applied as needed, e.g., application of Talstar and Azatin for whitefly, thrips and fungus gnats; application of Gnatrol for fungus gnats, biological control (e.g. mites, for gnat larvae) and safer soap.

Transformation was accomplished via a floral dip method wherein floral tissues were dipped into a solution containing Agrobacterium tumefaciens, 5% sucrose and a surfactant Silwet L-77, as described in Cough, SJ and Bent, AF, 1998. Briefly, above-ground parts of 2,000-3,000 plants were dipped (dunked) into an

Agrobacterium culture (GV3101 with pMP90RK, helper plasmid) carrying ACTTAG

(binary plasmid pSKI.015), 2-3 days after clipping for 15 minutes, with gentle agitation, then placing plants on their sides under a humidity dome or cover for 16-24 hours to maintain high humidity. A second dunking was carried out 6 days after removing the humidity domes, as described above. Plants were watered regularly until seeds were mature, at which time watering was stopped.

C. Selection Of Transgenic Plants

Dry Ti seed was harvested from transformed plants and stored at 4°C in Eppendorf tubes with desiccant. Transformants were selected at the Ti stage by sprinkling Ti seed on a flat, cold treating the flats for 2 to 3 days and spraying plants as soon as they germinated with Finale (Basta, glufosinate ammonium), diluted at 1:1000 of an 11.33% solution, followed by subsequent sprayings a day or two apart.

Following sprayings, non-transgenic seedlings produced chlorotic primary leaves and their hypocotyls dehydrated and collapsed, killing the plant. The survivors were counted and segregation data calculated after the non-transgenic plants had died (within two-three weeks following the sprayings). Survivors were transplanted into individual pots for further monitoring.

Images of each pool of plants were recorded using a Digital camera (DC-260), and morphology observations were taken from plants that exhibited an interesting phenotype. These plants were grown until seed was produced, which was collected and sown to yield T₂ plants. The ACTTAG™ line, W000006568 C'WCLl") was originally identified as having waxy leaf epidermis, high chlorophyll content, no leaf petiole, reduced stem height, and late flowering in the Ti plants.

Interesting Ti plants were grown until they produced T₂ seed, which was collected and planted. The phenotype of the T₂ plants was described as exhibiting waxy leaf epidermis, curled leaf margins, no leaf serration, no petiole, reduced stem height, compact stature and late flowering relative to wild type Arabidopsis plants.

EXAMPLE 2 Characterization of Plants That Exhibit the WCLl Phenotype. A. Genomic DNA Extraction and Analysis.

Nucleon PhytoPure systems from Amersham were used to extract genomic DNA from T plant tissue. Methods were essentially as follows: l.Og of fresh plant tissue was ground in liquid nitrogen to yield a free flowing powder, then transferred to a 15-ml polypropylene centrifuge tube. 4.6 ml of Reagent 1 from the Nucleon Phytopure kit was added with thorough mixing followed by addition of 1.5 ml of Reagent 2 from the Nucleon Phytopure kit, with inversion until a homogeneous mixture was obtained. The mixture was incubated at 65°C in a shaking water bath for 10 minutes, and placed on ice for 20 minutes. The samples were removed from the ice, 2 ml of - 20°C chloroform added, mixed and centrifuged at 1300g for 10 minutes. The supernatant was transferred to a fresh tube, 2 ml cold chloroform, 200 μl of Nucleon PhytoPure DNA extraction resin suspension added and the mixture shaken on a tilt shaker for 10 minutes at room temperature, then centrifuged at 1300g for 10 minutes. Without disturbing the Nucleon resin suspension layer, the upper DNA containing phase was transferred to a fresh tube, centrifuged at 9500 rpm for 30 minutes to clarify the transferred aqueous phase, an equal volume of cold isopropanol added, the tube gently inverted until the DNA precipitated and then it was pelleted by centrifugation, washed with cold 70% ethanol, pelleted again and air-dried.

DNA extracted from plants with the WCLl phenotype (WCLl) and from wild type plants (COL-0) was PCR amplified using primers that amplify a 35S enhancer sequence, and primers that amplify a region of the pBluescript vector sequence in pSKI015. Amplification using primers that span the 35S enhancer region resulted in a ladder of products, indicating that all four copies of the 35S enhancer were present. Amplification using primers to the pBluescript vector was done primarily to detect the T-DNA insert(s) in transformed plants and has been optimized for the following conditions: annealing temp: 57°C, 30 cycles [94°C, 30sec; 57°C, 1 min; 72°C, 1 min] 1 cycle [72°C, 7 min]. The ACTTAG™ line, W000006568 (WCLl), was confirmed as positive for the presence of 35S enhancer and pSKI015 vector sequences by PCR, and as positive for Southern hybridization verifying genomic integration of the ACTTAG DNA and showing the presence of two T-DNA insertions in the transgenic line (T-DNA 1 and T-DNA2). In order to identify single insertion lines and to determine which insertion was responsible for the phenotype, T₂ mutant plants were crossed with Wild Type plants. PCR primers specific to the T-DNA1 insertion site were designed to test the correlation of phenotype to genotype in F2 backcross populations. Ten mutant individuals that segregated from the F2 population were then screened by PCR with primers designed to span the T-DNAl/genomic-DNA junction, in addition to primers amplifying the 35S enhancer region and generic genomic primers. Four single insertion lines were identified, and it was confirmed that the mutant phenotype did not correlate with the T-DNA 1 insertion. Inverse PCR from individuals in the segregating F2 population was used to identify sequence flanking the T-DNA2, and the phenotype was correlated to the T-DNA2 genotype.

B. Plasmid Rescue Genomic DNA from T2 plants of the insertion line W000006568 ("WCLl") was digested by the restriction enzymes used in Southern Hybridization. The restriction fragments were self-ligated and used to transform the E. coli cells. The plasmids that contained a full-length pBluescript vector, 4X 35S enhancer, and a right border T-DNA flanking genomic DNA fragment were rescued. More specifically, genomic DNA was digested with HindHI, Pstl and or Xhol under standard reaction conditions at 37°C overnight. Briefly, each restriction enzyme was heat inactivated at 65°C for 20 minutes, phenol/ chloroform and chloroform isoamyl (24:1) extracted once with each, and the ligation reactions were set up containing the reagents set forth below and left at 16°C overnight.

Digested Genomic DNA 40 μl

5X Ligation Buffer 50 μl

Ligase (Gibcol, lU/μl) 10 μl ddH₂O 150 μl

The ligated DNA was precipitated, resuspended in ddH2O and used to transform E. coli SURE cells (Stratagene) via electroporation, with 10 pg of pUC18 plasmid as a control.

The transformation mixture was spread on two LB -plates containing 100 μg/ml ampicillin and incubated overnight at 37°C. Single colonies were picked from the plates and used to start a 5 ml LB-ampicillin broth culture from each colony by culturing overnight at 37°C. The plasmid was extracted from the culture and restriction digested to confirm the size of genomic insertion.

Plasmid rescue was used to identify sequence flanking the T-DNA 1 insertion.

C. Inverse PCR

Genomic DNA was extracted as described above from plant tissue from individuals with single T-DNA insertions corresponding to T-DNA2, and inverse PCR was performed. Genomic DNA was digested with restriction enzymes, fragments were self-ligated, and nested inverse PCR was used to amplify the sequence flanking T-DNA2. More specifically, lμg of genomic DNA was digested overnight with individual restriction enzymes Alul, Nlalll, and/or Hhal using standard reaction conditions in a total volume of 50 μl. The restriction enzymes were heat inactived at 65°C for 20 minutes, and the reaction was extracted once with phenol/Chloroform and once with Chloroform isoamyl (24:1).

Ligation reactions were set up in 100 μl total volume using 30 μl of the Chloroform-extracted reaction. Reactions used standard conditions for NEB (Beverly, MA) Ligase and were left overnight at 16°C, followed by heat inactivation.

Two rounds of nested PCR were performed to amplify the genomic sequence flanking the left (for Hhal digestion) or right (for AM and Nlalll digestions) T-DNA border. The primary PCR reactions used outward-facing primers positioned between the restriction enzyme site used for digestion and the T-DNA border. Nested PCR reactions used outward-pointing nested primers positioned between one of the primary PCR primers and either the T-DNA border or the relevant restriction site. The primary PCR reaction used 2μl of the ligation reaction, while the nested PCR reaction used 2μl of a 1 : 100 dilution of the primary PCR reaction product. Both reactions were set up in a 50μl volume using Amplitaq Gold polymerase (Perkin Elmer) and the following PCR conditions: 1 cycle [95°C, 10 min]; 35 cycles of [94°C, 30sec; 54°C, 1 min; 72°C, 1 min]; 1 cycle [72°C, 10 min]. PCR products were run in a 1.0% agarose gel and gel-purified for sequencing

D. Sequencing Of Rescued Plasmids and Inverse PCR Products Sequencing was accomplished using a ABI Prism BigDyeTM Terminator Cycle Sequencing Ready Reaction Kit (PE Applied Biosystem), AmpliTaq DNA Polymerase (Perkin-Elmer), an ABI PrismTM 310 Genetic Analyzer (Perkin-Elmer) and sequence analysis software, e.g., Sequencer^M 3 1 1 _or MacVector 6.5.3.

An ABI Prism BigDye^"^- Terminator Cycle Sequencing Ready Reaction Kit was used to sequence a rescued plasmid using an ABI PrismTM 310 Genetic Analyzer following the protocols from the manufacturer. The left ends of plasmids rescued were sequenced across the right T-DNA border.

The inverse PCR products were sequenced with the nested PCR primer pointing toward the T-DNA/genomic DNA border. The sequence from the inverse PCR product corresponding to sequence flanking T-DNA2 was subjected to a basic BLASTN search using the sequence comparison program available at the www.ncbi.nlm.nih.gov/BLAST website and a search of the Arabidopsis Information Resource (TAIR) database, available at the Arabidopsis.org website, which revealed sequence identity to BAG clone F21J9. This BAC is mapped to chromosome 1. Sequence analysis revealed that the T-DNA had inserted in the vicinity (i.e., within about 5-10 kb) of the gene whose nucleotide sequence is presented as SEQ ID NO: 1 and GI 9502365, complement of nucleotides 76139-75219. Predicted genes in the vicinity of the T-DNA insertion were subjected to further characterization by RT-PCR. The RT-PCR results show that the gene presented as SEQ ED NO:l was specifically overexpressed in tissue from plants having the WCLl phenotype. Specifically, RNA was extracted from tissues derived from plants exhibiting the

WCLl phenotype and from wild type COL-0 plants. RT-PCR was performed using primers specific to the sequence presented as SEQ ID NO:l and a constitutively expressed protein kinase (positive control). The results showed that plants displaying the WCLl phenotype over-expressed the mRNA for the WCLl gene, indicating the enhanced expression of the WCLl gene is correlated with the WCLl phenotype.

The amino acid sequence predicted from the WCLl nucleic acid sequence was determined using GENSCAN and is presented in SEQ D NO:2. A Basic BLASTP 2.0.11 search using the ncbi.nlm.nih.gov/BLAST website, was conducted and revealed identity to the protein of GI 9743351. The BLASTP analysis revealed that the WCLl gene encodes a putative AP2-like transcription factor.

The BLAST search results suggest that WCLl represents a newly discovered phenotype and function associated with a known DNA sequence found in the Arabidopsis BAC clone F21J9. These results suggest that WCLl is associated with altered leaf morphology, late flowering, and compact stature in Arabidopsis.

EXAMPLE 3 Confirmation of Phenotype/Genotype Association

The dominant inheritance pattern of the WCLl phenotype is confirmed through genetic analysis. In general, genetic analysis involves the production and analysis of Fl hybrids. Typically, Fl crosses are carried out by collecting pollen from T₂ plants, which is used to pollinate wild type plants. Such crosses are carried out by taking approximately 4 flowers from each selected individual plants, and using the T₂ flower as the male pollen donor and flowers of the wild type plants as the female. 4-5 crosses are done for an individual of interest. Seed formed from crosses of the same individual are pooled, planted and grown to maturity as Fl hybrids.

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Claims

IT IS CLAIMED:

1. A method of producing a WCLl phenotype in a plant, said method comprising a) introducing into progenitor cells of the plant a plant transformation vector i comprising a nucleotide sequence that encodes or is complementary to a sequence that encodes a WCLl polypeptide, and b) growing the transformed progenitor cells to produce a transgenic plant, wherein said polynucleotide sequence is expressed and said transgenic plant exhibits a WCLl phenotype.

2. The method of Claim 1 wherein the WCLl polypeptide has at least 50% sequence identity to the amino acid sequence presented as SEQ ID NO: 2 and comprises an AP2 domain.

3. The method of Claim 1 wherein the WCLl polypeptide has at least 80% sequence identity to the amino acid sequence presented as SEQ ED NO:2.

4. The method of Claim 1 wherein the WCLl polypeptide has at least 90% sequence identity to the amino acid sequence presented as SEQ ED NO:2.

5. The method of Claim 1 wherein the WCLl polypeptide has the amino acid sequence presented as SEQ ED NO:2.

6. The method of Claim 1 wherein a WCLl polypeptide is over-expressed in the transgenic plant.

7. A plant obtained by a method of Claim 1.

8. A plant part obtained from a plant according to Claim 1.