WO2002050292A2

WO2002050292A2 - Modulation of plant cyclin-dependent kinase inhibitor activity

Info

Publication number: WO2002050292A2
Application number: PCT/CA2001/001825
Authority: WO
Inventors: Hong Wang; Yongming Zhou; Larry C. Fowke
Original assignee: Her Majesty In Right Of Canada As Represented By The Minister Of Agriculture And Agrifood Canada; University Of Saskatchewan Technologies Inc.
Priority date: 2000-12-08
Filing date: 2001-12-18
Publication date: 2002-06-27
Also published as: WO2002050292A3

Abstract

In various aspects, the invention provides methods of modifying plant development using cyclin-dependent kinase (CDK) inhibitors, cyclins and proteins that bind to CDK inhibitors, including methods of genetic modification utilizing nucleic acids encoding such proteins. In alternative aspects, the invention may provide methods of plant breeding that facilitate the modulation of plant phenotype in alternative generations. The invention may also provide plants produced by the methods of the invention, including transgenic plants having recombinant genomes that include heterologous sequences encoding CDK inhibitors, cyclins and proteins that bind to CDK inhibitors.

Description

MODULATION OF PLANT CYCLIN-DEPENDENT KINASE INHIBITOR ACTIVITY

FIELD OF THE INVENTION

The invention relates to the modification of growth and development of plants through transgenic modification.

BACKGROUND OF THE INVENTION

In eukaryotes including plants, the progression of cell cycle events is regulated by a network of gene products and factors to ensure that this crucial process is initiated as an integral part of the growth and the developmental program, and modulated in response to the external environment. These factors influence the cell cycle machinery via various pathways. An important component of the cell cycle machinery is an enzyme complex consisting of a catalytic subunit, cyclin-dependent protein kinase (CDK), and a regulatory subunit, cyclin. CDKs are a group of related serine/threonine kinases having an activity that generally depends on their association with cyclins (Pines, 1995).

Early work disclosed the existence of CDKs in yeast. A CDK called Cdc2 (p34^{c a}, or CDK1) was identified in fission yeast Schizosaccharomyces pombe (Hindley and Phear, 1984) and a Cdc2 homolog called CDC28 was identified in budding yeast Saccharomyces cerevisiae (Lδrincz and Reed, 1984). In yeast, Cdc2 (or CDC28) kinase appears to be solely responsible for regulating the progression of the cell cycle.

Animal cells appear to have evolved several Cdc2 -related CDKs in order to achieve more complex regulation at multiple levels. In mammalian cells, seven distinct CDKs and eight types of cyclins have been identified (see review by Pines, 1995). Complexes of these CDKs and cyclins appear to act sequentially at different checkpoints during the cell cycle, while incorporating the input of different developmental and environmental cues.

Plants, like higher animals, have multiple CDKs (Francis and Halford, 1995; Jacobs,

1995) and cyclins (Renaudin et al., 1996). In Arabidopsis thaliana, several CDK-related genes and fifteen cyclin genes have been isolated (reviewed by Renaudin et al, 1996; Stals et al., 2000). Of the CDKs, Cdc2a (also known as Cdc2aAt or Arath;CDKA;l) resembles more closely Cdc2 homologues from other species because it has a conserved PSTAIRE motif and is able to genetically complement yeast cdc2 or CDC28 mutants (Ferreira et al., 1991; Hirahama et al., 1991), indicating some functional homology of A. thaliana Cdc2a with the yeast Cdc2 kinase. Expression analyses showed that A. thaliana Cdc2a expression was correlated with the "competence" of a cell to divide and preceded the re-entry of differentiated cells into the cell division cycle (Martinez et al., 1992; Hemerly et al., 1993), and expression of a dominant negative Cdc2a mutant resulted in cell cycle arrest (Hemerly et al., 1995). A. thaliana Cdc2b is atypical in that it has a PPTALRE motif in place of the PSTAIRE motif. Like Cdc2a, Cdc2b is also expressed in dividing plant cells. While Cdc2a is expressed constitutively throughout the cell cycle, Cdc2b is reportedly expressed preferably in S and G2 phases (Segers et al., 1996). In addition to Cdc2a and Cdc2b, other CDK-related genes have been isolated from Arabidopsis and other species (Joubes et al., 2000).

The activity of CDKs in controlling the cell cycle appears to be regulated by several other factors. Results from yeast and mammalian studies have demonstrated multiple pathways, both positive and negative, by which CDK activity can be modulated (Lees, 1995). In addition to binding by a cyclin, for example, activation of CDKs may also involve a CDK-activating kinase (CAK) which itself is a CDK, and CDC25 protein phosphatase.

The activity of CDKs may be regulated by CDK inhibitors (see reviews by Pines,

1995; Sherr and Roberts, 1995; Harper and Ellege, 1996). These small molecular weight proteins are understood to bind stoichiometrically to negatively regulate the activity of CDKs. It has been suggested that these inhibitors may be involved in animal development and cancer, in addition to their role in cell cycle regulation (Harper and Elledge, 1996). A plant CDK inhibitor activity was observed and was suggested to be involved in endosperm development in maize (Grafi and Larkins, 1995).

The activity of CDK inhibitors has been studied in animals. Transgenic mice have been generated lacking p21, p27 and p57 CDK inhibitor genes. The p21 knockout mice are reported to develop normally although they are deficient in Gl checkpoint control, such as cell cycle arrest in response to DNA damage (Deng et al., 1995). Analysis of p27 knockout mice from three independent studies shows that transgenic mice lacking p27 display larger body size than control mice (Fero et al., 1996; Kiyokawa et al., 1996; Nakayama et al., 1996). The enhanced growth is reportedly due to an increase in cell number (Kiyokawa et al., 1996) and is gene dose-dependent (Fero et al., 1996). In comparison, none of p21 or p57 knockout mice display enhanced growth. The transgenic mice lacking p57 show a range of developmental defects such as defective abdominal muscles, cleft palate and renal medullary dysplasia (Yan et al., 1997; Zhang et al., 1997). Developmental defects were observed in p27-/- mice, including impaired ovarian follicles (thus female sterility), impaired luteal cell differentiation and a disordered estrus cycle. These results may reflect a disturbance of the hypothalamic-pituitary-ovarian axis. In comparison, transgenic mice lacking p21 appear to develop normally at both gross anatomic and histologic levels (Deng et al., 1995). In addition, an increase in apoptosis is observed in mice lacking p57. When the CDK inhibitor p27 was over-expressed in mouse hepatocytes (Wu et al., 1996), the result was a general decrease in overall number of adult hepatocytes leading to aberrant tissue organization, body growth and mortality.

Despite some conservation of basic cell cycle machinery in eukaryotes, the role of cell division during growth and development is different in plants than in other eucaryotes. This different role is reflected in the many ways in which regulation of plant cell division and growth is distinct from regulatory mechanisms in other eucaryotic cells. For example, plant cells are not mobile during morphogenesis. Different sets of hormones are involved in modulating plant growth and development than animal growth and development. Plant cells are remarkable for their ability to re-enter the cell cycle following differentiation. Cell division in plants is continuous, along with organ formation, and plant body size (the number of total cells and size of the cells) can vary dramatically under different conditions. Plants also have an inherent ability to incorporate additional growth into normal developmental patterns, as is illustrated by a study showing that ectopic expression of a mitotic cyclin driven by the Cdc2a promoter resulted in a larger but normal root system (Doerner et al., 1996). At this time, relatively little is known about the connections in plants between the regulatory genes controlling cell division patterns and cell cycle regulators such as CDKs and cyclins (Meyerowitz, 1997). A few studies of transgenic expression of cell cycle genes in plants are documented using various cell cycle genes other than CDK inhibitors. A heterologous yeast cdc25 coding sequence, a mitotic inducer gene, was introduced into tobacco plants under the control of a constitutive CaMV 35s promoter (Bell et al., 1993). The transgenic tobacco plants showed abnormal leaves (lengthened and twisted lamina, pocketed interveinal regions), abnormal flowers, and also precocious flowering. Analysis of cell size in the root meristem revealed that the transgenic plants expressing the yeast cdc25 had much smaller cells (Bell et al., 1993). The wild type Cdc2a gene and variants of dominant negative mutations under the control of CaMN 35s promoter have been used to transform tobacco and Arabidopsis plants (Hemerly et al., 1995). Constitutive expression of wild-type and mutant Cdc2a did not significantly alter the development of the transgenic plants. For the dominant negative Cdc2a mutant, it was not possible to regenerate Arabidopsis plants. Some tobacco plants expressing this construct were obtained and they had considerably fewer but much larger cells. These cells, however, underwent normal differentiation. Moφhogenesis, histogenesis and developmental timing were unaffected (Hemerly et al., 1995). As mentioned above, ectopic expression of an Arabidopsis mitotic cyclin gene, CycBl (Arat ;CycBl;l, formerly CyclAt), under the control of the Cdc2α promoter increases growth without altering the pattern of lateral root development in Arabidopsis plants (Doerner et al., 1996).

The yeast two-hybrid system has been used to identify the cyclin-dependent kinase inhibitor gene ICK1 from a plant (Wang et al., 1997). ICK1 is different in sequence, structure and inhibitory properties from known mammalian CDK inhibitors. It has been shown that the recombinant protein produced from this gene in bacteria is able to inhibit plant Cdc2-like kinase activity in vitro (Wang et al., 1997).

Cytotoxin genes, i.e. genes encoding a protein which will cause cell death, have been tested in transgenic plants for genetic ablation of specific cells or cell lines during development, including RΝase (Mariani et al., 1990), DTT (diphitheria toxin) chain A (Thorsness et al., 1991; Czako et al., 1992), Exotoxin A (Koning et al., 1992) and ribosomal inhibitor proteins (United States Patent No. 5,723,765 issued 3 March 1998 to Oliver et al). Several disadvantages may be associated with the use of cytotoxin genes for modification of transgenic plants, particularly plants of agronomic importance. The action of the cytotoxin may not be specific and may result in non-specific destruction of plant cells. This effect may be the result of diffusion of the cytotoxin, or of non-specific expression of the cytotoxin gene in non-target tissues. Non-specific low-level expression of the cytotoxin may be a difficult problem to overcome, since most tissue-specific promoters have some levels of expression in other tissues in addition to a high level of expression in a particular tissue. Expression of a potent cytotoxin gene even at a low concentration may have a negative impact on growth and development in non-target tissues. The presence of cytotoxic proteins of transgenic origin may also have a negative effect on the marketability of an edible plant, or plant product, even if the cytotoxin is demonstrably benign to consumers. Recently, alternative methods have been disclosed for phenotypic modification of plants using, for example, CDK inhibitors (International Patent Publication No. WO 99/64599 of Wang et al, published 16 December 1999), using D-rype cyclins (International Patent Publication No. WO 98/42851 of Murray, published 1 October 1998) and using interacting CDKs and cyclins (International Patent Publication No. WO 00/56905 of De Veylder et al, published 28 September 2000).

SUMMARY OF THE INVENTION

In one aspect, the invention provides methods of modifying plant or plant cell development using heterologous proteins that bind to or interact with CDK inhibitors. The proteins that bind to CDK inhibitors may for example be used to counteract the effects of CDK inhibitors on plant growth and development. In one aspect, the methods of the invention involve introducing into a plant cell a nucleic acid encoding a protein that binds to a cyclin-dependent kinase inhibitor, wherein the plant cell or an ancestor of the plant cell has been transformed with a nucleic acid encoding a cyclin-dependent kinase inhibitor polypeptide; and, growing the plant cell, or progeny of the plant cell, under conditions wherein the protein that binds to the cyclin-dependent kinase inhibitor is co-expressed with the cyclin-dependent kinase inhibitor in the plant cell or in the progeny of the transformed plant cell. The step of introducing into the plant cell the nucleic acid encoding the protein that binds to the cyclin-dependent kinase inhibitor may for example be carried out by transforming the plant cell or by cross breeding. The step of growing the plant cell or progeny of the plant cell may be carried out to produce a plant, and the protein that binds to the cyclin-dependent kinase inhibitor may be expressed in such a way that the development of the plant or progeny of the plant is altered. The alteration may be to counteract the effect on the development of the plant that the cyclin-dependent kinase inhibitor would otherwise have had. In the context of the invention, the word 'development' may encompass a wide variety of biological process, including growth, moφhogenesis, multiplication, enlargement, differentiation or maturation of a cell.

In alternative embodiments, the cyclin-dependent kinase inhibitor may for example be ICK1, ICK 2, ICN2 (which may also be known as ICK4), ICN6 (which may also be known as ICK5), ICN7 (which may also be known as ICK6), ICN8 (which may also be known as ICK7), ICDK (which may also be known as ICKCr) or homologues thereof. The protein that binds to the CDK inhibitor may be a cyclin, such as cyclin D3. In particular embodiments, the plant may for example be A. thaliana, or a member of the Brassica genus, or a canola variety. The cyclin-dependent kinase inhibitor and the protein that binds to the CDK inhibitor may be expressed using constitutive, tissue-specific or inducible promoters. For example, tissue-specific promoters could be an NTM19 promoter, a promoter homologous to NTM19, an AP 3 promoter or a promoter homologous to AP3.

In one aspect, the invention provides methods of plant breeding that may include modulating phenotypic modification of plants over successive generations, including methods that may be used to at least partially reverse phenotypic modifications in alternative plant generations. In such methods, for example, one generation of a plant may express a CDK inhibitor so that the development of the plant is modified by the expressed CDK inhibitor to provide a plant having an altered phenotype. The plant having the altered phenotype may be crossed with a plant encoding a protein that binds to the CDK inhibitor. The progeny of the cross may be selected to include plants wherein the altered phenotype, caused by the CDK inhibitor, is further altered by the expression of the protein that binds to the CDK inhibitor. For example, the phenotype alteration caused by the CDK inhibitor may be at least partially reversed by the protein that binds to the CDK inhibitor. In some embodiments, hybrid plants may be provided by the methods of the invention, wherein a first parent plant is homozygous for a sequence encoding a CDK inhibitor and a second parent plant is homozygous for a sequence encoding a protein that binds to the CDK inhibitor, so that the hybrid offspring of the first and second parent plants is heterozygous, having a copy of the sequence encoding the CDK inhibitor and a copy of the sequence encoding the protein that binds to the CDK inhibitor. For example, a male-sterile first parent plant may be provided which is homozygous for a sequence encoding a CDK inhibitor that confers the male-sterile phenotype. The male-sterile parent may be crossed with pollen from a second parent plant that is homozygous for a sequence encoding a protein that binds to the CDK inhibitor, so that in the hybrid offspring the CDK inhibitor and the protein that binds to the CDK inhibitor are co-expressed and the hybrid FI plant is not male-sterile.

In one aspect, the invention also provides methods of modifying plant architecture and moφhology for desired size and shape according to alternative embodiments of this invention. Plant architecture, size and moφhology may be modified by expressing a CDK inhibitor and a protein that binds to the CDK inhibitor. Specific expression patterns conferred by a variety of promoters may be used to modify a specific tissue or organ or two or more tissues or organs. Therefore, plants of various shapes, sizes and appearances may be produced from plants of the same type. Specific modifications may be made for agriculture crops, horticultural plants or trees.

Another aspect of the invention provides transgenic plants comprising (i.e. having or including, but not limited to) an expressible heterologous nucleic acid encoding a CDK inhibitor and a heterologous nucleic acid encoding a protein that binds to the CDK inhibitor, wherein the heterologous nucleic acids are introduced into the plant, or an ancestor of the plant, by transgenic or classical methods. Plants of the invention may accordingly have a recombinant genome, with heterologous nucleic acids integrated into the recombinant genome. The invention encompasses plant tissues, such as seeds, comprising heterologous nucleic acids encoding a CDK inhibitor and a protein that binds to the CDK inhibitor.

In some aspects, the invention may also provide methods of identifying proteins that bind to selected CDK inhibitors, and corresponding methods of identifying CDK inhibitors that bind to selected proteins, such as selected cyclins or cyclin fragments. Methods of the invention may also be used to identify interacting pairs of CDK inhibitors and proteins that bind to CDK inhibitors, so that such pairs of interacting proteins may be used in reversible methods of phenotypic modification in accordance with other aspects of the invention. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows cDNA (Wang et al., 1997) and genomic sequences of ICKl, wherein: (A) shows the genomic organization ICKl (SEQ ID NO: 1). Open bars represent exons and filled bars represent introns; (B) shows features of the ICKl cDNA sequence (SEQ ID NO: 3) and ICKl deduced amino acid sequence (SEQ ID NO: 2). ^*

Figure 2 shows a CLUSTAL W alignment of an ICKl CDNA sequence (SEQ ID NO: 3) with alternative cDNAs: ICKlb (SEQ ID NO: 4) and ICKc (SEQ ID NO: 5).

Figure 3A shows the partial cDNA sequence of ICK2 (SEQ ID NO: 6).

Figure 3B shows the full-length cDNA sequence of ICK2 (SEQ ID NO: 18).

Figure 4 shows the cDNA sequence of ICN2 (SEQ ID NO: 7).

Figure 5 shows the cDNA sequence 0ΪICN6 (SEQ ID NO: 8).

Figure 6A shows the partial cDNA sequence of ICN7 (SEQ ID NO: 9).

Figure 6B shows the full-length cDNA sequence of ICN7 (SEQ ID NO: 20).

Figure 7A shows the amino acid sequence of ICK2 (SEQ ID NO.: 10) deduced from the partial cDNA.

Figure 7B shows the amino acid sequence of ICK2 (SEQ ID NO.: 19) deduced from the full-length cDNA.

Figure 8 A shows the deduced amino acid sequence of ICN2 (SEQ ID NO.: 11) as part of a functional fusion protein in the yeast two-hybrid system. Figure 8B shows the amino acid sequence of ICN2 (SEQ ID NO.: 22), deduced from

ICN2 cDNA sequence taking into account the most probable translation start site yielding a sequence with 16 amino acid residues removed from the N-terminus compared to the sequence shown in Figure 8A.

Figure 9 A shows the deduced amino acid sequence of ICN6 (SEQ ID NO.: 12) as part of a functional fusion protein in the yeast two-hybrid system. Figure 9B shows the amino acid sequence of ICN6 (SEQ ID NO.: 23), deduced from ICN6 cDNA sequence taking into account the most probable translation start site yielding a sequence with 12 amino acid residues removed from the N-terminus compared to the sequence shown in Figure 9A.

Figure 10A shows the amino acid sequence of ICN7 (SEQ ID NO.: 13) deduced from the partial cDNA.

Figure 10B shows the amino acid sequence of ICN7 (SEQ ID NO.: 21) deduced from the full-length cDNA.

Figure 11 shows the nucleic acid sequence of the ICDK cDNA (GenBank AJ002173; SEQ ID NO.: 14).

Figure 12 shows the deduced amino acid sequence of ICDK (SEQ ID NO.: 15).

Figure 13 shows the cDNA sequence of ICN8 (SEQ ID NO.: 16).

Figure 14 shows the deduced amino acid sequence of ICN8 (SEQ ID NO.: 17).

Figure 15A shows a ClustalW alignment of deduced amino acid sequences of: ICKl (SEQ ID NO: 2), ICK 2 (SEQ ID NO: 10), ICN2 (SEQ ID NO: 11), ICN6 (SEQ ID NO: 12), ICN7 (SEQ ID NO: 13), ICN8 (SEQ ID NO: 17), and ICDK (SEQ ID NO: 15).

Figure 15B shows an alternative alignment of deduced amino acid sequences of: ICKl (SEQ ID NO: 2), ICK 2 (SEQ ID NO: 19), ICN2 (SEQ ID NO: 22), ICN6 (SEQ ID NO: 23), ICN7 (SEQ ID NO: 21), ICN8 (SEQ ID NO: 17), and ICDK (SEQ ID NO: 15).

BRIEF DESCRIPTION OF THE SEQUENCE LISTING

The nucleic and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and three letter codes for amino acids. Only one strand of each nucleic acid sequence is shown, but the complementary strand is hereby included by any reference to the displayed strand. In the accompanying sequence listing:

SEQ ID NO.: 1 shows the nucleic acid sequence of ICKl. SEQ ID NO.: 2 shows the deduced amino acid sequence of ICKl.

SEQ ID NO.: 3 shows the nucleic acid sequence of the ICKl cDNA.

SEQ ID NO.: 4 shows the nucleic acid sequence of the ICKlb cDNA.

SEQ ID NO.: 5 shows the nucleic acid sequence of the ICKlc cDNA. SEQ ID NO.: 6 shows the nucleic acid sequence of the ICK2 partial cDNA.

SEQ ID NO.: 7 shows the nucleic acid sequence of the ICN2 cDNA.

SEQ ID NO.: 8 shows the nucleic acid sequence of the 7CN6^' cDΝA.

SEQ ID NO.: 9 shows the nucleic acid sequence of the ICN7 partial cDNA.

SEQ ID NO.: 10 shows the amino acid sequence of ICK2 deduced from the partial cDNA. SEQ ID NO.: 11 shows the deduced amino acid sequence of ICN2 as part of a functional fusion protein in the yeast two-hybrid system.

SEQ ID NO.: 12 shows the deduced amino acid sequence of ICN6 as part of a functional fusion protein in the yeast two-hybrid system.

SEQ ID NO.: 13 shows the amino acid sequence of ICN7 deduced from the partial cDNA. SEQ ID NO. : 14 shows the nucleic acid sequence of the ICDK cDNA (GenBank

AJ002173).

SEQ ID NO.: 15 shows the deduced amino acid sequence of ICDK.

SEQ ID NO.: 16 shows the cDNA sequence of ICN8.

SEQ ID NO.: 17 shows the deduced amino acid sequence of ICN8. SEQ ID NO.: 18 shows the nucleic acid sequence of the ICK2 full-length cDNA.

SEQ ID NO.: 19 shows the amino acid sequence of ICK2 deduced from the full-length cDNA.

SEQ ID NO.: 20 shows the nucleic acid sequence of the ICN7 full-length cDNA.

SEQ ID NO.: 21 shows the amino acid sequence of ICN7 deduced from the partial cDNA. SEQ ID NO.: 22 shows the amino acid sequence of ICN2 deduced from ICN2 cDNA sequence.

SEQ ID NO.: 23 shows the amino acid sequence of ICN6 deduced from 7CN6^' cDΝA sequence. DETAILED DESCRIPTION OF THE INVENTION

In the following detailed description, various examples are set out of particular embodiments of the invention, together with experimental procedures that may be used to implement a wide variety of modifications and variations in the practice of the present invention. The invention is however not limited to the disclosed exemplary embodiments, nor to the disclosed procedures for implementing such embodiments. Similarly, various embodiments and aspects of the invention are disclosed to provide alternative prospective advantages, which may not be available with other aspects or embodiments of the invention.

In one aspect, the invention provides methods of modifying plant or plant cell development. In the context of the invention, the word 'development' encompasses a very wide variety of biological process, including growth, moφhogenesis, multiplication, enlargement, differentiation or maturation of a cell or plant. In one aspect, the invention provides methods of modifying plant development that include introducing into a plant cell a nucleic acid encoding a protein that binds to a cyclin-dependent kinase inhibitor polypeptide, where the plant cell or an ancestor of the plant cell has been transformed with a nucleic acid encoding the cyclin-dependent kinase inhibitor polypeptide. The plant cell, or the progeny of the plant cell, may then be grown under conditions so that the protein that binds to the CDK inhibitor and the CDK inhibitor are expressed in the plant cell or in the progeny of the plant cell during development of the plant. It has unexpectedly been discovered that this allows one to modulate the effect that the CDK inhibitor would otherwise have on development of the plant. In some embodiments, the effect of the CDK inhibitor may be at least partly reversed by the protein that binds to the CDK inhibitor.

In various aspects, the invention relates to CDK inhibitor polypeptides, such as ICKl. A 'CDK inhibitor polypeptide' is any polypeptide capable of inhibiting a CDK, such as CDKs active during development of a plant or plant cell. Proteins that bind to CDK inhibitors in the context of the invention are those proteins that show a sufficiently strong interaction with the CDK inhibitor of interest so that they are capable of modulating the desired effects of the invention. In some embodiments, the protein-protein interactions that mediate such binding will be sufficiently strong to be detected using a two-hybrid system, or a similar system for characterizing protein-protein interactions. Accordingly, in some embodiments, proteins that bind to a CDK inhibitor are identifiable as proteins that have at least one segment that, when used as 'prey' in a yeast two-hybrid assay, bind to at least a segment of a CDK inhibitor, to produce a positive signal in the two-hybrid assay, or an alternative assay of protein-protein interaction.

The yeast two-hybrid and interaction trap systems may, for example, be used to identify proteins or protein fragments that bind to CDK inhibitors, or to characterize protein-protein interactions in various aspects of the present invention. Both the two-hybrid and interaction trap systems exploit the fact that the transcriptional activation and DNA binding domains of most eukaryotic transcriptional activators function when expressed as fusions with heterologous proteins (Brent and Ptashne, 1985, Cell 43 J29-36), and can transactivate when brought together by specific protein-protein interaction between separate fusion proteins (Chien, et al., 1991, Proc Natl Acad Sci U.S.A. 88:9578-82; Fields and Song, 1989, Nature 340:245-6). In the standard two-hybrid system a protein of interest is fused to the DNA-binding domain of GAL4 to create a "bait" fusion protein. Proteins that interact with the bait protein, termed the "prey" can be identified by their ability to cause transactivation of a GAL4-dependent reporter gene when expressed as a fusion to the C- terminal transactivation domain of GAL4 (Chien et al., 1991, Proc Natl Acad Sci U.S.A. 88:9578-82; Fields and Song, 1994, U.S. Pat. No. 5,283,173; Fields and Song, 1995, U.S. Pat. No. 5,468,614). The "interaction trap" system employs the identical principle of using separate fusions with DNA-binding and transactivation domains, except that the bait is fused to LexA, which is a sequence-specific DNA binding protein from E. coli, and an artificial transactivation domain known as B42 (Ma and Ptashne, 1987, Cell 51 :113-9) is used for the "prey" fusions. Interaction between the bait and prey fusions is detected by expression of a LexA-responsive reporter gene (Brent et al., 1996, U.S. Pat. No. 5,580,736). More recently, an alternative to the two-hybrid system has been disclosed, called the repressed trans-activator system, that may be used for characterization of protein-protein interactions in the context of the present invention (Sadowski et al., 1999, U.S. Pat. No. 5,885,779).

As an example of the use of yeast two-hybrid cloning and assay techniques, a cDNA library may be made using poly (A) mRNA isolated from whole plants at different stages of development and cloned in a suitable vector, such as Gal4 TA- (transcription-activation domain) pPC86 (Chevray and Nathans, 1992; available from GIBCO/BRL Life Technologies) or a similar vector (Koholmi et al., 1997). The cDNA of the gene (such as Arabidopsis Cdc2a, cyclin D2 and cyclin D3) to be used for screening the library may be cloned in a suitable vector, such as the Gal4 DB- (DNA-binding domain) vector. The yeast strain, such as MaV203, harboring the construct may be transformed using the library DNA.

In one example, for analysis of Cdc2a interactions, a total of 1.8 X 10 transformants were subjected to two-hybrid selection on supplemented synthetic dextrose medium lacking leucine, tryptophan and histidine but containing 5 mM 3-amino-l,2,4-triazole. The selected colonies were assayed for β-galactosidase activity using standard methods. DNAs were isolated from positive clones and used to transform E. coli. Clones harboring the TA-fusion cDNAs were identified by PCR and plasmids were then isolated for DNA sequencing (Wang et al., 1997).

Interactions in the yeast two-hybrid system may, for example, be analyzed by either filter assay (Chevray and Nathans, 1992) using X-gal as the substrate or by a quantification assay using ONPG (ortho-nitrophenyl-beta-D-galactoside) as the substrate (Reynolds and Lundlad, 1994). Three or more independent transformants may be used for each interaction.

In various aspects of the invention, a plant cell or progeny of the plant cell may be grown to produce a plant, such as by regenerating a plant from a transformed cell or cell culture, or by propagating or growing whole plants from transformed plant parts. The term 'progeny', with reference to a plant, includes progeny produced sexually or asexually (for example by tissue culture-based propagation). The term 'growing' with reference to the transformed cells or plants includes all methods for growing and propagating cells or plants, such as tissue culture or horticultural means of propagating plants or plant parts.

The growth of a plant cell, or progeny of a plant cell, in accordance with various aspects of the invention may be carried out so that an effect of a cyclin-dependent kinase inhibitor polypeptide on development of the plant is at least partly reversed by a protein that binds to the cyclin-dependent kinase inhibitor polypeptide. In this context, the 'effect' that is at least partially reversed may be any detectable physiological or genetic change caused by the expression of the CDK inhibitor. By 'reversal' it is meant that a measurable parameter of such a change is made to revert to a value that is closer to the value of the parameter in plants that do not manifest the change caused by the CDK inhibitor. For example, the size or weight of a plant may be reduced by a CDK inhibitor, compared to wild type plants, and a cyclin or a protein that binds to the CDK inhibitor may be used to increase the size or weight of the plant that expresses the CDK inhibitor. In some embodiments the effect may relate to the modification of a plant tissue, such as the total or partial ablation of a developmental cell line by a CDK inhibitor, which may result in a phenotypic modification of the tissue in the plant, which may be at least partially restored by a cyclin or a protein that binds to the CDK inhibitor.

Nucleic acid coding sequences may be used in various aspects of the invention. Such sequences may be operatively linked to promoters, terminators, signal sequences, polyadenylation sequences, splice sites or alternative control sequences that may be used to facilitate expression of a coding sequence. In the context of the present invention, "promoter" means a sequence sufficient to direct transcription of a gene when the promoter is operably linked to the gene. The promoter is accordingly the portion of a gene containing DNA sequences that provide for the binding of RNA polymerase and initiation of transcription. Promoter sequences are commonly, but not universally, located in the 5' non- coding regions of a gene. A promoter and a gene are "operably linked" when such sequences are functionally connected so as to permit gene expression mediated by the promoter. The term "operably linked" accordingly indicates that DNA segments are arranged so that they function in concert for their intended puφoses, such as initiating transcription in the promoter to proceed through the coding segment of a gene to a terminator portion of the gene. Gene expression may occur in some instances when appropriate molecules (such as transcriptional activator proteins) are bound to the promoter. Expression is the process of conversion of the information of a coding sequence of a gene into mRNA by transcription and subsequently into polypeptide (protein) by translation, as a result of which the protein is said to be expressed. As the term is used herein, a gene or nucleic acid is "expressible" if it is capable of expression under appropriate conditions in a particular host cell.

For the present invention, promoters may for example be used that provide for preferential gene expression within a specific organ or tissue, or during a specific period of development. For example, promoters may be used that are specific for leaf (Dunsmuir, et al Nucleic Acids Res, (1983) 11:4177-4183), root tips (Pokalsky, et al Nucleic Acids Res, (1989) 17:4661-4673), fruit (Peat, et al Plant Mol. Biol, (1989) 13:639-651; United States Patent No. 4,943,674 issued 24 July, 1990; International Patent Publication WO-A 8 809 334; United States Patent No. 5,175,095 issued 29 December, 1992; European Patent Application EP-A 0 409 629; and European Patent Application EP-A 0 409 625) embryogenesis (U.S. Patent No. 5,723,765 issued 3 March 1998 to Oliver et al), or young flowers (Nilsson et al. 1998). Such promoters may, in some instances, be obtained from genomic clones of cDNAs. Depending upon the application of the present invention, those skilled in this art may choose a promoter for use in the invention which provides a desired expression pattern. Promoters demonstrating preferential transcriptional activity in plant tissues are, for example, described in European Patent Application EP-A 0 255 378 and International Patent Publication WO-A 9 113 980. Promoters may be identified from genes which have a differential pattern of expression in a specific tissue by screening a tissue of interest, for example, using methods described in United States Patent No. 4,943,674 and European Patent Application EP-A 0255378. Non-dividing plant cells may tolerate low level expression of CDK inhibitors without detectable effect. Thus, the invention may be practiced in some embodiments using tissue specific promoters operably linked to CDK inhibitor encoding sequences, to give rise effects that may be reversed with a cyclin or protein that binds to the CDK inhibitor, even when the promoter mediates a tolerable basal level of CDK inhibitor expression in other tissues.

Various aspects of the present invention encompass nucleic acid or amino acid sequences that are homologous to other sequences. As the term is used herein, an amino acid or nucleic acid sequence is "homologous" to another sequence if the two sequences are substantially identical and the functional activity of the sequences is conserved (for example, both sequences function as or encode a cyclin-dependent kinase inhibitor; as used herein, sequence conservation or identity does not infer evolutionary relatedness). Nucleic acid sequences may also be homologous if they encode substantially identical amino acid sequences, even if the nucleic acid sequences are not themselves substantially identical, for example as a result of the degeneracy of the genetic code.

Two amino acid or nucleic acid sequences are considered substantially identical if, when optimally aligned, they share at least about 70% sequence identity. In alternative embodiments, sequence identity may for example be at least 75%, at least 90% or at least 95%o. Optimal alignment of sequences for comparisons of identity may be conducted using a variety of algorithms, such as the local homology algorithm of Smith and Waterman (1981) Adv. Appl. Math 2: 482, the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, the search for similarity method of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85: 2444, and the computerized implementations of these algorithms (such as GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, Madison, WI, U.S.A.). Sequence identity may also be determined using the BLAST algorithm, described in Altschul et al (1990), J. Mol. Biol. 215:403-10 (using the published default settings). Software for performing BLAST analysis may be available through the National Center for Biotechnology Information (through the internet at http://www.ncbi.nlm.nih.gov/). The BLAST algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold. Initial neighborhood word hits act as seeds for initiating searches to find longer HSPs. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extension of the word hits in each direction is halted when the following parameters are met: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The BLAST program may use as defaults a word length (W) of 11 , the BLOSUM62 scoring matrix (Henikoff and Henikoff (1992) Proc. Natl Acad. Sci. USA 89: 10915-10919) alignments (B) of 50, expectation (E) of 10, M=5, N=4, and a comparison of both strands. One measure of the statistical similarity between two sequences using the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. In alternative embodiments of the invention, nucleotide or amino acid sequences are considered substantially identical if the smallest sum probability in a comparison of the test sequences is less than about 1- preferably less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.

An alternative indication that two nucleic acid sequences are substantially identical is that the two sequences hybridize to each other under moderately stringent, or preferably stringent, conditions. Hybridization to filter-bound sequences under moderately stringent conditions may, for example, be performed in 0.5 M NaHPO₄, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65 °C, and washing in 0.2 x SSC/0.1% SDS at 42 °C (see Ausubel, et al. (eds), 1989, Current Protocols in Molecular Biology, Vol. 1, Green Publishing Associates, Inc., and John Wiley & Sons, Inc., New York, at p. 2.10.3). Alternatively, hybridization to filter-bound sequences under stringent conditions may, for example, be performed in 0.5 M NaHPO , 7% SDS, 1 mM EDTA at 65 °C, and washing in 0.1 x SSC/0.1% SDS at 68 °C (see Ausubel, et al. (eds), 1989, supra). Hybridization conditions may be modified in accordance with known methods depending on the sequence of interest (see Tijssen, 1993, Laboratory Techniques in Biochemistry and Molecular Biology — Hybridization with Nucleic Acid Probes, Part I, Chapter 2 "Overview of principles of hybridization and the strategy of nucleic acid probe assays", Elsevier, New York). Generally, stringent conditions are selected to be about 5°C lower than the thermal melting point for the specific sequence at a defined ionic strength and pH.

An alternative indication that two amino acid sequences are substantially identical is that one peptide is specifically immunologically reactive with antibodies that are also specifically immunoreactive against the other peptide. Antibodies are specifically immunoreactive to a peptide if the antibodies bind preferentially to the peptide and do not bind in a significant amount to other proteins present in the sample, so that the preferential binding of the antibody to the peptide is detectable in an immunoassay and distinguishable from non-specific binding to other peptides. Specific immunoreactivity of antibodies to peptides may be assessed using a variety of immunoassay formats, such as solid-phase ELISA immunoassays for selecting monoclonal antibodies specifically immunoreactive with a protein (see Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York). The cyclin-dependent kinase inhibitors of the present invention, the proteins that bind to the CDK inhibitors and the genes encoding these proteins, may include non- naturally occurring sequences, such as functionally active fragments of naturally occurring sequences. For example, fragments of ICKl, or amino acid sequences homologous to those fragments, that have cyclin-dependent kinase inhibitory activity may be used in some embodiments of the invention. The invention provides methods for identifying such fragments, for example by deletion mapping of active cyclin-dependent kinase inhibitors or cyclins. As used herein the terms "cyclin-dependent kinase inhibitor" or "cyclin" or "protein that binds to a CDK inhibitor" includes any polypeptide capable of having the relevant functioning, i.e. respectively inhibiting a cyclin-dependent kinase or at least partially reversing an effect of the CDK inhibitor. The invention encompasses nucleic acid sequences encoding such alternative polypeptides.

As used herein to describe nucleic acid or amino acid sequences the term "heterologous" refers to molecules or portions of molecules, such as DNA sequences, that are artificially introduced into a particular host cell or genome. Heterologous DNA sequences may for example be introduced into a host cell by transformation. Such heterologous molecules may include sequences derived from the host cell, and thereafter reintroduced into the host cell or a cell from the same cell line or species. Heterologous DNA sequences may become integrated into the host cell genome, either as a result of the original transformation of the host cells, or as the result of subsequent recombination events, but they remain heterologous because of their different 'artificial' origin.

In an alternative aspect of the invention, reversal of an effect of a CDK inhibitor, such as ICKl, may be used to enhance growth during plant development. Such growth enhancement may be tissue-specific. The expression of cyclins or proteins that bind to CDK inhibitors may be made to be tissue-specific by operably linking the relevant coding sequences to tissue-specific promoters.

A variety of plants can be used for identifying proteins that are useful in various embodiments of the present invention, such as CDK inhibitors, cyclins and proteins that bind to CDK inhibitors. Arabidopsis thaliana "Columbia" may be used as a convenient model system for identifying proteins that are useful in various embodiments of the present invention. Arabidopsis plants are generally grown in pots placed in growth chambers. Other plants may also of course be used in various embodiments of the invention in accordance with known growth and transformation techniques.

Standard methods are available for cloning genomic and cDNA nucleotide sequences for use in identifying and characterizing proteins and nucleic acids useful in various embodiments of the invention. General molecular techniques may for example be performed by procedures generally described by Ausubel et al. (1995). Sequence analyses, including determination of sequence homology, may be performed using a variety of software, such as LASERGENE (DNASTAR). Database searches may also use a variety of software tools, such as the BLAST program (NCBI). Alternative equivalent methods or variations thereof may be used in accordance with the general knowledge of those skilled in this art.

As an example of cloning methods that may be used in accordance with the invention, to clone the tobacco NTM19 promoter sequence (Oldenhof et al., 1996), genomic DNA was isolated from leaf tissue of Nicotiana tabacum CV "Xanthi" based on a described procedure (Dellaporta et al., 1983). The promoter sequence was amplified by 30 cycles of PCR using sequence-specific primers with incoφorated restriction sites. Pfu DNA polymerase (Stratagene), which has a higher replication fidelity than the Taq DNA polymerase, may be used. The amplified DNA fragment may be cloned into a suitable vector, such as pGEM3Zf(+) (Promega). Plasmids may then be purified and the cloned DNA fragment sequenced.

As another example, a cDNA clone ICDK (Fountain et al., 1999) (GenBank Accession AJ002173, SEQ ID No. 15 and SEQ ID No.16) was identified from Chenopodium rubrum, which encodes a protein sharing sequence similarity with ICKl and thus also with ICK2, ICN2, ICN6 and ICN7 (Table 1). RNA was isolated from seedlings and leaves of Chenopodium rubrum. The full-length coding region of ICDK cDNA was cloned by RNA RT-PCR (e.g. using ThermoScript RT-PCR System, GIBCO/BRL Life Technologies) with sequence-specific primers. The amplified fragment was cloned and sequenced. The sequence data showed that the cloned cDNA was identical to ICDK of C. rubrum. As yet another example, a putative protein is identified encoded by a sequence BAC clone F24L7 (GenBank AC003974), which share some homology with ICKl (Wang et al., 1997). Specific primers were designed and used to clone the cDNA corresponding to the genomic sequence by RNA RT-PCR. This cDNA was found to encodes a protein able to interact with Arabidopis CycD3, it is designated as ICN8.

As a further example, ICKl cDNA (SEQ ID NO: 1 ; Wang et al., 1997) was amplified by PCR and transcriptionally linked with the 35S promoter in a binary vector pBI121 (Clontech). The chimeric gene ends with a nopaline synthase terminator. As another example, the Arabidopsis CycD2 (Arath; CycD2;l, GenBank accession number X83370), CycD3 (Arath;CycD3;7, GenBank accession number X83371) and Cdc2a (Cdc2aAt or Arath;CDKA; 1 , GenBank accession number M59198 or X57839) were cloned by polymerase chain reaction (PCR) using Arabidopsis cDNA based on published sequence information (Hirayama et al, 1991 for Cdc2a, GenBank X57839; Soni et al., 1995 for CycD2 and CycD3, X83370 and X83371). They were transcriptionally linked to the 35S promoter. As another example, the Arabidopsis AP3 promoter was cloned by PCR from Arabidopsis thaliana "Columbia" genomic DNA, on the basis of the published sequence (Irish and Yamamoto, 1995; GenBank U30729). The promoter was linked transcriptionally with ICKl cDNA. As yet another example, the tobacco NTM19 promoter was linked transcriptionally with ICKl cDNA. The NTM19 promoter from tobacco has been shown to activate gene expression at early stages of microspore development (Custers et al., 1997; Oldenhof et al., 1996). The resulting plasmids were introduced into Agrobacterium tumefaciens strain GV3101 (bearing helper plasmid pMP90; Koncz and Schell 1986).

In accordance with various aspects of the invention, plant cells may be transformed with heterologous nucleic acids. In this context, "heterologous" denotes any nucleic acid that is introduced by transformation, or a sequence that is descended from a sequence introduced by transformation into a progenitor cell. Transformation techniques that may be employed include plant cell membrane disruption by electroporation, microinjection and polyethylene glycol based transformation (such as are disclosed in Paszkowski et al. EMBO J. 3:2717 (1984); Fromm et al, Proc. Natl Acad. Sci. USA 82:5824 (1985); Rogers et al, Methods Enzymol 118:627 (1986); and in U.S. Patent Nos. 4,684,611; 4,801,540; 4,743,548 and 5,231 ,019), biolistic transformation such as DNA particle bombardment (for example as disclosed in Klein, et al, Nature 327: 70 (1987); Gordon-Kamm, et al. "The Plant Cell" 2:603 (1990); and in U.S. Patent Nos. 4,945,050; 5,015,580; 5,149,655 and 5,466,587); Agrobacterium-mediated transformation methods (such as those disclosed in Horsch et al Science 233: 496 (1984); Fraley et al, Proc. Nat'l Acad. Sci. USA 80:4803 (1983); and U.S. Patent Nos. 4,940,838 and 5,464,763).

A wide variety of transformation techniques may be used in accordance with the invention to introduce nucleic acids into plants. For example, in one embodiment, transformation may be caried out by infiltration. For example, seeds (Tl generation) collected from infiltrated Arabidopsis plants may be surface-sterilized and placed onto MS medium containing 50 μg/ml kanamycin. The antibiotic timentin may also be included in the medium to prevent any bacterial growth, which could occur due to carrier-over from the infiltration. The vast majority of germinating seedlings will typically not be transformed, and will became pale and eventually stop growing, transformed seedlings will be green and display normal growth due to the presence of the selectable marker gene. After 4-5 weeks in the selection medium, transformants may be transferred to soil in pots. In the exemplary embodiment, the presence of the DNA insertion encoding a CDK inhibitor (ICKl) was confirmed by extracting the genomic DNA and then using it for PCR amplification. In one example, while the non-transformed wild-type plant gave a negative signal, all twelve (12) plants selected for their resistance to kanamycin were positive for transforming DNA.

Transformed plant cells may be cultured to regenerate whole plants having the transformed genotype and displaying a desired phenotype, as for example modified by the expression of a heterologous CDK inhibitor during growth or development. A variety of plant culture techniques may be used to regenerate whole plants, such as are described in Gamborg and Phillips, "Plant Cell, Tissue and Organ Culture, Fundamental Methods", Springer Berlin, 1995); Evans et al. "Protoplasts Isolation and Culture", Handbook of Plant Cell Culture, Macmillian Publishing Company, New York, 1983; or Binding, "Regeneration of Plants, Plant Protoplasts", CRC Press, Boca Raton, 1985; or in Klee et al, Ann. Rev. of Plant Phys. 38:467 (1987).

Standard techniques may be used for plant transformation, such as transformation of Arabidopsis. In one example, the 35S-ICK1, 35S-CycD2, 35S-CycD3 and 35S-GUS constructs were tested in A. thaliana by in planta transformation techniques. Wild type (WT) A. thaliana seeds of ecotype "Columbia" were planted in 4" pots containing soil and plants grown in a controlled growth chamber or greenhouse. The vacuum infiltration method of in planta transformation (Bechtold et al., 1993) was used to transform .4. thaliana plants with overnight culture of A. tumefacian strain GV3101 bearing both the helper nopoline plasmid and the binary construct containing the described chimeric gene. pMP90 is a disarmed Ti plasmid with intact vir region acting in trans, gentamycin and kanamycin selection markers as described in Koncz and Schell (1986). Following infiltration, plants were grown to maturity and seeds (Tl) were collected from each pod individually. Seeds were surface-sterilized and screened on selective medium containing 50 mg/L kanamycin with or without 200-300 mg/L timentin. After about four weeks on selection medium, the non-transformed seedlings died. The transformed seedlings were transferred to soil in pots. Leaf DNA was isolated (Edwards et al., 1991) and analyzed by PCR for the presence of the DNA insertion. Genomic DNA was also isolated and used in Southern hybridization (Southern, 1975) to determine the copy number of the inserted sequence in a given transformant. To determine the segregation, T2 seeds were collected from Tl plants.

Wherever the Tl plant was male sterile, crosses were made using the WT A. thaliana pollen to obtain seeds. As described, T2 seeds were surface-sterilized and screened on selective medium.

Alternative embodiments of the invention may make use of techniques for transformation of Brassica. Such as transformation of B. napus cv. Westar and B. carinata cv. Dodolla by co-cultivation of cotyledonary petioles or hypocotyl explants with A. tumefaciens bearing the plasmids described herein. Transformation of 5. napus plants may, for example, be performed according to the method by Moloney et al. (1989). Modifications of that method may include the introduction of a 7-day explant-recovery period following co-cultivation, on MS medium with the hormone benzyladenine (BA), and the antibiotic timentin for the elimination of Agrobacterium. Transformation of B. carinata plants may be performed according to the method by Babic et al. (1998). Cotyledonary petiole explants may be dipped in suspension of Agrobacterium bearing the desired constructs and placed on 7-cm filter paper (Whatman no. 1) on top of the regeneration medium for 2 days. After co- cultivation, explants may be transferred onto the selection medium containing 50 mg/L kanamycin. Regenerated green shoots may first be transferred to a medium to allow elongation and then to a rooting medium all containing 50 mg/L kanamycin. Putative transformants with roots (TO) may be transferred to soil. Genomic DNA may be isolated from developing leaves for PCR and Southern analyses. Seeds (Tl) from transgenic plants may then be harvested. Other techniques known in the art may be used to transform plants of other species such as tobacco.

Transgenic plants may be analysed for changes in growth and development. For example, seeds of T2 transgenic 35S-ICK1 lines were planted in soil. Wild type plants and transgenic plants carrying the 35S-GUS construct were used as controls. At the three-week stage, the above ground portion (including cotyledons, leaves and shoot) was removed and the fresh weight determined. The number of days to flower and leaf number (rosette plus inflorescence leaves on the primary axis) were obtained. Changes in the development of a particular organ can also be analysed. For instance, the leaf moφhology and width/length ratio were determined. Similarly, the length of roots and shoots could be determined. Transgenic plants may be observed and characterized for alteration of traits such as petals, male sterility and ability to set seeds. For example, to determine the development of floral organs, flowers at different stages of development may be dissected and examined under a stereomicroscope.

RNA isolation and northern blotting analysis may be useful in various embodiments of the invention. For example, to analyze ICKl expression during plant development, various tissues may be taken from Arabidopsis plants (Wang et al., 1995). Total RNA was isolated using TRIzol reagent (GIBCO BRL). For northern analysis, the indicated amount of RNA was fractionated in a 1.2% agarose gel and transferred onto Hybond-N+ nylon membrane (Amersham). The RNA was crosslinked to the membrane by UV-light (Stratalinker, Stratagene) and hybridized with P-labeled probes. The membranes may be wrapped and used to expose Hyperfilm MP (Amersham) film. For re-probing, membranes may be stripped by treating with a boiling solution of 0.1X SSC and 0.1% SDS for 5 min. Quantification of hybridized signal was performed using a phosphoimager and the accompanying software.

Kinase assays may be useful in some aspects of the invention, for example to assay the function of CDK inhibitors on particular kinases. For example, kinases may be purified from A. thaliana tissues or cultured B. napus cells. Plant materials may be homogenized in 2 mis per gram tissue of ice cold extraction buffer consisting of 25 mM Tris pH 8.0, 100 mM NaCl, 10 mM DTT, 5 mM NaF, 1 mM Na₃VO , 1 mM β-glycerophosphate, 2.5 mM EDTA, 400 μg/ml AEBSF [4-(2-aminoethyl)-enzensulfonyl fluoride], 1 μg/ml leupeptin and 1 μg/ml pepstatin. The homogenate was centrifuged at 12,000g at 4°C for 30 min. The supernatants may be used to purify Cdc2-like protein kinases using pl3^suc -conjugated agarose beads (Oncogene Sciences). The required amount of supernatant (150 μg protein for each reaction) was added to the beads and tumbled at 4°C for 2 h. The beads may be washed twice in a washing buffer consisting of 50 mM Tris pH 7.4, 250 mM NaCl, 0.1% NP-40, 2.5 mM EDTA, 1 mM DTT and inhibitor cocktail of (in final concentrations) 10 μg/ml apotinin, 10 μg/ml antipain, 10 μg/ml soybean trypsin inhibitor, 10 mM β- glycerophosphate, 1 mM NaF and 0.2 mM Na₃VO₄. Beads may then be washed twice in the kinase assay buffer (50 mM Tris pH 7.4, 10 mM MgCl, 2 mM EGTA, 2 mM DTT and the inhibitor cocktail). For inhibition assays, the recombinant protein was added to the reactions and incubated (tumbling slowly) for 1.5 h at 4°C. The kinase reaction was initiated by adding 1 μg/μl histone HI (Sigma), 25 μM ATP and 0.05 μCi/ul ³²P-γ-ATP (final concentrations), and stopped after 20 min incubation by adding the sample buffer. Denatured supernatant was resolved by SDS-PAGE.

The structure of the transgenic plants may be characterized by scanning electron microscopy (SEM) and light microscopy. In one example, plant tissue samples were taken from 35S-ICK1 and control plants grown under the same conditions. For SEM of tissues such as leaves and hypocotyls, the epoxy replica method (Green and Linstead, 1990; Fowke et al., 1994) was used. The impression moulds were prepared using dental impression material (GC Exaflex vinyl silicone). The moulds were then used to make replicas of original samples with epoxy cement. Some tissues (e.g. flowers) were fixed and critical point dried as described previously (Fowke et al., 1994). The epoxy replicas and critical point-dried specimens were mounted on SEM stubs, coated with gold in an Edwards Sputter Coater (Model S150B) and then examined in a Philips 505 Scanning Electron Microscope. For light microscopy of methacrylate sections, samples (leaf tissues) were fixed, dehydrated and embedded in methacrylate according to the method of Baskin et al. (1992). 1.5-2.5 μm sections were stained with toluidine blue before examination. Determination of organ size and cell size may be useful in various embodiments of the invention. In one example, the leaf size and cell size of Arabidopsis 35S-ICK1 transgenic and controls plants were analysed. Leaves (number 5-8) of 30-day plants grown in a growth chamber were used, as leaves of younger plants (e.g. 15-day) may continue to expand. At this stage, both the 35S-ICK1 and control plants were flowering. Leaves were excised and scanned first on an Epson flat bed scanner (model 1200S) to determine the leaf size. Epoxy replicas of the adaxial surface were prepared and SEM photographs taken. Two non-marginal sectors from the widest part of each leaf were photographed and about twenty pavement cells of each photographed sector were chosen at random. Guard cells were excluded from measurements. The surface area of each cell was determined by Image J (http:/rsb.Mo.rim.gov/ij/docs/intro.html). Results from the same transformed lines were pooled for each leaf number. To determine the leaf size, the margins of scanned individual leaf images were traced and areas were determined using ImageJ.

In vitro binding assays may be useful in various aspects of the invention, for example to assay the interaction of a CDK inhibitor, or fragments of a CDK inhibitor, and a particular kinase, cyclin or protein that binds to the CDK inhibitor. As an example of such an approach, S-Met labeled ICKl protein may be expressed from a T7 promoter construct using an in vitro coupled rabbit reticulocyte transcription/translation system ('TNT', Promega). Ni⁺-NTA beads (Qiagen) may be equilibrated and blocked in NETN buffer lacking EDTA (NTN) (Bai et al., 1996), and supplemented with 2 mg/ml BSA. Equilibrated beads may be incubated with His₆-CycD3 protein NTN buffer for 2 h followed by washing with 2 X 1 ml NTN buffer. Binding experiments may be carried out in NTN containing 10 μl beads, plus 5 μl S-Met labeled protein. The binding reaction was incubated at 10°C for 2 h, followed by washing with 3 X 0.5 ml NTN buffer. Washed beads may be eluted with 10 μl SDS-containing denaturing buffer at 100°C for 5 min. The bound ⁵S-Met labeled proteins may be analyzed by SDS-PAGE and autoradiography. In the present invention, 'proteins that bind to a CDK inhibitor' include proteins identifiable by such assays.

Deletion constructs may be useful for domain mapping to determine the functional domains of a CDK inhibitor, cyclin or protein that binds to a CDK inhibitor. For example, N-terminal deletion constructs of ICKl were made using cDNAs with deletions of various lengths from the N-terminal end. The C-terminal deletion constructs were prepared by PCR using Pfu DNA polymerase with sequence-specific primers and the resulting DNA fragments were cloned into the yeast two-hybrid activation-domain (AD) vector (Fields and Song, 1989). The deletion clones may be verified by DNA sequencing. The constructs may be used to transform a suitable yeast strain. The yeast strain harbouring a deletion construct was further transformed with either Cdc2a or CycD3 cloned in a BD- (binding domain) vector. Interactions in the yeast two-hybrid system may then, for example, be analyzed by X-gal filter assay (Chevray and Nathans, 1992) and by liquid culture assays for relative β- galactosidase activity (Reynolds and Lundlad, 1994). Three or more independent transformants may be used for each interaction.

In some aspects of this invention, it may be useful to determine and verify the expression pattern of a particular promoter-gene construct. Tissue-specific or inducible expression may for example be determined by activity of a reporter gene/protein, by northern hybridization or by in situ hybridization. For example, the activity of the GUS reporter gene was analysed by histochemical staining following the method as described (Jefferson, 1987) using 5-bromo-4-chloro-3-indolyl glucuronidase (X-gluc) as the substrate. Fresh tissues were removed from plants. Tissues were placed in a staining solution consisting of 1 mg ml^"1 X-gluc, 100 mM PO (pH 7.0), 0.5 mM K₃[Fe(CN)₆], 0.5 mM K4[Fe(CN)₆]- 10 mM Na₂EDTA and 0.02% Triton X-100, and were infiltrated under partial vacuum (50-64 cm Hg). They were then incubated in the staining solution at 37°C. After the staining, tissues may be cleared with 70% ethanol.

The cyclin-dependent kinase inhibitors may be used to modify plant development. For example, Arabidopsis thaliana plants were used as hosts to express the plant cyclin- dependent kinase inhibitor ICKl. Fifty independent transgenic 35S-ICK1 plants were analysed. Majority of transgenic lines expressing ICKl displayed modifications in plant growth and development. These plants were smaller in size. Most organs were smaller. The moφhology of leaves and floral organs were also altered. These plants also showed reduced number of leaves and early flowering in comparison to control plants. These modifications of size and moφhology in transgenic 35S-ICK1 plants made it easy to distinguish them from the wild type plants.

The present invention discloses that these modifications can be at least partially restored by the expression of a protein that binds to a cyclin-dependent kinase inhibitor. In one example, transgenic plants were obtained expressing CycD3 or CycD2 of Arabidopsis. The homozygous 35S-ICK1 lines were crossed with 35S-CycD lines. The FI plants from such crosses displayed characteristics similar to wild type plants rather than to the parental 35S-ICK1 plants. For instance, the FI plants were much bigger than 35S-ICK1 parental plants. In 35S-ICK1 parental plants, there was strong serration in leaves while FI plants displayed leaf moφhology as the wild type plants. The FI plants also showed normal flower moφhology rather than the modified flower moφhology observed in the 35S-ICK1 plants. These results suggest that the expression of CycD3 or CycD2 had at least partially restore the modifications resulted from the expression of ICKl in these plants. In another example at least partial restoration may be achieved by the expression of a CDK. Transgenic Arabidopsis plants expressing Cdc2a were obtained. The homozygous 35S-ICK1 lines were crossed with 35S-Cdc2a lines. The FI plants from such crosses were analysed similarly.

In alternative embodiments of this invention, restoration of the phenotypic effects as a result of expressing a plant CDK inhibitor may be accomplished by expression of a protein that mediates specific degradation of the CDK inhibitor. It is generally known that many cell cycle regulators are controlled by protein degradation and more specifically through ubiquitin-mediated proteolysis (King et al., 1996; Pagano et al., 1997). The ubiquitin- mediated proteolysis pathway is conserved in plants (Callis, 1997). Published results suggest that plant cell cycle regulators may also be degradated by the ubiqutin-proteasome pathway (Genschik et al., 1998). The attachment of aubiquitin to a protein, a process called ubiquitination, involves sequential transfer of an activated ubiquitin to the protein through a cascade of three different classes of enzymes El, E2 and E3. E3s also collectively known as ubiqutin ligase transfer ubiquitin to specific substrates. In one aspect of this invention, at least partial restoration of the phenotypic effects due to expression of a CDK inhibitor may be restored by expressing in the plant an E3-like protein that interacts with and transfers the activated ubiquitin to the CDK inhibitor.

In alternative embodiments of this invention, restoration of the phenotypic effects as a result of expressing a plant CDK inhibitor may be accomplished by expression of a recombinant antibody that specifically recognizes the CDK inhibitor or a recombinant peptide that specifically interacts with the CDK inhibitor. Techniques are available for selecting such a recombinant antibody or peptide. For instance, bacterial phage can be used to display recombinant antibodies (e.g. McCafferty et al., 1990; Winter et al., 1994; Gavilondo and Larrick, 2000) or recombinant peptides (e.g. Cwirla et al., 1990; Burritt et al., 1996). The immunoglobulin variable (V) domain genes of both heavy (H) and light (L) chains, which determine the functional structure of the antigen-binding site, can be amplified by PCR and reconstructed into large single-chain Fv antibody (scFv) libraries. Single chain antibodies that specifically recognize a plant CDK inhibitor can be selected by affinity binding of filamentous phage that display the recombinant antibodies. Alternatively, recombinant antibodies may be displayed on bacteria (Daugherty et al., 1998), yeast (Kieke et al., 1997) and ribosomes (Hanes et al., 1997). Specific peptides that interact with a plant CDK inhibitor may be selected from recombinant peptide libraries (Burritt et al., 1996). The DNA sequences encoding the recombinant antibodies or peptides may be expressed in plants (e.g. Hiatt et al, 1989; Tavladoraki et al., 1993; Artsaenko et al., 1995) to modulate the function of the target CDK inhibitor.

In accordance with alternative embodiments of the invention, a wide variety of plant species may be modified. As an example, transgenic Nicotiana tobaccum plants (Tl) were obtained with 35S-ICK1 construct. Twenty one independent transformants were analysed. The insert number was analysed by Southern hybridization. ICKl expression was anlaysed by northern hybridization. Results show that at least twelve plants had significant levels of ICKl expression. Some transgenic plants showed modified development including smaller plant size and smaller organs such as leaves. The progeny plants may be further analysed for modifications. For instance, five tobacco 35S-ICK1 Tl lines were used to determine and verify modifications. The non-transformed tobacco plants and two Tl lines carrying 35S- GUS construct were used as controls. For each line, five progeny plants were grown in pots. At about ten weeks, the length of three longest leaves on each plant was measured. The average length of these three leaves for the Wt and two 35S-GUS Tl lines were 23.2, 23.8 and 23 J cm respectively. In comparison, the average leaf length for four 35S-ICK1 Tl lines was 17.5, 19.5, 14.6 and 12.1 cm respectively. T2 plants of the Tl transgenic 35S-ICK1 lines were also shorter on average than the control plants when measured at the same time. Plant moφhology of the 35S-ICK1 tobacco plants was also modified. For instance, leaf may be mottled in color. Similarly, the expression of a CDK inhibitor and a protein that can bind to the CDK inhibitor can be achieved in a variety of plant species. This is accomplished for instance by transforming a plant that expresses the CDK inhibitor with a construct containing a gene encoding the protein that binds to the CDK inhibitor or by crossing the plant expressing the CDK inhibitor with another plant expressing the protein that binds to the CDK inhibitors. In one example, tobacco plants were transformed with 35S-CycD2 and 35S-CycD3 construsts. The insert number was analysed by Southern hybridization. Northern analysis using RNA isolated from leaf tissues showed that many of these plants had a significant level of cyclin expression while controls (non-transformed plants and plants carrying 35S-GUS construct) had no detected level of transcript. Transgenic lines carrying 35S-CycD2 or 35S-CycD3 construct were crossed with homozygous 35S-ICK1 lines. FI seeds from these crosses were planted. At least partial restoration of plant development modifications as described above due to ICKl expression was observed.

As yet another example, transgenic Brassica napus plaints expressing ICKl were obtained. The presence of heterologous transgene was verified by PCR and southern hybridization. Modifications of plant development were observed in the original (Tl) and subsequent generations of plants. Thus a variety of plant species may be transformed by techniques known in the art to express a CDK inhibitor, a cyclin or a protein that binds to a CDK inhibitor.

Plants (e.g. T2) derived from the original transformants (Tl) may be studied to determine the segregation of the inserted gene and also to verify whether the particular phenotype is co-inherited with the inserted gene. For example, T2 seeds of four transgenic Arabidopsis 35S-ICK1 lines and seeds of control Arabidopsis plants were sterilized and placed onto the selective medium containing 50mg/L kanamycin. Four duplicate plates were used for each type of plant. The number of resistant susceptible seedlings for four independent Arabidopsis 35S-ICK1 lines (line# 12, 13, 15 and 16) was 103/27, 89/29, 116/36 and 108/33, indicating that there was one insert in each of these transgenic lines. Seedlings of non-transformed control were all susceptible while seedlings of a homozygous control transgenic line were all resistant. As another example, seven transgenic tobacco lines were determined for segregation and five showed a segregation ratio close to 3 : 1. Genomic DNA may be isolated and the copy number of the transgene may be determined by Southern hybridization. For instance, of the four transgenic Arabidopsis 35-ICK1 lines that showed 3:1 segregation, two had a single copy of the transgene, one had two copies and one had three copies.

The expression of a CDK inhibitor, cyclin or protein that binds to a CDK inhibitor in particular plant tissues may be assayed to determine, for example, whether that CDK inhibitor will have utility as a division or growth modulator when expressed in such tissues. For instance, the expression of ICKl was analyzed in independent transgenic Arabidopsis 35S-ICK1 plants. The ICKl expression level increased significantly in transgenic 35S-ICK1 Arabidopsis plants as shown by several independent analyses. Increased ICKl expression was observed in original Tl transformants and was similarly observed in the progeny T2 plants, indicating that the increased level was due to transgene integration. The increased expression was detected in different tissues analysed, as expected since the 35S promoter activates gene expression in most tissues. Expression of the CDK gene Cdc2a did not decrease and perhaps showed a slight increase. For comparison, a ubiquitin gene UBQ11 (Callis et al., 1993) remained more consistent.

To verify the functional role of a putative CDK inhibitor in such tissues, the CDK activity may also be assayed. For example, the pi 3 ^uc -associated Cdc2-like histone HI kinase activity was analysed with the same source tissues that were used in gene expression analyses as described above. Results show that, coinciding with increased ICKl expression, the Cdc2-like kinase activity decreased significantly in comparison to control plants. This decrease was observed in independent transgenic 35S-ICK1 plants and also in different tissues. As there was no decrease in the level of expression for positive cell cycle regulators such as Cdc2a, it is concluded that the decreased Cdc2 kinase activity is directly due to inhibition by increased ICKl expression in these 35S-ICK1 plants.

In vitro kinase assays may be used to demonstrate that a recombinant putative CDK inhibitor, such as ICKl protein, is an effective inhibitor of plant Cdc2-like kinases. Plant CDK inhibitors may not inhibit CDK from mammalian and yeast cells (Wang et al., 1997). For example, recombinant ICKl is effective in vitro in inhibiting the histone HI kinase activity of pl3^suc -associated kinases from A. thaliana and heterologous Brassica napus (Wang et al., 1997; 1998) CDK inhibitor-binding proteins for use in various aspects of the invention may be identified using a yeast two hybrid screening protocol with a variety of bait fusion protein sequences. For example, ICKl may be used as a bait to identify cyclin homologs or proteins that interact with ICKl . Interactions in the yeast two-hybrid system may then, for example, be analyzed by X-gal filter assay (Chevray and Nathans, 1992) and by liquid culture assays for relative activity (Reynolds and Lundlad, 1994). To provide evidence confirming the interaction of a CDK inhibitor with a target protein of interest, further in vitro binding assays may be conducted as described (Wang et al., 1998). Similar assays may be used to identify CDK inhibitors capable of interacting with other cellular targets.

The regions of a proteins that are functionally involved in interactions with other proteins in various aspects of the invention may be mapped by deletion mapping using a variety of techniques, such as the yeast two-hybrid system and variations thereof. Such in vitro assay results may be verified by in vivo tests, since the persistence of interactions in the two-hybrid system may be affected by possible alterations in functionality of plant proteins expressed in yeast. As an example of an in vitro assay, to determine the functional significance of the C-terminal domain and other regions of ICKl, three N-terminal and three C-terminal deletion mutants were assessed for their interactions with Cdc2a and CycD3 in the two-hybrid system. Major shifts in β-galactosidase activity were observed when amino acid regions 3-72, 109-153 and 176-191 were deleted. The reporter activity for interactions of ICKl with Cdc2a and CycD3 was both increased upon deletion of amino acids 3-72, suggesting that this region may have a regulatory role on the interactions of ICKl with the other proteins. In pairwise comparison, the deletion of amino acid regions 3-72, 73-108, 163-175 or 153-162 had comparable effects on the interactions of ICKl with Cdc2a versus CycD3, as reflected by the marker gene expression, while the deletions of amino acid regions 109-153 and 176-191 had clearly differential effects. The most significant reduction in β-galactosidase activity for the interaction of ICKl with CycD3 resulted from the deletion of amino acids 109-153, whereas the deletion of amino acids 176-191 had a more detrimental effect on the interaction with Cdc2a. As another example, the functional importance of a portion of a CDK inhibitor may also be assayed by analyzing transgenic plants expressing a modified version of the inhibitor. Deletion constructs of ICKl may be used to transform Arabidopsis plants. The changes in these transgenic plants expressing variants of ICKl may be compared with plants expressing an ICKl with unmodified functionality. As another example, a series of deletions may also be made to map the regions in CycD3 that is responsible for interacting with ICKl and Cdc2a.

One aspect of the invention utilizes functionally important regions of a protein. For example, the functionally important regions of a CDK inhibitor, cyclin or protein that binds to a CDK inhibitor may be determined through routine assays. Randomly selected portions of a protein, such as a CDK inhibitor, may be selected for use in assays to determine whether the selected region is capable of functioning as required in the context of the present invention. For example, in various embodiments regions of ICKl may be used, such as the 109-153 region and/or the 163-191 region, with or without additional regions from ICKl or other CDK inhibitors, provided the recombinant protein meets the functional requirements of the present invention (which may be determined through routine screening of functionality) .

The modifications of plant development by expression of a CDK inhibitor, a cyclin or a protein that binds to a CDK inhibitor, may involve modification of cell number and/or cell size. For example, analyses of the structure and cell size in 35S-ICK1 and control plants by scanning electron microscopy and light microscopy showed that the cells of 35S-ICK1 plants were on average larger than the corresponding cells in control plants in different tissues examined (leaves, hypocotyl, root and flower organs). In another example, the leaf and cell size of35S-ICKl and control plants were quantified using fully expanded leaves (the 5 to 8 leaves) of 30-day plants. The average leaf size of the 35S-ICK1 plants (lines) used was between 3.4% to 57.1% of the leaf size of the wild type plants. Pavement cells on the adaxial surface in similar areas of leaves were measured. Cells in leaves of different 35S-ICK1 lines were 1J-2J times larger than the cells of control plants.

Various aspects of the invention may be used to obtain a wide variety of phenotypic variations in plant moφhology or other characteristics, which are at least partially reversible effects. For instance, various promoters with tissue-specific expression patterns or with inducible expression may be used. For example, Arabidopsis plants were transformed with NTM19-ICK1, NTM19-CycD3 and NTM19-GUS constructs respectively. The pattern of gene expression directed by the NTM19 promoter (Oldenhof et a., 1996) was confirmed by histochemical staining of the GUS reporter gene in transgenic NTM19-GUS plants. These plants including NTM19-ICK1 plants developed normally before fertilization and silique (pod) development. However, pod development was affected in a large proportion of NTM19-ICK1 plants. These results are consistent with the expression pattern of the NTM19 promoter as it is specifically expressed during microspore development. In an example of analysing transformants growing under identical conditions, five (5) out of thirty (30) NTM19-ICK1 transformants showed significantly reduced pod development (defined as "less than half pods are developed"). In contrast, only one (1) of fourteen (14) NTM19-GUS transformants and none (0) of thirty (30) NTM19-CycD3 transformants had a similar phenotype. These results demonstrate that tissue-specific expression of ICKl may be used to produce plants with modified male sterility, which may be at least partially reversed with cyclins or proteins that bind to the CDK inhibitor. In some embodiments, the transgenic plants with male sterility may set seeds after pollination, using pollen from non-transformed plants, indicating that the female reproduction system is unaffected in these male sterile plants. Apart from these specific modifications, these transgenic plants otherwise grew and developed normally.

As yet another example, transgenic B. napus and Arabidopsis plants were obtained with a chimeric gene construct consisting of the Bgpl promoter and ICKl. The Bgpl promoter has previously been shown to direct strong exogenous GUS reporter gene expression in pollen of Arabidopsis and Nicotiana tobacum plants (Xu et al., 1993). Of over forty putative Brassica transformants obtained, some showed reduced seed setting with four transformants showing a greater reduction. The degree of reduction varied from an amount of about half the seed-setting in normal plants (in term of number of seeds per pods) to nearly complete sterility. All of over twenty transgenic Arabidopsis plants were normal and showed no significant reduction in seed-setting. Northern analysis was performed using RNA samples isolated from anthers of the transgenic Brassica plants and mature buds of transgenic Arabidopsis plants and results showed that most of these plants showed a prominent level of ICKl expression. The absence of sterility among Arabidopsis transformants could be attributed to the difference in gene expression mediated by the Bgpl promoter in different species, indicating that routine experimentation may be necessary to identify suitable promoters, and other control elements, for use in alternative embodiments of the invention. For example, the Bgpl promoter, which is from B. rapa (B. campestris) (Xu et al., 1993), may be more effective in activating transgenic ICKl expression in B. napus than in A. thaliana plants. The results in Brassica are indicative of the fact that it may be desirable in alternative embodiments to select promoters that would be suitable for a particular aspect of this invention.

As an example, transgenic Brassica napus plants were obtained with AP3-ICK1 construct. Some of the plants showed much reduced petal size and significant reduction in seed-setting, with one plant showing almost complete sterility. The transgenic Brassica phenotypes were consistent with the pattern of AP3 promoter-directed gene expression, i.e. stronger expression in petal and stamen primordia and possibly low levels of expression in the inner integument or ovule (Day et al., 1995). Of fifty-two primary transformants, some transformants showed changes in petal moφhology and development with four transformants displaying significant alterations. Plants showing a strong phenotype in modification of petal moφhology also had reduced seed-setting. For plants showing poor seed-setting, seeds were obtained by crosses with non-transformed wild-type B. napus plants. The inheritance of the altered phenotype was evident in subsequent progeny plants. Transgenic B. carinata plants were also obtained, and phenotypic changes in petal development, similar to transgenic B. napus plants, were observed.

The specific expression patterns of various promoters used in various embodiments of this invention may be determined and verified by techniques known in the art, including northern hybridization, histochemical staining of a reporter gene or protein such as GUS and in situ hybridization. For example, the expression of ICKl in transgenic Bgpl -ICKl B. napus plants was analysed by northern hybridization using P-labeled ICKl cDNA as probe. RNA samples were isolated from the leaf and mature anthers of a transgenic plant showing sterility phenotype and the control B. napus (Westar) plant. For each sample, 15 μg of RNA was loaded and separated by electrophoresis. RNA transfer and hybridization were performed as described. There were no significant levels of ICKl expression in leaves of both transgenic and control plants as well as in the pollen of the control plant. However, as expected, a strong level of ICKl expression was observed in anthers of the transgenic plant. As another example, ICKl expression was analysed in plants transformed by AP3-ICK1 construct. RNA samples were isolated from the leaf, sepal, petal, anther and whole young flower of the transgenic plant and the control plant. The highest level of ICKl expression was shown to be in the petals of the transgenic plants. There was no detectable signal under the conditions used for the tissues from the control plant.

In alternative embodiments, a variety of plant CDK inhibitors may be used in the invention. They include ICKl , ICK2, ICN2, ICN4, ICN6, ICN7, ICN8 and ICDK. For example, a putative CDK inhibitor gene ICDK (AJ002173, SEQ ID No. 15 and SEQ ID No.16) was identified from Chenopodium rubrum (Fountain et al., 1999), as it shares some similar properties as ICKl and thus with ICK2, ICN2, ICN6 and ICN7 as well (Table 1). C. rubrum seeds were collected in Saskatchewan, Canada. RNA was isolated from seedlings and leaves. The full-length coding region of ICDK cDNA was cloned using RNA RT-PCR. The sequence data showed that the cloned cDNA was identical to ICDK of C. rubrum in the database. A construct consisting of the 35S promoter and ICDK was prepared. The Agrobacterium strain harboring the 35S-ICDK was used to transform Arabidopsis. Selection for transformants was performed as described elsewhere herein. Of thirty eight (38) independent transgenic 35S-ICDK plants, twelve (12) showed serrated leaves and twenty two (22) showed modified flowers, which were observed in 35S-ICK1 plants. Expression of ICDK in these plants was confirmed by northern analysis. A construct consisting of 35S promoter and ICN2 was prepared and used to transform Arabidopsis plants. Among forty six (46) independent 35S-ICN2 transformants, twelve (12) showed serrated leaves and nine (9) showed modified flowers, as observed in 35S-ICK1 plants. Results from the 35S-ICN2 transformants also indicate that different CDK inhibitors from the same species may be used in various aspects of the present invention. Further, Chenopodium and Arabidopsis are phylogenetically rather distant species with Chenopodium belonging to the subclass of Caryophyllidae and Arabidopsis to the subclass Dilleniidae. The observation that a Chenopodium CDK inhibitor functions in Arabidopsis in the context of the present invention indicates that plant CDK inhibitors of different species may be used in various aspects of the present invention. Thus, diverse CDK inhibitors may be used in accordance with various aspects of the invention. Table 1: Percent Identity, using Clustal method with PAM250 residue weight table

Other plant CDK inhibitors and CDK inhibitor genes sharing functional and sequence similarity with ICKl may be identified using an approach similar to the approach used to isolate ICKl, based for example on their interactions with either Arabidospis Cdc2a or a D-class cyclin (e.g. cyclin D3 or cyclin D2). The sequences of ICK2 (SEQ ID NO: 6), ICN2 (SEQ ID NO: 7), ICN6 (SEQ ID NO: 8), and ICN7 (SEQ ID NO: 9) are shown in Figs 2 through 6. Additional CDK inhibitors may identified by other techniques known in the art. For instance, other plant inhibitors may be identified by comparing the sequences of cloned cDNAs and genes with the nucleotide or amino acid sequences of ICKl and other known plant CDK inhibitors. The properties of additional plant CDK inhibitors may be further verified. For example, a segment of Arabidopsis BAC clone F24L7 (GenBank Accession AC003974) encodes a putative protein that shares some similarity with the ICK/ICN proteins. A corresponding cDNA clone to this segment of genomic DNA was identified and sequenced. The protein encoded by this clone interacted with CycD3 as the ICN proteins do by yeast two-hybrid assay and thus it is designated as ICN8. ICN8 cDNA sequence is given in Figure 7. Similarly, a BAC clone from Oryza sativa contains a segment that encodes a protein (GenBank AAG16867) sharing similarity with ICK/ICN proteins.

In some embodiments, partial cDNA sequences may be identified first. Full-length cDNA sequences can then be identified using routine techniques known to the art. For instance, partial cDNAs can be used as probes to screen cDNA libraries to obtain full-length cDNA sequences. If the 5'-part or 3'-part of the full cDNA is missing, particular techniques of polymerase chain reaction such as 5' RACE and 3' RACE methods can be used to identify the sequences. Alternatively, other techniques can also be used to identify full-length cDNA sequences for instance by searching sequences databases using the partial cDNA sequences. Originally, for example, the cDNA sequences for ICK2 and ICN6 were partial sequences. Subsequently, full-length cDNA sequences were identified.

These genes share at least two functional properties with ICKl: First, all of these genes encode proteins able to interact with either Cdc2a or a D-class cyclin or both. Such interactions may enable them to regulate the activity of plant CDKs in alternative embodiments of the invention. Second, these ICK/ICN proteins all share some sequence similarity in the region of ICKl that is functionally important in some embodiments for its interaction with Cdc2a and cyclin D3 (discussed above). These homologous genes or proteins may be used in some embodiments, in a manner similar to ICKl, to modulate plant growth and development. One or more such genes or proteins may be used in some embodiments alone or in combination to provide temporal and spatial regulation of cell cycle initiation and progressing during plant development in accordance with this invention.

In one aspect of the invention, an assay is provided to determine if a CDK inhibitor interacts with a known protein, which are thereby identified as proteins that bind to the CDK inhibitor. For example, the full-length cDNA of the gene to be analyzed may be cloned in a GAL4-binding domain vector using PCR and gene specific primers with flanking restriction sites. Such constructs may be used to transform the yeast carrying the CDK inhibitor of interest, such as ICKl in a GAL4-activation domain vector. Using this approach, for example, the interactions of ICKl with a number of cell cycle-related genes from A. thaliana were examined in accordance with the invention (Table 2). In these examples, the yeast two-hybrid assay results indicate that in particular embodiments of the invention, ICKl protein may interact with Cdc2a but not with Cdc2b. Similarly, ICKl may interact with D-type cyclins, CycDl, CycD2 and CycD3, while not interacting with A/B- class mitotic cyclins, CycA2, CycBl and CycB2 (Table 2). The yeast two-hybrid assay results also indicate that ICKl may not interact in some embodiments with PCNA, also a cell cycle protein, and ATMAP2, a kinase sharing some similarity with Cdc2 kinase. Table 2. Analyses of ICKl interactions with other proteins in the yeast two-hybrid system

The interactions of additional plant CDK inhibitors can also be determined using similar methods. For instance, the interactions of ICKl, ICK2, ICN2, ICN6, ICN7, ICN8 and ICDK with Arabidopsis CDKs (Cdc2a and Cdc2b) and the D-type cyclins were analysed using the yeast two-hybrid system. As summarized in Table 3, CKS1 At (De Veylder et al. 1997), one of the controls used, was able to interact with Arabidopsis Cdc2a and Cdc2b. ICKl, ICK2, ICN7 and ICN8 interacted with Cdc2a but not with Cdc2b, and interacted with the three D-type cyclins (Dl, D2 and D3) tested. On the other hand, ICN2 and ICN6 interacted only with D-type cyclins but not with Cdc2a and Cdc2b. Similar to the results for ICN2 and ICN6, the Chenopodium ICDK interacted only with the D-type cyclins. As a comparison, none of the ICKs interacted with a cell cycle-related Arabidopsis PCNA. In the absence of interacting partners, only CycD3 had a low level of background activity (Table 3). However, interaction of CycD3 with ICKs increased the β-galactosidase activity greatly. These results indicate that one may segregate the ICKl -related inhibitor proteins into two groups, which can be distinguished by the differences in their two-hybrid interactions with Cdc2a and D-type cyclins. The A-group (ICKl, ICK2, ICN7 and ICN8) is able to interact with both Cdc2a and D-type cyclins, while the B-group (ICN2, ICN6 and ICDK) is only able to interact with D-type cyclins.

Table 3. Summary of X-Gal assays for determining the interactions of ICKs with Cdc2a, Cdc2b and D-type cyclins.

For each interaction test, yeast strain (MaV203) was transformed first with AD constructs listed in the first row, and then with BD constructs listed in the first column of the table following standard protocol. Three independent colonies from each interaction combination were used in X-Gal filter assay for β-galactosidase activity. The results of X-Gal assays were recorded at 2, 4, and 20 hours (overnight). Color intensity determined by visual inspection: "-" = none; "+" = weak; "++" = moderate; "+++" = strong.

Faint color was visible after overnight incubation.

EXAMPLE 1

Transgenic Arabidopsis plants were used as hosts for a CDK inhibitor expression construct designated 35S-ICK1 and for two alternative cyclin D expression constructs designated 35S-CycD2 and 35S-CycD3 (for expression respectively of cyclin D2 and cyclin D3). Methods

The ICKl cDNA was linked to the CaMV 35S promoter in a vector (pBI121) for plant expression. Transformation of Arabidopsis was performed based on the infiltration method (Bechtold et al., 1993) except the surfactant Silwet-40 (0.01-0.05%) (Clough et al, 1998) was added to the final suspension for infiltration. Seeds (Tl) were harvested and selected on Vi MS basal medium (Sigma) containing 50 mg/1 kanamycin and 300 mg/1 Timentin. Transformants were transferred to soil and grown in growth chambers.

Results

Plant growth was significantly inhibited by ICKl expression Among fifty independent ICK1-35S transformants examined, the majority displayed significant growth and moφhological changes. One striking change was the much smaller size of transformants compared to control plants. The smaller size of 35 S-ICK1 plants persisted through all stages of plant development and was reflected by the size of most organs including leaf, stem, root and floral organs. It was consistent from original Tl to subsequent generations (e.g. T2 and T3), indicating that it is genetically stable. There was a range in the extent of phenotypic alterations among independent transformants. Consistent with this observation, the level of ICKl expression varied among different transformants. It is well established that there exists a wide range of variation in the level of expression for a given gene introduced into independent transgenic plants.

The inhibition of growth was initially assessed by fresh weight of transgenic T2 and control seedlings grown in Petri plates. Results indicated that plants of independent 35S- ICK1 lines had lower fresh weight than wild type plants. For quantification of growth inhibition under typical physiological conditions, 35S-ICK1 and control plants were grown in soil. The fresh weight (at 3 -weeks) was significantly lower in a number of independent 35S-ICK1 lines in comparison to plants without 35S-ICK1 or plants harboring 35S-GUS construct (Table 4). Transgenic 35S-ICK1 plants were smaller than control plants. In some lines, 35S-ICK1 plants were less than 1/10 of the control plants that did not carry 35S-ICK1 (Table 4). The finding that the smaller size (and lower fresh weight) of 35S-ICK1 plants was consistent for plants grown under different growth conditions suggests that the inhibition of growth was not due to variations in growth conditions. These data clearly show that growth was significantly inhibited by ICKl over-expression.

Table 4 shows growth and development of transgenic 35S-ICK1 Arabidopsis plants. 35S-ICK1 and control plants were grown in growth chamber. For growth evaluation, shoot (above-ground tissues) fresh weight of 21 -day plants was determined. For development evaluation, flowering time and leaf number (rosette plus inflorescence leaves on the primary axis) were obtained. Table 4

SD = standard deviation. ** indicates significance (t-test) from the Wt control plants at PO.001 level and * indicates significance at PO.05 level.

Altered aspects of plant morphology

The 35S-ICK1 plants showed profound changes in moφhology of organs such as leaves. Depending on transgenic lines, there was a range of changes in leaf shape, in addition to a reduction in size. In some lines, leaves were significantly serrated. In wild type plants, only slight serration occurred in adult leaves. The expression of ICKl resulted in much more prominent serration of the leaves and this characteristic was observed in almost all leaves in 35S-ICK1 plants with strong phenotype of growth inhibition. The smaller leaves and shorter leaf petioles gave 35S-ICK1 plants a more compact appearance. Root growth of 35S-ICK1 plants was similarly affected.

Striking changes also occurred in floral organs. In wild type plants, the fully-opened flowers were spread and the top of flowers often exceeded the inflorescence apex. In 35S- ICK1 plants, flowers stayed closer and usually were at the same level or below the inflorescence apex. On the inflorescence of 35S-ICK1 plants, the reduced distance between flowers was likely due to reduced growth of inflorescence stem and flower pedicels. Thus the flowers appeared as a compact cluster when viewed from the top. Changes in size and moφhology were also evident in individual flowers. The flowers of 35S-ICK1 plants had smaller or shorter sepals, petals and stamens. Mature petals of normal Arabidopsis flowers bent at the halfway point horizontally above sepals while those of 35S-ICK1 plants were straight upward. Petals of 35S-ICK1 plants were also narrower with serration along the top edge. These changes were so profound that transgenic 35S-ICK1 plants bore little resemblance to the wild type Arabidopsis plants.

Most of the above changes could theoretically be attributed to reduction of cell division and thus organ growth. However, there were also developmental changes beyond this simple explanation: (1) 35S-ICK1 plants flowered earlier and had significantly fewer leaves at flowering time than control plants (Table 4). Precocious flowering of 35S-ICK1 plants was similarly observed when grown in Petri plates (data not shown). (2) Transgenic 35S-ICK1 plants showed reduced apical dominance. This reduced apical dominance was evident in two ways: reduced prominence of the primary inflorescence, and a large number of lateral branches.

Increased expression of ^'ICKl resulted in reduction of CDK activity in plant cells

The ICKl expression level increased significantly in transgenic 35S-ICK1 Arabidopsis plants as shown from several independent northern analyses. Increased ICKl expression was observed in original Tl transformants and was similarly observed in the progeny T2 plants, indicating that the increased level was due to transgene integration. The increased expression was detected in tissues analysed including shoots, roots, leaves, stems and flowers, as expected since the 35S promoter activates gene expression in most tissues.

The pl3 -associated Cdc2-like histone HI kinase activity was analysed with the same source tissues that were used in gene expression analyses. Results show that, coinciding with increased ICKl expression, the Cdc2-like kinase activity decreased significantly in comparison to control plants. This decrease was observed in independent 35S-ICK1 tranformants and different tissues. Results also show that there was no decrease in the expression level of positive cell cycle regulators such as Cdc2a. It is concluded that the decreased Cdc2 kinase activity is directly due to inhibition by increased ICKl expression in these 35S-ICK1 plants. Cell number and cell size were affected

The structure and cell size of 35S-ICK1 and control plants were examined by scanning electron microscopy and light microscopy. It was consistently observed that the cells of 35S-ICK1 plants in all tissues examined (leaves, hypocotyl, root and flower organs) were on average slightly larger than the corresponding cells in control plants.

An initial quantitative analysis of cell size was made using pavement cells of the first pair of true leaves on 15-day plants growing in Petri plates. Data showed that cells of 35S-ICK1 plants were clearly larger than control plants. To better quantify the cell size difference, fully expanded leaves (the 5 to 8 leaves) of 30-day plants grown in soil were used for determining leaf and epidermal cell size. The average leaf size of the 35S-ICK1 plants (lines) used was between 3.4% to 57.1% of the leaf size of the wild type plants (data not shown). Pavement cells on the adaxial surface in similar areas of leaves were measured. Cells in leaves of different 35S-ICK1 lines were 1J-2J times larger than the cells of control plants. The cell size was similar for the four different leaves of each type of plant surveyed, indicating that cells were at their mature size in these leaves.

Transgenic expression of other plant CDK inhibitors in Arabidopsis plants Other plant CDK inhibitors can also be expressed in plants in a manner similar to ICKl . For instance, the Arabidopsis CDK inhibitor gene ICN2 (ICK4) and the

Chenopodium rubrum CDK inhibitor gene ICDK (ICKCr) were introduced into Arabidopsis plants and the transformed plants were characterized, using the 35S-ICK1 transformants as comparisons for determining the effects of other plant CDK inhibitor genes.

Similar phenotypic changes in leaf and flower moφhology were observed in plants expressing 35S-ICN2 or 35S-ICDK as observed in plants expressing 35S-ICK1, but not in plants transformed with 35S-GUS. Leaf serration and modifications of flower moφhology were easily distinguished in independent transformants. The frequency of transformants showing such phenotypic changes differed among the three CDK inhibitor constructs. For example, the frequency of plants showing modified flower moφhology was significantly higher in transformants with 35S-ICK1 (70% of 45 independent transgenic lines) and 35S- ICDK (51%) of 40 independent transgenic lines) than in transformants with 35S-ICN2 (16% of 48 independent transgenic lines).

RNA samples were prepared from transgenic and control plants. Northern analyses showed that specific transgenes were overexpressed respectively in the plants transformed with 35S-ICK1, 35S-ICN2 and 35S-ICDK constructs compared to the controls. There was a low background level of ICKl and ICN2 in wild type Arabidopsis plants but there was no signal for ICDK in Arabidopsis plants except those transformed with 35S-ICDK.

Based on a phenotypic survey of transformed plants and Southern analysis (data not shown) from the Tl generation, seeds harvested from plants with a single insert (1-2 copies of transgenes) were planted to obtain T2 plants for detailed evaluation and for confirmation of true breeding of the novel alterations observed in the Tl generation. Most 35S-ICN2, and 35S-ICDK lines showed segregation of the mutant phenotype (modified leaf and/or flower moφhology) distinguishable from the wild type plants. PCR using marker gene primers was used to confirm the transformants within a population of each line. The phenotypic changes in T2 populations were consistent with the changes observed in Tl plants. Inhibition of plant growth was observed starting from the emergence of the seedling and lasted throughout plant growth and development. The 35-ICDK or 35S-ICN2 plants with phenotypic changes were smaller in size than the wild type plants and 35S-GUS plants. As for 35S-ICK1 transformants described above, the major changes in moφhology for plants carrying 35S-ICN2 and 35S-ICDK included leaf serration and modified flowers. Transformed plants showing these phenotypes flowered earlier than control plants.

Quantitative analysis showed that the plant fresh weight was reduced in transgenic lines compared with wild type and 35S-GUS plants. There was variation in biomass among different lines for both 35S-ICN2 and 35S-ICDK constructs. Statistical analysis (single factor variance analysis and LSD test) showed that all transgenic lines had significantly lower fresh weight than wild type and 35S-GUS plants. The average reduction of fresh weight was 61% for 35S-ICN2 and 73%) for 35S-ICDK as compared with wild type.

Reduction ofploidy level in plants expressing plant CDK inhibitors

Mature rosette leaves from four-week wild type and transgenic T3 plants were analysed for DNA content (ploidy level) of isolated nuclei by flow cytometry. Wild type Arabidopsis leaf tissue showed a similar profile of nuclear DNA content to that described by Galbraith et al (1991) with four major peaks at 2C, 4C, 8C and 16C levels and a minor peak at the 32C level occasionally. In contrast, decreased ploidy level was observed in transgenic lines expressing one of the plant CDK inhibitors. The extent of decrease varied with different transgenic lines. Quantitative analyses were performed using transgenic 35S-ICK1, 35S-ICN2 and 35S-ICDK lines with a strong phenotype for the respective construct as well as controls. The average peak values from 5-8 individual plants of representative transgenic lines are presented in Table B. In plants of a 35S-ICK1 line, only 2C and 4C peaks were observed. Plants of 35S-ICN2 and 35S-ICDK lines showed an 8C peak in addition to 2C and 4C peaks, with 35S-ICN2 plants having a higher 8C peak (Table 5). The data indicate that endoreduplication was inhibited in these transgenic plants and most severely in 35S-ICK1 plants.

Table 5. Ploidy levels in wild type and transgenic plants.

In this example, DNA content was determined using nuclei isolated from comparable leaves of wild type and transgenic plants expressing plant CDK inhibitors as described. Each datum for a particular DNA content peak represents the average of 5-8 individual plants measured and is expressed relatively in percentage with a total value for all peaks to be 100%.

EXAMPLE 2

Transgenic Arabidopsis plants were used as hosts for a CDK inhibitor expression construct designated 35S-ICK1, for two alternative cyclin D expression constructs designated 35S-CycD2 and 35S-CycD3 (for expressing respectively of Arabidopsis cyclin D2;l and cyclin D3;l), and for a CDK expression construct designated 35S-Cdc2a (for expressing Arabidopsis Cdc2a). Crosses were made between 35S-ICK1 lines, expressing the CDK inhibitor ICKl, and the cyclin expressing lines. FI plants were analyzed, demonstrating that cyclin D2 (CycD2) and cyclin D3 (CycD3) can reverse the inhibition of cell division and plant growth that is otherwise mediated by ICKl over-expression.

Methods

Transgenic Arabidopsis plants were obtained as described. Crosses were made between homozygous 35S-ICK1 (200-13 female parent) and the rest of plant lines (male parent).

Table 6: List of crosses

Parent Plant line* Description .

Female 200-13-1 Homozygous 35S-ICK1 transgenic line (with a single copy).

Expression of ICKl resulted in inhibition of cell division, inhibition of plant growth and distinct changes in plant moφhology

Male Wt Wild type Arabidopsis

292-11 Tl heterozygous line expressing CycD2

292-15 Tl heterozygous line expressing CycD2

294-2** Tl heterozygous line expressing CycD3

297-18 Tl heterozygous line expressing CycD3

* All transgenic lines carry a single insert and all has one copy of the transgene except 297-18 with two copies. Wt plant has no transgene present. The insert and copy numbers were determined by segregation and Southern analyses respectively.

** Resistant to kanamycin weakly but grows normally in soil.

Plant growth was anayzed in Petri plates. FI and transformed parent seeds were sterilized and plated on medium (1/2 MS, 1% sucrose, pH5.6, 0.8% agar) containing 300 mg/L timentin and 50mg/L kanamycin, and Wt seeds were plated on the same medium but without kanamycin. After 14 days, the fresh weight of seedlings was determined. Results

All seedlings from FI seeds and the 200-13-1 female parent were resistant to kanamycin and grew normally on the selection medium. Seedlings from the heterozygous male CycD parent lines were segregated in resistance, as expected. Line 294-2 had weaker resistance to kanamycin than other lines, and 294-2 seedlings became albino a week after plating and did not grow normally, being much smaller in size than other CycD transformed lines at the 14 day.

Modulation of plant growth and development in FI plants

The fresh weight of seedlings for the crosses and parents are presented in Table 7.

Table 7: Fresh weight of FI Arabidopsis plants from crosses between 35S-ICK1 female arent line and C cD male arent lines³

a) Seedlings from each plate were separated according to distinct phenotypes at 14 day after plating. Those of the same type were pooled and weighted. Statistics calculated based on plate means. b) Type 1 refers to the plants with the phenotype of 200-13-1 which have changed leaf shape compared with wild type, such as narrowed and serrated leaves at the investigation time (see example 1).

c) Type 2 refers to the plants with wild type moφhology although their size may vary. In general type 2 plants are larger than type 1 plants. d) Wt seedlings were grown on medium without kanamycin. As shown in Table 7, there were two distinct phenotypes in crosses involving CycD3 expressing male parent lines: i.e. 200-13-1 X 294-2 and 200-13-1 X 297-18. The two phenotypes were distinguishable by both moφhology and seedling size even before weighing. The two types had a segregation ratio close to 1:1. These results indicate that plants with genotype 35S-ICKl/ t had the typical phenotype of ICKl overexpression while plants with genotype 35S-ICKl/35S-CycD3 showed much reduced growth inhibition and reversal of altered leaf shape, demonstrating that the phenotypic effect of ICKl expression in these plants is significantly decreased by the co-expression of a cyclin (CycD3).

The moφhology of seedlings from crosses involving male parent lines expressing CycD2 was catagorized as type 1, i.e. 200-13-1 X 292-15 and 20-13-1 X 292-11. However, the values for fresh weights of these FI seedlings were higher than the type 1 seedlings from the 200-13-1 female parent line and higher than the type 1 seedlings from crosses made with CycD3 expressing male parent lines. These results showed that there was an alternative reversion of plant moφhology mediated by CycD2 compared to the moφhological reversion demonstrated in the CycD2 expressing FI plants. These results show that alternative proteins (CycD2 and CycD3) that bind to a CDK inhibitor (ICKl) may be used to mediate alternative effects on plant development.

Alternatively, transgenic Arabidopsis plants were obtained expressing 35S-Cdc2a (Cdc2a from Arabidopsis). Homozygous lines were selected. Crosses were made between the homozygous 35S-ICK1 and 35S-Cdc2a lines. The FI plants were analysed as above. The data indicate that the modified plant phenotype due to ICKl expression is at least partially restored by the expression of Cdc2a.

As comparisons, the 35S-ICK1 line was also crossed with a transgenic line transformed with 35S-GUS and a transgenic line transformed with 35S-antisense-ICKl . The FI plants from the cross [35S-ICK1 X 35S-GUS] displayed the typical phenotype of ICKl overexpression i.e. smaller plants, serrated leaves and modified flowers. In contrast, the FI plants from the cross [35S-ICK1 X 35S-antisense-ICKl] were indistinguishable from the Wt plants. These results indicate that the effects of ICKl overexpression was not reversed by concurrent expression of GUS, but almost completely reversed by the expression of antisense ICKl.

It is evident from these results that in alternative aspects, by expression of a gene that encodes a protein that antagonizes the function of the CDK inhibitor, the invention facilitaties modulation of the effects of the CDK inhibitor to different extents. Differing extents of modulation may for example be accomplished by various levels of expression of the gene encoding the modulatory protein. In alternative embodiments of the present invention, the plant CDK inhibitor and the modulatory protein may be regulated differently (for instance by using different promoters), and the their transgenic expression may be optimized (for instance by selecting transgenic lines) so that desired effects on growth may be achieved for whole plants or for certain plant organs.

Modulation of cell size in FI plants

Transgenic Arabidopsis plants overexpressing ICKl that displayed a much smaller plant size had larger cells than Wt plants (Example #1). The cell size was thus examined in

FI plants from these crosses using scanning electron microscopy (SEM) and light microscopy. There was no apparent difference in cell size between the Wt and 35S-GUS plants. A normal range of variation in leaf cell size was observed in both plant types.

The FI plants from the crosses [35S-ICK1 X Wt] and [35S-ICK1 X 35S-GUS] had larger cells than control plants, as observed for ICKl -overexpressing plants. The FI plants from the cross [35S-ICK1 X 35S-antisense-ICKl] had leaf cells similar in size to control plants. The FI plants from the cross [35S-ICK1 X 35S-CycD3] had smaller cells than the control plants, despite the fact that these FI plants were smaller than control plants. The SEM results of leaf epidermal cells and light microscopy results of leaf transverse sections are consistent with each other. The present results show that expression of CycD3 in these FI plants had modified the cell size in comparison to Wt plants and plants expressing ICKl alone.

Modulation of ploidy level in FI plants Overexpression of a plant CDK inhibitor such as ICKl inhibited endoreduplication and thus decreased the ploidy level in Arabidopsis leaves, which normally have cells of mixed polyploidy. Therefore the ploidy level in the FI plants was determined using comparable leaf tissues (leaves no. 7 and 8; see Example #1) from plants grown under identical conditions. As shown in Table 8, the FI plants from the cross [35S-ICK1 X 35S- antisense-ICKl] has similar ploidy profile as the Wt plants with four peaks of nuclear contents at 2C, 4C and 8C peaks and 16C. In comparison to Wt plants, the FI plants from the crosses [35S-ICK1 X Wt] and [35S-ICK1 X 35S-GUS showed a significant reduction in ploidy level with essentially two peaks and mostly at 2C level. The FI plants from the crosses [35S-ICK1 X 35S-CycD2] and [35S-ICK1 X 35S-CycD3] had a ploidy profile between the [35S-ICK1 X Wt] type and the [35S-ICK1 X 35S-antisense-ICKl] type. They had major 2C and 4C peaks with a minor 8C peak. The data on ploidy level were consistent with the phenotype of these plants, indicating that the effect on endoreduplication from ICKl overexpression was partially reversed by the expression of Arabidopsis CycD3 or CycD2.

Table 8. Ploidy levels in FI plants

Parent/Fl Transgene % of area value for different DNA contents 2C 4C 8C 16C_

Wt None 25.3±1.6 2277..88±±33..00 3377..11±±33..00 9.9±4.4

13-1 X Wt ICKl 82.6±2.0 17.4±2.0

13-1 X 245 GUS + ICKl 82.4±9.0 16.2±8.1 1.4±1.3 13-1 X 292 ICKl + CycD2 71.2±14J 25J±11.0 3.2±4.4 13-1 X 297 ICKl + CycD3 52J±11.5 38.1±6.0 9.1±6.4

13-1 X A52 ICK1 + 24.0±3.1 2288±±55JJ 3388..11++11.-66 9.9±4.0 antisense/C ^"i

DNA content was determined using nuclei isolated from the control and FI plants containing corresponding genes as indicated. An average for each peak was obtained from 5-6 individual plants measured and expressed relatively in percentage with a total value for all peaks to be 100%). The data presented are means plus standard deviation.

Expression of transgenes in FI plants

To exemplify alternative aspects of the invention, gene expression was determined in FI plants. High levels of ICKl expression were observed in FI plants from crosses of

[35S-ICK1 X Wt], [35S-ICK1 X 35S-GUS], [35S-ICK1 X 35S-CycD2] and [35S-ICK1 X 35S-CycD3]. ICKl expression was much weaker in FI plants from the cross of [35S-ICK1 X 35S-antisense-ICKll. The gene GUS, CycD2 or CycD3 was also expressed in the perspective FI plants.

EXAMPLE 3

Transgenic Nicotiana tobacum plants were used as hosts for a CDK inhibitor expression construct designated 35S-ICK1 and for two alternative cyclin D expression constructs designated 35S-D2 and 35S-D3 (for expressing respectively of Arabidopsis cyclin D2;l and cyclin D3;l). Phenotypic effects were determined in these transgenic plants expressing one of these genes. These analyses were performed for the puφose of determining the effects of interaction in the plant of a CDK inhibitor and a protein that binds to the inhibitor.

In this example, a number of independent tobacco transformants (Tl) were obtained harboring either of 35S-ICK1, 35S-CycD2 and 35S-CycD3 constructs. Gene expression in these transgenic plants was analysed by northern hybridization. The transgenic 35S-ICK1 tobacco plants shown several major changes. The plants were smaller in comparison to same-aged control plants. They had smaller leaves and shorter internodes. Thus, they appeared stockier that control plants. On the other hand, the CyαD-expressing transgenic plants displayed a ranged of other modifications. These modifications were similar in types between CycD2 and CycD3- expressing plant, but were different from the changes observed in 35S-ICK1 plants. The main changes include faster growth, taller plants and bigger leaves. The leaves curled downward, and stems were curled or twisted. These modifications were observed in the first and subsequent generations of transgenic plants.

In one example, the leaf length for three longest leaves of individual T2 plants (10 weeks) was measured. Five to six plants were per line of plant were used for the measurement. The mean value and standard deviation for each line of plant are given as below. Table 9: Average leaf length (cm) of three longest leaves of T2 plants (10 weeks). For each line, 5-6 plants were used for measurement.

*SD= standard deviation

In another example, the T3 plants of homozygous T2 35S-ICK1 tobacco lines were grown in soil in greenhouse. Transgenic 35S-ICK1 plants were much smaller. For instance, after 45 days, individual plants were removed from pots and the fresh weight of the plant (above ground) was determined. Typically, 6-8 plants were used for each type and the data are summarized below in Table 10.

Table 10

Plant type Tl lines Average ± SD

(g/plant) t Wt 37.55 ± 8.35

35S-GUS 5C2C 38.87 ± 5.87

35S-GUS 5C3B 34.33 ± 5.25

35S-ICK1 6A13D 9.18 ± 3.58

35S-ICK1 6A2E 14.79 ± 6.25

Cellular structure and cell size can also be analysed as described in Example #1. For instance, tobacco plants of Wt, 35S-GUS and 35S-ICK1 lines were grown. Leaf samples were taken from comparable mature leaves of 14- week plants. The samples were fixed, dehydrated and embedded as described. Examination by light microscopy and electron microscopy revealed that cells in 35S-ICK1 plants were larger, as observed in Arabidopsis plants overexpressing ICKl. The results show that cells were larger in 35S-ICK1 tobacco plants than control plants. This effect on cell size in tobacco plants from expression of a plant CDK inhibitor was similar to what observed in Arabidopsis plants. EXAMPLE 4

Transgenic Nicotiana tobacum plants were used as hosts for a CDK inhibitor expression construct designated 35S-ICK1 and for two alternative cyclin D expression constructs designated 35S-D2 and 35S-D3 (for expression respectively of cyclin D2 and cyclin D3). Crosses were made between 35S-ICK1 lines, expressing the CDK inhibitor ICKl, and the cyclin expressing lines. FI plants were analyzed, demonstrating that cyclin D2 (CycD2) and cyclin D3 (CycD3) can reverse the inhibition of cell division and plant growth that is otherwise mediated by ICKl over-expression.

Methods

Two sets of crosses were made between two homozygous 35S-ICK1 tobacco plants (6A13D and 6A2E, female parents) and the rest of plant lines (male parent) as follows

Females Males.

Construct Plant line Construct Plant line

35S-ICK1 6A13D X None Wt2

X 35S-GUS 5C3B

X 35S-CycD2 6B3B

X 35S-CycD2 6B9A

X 35S-CycD3 6C6A

X 35S-CycD3 6C6B

35S-ICK1 6A2E X None Wt2

X 35S-GUS 5C3B

X 35S-CycD2 6B3B

X 35S-CycD2 6B9A

X 35S-CycD3 6C6A

X 35S-CycD3 6C6B

Plants were grown in greenhouse and analysed.

Results

FI plants from crosses between 35S-ICK1 and Wt lines showed consistently the phenotype similar to the 35S-ICK1 parent. For example, they had smaller leaves, and shorter in height. Overall, they were smaller than Wt plants. In contrast, FI plants from crosses between 35S-ICK1 and 35S-CycD lines showed significant restoration of the modified phenotype that is observed the 35S-ICK1 plants. For instance they were taller and had larger leaves than 35S-ICK1 plants. However, some FI plants were not entirely similar to CycD parents but displayed intermediate characteristics between ICKl and CycD parents. Crosses were made between a homozygous 35S-ICK1 line and a heterozygous 35S-CycD3 line. PCR was used to verify the presence of the particular transgenes in the FI plants. Plants that only harbored 35S-ICK1 showed similar phenotype to the 35S-ICK1 parent, while plants harboring 35S-ICK1 and 35S-CycD3 were bigger than the 35S-ICK1 parent. These results show that the expression of CycD3 that interacts with ICKl in regulating CDK activity could at least partially restore some of the modifications due to ICKl expression. In addition, either due to a partial restoration or due to novel effects of over-expression of both ICKl and CycD3, alternative modifications may be achieved.

EXAMPLE 5 Transgenic Arabidopsis plants were used as hosts for a CDK inhibitor expression during early stages of microspore development using construct designated NTM19-ICK1. The promoter NTM19 from tobacco has been shown to activate gene expression at early stages of microspore development (Custers et al., 1997; Oldenhof et al., 1996).

Methods

The NTM19 promoter was cloned from tabacco genomic DNA using PCR with primers sequence-specific to the NTM19 promoter (Oldenhof et al., 1996). The ICKl cDNA was linked to the NTM19 promoter in a vector for plant expression. Similarly, NTM19-GUS, and NTM19-CycD3 (from Arabidopsis) constructs were also prepared. Transformation of Arabidopsis plants was as described. The specificity of NTM19 promoter-directed gene expression was verified using NTM19-GUS plants. GUS activity was detected histochemically using 5-bromo-4-chloro-3-indolyl glucuronidase (X-gluc) as substrate following the method as described (Jefferson et al., 1987).

Results

The histochemical staining of NTM19-GUS expressing plants confirmed that NTM19 activated gene expression during early stage of microspore development (Custers et al., 1997; Oldenhof et al., 1996).

The impact of gene expression on plant growth and fertility was determined for Tl plants carrying the described construct. The results are summarized in Table 11. All these transformants showed similar growth to WT plants. In terms of fertility, reduction of pod development by at least a half was defined as fertility being significantly impaired. Five independent transformants of NTM19-ICK1 construct showed significantly reduced number of pods. Such result indicates that expression of ICKl directed by NTM19 affected either microspore development viability in these plants.

Table 11: Summary of plant growth and seed setting for Tl transformants

EXAMPLE 6 Transgenic Arabidopsis plants were used as hosts for a CDK inhibitor expression construct designated NTM19-ICK1 and for two alternative expression constructs designated NTM19-GUS and NMT19-CycD3 (for expression respectively of the GUS and Arabidopsis cyclin D3). Crosses were made between NTM19-ICK1 lines, expressing the CDK inhibitor ICKl, and the other lines.

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CONCLUSION

Although various embodiments of the invention are disclosed herein, many adaptations and modifications may be made within the scope of the invention in accordance with the common general knowledge of those skilled in this art. Such modifications include the substitution of known equivalents for any aspect of the invention in order to achieve the same result in substantially the same way. For example, additional CDK inhibitors, cyclins and proteins that bind to CDK inhibitors may be disclosed using the screening methods of the invention. The examples herein are illustrative only of various aspects or embodiments of the invention. Numeric ranges are inclusive of the numbers defining the range. The word "comprising" is used as an open-ended term, substantially equivalent to the phrase "including, but not limited to", and the word "comprises" has a corresponding meaning. Citation of references herein shall not be construed as an admission that such references are prior art to the present invention. All publications, including but not limited to patents and patent applications, cited in this specification are incoφorated herein by reference as if each individual publication were specifically and individually indicated to be incoφorated by reference herein and as though fully set forth herein. The invention includes all embodiments and variations substantially as hereinbefore described and with reference to the examples and drawings.

Claims

What is claimed is:

1. A method of modifying development of a plant comprising introducing into a plant cell a nucleic acid encoding a protein that binds to or interacts with a cyclin-dependent kinase inhibitor polypeptide, wherein the plant cell or an ancestor of the plant cell has been transformed with a nucleic acid encoding the cyclin-dependent kinase inhibitor polypeptide; and, growing the plant cell, or a progeny of the plant cell, under conditions wherein the protein that binds to the cyclin-dependent kinase inhibitor polypeptide and the cyclin-dependent kinase inhibitor are expressed in the plant cell or in the progeny of the plant cell during development of the plant, so that an effect of the cyclin-dependent kinase inhibitor polypeptide on development of the plant is at least partly reversed by the protein that binds to the cyclin-dependent kinase inhibitor polypeptide.

2. The method of claim 1, wherein the effect of the cyclin-dependent kinase inhibitor polypeptide on development of the plant is to inhibit development of a differentiated tissue in the plant, and the inhibition of development of the differentiated tissue is at least partly reversed by the protein that binds to the cyclin-dependent kinase inhibitor polypeptide.

3. The method of claim 2, wherein the cyclin-dependent kinase inhibitor polypeptide is expressed in the proliferative tissue of the plant.

4. The method of claim 2 or 3, wherein the protein that binds to the cyclin-dependent kinase inhibitor polypeptide is expressed in the proliferative tissue of the plant

5. The method of any one of claims 1 through 4 wherein the cyclin-dependent kinase inhibitor polypeptide is a plant cyclin-dependent kinase inhibitor polypeptide.

6. The method of any one of claims 1 through 5, wherein the nucleic acid encoding the cyclin-dependent kinase inhibitor is at least 80% identical, when optimally aligned, to a sequence encoding a cyclin-dependent kinase inhibitor selected from the group consisting of ICKl, ICK2, ICN2 (ICK4), ICN6 (ICK5), ICN7 (ICK6) and ICN8 (ICK7).

7. The method of any one of claims 1 through 5, wherein the nucleic acid encoding the cyclin-dependent kinase inhibitor is at least 80% identical, when optimally aligned, to a coding sequence selected from a group consisting of the coding sequences of ICKl, ICK2, ICN2, ICN6, ICN7 and ICN8.

8. The method of any one of claims 1 through 5, wherein the cyclin-dependent kinase inhibitor polypeptide comprises a peptide sequence that is at least 80% identical, when optimally aligned, to a peptide sequence selected from the group consisting of the peptide sequences of ICKl, ICK2, ICN2, ICN6, ICN7 and ICN8.

9. The method of any one of claims 1 through 5, wherein the cyclin-dependent kinase inhibitor polypeptide is selected from a group consisting of ICKl, ICK2, ICN2, ICN6, ICN7 and ICN8.

10. The method of any one of claims 1 through 9, wherein the protein that binds to the CDK inhibitor is a cyclin or a polypeptide fragment of the cyclin.

11. The method of claim 10, wherein the cyclin is a D-type cyclin, cyclin D2 or cyclin D3.

12. The method of claim 10 or 11, wherein the polypeptide fragment of the cyclin comprises at least 10 amino acids.

13. The method of any one of claims 1 through 9, wherein the protein that binds to the CDK inhibitor is a cyclin-dependent kinase (CDK) or a polypeptide fragment of the CDK.

14. The method of claim 13, wherein the CDK is Arabidopsis Cdc2a.

15. The method of claim 13 or 14, wherein the polypeptide fragment of the CDK comprises at least 10 amino acids

16. The method of any one of claims 1 through 15, wherein the plant is a dicotyledon or monocotyledon plant

17. The method of any one of claims 1 through 15, wherein the plant is a member of the Cruciferae family.

18. The method of any one of claims 1 through 15, wherein the plant is a member of the Brassica genus.

19. The method of any one of claims 1 through 15, wherein the plant is a member of the tobacco family.

20. The method of any one of claims 1 through 19, wherein the nucleic acid encoding the cyclin-dependent kinase inhibitor polypeptide is operably linked to a constitutive promoter.

21. The method of any one of claims 1 through 19, wherein the nucleic acid encoding the protein that binds to the cyclin-dependent kinase inhibitor polypeptide is operably linked to a constitutive promoter.

22. The method of any one of claims 1 through 19, wherein the nucleic acid encoding the cyclin-dependent kinase inhibitor polypeptide is operably linked to a tissue-specific promoter.

23. The method of any one of claims 1 through 19, wherein the nucleic acid encoding the protein that binds to the cyclin-dependent kinase inhibitor polypeptide is operably linked to a tissue-specific promoter.

24. The method of claim 22 or 23, wherein the tissue-specific promoter is an AP 3 promoter.

25. The method of claim 22 or 23, wherein the tissue-specific promoter is anNTM19 promoter.

26. The method of any one of claims 2 through 25 wherein the differentiated tissue in the plant is a male reproductive tissue.

27. The plant produced by the method of any one of claims 1 through 26.

28. A method of modifying development of a plant comprising introducing into a plant cell a nucleic acid encoding a cyclin polypeptide, wherein the plant cell or an ancestor of the plant cell has been transformed with a nucleic acid encoding a cyclin-dependent kinase inhibitor polypeptide; and, growing the plant cell, or a progeny of the plant cell, under conditions wherein the cyclin polypeptide and the cyclin-dependent kinase inhibitor are expressed in the plant cell or in the progeny of the plant cell during development of the plant, so that an effect of the cyclin-dependent kinase inhibitor polypeptide on development of the plant is at least partly reversed by the cyclin polypeptide.

29. The method of claim 28, wherein the cyclin polypeptide is a full length cyclin or a polypeptide fragment of the full length cyclin.

30. The method of claim 29, wherein the full length cyclin is selected from the group consisting of cyclin D2 and cyclin D3.

31. The method of claim 29 or 30, wherein the polypeptide fragment of the cyclin comprises at least 10 amino acids.

32. A plant having a recombinant genome comprising:

a) a heterologous nucleic acid encoding a cyclin-dependent kinase inhibitor; and,

b) a heterologous nucleic acid encoding a protein that binds to the cyclin-dependent kinase inhibitor.

33. The plant of claim 32, wherein the protein that binds to the cyclin-dependent kinase inhibitor is a cyclin or a polypeptide fragment of the cyclin.

34. A plant having a recombinant genome comprising:

b) a heterologous nucleic acid encoding a cyclin or a polypeptide fragment of the cyclin.

35. The plant of claim 33 or 34, wherein the cyclin is selected from the group consisting of cyclin D2 and cyclin D3.

36. A plant tissue derived from the plant of any one of claims 27 or 32 through 35.

37. The plant tissue of claim 36 wherein the tissue is a seed.

38. A method of growing a plant, wherein the plant comprises a heterologous nucleic acid encoding a cyclin-dependent kinase inhibitor polypeptide and a heterologous nucleic acid encoding a protein that binds to the cyclin-dependent kinase inhibitor polypeptide, the method comprising growing the plant under conditions so that an effect of the cyclin-dependent kinase inhibitor polypeptide on development of the plant is at least partly reversed by the protein that binds to the cyclin-dependent kinase inhibitor polypeptide.

39. A method of growing a plant, wherein the plant comprises a heterologous nucleic acid encoding a cyclin-dependent kinase inhibitor polypeptide and a heterologous nucleic acid encoding a cyclin polypeptide, the method comprising growing the plant under conditions so that an effect of the cyclin-dependent kinase inhibitor polypeptide on development of the plant is at least partly reversed by the cyclin polypeptide.

40. A method of plant breeding comprising:

a) providing a first parent plant having a heterologous nucleic acid sequence encoding a cyclin-dependent kinase inhibitor polypeptide;

b) providing a second parent plant having a heterologous nucleic acid sequence encoding a protein that binds to the cyclin-dependent kinase inhibitor polypeptide;

c) crossing the first and second parent plants to obtain a hybrid offspring plant having a copy of the heterologous sequence encoding the cyclin-dependent kinase inhibitor and a copy of the heterologous sequence encoding the protein that binds to the cyclin- dependent kinase inhibitor.

41. A method of plant breeding comprising:

b) providing a second parent plant having a heterologous nucleic acid sequence encoding a cyclin polypeptide;

c) crossing the first and second parent plants to obtain a hybrid offspring plant having a copy of the heterologous sequence encoding the cyclin-dependent kinase inhibitor and a copy of the heterologous sequence encoding the cyclin polypeptide.

42. A method of plant breeding comprising:

b) providing a second parent plant having a heterologous nucleic acid sequence encoding a cyclin-dependent kinase polypeptide;

c) crossing the first and second parent plants to obtain a hybrid offspring plant having a copy of the heterologous sequence encoding the cyclin-dependent kinase inhibitor and a copy of the heterologous sequence encoding the cyclin-dependent kinase polypeptide.

43. A method of modifying development of a plant comprising introducing into a plant cell a nucleic acid encoding a cyclin-dependent kinase, wherein the plant cell or an ancestor of the plant cell has been transformed with a nucleic acid encoding a cyclin-dependent kinase inhibitor polypeptide; and, growing the plant cell, or a progeny of the plant cell, under conditions wherein the cyclin-dependent kinase and the cyclin-dependent kinase inhibitor are expressed in the plant cell or in the progeny of the plant cell during development of the plant, so that an effect of the cyclin-dependent kinase inhibitor polypeptide on development of the plant is at least partly reversed by the cyclin- dependent kinase.

44. A method of modifying development of a plant comprising introducing into a plant cell a nucleic acid encoding a recombinant polypeptide, wherein the plant cell or an ancestor of the plant cell has been transformed with a nucleic acid encoding a cyclin-dependent kinase inhibitor polypeptide; and, growing the plant cell, or a progeny of the plant cell, under conditions wherein the recombinant polypeptide and the cyclin-dependent kinase inhibitor are expressed in the plant cell or in the progeny of the plant cell during development of the plant, so that an effect of the cyclin-dependent kinase inhibitor polypeptide on development of the plant is at least partly reversed by the recombinant polypeptide.

45. The method of claim 44 wherein the polypeptide is an antibody such as single-chain antibody.

46. The method of claim 44 wherein the polypeptide is a peptide selected from a peptide library.

47. A method of modifying development of a plant comprising introducing into a plant cell a nucleic acid encoding a polypeptide that modulates the degradation of a cyclin- dependent kinase inhibitor polypeptide, wherein the plant cell or an ancestor of the plant cell has been transformed with a nucleic acid encoding the cyclin-dependent kinase inhibitor polypeptide; and, growing the plant cell, or a progeny of the plant cell, under conditions wherein the polypeptide that modulates the degradation of the cyclin- dependent kinase inhibitor and the cyclin-dependent kinase inhibitor polypeptide are expressed in the plant cell or in the progeny of the plant cell during development of the plant, so that an effect of the cyclin-dependent kinase inhibitor polypeptide on development of the plant is at least partly reversed by the polypeptide that modulates the degradation of the cyclin-dependent kinase inhibitor.