CA2433048A1 - Modulation of plant cyclin-dependent kinase inhibitor activity - Google Patents

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°d°2, 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 (Lorincz 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;I) 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 (Graft 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 G1 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 1 S 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 CaMV 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.
Morphogenesis, histogenesis and developmental timing were unaffected (Hemerly et al., 1995). As mentioned above, ectopic expression of an Arabidopsis mitotic cyclin gene, CycBl (Arath;CycBl; l, formerly CyclAt), under the control of the Cdc2a 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 ICKI 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 RNase (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-type 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, morphogenesis, 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 ICKS), 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 NTMl9, an AP3 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 F1 plant is not male-sterile.
In one aspect, the invention also provides methods of modifying plant architecture and morphology for desired size and shape according to alternative embodiments of this invention. Plant architecture, size and morphology 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 ICKI (SEQ ID NO: 1 ). Open bars represent exons and filled bars represent introns; (B) shows features of the ICKI cDNA
sequence (SEQ ID NO: 3) and ICK1 deduced amino acid sequence (SEQ ID NO: 2). ' Figure 2 shows a CLUSTAL W alignment of an ICKI cDNA sequence (SEQ ID
NO: 3) with alternative cDNAs: ICKl b (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 of ICN6 (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 8A 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 9A 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.
-g_ 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 lOB 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: ICKI
(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: 1 S).
Figure 15B shows an alternative alignment of deduced amino acid sequences of:
ICK1 (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 ICK1.

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

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

SEQ ID NO .: 5 shows the nucleic acid sequence of the ICKIc cDNA.

SEQ ID .: 6 shows the nucleic acid sequence of the ICK2 NO 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 ICN6 cDNA.

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 .: 11 shows the deduced amino acid sequence of ICN2 NO 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.: 1 S shows the deduced amino acid sequence of ICDK.
SEQ ID NO.: 16 shows the cDNA sequence of ICNB.
SEQ ID NO.: 17 shows the deduced amino acid sequence of ICNB.
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 ICN6 cDNA
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, morphogenesis, 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 ICK1. 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 43:729-36), 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 S 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-1,2,4-triazole.
The selected colonies were assayed for (3-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 polymerise 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 purposes, 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-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.5. 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%. 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 NaHPOa, 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 NaHPOa, 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 Technigues 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 ICK1, 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'artificiaf origin.
In an alternative aspect of the invention, reversal of an effect of a CDK
inhibitor, such as ICK1, 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 incorporated 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.l6) was identified from Chenopodium rubrum, which encodes a protein sharing sequence similarity with ICK1 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 ICK1 (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 ICNB.
As a further example, ICKI cDNA (SEQ ID NO: l; Wang et al., 1997) was amplified by PCR and transcriptionally linked with the 355 promoter in a binary vector pBIl21 (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; l, GenBank accession number X83371) and Cdc2a (Cdc2aAt or Arath;CDKA; l, 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 promoter. As another example, the Arabidopsis AP3 promoter was cloned by PCR
from Arabidopsis thaliaha "Columbia" genomic DNA, on the basis of the published sequence (Irish and Yamamoto, 1995; GenBank U30729). The promoter was linked transcriptionally with ICKI cDNA. As yet another example, the tobacco NTM19 promoter was linked transcriptionally with ICKI cDNA. The NTMl9 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 Enrymol. 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 caned out by infiltration. For example, seeds (T 1 generation) collected from infiltrated Arabidopsis plants may be surface-sterilized and placed onto MS
medium containing 50 pg/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 355-ICKl, 35S-CycD2, 355-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 A. thaliana plants with overnight culture ofA. 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 traps, gentamycin and kanamycin selection markers as described in Koncz and Schell (1986). Following infiltration, plants were grown to maturity and seeds (T1) 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 T1 plants.
Wherever the T1 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 B. 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 SO mg/L
kanamycin. Regenerated green shoots may first be transferred to a medium to allow 2:603 (1990); and in U.S. Patent elongation and then to a rooting medium all containing SO 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 (T1) 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-ICKl 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 morphology 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 ICKI expression during plant development, various tissues may be taken from Arabidopsis plants (Wang et al., 1995).
Total RNA was isolated using TRIzoI 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 32P-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 O.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 mls per gram tissue of ice cold extraction buffer consisting of 25 mM Tris pH

8.0, 100 mM
NaCI, 10 mM DTT, S mM NaF, 1 mM Na3VOa, 1 mM (3-glycerophosphate, 2.5 mM EDTA, 400 p,g/ml AEBSF [4-(2-aminoethyl)-enzensulfonyl fluoride], 1 pg/ml leupeptin and 1 pg/ml pepstatin. The homogenate was centrifuged at 12,OOOg at 4°C for 30 min. The supernatants may be used to purify Cdc2-like protein kinases using pl3s°°1-conjugated agarose beads (Oncogene Sciences). The required amount of supernatant (150 p,g 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 NaCI, 0.1 %
NP-40, 2.5 mM EDTA, 1 mM DTT and inhibitor cocktail of (in final concentrations) 10 ~,g/ml apotinin, 10 ~g/ml antipain, 10 pg/ml soybean trypsin inhibitor, 10 mM (3-glycerophosphate, 1 mM NaF and 0.2 mM Na3VOa. Beads may then be washed twice in the kinase assay buffer (50 mM Tris pH 7.4, 10 mM MgCI, 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 pg/pl histone H1 (Sigma), 25 ~M ATP and 0.05 ~Ci/ul 32P-y-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-ICKl 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 ~n 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-ICKI
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-ICKI 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) (http:/rsb.info.nih.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 Image).
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, 35S-Met labeled ICK1 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 ~,1 beads, plus 5 p1 35S-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 ~,1 SDS-containing denaturing buffer at 100°C for 5 min. The bound 35S-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 ICK1 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 S 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 ~3-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 S-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-~ X-gluc, 100 mM PO4 (pH 7.0), 0.5 mM K3 [Fe(CN)6], 0.5 mM
K4[Fe(CN)6], 10 mM NazEDTA 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 ICK1. Fifty independent transgenic 35S-ICKl plants were analysed. Majority of transgenic lines expressing ICKI displayed modifications in plant growth and development. These plants were smaller in size. Most organs were smaller. The morphology 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 morphology in transgenic 35S-ICKl 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-ICKl lines were crossed with 35S-CycD lines. The F 1 plants from such crosses displayed characteristics similar to wild type plants rather than to the parental 35S-ICKI plants. For instance, the F 1 plants were much bigger than 35S-ICKI
parental plants. In 35S-ICKI parental plants, there was strong serration in leaves while F 1 plants displayed leaf morphology as the wild type plants. The F1 plants also showed normal flower morphology rather than the modified flower morphology observed in the 35S-ICKI
plants.
These results suggest that the expression of CycD3 or CycD2 had at least partially restore the modifications resulted from the expression of ICKI 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-ICKI
lines were crossed with 35S-Cdc2a lines. The F 1 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 E1, 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.
1 S In accordance with alternative embodiments of the invention, a wide variety of plant species may be modified. As an example, transgenic Nicotiana tobaccum plants (T1) were obtained with 35S-ICKI construct. Twenty one independent transformants were analysed.
The insert number was analysed by Southern hybridization. ICKI expression was anlaysed by northern hybridization. Results show that at least twelve plants had significant levels of ICKI 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-ICKI T 1 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 T1 lines were 23.2, 23.8 and 23.7 cm respectively. In comparison, the average leaf length for four 35S-ICKI T1 lines was 17.5, 19.5, 14.6 and 12.1 cm respectively. T2 plants of the T1 transgenic lines were also shorter on average than the control plants when measured at the same time.
Plant morphology of the 35S-ICKl 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 constructs.
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-ICKI lines. F1 seeds from these crosses were planted. At least partial restoration of plant development modifications as described above due to ICKI expression was observed.
As yet another example, transgenic Brassica napus plants expressing ICKI were obtained. The presence of heterologous transgene was verified by PCR and southern hybridization. Modifications of plant development were observed in the original (T1) 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 (T1) 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-ICKI lines and seeds of control Arabidopsis plants were sterilized and placed onto the selective medium containing SOmg/L kanamycin. Four duplicate plates were used for each type of plant. The number of resistant/susceptible seedlings for four independent Arabidopsis 35S-ICKI 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-ICKI 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 ICKI was analyzed in independent transgenic Arabidopsis 35S-ICKI plants. The ICKl expression level increased significantly in transgenic 35S-ICKI
Arabidopsis plants as shown by several independent analyses. Increased ICKI
expression was observed in original T1 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 UBQll (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 pl3s°°1-associated Cdc2-like histone Hl 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 ICKI
expression, the Cdc2-like kinase activity decreased significantly in comparison to control plants. This decrease was observed in independent transgenic 35S-ICKI 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 ICKI expression in these 35S-ICKl plants.
In vitro kinase assays may be used to demonstrate that a recombinant putative CDK
inhibitor, such as ICK1 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 ICK1 is effective in vitro in inhibiting the histone H1 kinase activity of pl3s°~1-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, ICKI may be used as a bait to identify cyclin homologs or proteins that interact with ICK1. 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 ICKI , 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 ~3-galactosidase activity were observed when amino acid regions 3-72, 109-153 and 176-191 were deleted. The reporter activity for interactions of ICK1 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 ICK1 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 ICK1 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 (3-galactosidase activity for the interaction of ICK1 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 ICK1 may be compared with plants expressing an ICKI 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 ICK1 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 ICK1 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-ICKI
and control plants by scanning electron microscopy and light microscopy showed that the cells of 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 of 35S-ICKI 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-ICKl 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-ICKI lines were 1.7-2.7 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 morphology 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 NTMI9-ICKI, NTMI9-CycD3 and NTMI9-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 NTMl9-GUS
plants. These plants including NTMl9-ICKI plants developed normally before fertilization and silique (pod) development. However, pod development was affected in a large proportion of NTM19-ICKI plants. These results are consistent with the expression pattern of the NTMl9 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) NTMl9-ICKI transformants showed significantly reduced pod development (defined as "less than half pods are developed"). In contrast, only one ( 1 ) of fourteen ( 14) NTMl9-G US
transformants and none (0) of thirty (30) NTM19-CycD3 transformants had a similar phenotype. These results demonstrate that tissue-specific expression of ICKI
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 ICKI. 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-ICKI
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 morphology and development with four transformants displaying significant alterations. Plants showing a strong phenotype in modification of petal morphology 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 ICKI in transgenic Bgpl -ICKI B.
napus plants was analysed by northern hybridization using 3zP-labeled ICKI
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 pg of RNA was loaded and separated by electrophoresis. RNA transfer and hybridization were performed as described. There were no significant levels of ICKI 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 ICKI expression was observed in anthers of the transgenic plant.
As another example, ICKl expression was analysed in plants transformed by AP3-ICKI
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 ICKI 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 ICK1, 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.l6) was identified from Chenopodium rubrum (Fountain et al., 1999), as it shares some similar properties as ICK1 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) 1 S independent transgenic 35S-ICDK plants, twelve ( 12) showed serrated leaves and twenty two (22) showed modified flowers, which were observed in 35S-ICKI 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-ICKI plants. Results from the 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 100 24.3 22.4 24.5 27.0 21.4 23.4 ICKl 100 20.3 19.2 21.9 15.8 20.3 ICK2 100 33.7 27.7 19.3 21.9 ICN2 100 30.7 21.2 23.5 ICN6 100 36.5 28.5 ICN7 100 21.9 ICN8 Other plant CDK inhibitors and CDK inhibitor genes sharing functional and sequence similarity with ICK1 may be identified using an approach similar to the approach used to isolate ICKI , 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 ICNB. 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 ICK1: 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 ICK1 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 ICK1, 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 ICK1 in a GAL4-activation domain vector.
Using this approach, for example, the interactions of ICK1 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, ICK1 protein may interact with Cdc2a but not with Cdc2b. Similarly, ICK1 may interact with D-type cyclins, CycDl, CycD2 and CycD3, while not interacting with A/B-class mitotic cyclins, CycA2, CycB 1 and CycB2 (Table 2). The yeast two-hybrid assay results also indicate that ICK1 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 Gene Group Gene in DB-Vector Interaction Examined with ICK1 Filter assa ~1~
Quantification~z~

Control vector alone - 0 CDK Cdc2a Cdc2aAt +++ 2.65 Cdc2b Cdc2bAt - 0 cyclin C cD 1 (Arath;C +++ 3 .13 cD 1;1 C cD2 Arath;C cD2;1++++ 14.80 C cD3 Arath;C cD3;1+++++ 22.70 C cA2 Arath;C cA2;2- 0.03 C cB 1 Arath;C - 0.06 cB 1;1 C cB2 (Arath;C - 0.05 cB2;2 PCNA PCNAAt - 0 MAP kinase ATMAP2 - 0 The interactions of additional plant CDK inhibitors can also be determined using similar methods. For instance, the interactions of ICK1, 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, CKS 1 At (De Veylder et al. 1997), one of the controls used, was able to interact with Arabidopsis Cdc2a and Cdc2b. ICK1, ICK2, ICN7 and ICN8 interacted with Cdc2a but not with Cdc2b, and interacted with the three D-type cyclins (D1, 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 (3-galactosidase activity greatly. These results indicate that one may segregate the ICK1-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 (ICK1, ICK2, ICN7 and ICNB) 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.

None CKS1 ICK1 ICK2 ICN7 ICN8 ICN2 ICN6 ICDK

None - - - - - - - - -Cdc2a - ++ ++ + + + - - -Cdc2 - ++ - - - - - - -b C cDl - - ++ +++ +++ +++ +++ +++ +++

C cD2 - - +++ +++ ++ +++ + ++ ++

C cD3 - +/- ++ +++ +++ +++ ++ +++ +++

PCNA - _ _ _ _ _ _ _ _ ~1~ 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 ~3-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.
~2~ Faint color was visible after overnight incubation.

Transgenic Arabidopsis plants were used as hosts for a CDK inhibitor expression construct designated 35S-ICKI 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 ICKI 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 (T1) were harvested and selected on'/2 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 ICK1 expression Among fifty independent ICKl -35S transformants examined, the majority displayed significant growth and morphological changes. One striking change was the much smaller size of transformants compared to control plants. The smaller size of 35S-ICKI
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 T1 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 ICKI 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-ICKI lines had lower fresh weight than wild type plants. For quantification of growth inhibition under typical physiological conditions, 35S-ICKl and control plants were grown in soil. The fresh weight (at 3-weeks) was significantly lower in a number of independent 35S-ICKI lines in comparison to plants without 35S-ICKI or plants harboring construct (Table 4). Transgenic 35S-ICKI plants were smaller than control plants. In some lines, 35S-ICKI plants were less than 1/10 of the control plants that did not carry 35S-ICKI
(Table 4). The finding that the smaller size (and lower fresh weight) of 35S-ICKl 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 ICKI over-expression.
Table 4 shows growth and development of transgenic 35S-ICKl Arabidopsis plants.
35S-ICKI 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 Growth Flowering Leaf in time number pots 21-da shoots Plant typeNo. MeanSD' No. Days No. No. of o mg/ lant o MeanSD1 o leaves plants plants plantsMeanSD1 Controls Wt 44 392.578.993 25.10.4 22 15.51.2 35S-GUS 21 362.662.647 25.30.7 10 15.91.0 35S-ICKl lines 5-3 52 35.526.2**83 20.92.5* 17 11.81.1**

12-3 39 57.133.4**35 22.12.3* 13 11.52.0**

13-Sb SO 32.732.0**63 19.42.3* 25 9.81.2**

15-2 43 95.236.4**56 22.31.5* 25 12.71.5**

1 SD = standard deviation. * * indicates significance (t-test) from the Wt control plants at P<0.001 level and * indicates significance at P<0.05 level.
Altered aspects of plant morphology The 35S-ICKI plants showed profound changes in morphology 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 ICKI
resulted in much more prominent serration of the leaves and this characteristic was observed in almost all leaves in 35S-ICKI plants with strong phenotype of growth inhibition. The smaller leaves and shorter leaf petioles gave 35S-ICKl plants a more compact appearance. Root growth of 35S-ICKI 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-ICKI plants, flowers stayed closer and usually were at the same level or below the inflorescence apex. On the inflorescence of 35S-ICKI 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 morphology were also evident in individual flowers. The flowers of 35S-ICKl 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-ICKI
plants were straight upward. Petals of 35S-ICKI plants were also narrower with serration along the top edge. These changes were so profound that transgenic 35S-ICKI 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-ICKI plants flowered earlier and had significantly fewer leaves at flowering time than control plants (Table 4). Precocious flowering of 35S-ICKI
plants was similarly observed when grown in Petri plates (data not shown). (2) Transgenic 35S-ICKl 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 ICK1 resulted in reduction of CDK activity in plant cells The ICKI expression level increased significantly in transgenic 35S-ICKI
Arabidopsis plants as shown from several independent northern analyses.
Increased ICKI
expression was observed in original T1 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 pl3s°~1-associated Cdc2-like histone H1 kinase activity was analysed with the same source tissues that were used in gene expression analyses. Results show that, coinciding with increased ICKI expression, the Cdc2-like kinase activity decreased significantly in comparison to control plants. This decrease was observed in independent 35S-ICKI 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 ICKI expression in these 35S-ICKI plants.

Cell number and cell size were affected The structure and cell size of 35S-ICKI and control plants were examined by scanning electron microscopy and light microscopy. It was consistently observed that the cells of 35S-ICKl 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-ICKl plants were clearly larger than control plants. To better quantify the cell size difference, fully expanded leaves (the 5~' to 8a' leaves) of 30-day plants grown in soil were used for determining leaf and epidermal cell size. The average leaf size of the 35S-ICKI
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-ICKI lines were 1.7-2.7 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-ICKI
transformants as comparisons for determining the effects of other plant CDK inhibitor genes.
Similar phenotypic changes in leaf and flower morphology were observed in plants expressing 35S-ICN2 or 35S-ICDK as observed in plants expressing 35S-ICKI , but not in plants transformed with 35S-GUS. Leaf serration and modifications of flower morphology 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 morphology was significantly higher in transformants with 35S-ICKl (70% of 45 independent transgenic lines) and 35S-ICDK (S 1 % 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-ICKl, 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 T1 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 T1 generation. Most 35S-ICN2, and 35S-ICDK lines showed segregation of the mutant phenotype (modified leaf and/or flower morphology) 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 T1 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-ICKI transformants described above, the major changes in morphology 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 expressingplant 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-ICKI , 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-ICKl 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-ICKI
plants.
Table 5. Ploidy levels in wild type and transgenic plants.
Plant % of a area value for different DNA
contents Wt 24.2 31.1 32.6 11.6 0.4 35S-ICKl 71.9 28.1 0 0 0 35S-ICN2 44.9 40.3 14.7 0 0 35S-ICDK 51.1 41.9 7.0 0 ~ 0 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%.

Transgenic Arabidopsis plants were used as hosts for a CDK inhibitor expression construct designated 35S-ICKI , for two alternative cyclin D expression constructs designated 35S-CycD2 and 35S-CycD3 (for expressing respectively of Arabidopsis cyclin D2;1 and cyclin D3;1 ), and for a CDK expression construct designated 35S-Cdc2a (for expressing Arabidopsis Cdc2a). Crosses were made between 35S-ICKl lines, expressing the CDK inhibitor ICK1, and the cyclin expressing lines. F1 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-ICKl (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-ICKI transgenic line (with a single copy).
Expression of ICKl resulted in inhibition of cell division, inhibition of plant growth and distinct changes in plant morphology Male Wt Wild type Arabidopsis 292-11 T1 heterozygous line expressing CycD2 292-15 T1 heterozygous line expressing CycD2 294-2* * T 1 heterozygous line expressing CycD3 297-18 T1 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. F1 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 SOmg/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 F1 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 F1 Arabidopsis plants from crosses between 35S-ICKI
female parent line and CvcD male parent linesa Pheno Avera a a weight m lant) Line/Cross Type Type Type Type Mean SD Mean SD

200-13-1 137 3.47 0.34 200-13-1 X Wt 92 3.54 0.50 200-13-1 X 294-2/C 77 70 3.45 0.28 8.79 1.83 cD3 200-13-1 X 297-18/CycD348 69 3.50 0.29 7.97 1.62 297-18 123 12.79 1.73 Wt 10 17.24 200-13-1 X 292-11/C128 6.43 0.67 cD2 292-11 90 10.07 3.59 200-13-1 X 292-15/C131 4.71 0.31 cD2 292-15 119 5.87 1.45 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.
1 S 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 morphology 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 morphology 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-ICKIlWt had the typical phenotype of ICKl overexpression while plants with genotype 35S-ICKIl35S-CycD3 showed much reduced growth inhibition and reversal of altered leaf shape, demonstrating that the phenotypic effect of ICKI expression in these plants is significantly decreased by the co-expression of a cyclin (CycD3).
The morphology 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 F 1 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 morphology mediated by CycD2 compared to the morphological reversion demonstrated in the CycD2 expressing F1 plants. These results show that alternative proteins (CycD2 and CycD3) that bind to a CDK inhibitor (ICK1) 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-ICKI and 35S-Cdc2a lines. The F 1 plants were analysed as above. The data indicate that the modified plant phenotype due to ICKI expression is at least partially restored by the expression of Cdc2a.
As comparisons, the 35S-ICKI line was also crossed with a transgenic line transformed with 35S-GUS and a transgenic line transformed with 35S-antisense-ICKI. The Fl plants from the cross [35S-ICKl X 35S-GUS] displayed the typical phenotype of ICKl overexpression i.e. smaller plants, serrated leaves and modified flowers. In contrast, the Fl plants from the cross [35S-ICKI X 35S-antisense-ICKI ] 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 Fl plants Transgenic Arabidopsis plants overexpressing ICKI that displayed a much smaller plant size had larger cells than Wt plants (Example # 1 ). The cell size was thus examined in Fl plants from these crosses using scanning electron microscopy (SEM) and light microscopy. There was no apparent difference in cell size between the Wt and plants. A normal range of variation in leaf cell size was observed in both plant types.
The F1 plants from the crosses [35S-ICKI X Wt] and [35S-ICKI X 35S-GUSJ had larger cells than control plants, as observed for ICKI-overexpressing plants.
The F1 plants from the cross [35S-ICKl X 35S-antisense-ICKl ] had leaf cells similar in size to control plants. The F1 plants from the cross [35S-ICKl X 35S-CycD3] had smaller cells than the control plants, despite the fact that these F 1 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 F 1 plants had modified the cell size in comparison to Wt plants and plants expressing ICKI
alone.
Modulation of ploidy level in Fl plants Overexpression of a plant CDK inhibitor such as ICK1 inhibited endoreduplication and thus decreased the ploidy level in Arabidopsis leaves, which normally have cells of mixed polyploidy. Therefore the ploidy level in the F 1 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 F1 plants from the cross [35S-antisense-ICKI ] 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 F1 plants from the crosses [35S-ICKl X Wt] and [35S-ICKI X 35S-GUS] showed a significant reduction in ploidy level with essentially two peaks and mostly at 2C level. The F1 plants from the crosses [35S-ICKI X 35S-CycD2] and [35S-ICKI X 35S-CycD3] had a ploidy profile between the [35S-ICKl X Wt] type and the [35S-ICKI X 35S-antisense-ICKI ]
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 ICKI overexpression was partially reversed by the expression of Arabidopsis CycD3 or CycD2.
Table 8. Ploidy levels in Fl plants Parent/F 1 Transgene % of area value for different DNA contents Wt None 25.31.6 27.83.0 37.13.0 9.94.4 13-1 X ICKI 82.62.0 17.42.0 Wt 13-1 X GUS + ICKI 82.49.0 16.28.1 1.41.3 13-1 X ICKI + CycD271.214.7 25.711.0 3.24.4 13-1 X ICKI + CycD352.711.5 38.16.0 9.16.4 13-1 X ICKI + 24.03.1 285.7 38.11.6 9.94.0 antisenseICKl DNA content was determined using nuclei isolated from the control and F1 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 Fl plants. High levels of ICKl expression were observed in F1 plants from crosses of [35S-ICKI X Wt], [35S-ICKI X 35S-GUS], [35S-ICKI X 35S-CycD2] and [35S-ICKI X
35S-CycD3]. ICKl expression was much weaker in F1 plants from the cross of [35S-ICKI

X 35S-antisense-ICKl ]. The gene GUS, CycD2 or CycD3 was also expressed in the perspective F 1 plants.

Transgenic Nicotiana tobacum plants were used as hosts for a CDK inhibitor expression construct designated 35S-ICKI and for two alternative cyclin D
expression constructs designated 35S-D2 and 35S-D3 (for expressing respectively of Arabidopsis cyclin D2;1 and cyclin D3;1). Phenotypic effects were determined in these transgenic plants expressing one of these genes. These analyses were performed for the purpose 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 (T 1 ) were obtained harboring either of 35S-ICKl , 35S-CycD2 and 35S-CycD3 constructs. Gene expression in these transgenic plants was analysed by northern hybridization. The transgenic 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 CycD-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-ICKI 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.
Plant type T1 line Mean SD*
cm cm Wt WT 23.2 3.6 35S-GUS SC2 23.8 3.8 35S-GUS SC3 23.7 3.0 35S-ICK1 SAS 17.5 3.0 3 5 S-ICK 6A2 19.5 1.8 35S-ICK1 6A10 14.6 3.9 3 5 S-ICK 6A 13 12.1 2.1 l * SD= standard deviation In another example, the T3 plants of homozygous T2 35S-ICKI tobacco lines were grown in soil in greenhouse. Transgenic 35S-ICKI 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 T1 lines Average SD

(g/plant) Wt Wt 37.55 8.35 35S-GUS SC2C 38.87 5.87 35S-GUS SC3B 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-ICKl 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-ICKI plants were larger, as observed in Arabidopsis plants overexpressing ICKl. The results show that cells were larger in 35S-ICKl 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.

Transgenic Nicotiana tobacum plants were used as hosts for a CDK inhibitor expression construct designated 35S-ICKl 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 ICK1, and the cyclin expressing lines. F1 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 ICK1 over-expression.
Methods Two sets of crosses were made between two homozygous 35S-ICKI 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-ICKI 6A13D X None Wt2 X 35S-CycD2 6B3B

X 35S-CycD2 6B9A

X 35S-CycD3 6C6A

X 35S-CycD3 6C6B

35S-ICKI 6A2E X None Wt2 X 35S-CycD2 6B3B

X 35S-CycD2 6B9A

X 35S-CycD3 6C6A

X 35S-CycD3 6C6B

Plants were grown in greenhouse and analysed.
Results F1 plants from crosses between 35S-ICKI and Wt lines showed consistently the phenotype similar to the 35S-ICKI parent. For example, they had smaller leaves, and shorter in height. Overall, they were smaller than Wt plants. In contrast, F1 plants from crosses between 35S-ICKI and 35S-CycD lines showed significant restoration of the modified phenotype that is observed the 35S-ICKI plants. For instance they were taller and had larger leaves than 35S-ICKI plants. However, some F1 plants were not entirely similar to CycD
parents but displayed intermediate characteristics between ICK1 and CycD
parents. Crosses were made between a homozygous 35S-ICKl line and a heterozygous 35S-CycD3 line. PCR
was used to verify the presence of the particular transgenes in the F 1 plants. Plants that only harbored 35S-ICKI showed similar phenotype to the 35S-ICK1 parent, while plants harboring 35S-ICKI and 35S-CycD3 were bigger than the 35S-ICKI parent. These results show that the expression of CycD3 that interacts with ICK1 in regulating CDK
activity could at least partially restore some of the modifications due to ICK1 expression. In addition, either due to a partial restoration or due to novel effects of over-expression of both ICKI and CycD3, alternative modifications may be achieved.

Transgenic Arabidopsis plants were used as hosts for a CDK inhibitor expression during early stages of microspore development using construct designated NTM19-ICKI.
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 NTMl9 promoter was cloned from tabacco genomic DNA using PCR with primers sequence-specific to the NTMl9 promoter (Oldenhof et al., 1996). The ICKI cDNA
was linked to the NTMl9 promoter in a vector for plant expression. Similarly, NTM19-GUS, and NTMl9-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 NTMl9-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 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 NTMl9-ICKI 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 T1 transformants Construct Seed lot No of Plant growthReduced pods transformants b at least NTM19-GUS 305-306 14 Normal 1 NTM19-ICKl 310-312 30 Normal 5 NTM19-C cD3 313-315 30 Normal 0 Transgenic Arabidopsis plants were used as hosts for a CDK inhibitor expression construct designated NTMl9-ICKl and for two alternative expression constructs designated NTMl9-GUS and NMTl9-CycD3 (for expression respectively of the GUS and Arabidopsis cyclin D3). Crosses were made between NTMl9-ICKI lines, expressing the CDK
inhibitor ICK1, and the other lines.
REFERENCES
The following documents are incorporated herein by reference:
Artsaenko O, Peisker M, zur Nieden U, Fiedler U, Weiler EW, Muntz K, Conrad U
(1995) Expression of a single-chain Fv antibody against abscisic acid creates a witty phenotype in transgenic tobacco. Plant J. 8: 745-750 Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Stuhl K.
(1995) Current Protocols in Molecular Biology, Vols 1, 2 and 3 Babic V, Datla RS, Scoles GJ, Keller WA (1998) Development of an efficient Agrobacterium-mediated transformation system for Brassica carinata. Plant Cell Rep 17:

Bai C, Sen P, Hofmann K, Ma L, Goebl M, Harper JW, Elledge SJ (1996) SKPI
connects cell cycle regulators to the ubiquitin proteolysis machinery through a novel motif, the F-box. Cell 86: 263-274 Baskin TI, Busby CH, Fowke LC, Sammut M, Gubler F (1992) Improvements in immunostaining samples embedded in methacrylate: localization of microtubules and other antigens throughout developing organs in plants of diverse taxa. Planta 187:

Bechtold N, Ellis J, Pelletier (1993) In planta Agrobacterium-mediated gene transfer by infiltration of adult Arabidopsis thaliana plants. C R Acad Sci Paris, Science de la vielLife ciences 316: 1194-1199 Bell MH, Halford NG, Ormrod JC, Francis D (1993) Tobacco plants transformed with cdc25, a mitotic inducer gene from fission yeast. Plant Mol Biol 23: 445-Brent R, Ptashne M (1985) A eukaryotic transcriptional activator bearing the DNA
specificity of a prokaryotic repressor. Cell 43: 729-36 Brent R, Gyuris J, Golemis E (1996) U.S. Pat. No. 5,580,736.
Burritt JB, Bond CW, Doss KW, Jesaitis AJ (1996) Filamentous phage display of oligopeptide libraries. Anal Biochem 238: 1-13 Callis J (1997) Regulation of protein degradation in plants. In: Setlow JK
(ed) Genetic Engineering, Vol 19. Plenum Press, New York, pp121-148 Callis J, Carpetnter T, Sun C-W, Vierstra R (1993) The intron of Arabidopsis thaliana polyubiquitin genes is conserved in location and is a quantitative determinant of chimeric gene expression. Plant Mol Biol 21: 895-906 Chevray PM, Nathans D (1992) Protein interaction cloning in yeast:
identification of mammalian proteins that react with the leucine zipper of Jun. Proc Natl Acad Sci USA 89:

Chien CT, Bartel PL, Sternglanz R, Fields S (1991) The two-hybrid system: a method to identify and clone genes for proteins that interact with a protein of interest. Proc Natl Acad Sci USA 88: 9578-9582 Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16: 735-743 Ousters JB, Oldenhof MT, Schrauwen JA, Cordewener JH, Wullems GJ, van Lookeren Campagne MM (1997) Analysis of microspore-specific promoters in transgenic tobacco. Plant Mol Biol 35: 689-699 Cwirla SE, Peters EA, Barrett RW, Dower WJ (1990) Peptides on phage: a vast library of peptides for identifying ligands. Proc Natl Acad Sci USA 87: 6378-Czako M, Jang JC, Herr JM Jr, Marton L (1992) Differential manifestation of seed mortality induced by seed-specific expression of the gene for diphtheria toxin A chain in Arabidopsis and tobacco. Mol Gen Genet 235: 33-40.
Daugherty PS, Chen G, Olsen MJ, Iverson BL, Georgiou G (1998) Antibody affinity maturation using bacterial surface display. Protein Eng 11: 825-832 Day CD, Galgoci BF, Irish VF (1995) Genetic ablation of petal and stamen primordia to elucidate cell interactions during floral development.
Development 121: 2887-Dellaporta SL, Wood J, Hicks JB (1983) Plant Molecular Biology Rep 1: 19-21.
Deng C, Zhang P, Harper JW, Elledge SJ, Leder P (1995) Mice lacking p21 CIP 1 /WAF 1 undergo normal development, but are defective in G 1 checkpoint control.
Cel182:675-684 Doerner P, Jorgensen J-E, You R, Steppuhn J, Lamb C (1996) Control of root growth and development by cyclin expression. Nature 380: 520-523 Dunsmuir, et al (1983) Nucleic Acids Res 11: 4177-4183 Edwards K, Johnstone C, Thompson C (1991) A simple and rapid method for the preparation of plant genomic DNA for PCR analysis. Nucleic Acids Res 19: 1349 European Patent Application EP-A 0 255 378 European Patent Application EP-A 0 409 625 European Patent Application EP-A 0 409 629 Fero ML, Rivkin M, Tasch M, Porter P, Carow CE, Firpo E, Polyak K, Tsai LH, Broudy V, Perlmutter RM, Kaushansky K, Roberts JM ( 1996) A syndrome of multiorgan hyperplasia with features of gigantism, tumorigenesis, and female sterility in p27(Kipl)-deficient mice. Cell 85: 733-744 Ferreira PCG, Hemerly AS, de Almeida Engler J, Van Montagu M, Engler G, Inze D (1994) Developmental expression of the Arabidopsis cyclin gene cyclAt. Plant Cell 6:

Ferreira PCG, Hemerly AS, Villarroel R, Van Montagu M, Inze D (1991) The Arabidopsis functional homolog of the p34~a°2 protein kinase. Plant Cell 3: 531-540 Fields S, Song O-K (1989) A novel genetic system to detect protein-protein interactions. Nature 340: 245-246 Fields S, Song O-K (1994) U.S. Pat. No. 5,283,173 Fields S, Song O-K (1995) U.S. Pat. No. 5,468,614 Fountain MD, Renz A, Beck E (1999) Isolation of a cDNA encoding a Gl-cyclin-dependent kinase inhibitor (ICDK) from suspension cultured photoautotrophic Chenopodium rubrum L. cells. Plant Physiol 120: 339 Fowke LC, Attree SM, Rennie PJ (1994) Scanning electron microscopy of hydrated and desiccated mature somatic embryos and zygotic embryos of white spruce (Picea glauca [Moench] Voss.). Plant Cell Rep 13: 612-618 Francis D, Halford NG (1995) The plant cell cycle. Physiol Plant 93: 365-374 Gavilondo JV, Larrick JW (2000) Antibody engineering at the millennium.
Biotechniques 29:128-138 Genschik P, Criqui MC, Parmentier Y, Derevier A, Fleck J (1998) Cell cycle -dependent proteolysis in plants. Identification Of the destruction box pathway and metaphase arrest produced by the proteasome inhibitor mg132. Plant Cell 10:

Grafi G, Larkins BA (1995) Endoreduplication in maize endosperm: involvement of M phase-promoting factor inhibition and induction of S phase-related kinases.
Science 269: 1262-1264 Green PB, Linstead P (1990) A procedure for SEM of complex shoot structures applied to the inflorescence of snapdragon (Antirrhinum). Protoplasma 158: 33-Hanes J, Pluckthun A (1997) In vitro selection and evolution of functional proteins by using ribosome display. Proc Natl Acad Sci USA 94: 4937-4942 Harper JW, Elledge SJ (1996) Cdk inhibitors in development and cancer. Curr Opin Genet Dev 6: 56-64 Hemerly A, de Almeida Engler J, Bergounioux C, Van Montagu M, Engler G, Inze D, Ferreira P (1995) Dominant negative mutants of the Cdc2 kinase uncouple cell division from iterative plant development. EMBO J 14: 3925-3936 Hemerly AS, Ferreira PCG, de Almeida Engler J, Van Montagu M, Engler G, Inze D (1993) cdc2a expression in Arabidopsis thaliana is linked with competence for cell division. Plant Cell 5: 1711-1723 Hiatt A, Cafferkey R, Bowdish K (1989) Production of antibodies in transgenic plants. Nature 342:76-78 Hindley J, Phear GA (1984) Sequence of the cell division gene CDC2 from Schizosaccharomyces pombe: patterns of splicing and homology to protein kinases. Gene 31: 129-134 Hirayama T, Imajuku Y, Anai T, Matsui M, Oka A (1991) Identification of two cell-S cycle-controlling cdc2 gene homologs in Arabidopsis thaliana. Gene 105: 159-International Patent Publication WO-A 8 809 334 International Patent Publication WO-A 9 113 980 Jacobs TW (1995) Cell cycle control. Annu Rev Plant Physiol Plant Mol Biol 46:

Jefferson RA (1987) Assaying chimeric genes in plants: the GUS gene fusion system. Plant Mol Biol Rep 5: 387-405 Joubes J, Chevalier C, Dudits D, Heberle-Bors E, Inze D, Umeda M, Renaudi JP
(2000) CDK-related protein kinases in plants. Plant Mol. Biol 43: 607-620 Kieke MC, Cho BK, Boder ET, Kranz DM, Wittrup KD (1997) Isolation of anti-T
cell receptor scFv mutants by yeast surface display. Protein Eng 10:1303-1310 King RW, Deshaies RJ, Peters JM, Kirschner MW (1996) How proteolysis drives the cell cycle. Science 274: 1652-1659 Kiyokawa H, Kineman RD, Manova-Todorova KO, Soares VC, Hoffman ES, Ono M, Khanam D, Hayday AC, Frohman LA, Koff A (1996) Enhanced growth of mice lacking the cyclin-dependent kinase inhibitor function of p27(Kipl). Cell 85: 721-732 Koncz C, Schell J ( 1986) The promoter of TL-DNA gene 5 controls the tissue-specific expression of chimeric genes carried by a novel type of Agrobacterium binary vector. Mol Gen Genet 204: 383-396 Koning A, Jones A, Fillatti JJ, Comai L, Lassner MW (1992) Arrest of embryo development in Brassica napus mediated by modified Pseudomonas aeruginosa exotoxin A.
Plant Mol Biol 18: 247-258 Lorincz AT, Reed SI (1984) Primary structure homology between the product of yeast cell division control gene CDC28 and vertebrate oncogenes. Nature 307:

Lees E (1995) Cyclin-dependent kinase regulation. Curr Opin Cell Biol 7: 773-Ma J, Ptashne M (1987) A new class of yeast transcriptional activators. Cell 51: 113-Malmqvist M, Karlsson R (1997) Biomolecular interaction analysis: affinity biosensor technologies for functional analysis of proteins. Curr Opin Chem Biol 1: 378-83 Mariani C, De Beuckeleer M, Truettner J, Leemans J, Goldberg RB (1990) Induction of Male sterility in plants by a chimeric ribonuclear gene. Nature 347: 737-Martinez MC, Jorgensen JE, Lawton MA, Lamb CJ, Doerner PW (1992) Spatial pattern of cdc2 expression in relation to meristem activity and cell proliferation during plant development. Proc Natl Acad Sci USA 89: 7360-7364 McCafferty J, Griffiths AD, Winter G, Chiswell DJ (1990) Phage antibodies:
filamentous phage displaying antibody variable domains. Nature 348: 552-554 Meyerowitz EM (1997) Genetic control of cell division patterns in developing plants. Cell 88: 299-308 Moloney MM, Walker JM, Sharma KK (1989) High efficiency transformation of Brassica napus using Agrobacterium vectors. Plant Cell Rep 8: 238-242 Nakayama K, Ishida N, Shirane M, Inomata A, moue T, Shishido N, Horii I, Loh DY, Nakayama K (1996) Mice lacking p27(Kipl) display increased body size, multiple organ hyperplasia, retinal dysplasia, and pituitary tumors. Cell 85: 707-720 Nilsson O, Wu E, Wolfe DS, Weigel D (1998) Genetic ablation of flowers in transgenic Arabidopsis. Plant J 15: 789-804 Oldenhof MT, de Groot PF, Visser JH, Schrauwen JA, Wullems GJ (1996) Isolation and characterization of a microspore-specific gene from tobacco. Plant Mol Biol 31: 213-Oliver et al., U.S. Patent No. 5,723,765 issued 3 March 1998.
Pagano M (1997) Cell cycle regulation by the ubiquitin pathway. FASEB J 11:

Peat, et al (1989) Plant Mol Biol 13:639-651 Phizicky EM, Fields S (1995) Protein-protein interactions: methods for detection and analysis. Microbiol Rev 59: 94-123 Pines J (1995) Cyclins and cyclin-dependent kinases: a biochemical view.
Biochem J
308: 697-711 Pokalsky et al (1989) Nucleic Acids Res 17: 4661-4673 1 S Renaudin J-P, Doonan JH, Freeman D, Hashimoto J, Hirt H, Inz D, Jacobs T, Kouchi H, Rouze P, Sauter M, Savour A, Sorrell DA, Sundaresan V, Murray JAH
(1996) Plant Cyclins: a unified nomenclature for plant A-, B- and D-type cyclins based on sequence organization. Plant Mol Biol 32: 1003-1018 Reynolds A, Lundblad V (1994) Yeast vectors and assays for expression of cloned genes. In Current Protocols in Molecular Biology (Ausubel, F.M. et al, eds), New York, Wiley & Sons, pp. 13.6.1-13.6.4 Russo AA, Jeffrey PD, Patten AK, Massague J, Pavletich NP (1996) Crystal structure of the p27Kip 1 cyclin-dependent-kinase inhibitor bound to the cyclin A-Cdk2 complex. Nature 382: 325-331 Sadowski et al. (1999) U.S. Pat. No. 5,885,779 Segers G, Gadisseur I, Bergounioux C, de Almeida Engler J, Jacqmard A, Van Montagu M, Inze D (1996) The Arabidopsis cyclin-dependent kinase gene cdc2bAt is preferentially expressed during S and G2 phases of the cell cycle. Plant J 10:

Sherr CJ, Roberts JM (1995) Inhibitors of mammalian G1 cyclin-dependent kinases.
Genes Dev 9: 1149-1163 Soni R, Carmichael JP, Shah ZH, Murray JAH (1995) A family of cyclin D
homologs from plants differentially controlled by growth regulators and containing the conserved retinoblastoma protein interaction motif. Plant Cell 7: 85-103 Southern EM (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. JMoI Biol 98: 503-517 Stals H, Casteels P, Van Montagu M, Inze D (2000) Regulation of cyclin-dependent kinases in Arabidopsis thaliana. Plant Mol Biol 43: 583-593 Tavladoraki P, Benvenuto E, Trinca S, De Martinis D, Cattaneo A, Galeffi P
(1993) Transgenic plants expressing a functional single-chain Fv antibody are specifically protected 1 S from virus attack. Nature 366: 469-472 Thorsness MK, Kandasamy MK, Nasrallah ME, Nasrallah JB (1991) A Brassica S-locus gene promoter targets toxic gene expression and cell death to the pistil and pollen of transgenic Nicotiana. Dev Biol 143: 173-184 United States Patent No. 4,943,674 issued 24 July, 1990 United States Patent No. 5,175,095 issued 29 December, 1992 Wang H, Datla R, Georges F, Loewen M, Cutler AJ (1995) Promoters from kinl and cor6.6, two homologous Arabidopsis thaliana genes: transcriptional regulation and gene expression induced by low temperature, ABA, osmoticum and dehydration. Plant Mol Biol 28: 605-617 Wang H, Fowke LC, Crosby WL (1997) A plant cyclin-dependent kinase inhibitor gene. Nature 386: 451-452 Wang H, Qi Q, Schorr P, Cutler AJ, Crosby WL, Fowke LC (1998) ICK1, a cyclin-dependent protein kinase inhibitor from Arabidopsis thaliana interacts with both Cdc2a and CycD3, and its expression is induced by abscisic acid. Plant J 15: 501-510 Winter G, Griffiths AD, Hawkins RE, Hoogenboom HR (1994) Making antibodies by phage display technology. Annu Rev Immunol 12: 433-455 Wu H, Wade M, Krall L, Grisham J, Xiong Y, Van Dyke T (1996) Targeted in vivo expression of the cyclin-dependent kinase inhibitor p21 halts hepatocyte cell-cycle progression, postnatal liver development and regeneration. Genes Dev 10: 245-Xu H, Davies SP, Kwan BY, O'Brien AP, Singh M, Knox RB (1993) Haploid and diploid expression of a Brassica campestris anther-specific gene promoter in Arabidopsis and tobacco. Mol Gen Genet 239: 58-65 Yan Y, Frisen J, Lee MH, Massague J, Barbacid M (1997) Ablation of the CDK
inhibitor p57Kip2 results in increased apoptosis and delayed differentiation during mouse development. Genes Dev 11: 973-983 Zhang P, Liegeois NJ, Wong C, Finegold M, Hou H, Thompson JC, Silverman A, Harper JW, DePinho RA, Elledge SJ (1997) Altered cell differentiation and proliferation in mice lacking p57KIP2 indicates a role in Beckwith-Wiedemann syndrome. Nature 387:

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 incorporated herein by reference as if each individual publication were specifically and individually indicated to be incorporated 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 (47)

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 ICK1, 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 ICK1, 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 ICK1, 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 ICK1, 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 AP3 promoter.

25. The method of claim 22 or 23, wherein the tissue-specific promoter is an 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:
a) a heterologous nucleic acid encoding a cyclin-dependent kinase inhibitor;
and, 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:
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 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:
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 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.