# Patent

## Title
Plants having improved growth characteristics and method for making the same

## Abstract

The present invention concerns a method for improving growth characteristics of plants by modulating activity of a mutant CDKA kinase or a homologue thereof in a plant and/or modulating expression of a nucleic acid encoding such mutant CDKA. One such method comprises introducing into a plant a mutant CDKA nucleic acid molecule or mutant functional variant thereof. The invention also provides an isolated CKA mutant protein and nucleic acids encoding such protein. The invention furthermore relates to transgenic plants having improved growth characteristics, which plants have modulated expression of a nucleic acid encoding a mutant CDKA kinase. The present invention also concerns constructs useful in the methods of the invention.

## Background
RELATED APPLICATIONS
This application is a national stage application (under 35 U.S.C. 371) of PCT/EP2005/056364 filed Nov. 30, 2005, which claims benefit of European application 04106225.8 filed Dec. 1, 2004 and U.S. Provisional Application 60/634,015 filed Dec. 7, 2004.


SUBMISSION ON COMPACT DISC
The contents of the following submission on compact discs are incorporated herein by reference in its entirety: two copies of the Sequence Listing (COPY 1 and COPY 2) and a computer readable form copy of the Sequence Listing (CRF COPY), all on compact disc, each containing: file name: Sequence list-14546-00018-US, date recorded: May 30, 2007, size: 130 KB.
The present invention relates generally to the field of molecular biology and concerns a method for improving plant growth characteristics. More specifically, the present invention concerns a method for improving plant growth characteristics by modulating expression in a plant of a plant nucleic acid encoding an A-type cyclin dependent kinase (CDKA) and/or by modulating activity in a plant of a plant CDKA protein, which CDKA protein comprises a T161D-type mutation or which CDKA nucleic acid encodes such protein. The present invention also concerns plants having modulated expression of a plant CDKA nucleic acid and/or modulated activity of a plant CDKA protein, which CDKA protein comprises a T161D-type mutation or which nucleic acid encodes such protein and which plants have improved growth characteristics relative to corresponding wild type plants. The invention also provides plant CDKs with a PSTAIRE motif and a T161D-type mutation, and nucleic acids encoding such proteins.
The ever-increasing world population and the dwindling supply of arable land available for agriculture fuel research towards improving the efficiency of agriculture. Conventional means for crop and horticultural improvements utilise selective breeding techniques to identify plants having desirable characteristics. However, such selective breeding techniques have several drawbacks, namely that these techniques are typically labour intensive and result in plants that often contain heterogeneous genetic components that may not always result in the desirable trait being passed on from parent plants. Advances in molecular biology have allowed mankind to modify the germplasm of animals and plants. Genetic engineering of plants entails the isolation and manipulation of genetic material (typically in the form of DNA or RNA) and the subsequent introduction of that genetic material into a plant. Such technology has the capacity to deliver crops or plants having various improved economic, agronomic or horticultural traits. A trait of particular economic interest is yield. Yield is normally defined as the measurable produce of economic value from a crop. This may be defined in terms of quantity and/or quality. Crop yield is influenced by the typical stresses to which plants or crops are subjected. Such stresses include environmental (abiotic) stresses (such as temperature stresses caused by atypical high or low temperatures; stresses caused by nutrient deficiency; stresses caused by lack of water (drought)) and biotic stresses (which can be imposed on plants by other plants (weeds), animal pests and pathogens). Crop yield may not only be increased by combating one or more of the stresses to which the crop or plant is subjected, but may also be increased by modifying the inherent growth mechanisms of a plant.
The inherent growth mechanisms of a plant reside in a highly ordered sequence of events collectively known as the ‘cell cycle’. The ability to influence the cell cycle in a plant (either using recombinant DNA technology or using non-recombinant means), and to thereby modify various growth characteristics of a plant, would have many applications in areas such as crop enhancement, plant breeding, production of ornamental plants, arboriculture, horticulture, forestry, the production of algae or plants (for example for use as bioreactors, for the production of substances such as pharmaceuticals, antibodies, or vaccines, or for the bioconversion of organic waste or for use as fuel in the case of high-yielding algae and plants).
Progression through the cell cycle is fundamental to the growth and development of all multicellular organisms and is crucial to cell proliferation. The major components of the cell cycle are highly conserved in yeast, mammals, and plants. The cell cycle is typically divided into the following sequential phases: G0-G1-S-G2-M. DNA replication or synthesis generally takes place during the S phase (“S” is for DNA synthesis) and mitotic segregation of the chromosomes occurs during the M phase (the “M” is for mitosis), with intervening gap phases, G1 (during which cells grow before DNA replication) and G2 (a period after DNA replication during which the cell prepares for division). Cell division is completed after cytokinesis, the last step of the M phase. Cells that have exited the cell cycle and that have become quiescent are said to be in the G0 phase. Cells in this phase may be stimulated to renter the cell cycle at the G1 phase. The “G” in G1, G2 and G0 stands for “gap”. Completion of the cell cycle process allows each daughter cell during cell division to receive a full copy of the parental genome.
Cell division is controlled by two principal cell cycle events, namely initiation of DNA synthesis and initiation of mitosis. Each transition to each of these key events is controlled by a checkpoint represented by specific protein complexes (involved in DNA replication and division). The expression of genes necessary for DNA synthesis at the G1/S boundary is regulated by the E2F family of transcription factors in mammals and plant cells (WO 96/25494; Muller et al., Genes and Development 15, 267-285, 2001; De Veylder et al., EMBO J. 21, 13602-1368, 2002). Entry into the cell cycle is regulated/triggered by an E2F/Rb complex that integrates signals and allows activation of transcription of cell cycle genes. The transition between the different phases of the cell cycle, and therefore progression through the cell cycle, is driven by the formation and activation of different heterodimeric serine/threonine protein kinases, generally referred to as cyclin-dependent kinases (CDK). A prerequisite for activity of these kinases is the physical association with a specific cyclin, the timing of activation being largely dependent upon cyclin expression. Cyclin-binding induces conformational changes in the N-terminal lobe of the associating CDK and contributes to the localisation and substrate specificity of the complex. Monomeric CDKs are activated when they are associated with cyclins and thus have kinase activity. Cyclin protein levels fluctuate in the cell cycle and therefore represent a major factor in determining timing of CDK activation. The periodic activation of these complexes containing cyclins and CDK during cell cycle mediates the temporal regulation of cell-cycle transitions (checkpoints). Other factors regulating CDK activity include CDK inhibitors (CKIs or ICKs, KIPs, CIPs, INKs), CDK activating kinase (CAK), CDK phosphatase (Cdc25) and CDK subunit (CKS) (Mironov et al. Plant Cell 11, 509-522, 1999; Reed, S. I. Progress in Cell Cycle Research 2, 5-27, 1996).
In plants, two major classes of CDKs, known as A-type and B-type CDKs, have been studied to date. The A-type CDKs regulate both the G1-to-S and G2-to-M transitions, whereas the B-type CDKs seem to control the G2-to-M checkpoint only (Hemerly et al., 1995; Magyar et al., 1997; Porceddu et al., 2001). In addition, the presence of C-type CDKs and CDK-activating kinases (CAKs) has been reported (Magyar et al., 1997; Umeda et al., 1998; Joubès et al., 2001), as has the presence of D-type, E-type and F-type CDKs (Vandepoele et al. Plant Cell 14, 903-916, 2002).
A-type CDKs are known to have a conserved tertiary structure (Goldsmith and Cobb, Curr. Opin. Struct. Biol. 4, 833-840), including a highly conserved PSTAIRE motif that is involved in cyclin binding. The catalytic core of a CDK is composed of an N-terminal and a C-terminal lobe. The C-terminal lobe encompasses a catalytic cleft (responsible for ATP and substrate binding) and further comprises a so-called T-loop, named after a threonine residue that is conserved in several kinase families. In human CDK2, this threonine residue is on position 161, whereas in Saccharomyces cerevisiae cdc28 and in Schizosaccharomyces pombe cdc2 it is located on position 169 and 167 respectively. Phosphorylation of this threonine residue is reported to cause a structural conformation change in the T-loop that is necessary for switching the kinase into an active state (Gu et al., EMBO J. 11, 3995-4005). Several studies describe mutations of the conserved threonine in the T-loop (Ducommun et al. EMBO J. 10, 3311-3319, 1991; Gould et al. EMBO J. 10, 3297-3309; Marcote et al. Mol. Cell. Biol. 13, 5122-5131, 1993; Ducommun et al. Mol Cell. Biol. 11, 6177-6184, 1991; Coleman et al. J. Biol. Chem. 272, 18869-18874, 1997; Martinez et al. EMBO J. 16, 343-354, 1997; Gould et al. Mol. Gen. Genet. 259, 437-448, 1998; Booher et al. Mol. Cell. Biol. 6, 3523-3530, 1986; Solomon et al. Mol. Biol. Cell 3, 13-27, 1992; Lim et al. Mol. Cell. Biol. 16, 4573-4583, 1996), all mutations tested were shown to have a serious impact on binding of ligands (such as cyclin or Suc1/ICK) and/or on kinase activity, resulting in defective or lethal phenotypes in yeast complementation experiments. Although the T169E mutation (according to the numbering for yeast cdc28), and by analogy also the T169D mutation, mimics a phosphorylation, it was demonstrated that none of the CDKs with such mutations were able to fully complement yeast. Other residues that play an important role in A-type CDK protein activity are threonine at position 14 and tyrosine at position 15. Upon phosphorylation of at least one of these amino acids, the CDK becomes inactivated. WO 99/54489 describes the use of a CDK with threonine 14 and tyrosine 15 substituted by alanine and phenylalanine respectively to increase the tolerance of plants to salt stress. WO 00/52171 describes a method of modifying one or more plant cytokinin-mediated morphological, biochemical and physiological properties or characteristics comprising expressing a Cdc25 phosphoprotein phosphatase in a plant.
It has now surprisingly been found that expression in a plant of an A-type cyclin dependent kinase (CDKA) with a T161D-type mutation gives plants having improved growth characteristics.
Therefore, according to one embodiment of the present invention there is provided a method for improving plant growth characteristics relative to corresponding wild type plants, comprising modulating activity in a plant of an A-type CDK having a T161D-type mutation and/or modulating expression of a nucleic acid encoding such A-type CDK, and optionally selecting plants having improved growth characteristics.
Advantageously, performance of the method according to the present invention results in plants having a variety of improved growth characteristics relative to corresponding wild type plants and which improved growth characteristics comprise at least increased yield relative to corresponding wild type plants.
The term “increased yield” as defined herein is taken to mean an increase in any one or more of the following, each relative to corresponding wild type plants: (i) increased biomass (weight) of one or more parts of a plant, particularly aboveground (harvestable) parts, increased root biomass or increased biomass of any other harvestable part; (ii) increased total seed yield, which includes an increase in seed biomass (seed weight) and which may be an increase in the seed weight per plant or on an individual seed basis; (iii) increased number of flowers (“florets”) per panicle (iv) increased number of (filled) seeds; (v) increased seed size, which may also influence the composition of seeds; (vi) increased seed volume, which may also influence the composition of seeds (including oil, protein and carbohydrate total content and composition); (vii) increased individual seed area; (viii) increased individual seed length and/or width; (ix) increased harvest index, which is expressed as a ratio of the yield of harvestable parts, such as seeds, over the total biomass; and (x) increased thousand kernel weight (TKW), which is extrapolated from the number of filled seeds counted and their total weight. An increased TKW may result from an increased seed size and/or seed weight. An increased TKW may result from an increase in embryo size and/or endosperm size.
Taking corn as an example, a yield increase may be manifested as one or more of the following: increase in the number of plants per hectare or acre, an increase in the number of ears per plant, an increase in the number of rows, number of kernels per row, kernel weight, TKW, ear length/diameter, among others. Taking rice as an example, a yield increase may be manifested by an increase in one or more of the following: number of plants per hectare or acre, number of panicles per plant, number of spikelets per panicle, number of flowers per panicle, increase in the seed filling rate, expressed (in %) as the proportion of the number of filled seeds over the number of florets (total number of seeds), increase in TKW, among others. An increase in yield may also result in modified architecture, or may occur as a result of modified architecture.
According to a preferred feature, performance of the methods according to the present invention results in plants having increased yield and more particularly, increased biomass and/or increased seed yield. Preferably, the increased seed yield comprises an increase in one or more of the following: number of (filled) seeds, total seed weight, seed size, seed volume, thousand kernel weight and harvest index, each relative to control plants.
Therefore, according to the present invention, there is provided a method for increasing plant yield relative to corresponding control plants, which method comprises modulating activity of a CDK or a homologue thereof in a plant, which CDK or homologue has a PSTAIRE motif and a T161D-type mutation, and/or modulating expression of a nucleic acid encoding such a CDKA or homologue thereof.
Since the plants according to the present invention have increased yield, it is likely that these plants exhibit an increased growth rate (during at least part of their life cycle), relative to the growth rate of corresponding wild type plants at a corresponding stage in their life cycle. The increased growth rate may be specific to one or more parts of a plant or cell types, including seeds, of a plant, or may be throughout substantially the whole plant. Plants having an increased growth rate may have a shorter life cycle. The life cycle of a plant may be taken to mean the time needed to grow from a dry mature seed up to the stage where the plant has produced dry mature seeds, similar to the starting material. This life cycle may be influenced by factors such as early vigour, growth rate, flowering time and speed of seed maturation. An increase in growth rate may take place at one or more stages in the life cycle of a plant or during substantially the whole plant life cycle. Increased growth rate during the early stages in the life cycle of a plant may reflect enhanced vigour. The increase in growth rate may alter the harvest cycle of a plant allowing plants to be sown later and/or harvested sooner than would otherwise be possible. If the growth rate is sufficiently increased, it may allow for the sowing of further seeds of the same plant species (for example sowing and harvesting of rice plants followed by sowing and harvesting of further rice plants all within one conventional growing period). Similarly, if the growth rate is sufficiently increased, it may allow for the further sowing of seeds of different plants species (for example the sowing and harvesting of rice plants followed by, for example, the sowing and optional harvesting of soy bean, potatoes or any other suitable plant). Harvesting additional times from the same rootstock in the case of some plants may also be possible. Altering the harvest cycle of a plant may lead to an increase in annual biomass production per acre (due to an increase in the number of times (say in a year) that any particular plant may be grown and harvested). An increase in growth rate may also allow for the cultivation of transgenic plants in a wider geographical area than their wild-type counterparts, since the territorial limitations for growing a crop are often determined by adverse environmental conditions either at the time of planting (early season) or at the time of harvesting (late season). Such adverse conditions may be avoided if the harvest cycle is shortened. The growth rate may be determined by deriving various parameters from growth curves plotting growth experiments, such parameters may be: T-Mid (the time taken for plants to reach 50% of their maximal size) and T-90 (time taken for plants to reach 90% of their maximal size), amongst others.
Performance of the methods of the invention gives plants having an increased growth rate. Therefore, according to the present invention, there is provided a method for increasing the growth rate of plants, which method comprises modulating activity of a CDK or a homologue thereof in a plant, which CDK or homologue has a PSTAIRE motif and a T161D-type mutation, and/or modulating expression of a nucleic acid encoding such a CDKA or homologue thereof.
An increase in yield and/or growth rate occurs whether the plant is under non-stress conditions or whether the plant is exposed to various stresses compared to control plants. Plants typically respond to exposure to stress by growing more slowly. In conditions of severe stress, the plant may even stop growing altogether. Mild stress on the other hand is defined herein as being any stress to which a plant is exposed which does not result in the plant ceasing to grow altogether without the capacity to resume growth. Due to advances in agricultural practices (irrigation, fertilization, pesticide treatments) severe stresses are not often encountered in cultivated crop plants. As a consequence, the compromised growth induced by mild stress is often an undesirable feature for agriculture. Mild stresses are the typical stresses to which a plant may be exposed. These stresses may be the everyday biotic and/or abiotic (environmental) stresses to which a plant is exposed. Typical abiotic or environmental stresses include temperature stresses caused by atypical hot or cold/freezing temperatures; salt stress; water stress (drought or excess water). Abiotic stresses may also be caused by chemicals. Biotic stresses are typically those stresses caused by pathogens, such as bacteria, viruses, fungi and insects. The term “non-stress conditions” as used herein are those environmental conditions that do not significantly go beyond the everyday climatic and other abiotic conditions that plants may encounter. Persons skilled in the art are aware of normal soil conditions and climatic conditions for a given geographic location.
The abovementioned growth characteristics may advantageously be modified in any plant.
The term “plant” as used herein encompasses whole plants, ancestors and progeny of the plants and plant parts, including seeds, shoots, stems, leaves, roots (including tubers), flowers, and tissues and organs, wherein each of the aforementioned comprise the gene/nucleic acid of interest or the specific modification in the gene/nucleic acid of interest. The term “plant” also encompasses plant cells, suspension cultures, callus tissue, embryos, meristematic regions, gametophytes, sporophytes, pollen, and microspores, again wherein each of the aforementioned comprise the gene/nucleic acid of interest.
Plants that are particularly useful in the methods of the invention include algae, ferns, and all plants which belong to the superfamily Viridiplantae, in particular monocotyledonous and dicotyledonous plants, including fodder or forage legumes, ornamental plants, food crops, trees, or shrubs selected from the list comprising Abelmoschus spp., Acer spp., Actinidia spp., Agropyron spp., Allium spp., Amaranthus spp., Ananas comosus, Annona spp., Apium graveolens, Arabidopsis thaliana, Arachis spp, Artocarpus spp., Asparagus officinalis, Avena sativa, Averrhoa carambola, Benincasa hispida, Bertholletia excelsea, Beta vulgaris, Brassica spp., Cadaba farinosa, Camellia sinensis, Canna indica, Capsicum spp., Carica papaya, Carissa macrocarpa, Carthamus tinctorius, Carya spp., Castanea spp., Cichorium endivia, Cinnamomum spp., Citrullus lanatus, Citrus spp., Cocos spp., Coffea spp., Cola spp., Colocasia esculenta, Corylus spp., Crataegus spp., Cucumis spp., Cucurbita spp., Cynara spp., Daucus carota, Desmodium spp., Dimocarpus longan, Dioscorea spp., Diospyros spp., Echinochloa spp., Eleusine coracana, Eriobotrya japonica, Eugenia uniflora, Fagopyrum spp., Fagus spp., Ficus carica, Fortunella spp., Fragaria spp., Ginkgo biloba, Glycine spp., Gossypium hirsutum, Helianthus spp., Hibiscus spp., Hordeum spp., Ipomoea batatas, Juglans spp., Lactuca sativa, Lathyrus spp., Lemna spp., Lens culinaris, Linum usitatissimum, Litchi chinensis, Lotus spp., Luffa acutangula, Lupinus spp., Macrotyloma spp., Malpighia emarginata, Malus spp., Mammea americana, Mangifera indica, Manihot spp., Manilkara zapota, Medicago sativa, Melilotus spp., Mentha spp., Momordica spp., Morus nigra, Musa spp., Nicotiana spp., Olea spp., Opuntia spp., Ornithopus spp., Oryza spp., Panicum miliaceum, Passiflora edulis, Pastinaca sativa, Persea spp., Petroselinum crispum, Phaseolus spp., Phoenix spp., Physalis spp., Pinus spp., Pistacia vera, Pisum spp., Poa spp., Populus spp., Prosopis spp., Prunus spp., Psidium spp., Punica granatum, Pyrus communis, Quercus spp., Raphanus sativus, Rheum rhabarbarum, Ribes spp., Rubus spp., Saccharum spp., Sambucus spp., Secale cereale, Sesamum spp., Solanum spp., Sorghum bicolor, Spinacia spp., Syzygium spp., Tamarindus indica, Theobroma cacao, Trifolium spp., Triticosecale rimpaui, Triticum spp., Vaccinium spp., Vicia spp., Vigna spp., Vitis spp., Zea mays, Zizania palustris, Ziziphus spp., amongst others.
According to a preferred feature of the present invention, the plant is a crop plant comprising soybean, sunflower, canola, alfalfa, rapeseed, cotton, tomato, potato or tobacco. Further preferably, the plant according to the present invention is a monocotyledonous plant such as sugar cane, most preferably a cereal, such as rice, maize, wheat, millet, barley, rye, oats or sorghum.
The activity of a CDKA protein may be modulated by modulating the levels of the CDKA protein. Alternatively, activity may also be modulated when there is no change in levels of a CDKA protein, this may occur when the intrinsic properties of the polypeptide are altered, for example by making a mutant. According to a preferred feature of the invention, modulated activity of the CDKA protein with a T161D-type mutation and/or modulated expression of a nucleic acid encoding this CDKA is introduced and/or increased activity of a CDKA protein with a T161D-type mutation and/or increased expression of a nucleic acid encoding this CDKA.
The terms “A-type CDK” or “CDKA” as defined herein may be used interchangeably and encompass any amino acid sequence having cyclin dependent kinase activity and which sequence when used in the construction of a CDK phylogenetic tree, such as the ones depicted in FIG. 1 and FIG. 2, clusters around the A-type CDKs rather than any of the other CDK groups and which amino acid sequence comprises a PSTAIRE amino acid sequence. A person skilled in the art could readily determine whether any amino acid sequence in question falls within the definition of an “A-type CDK” using known techniques and software for the making of such a phylogenetic tree, such as a GCG, EBI or CLUSTAL package, using default parameters (see for example Vandepoele et al. 2002). Upon construction of such a phylogenetic tree, sequences clustering in the A-type CDK group will be considered to fall within the definition of an “A-type CDK” or “CDKA”, and will therefore be useful in performing the methods of the invention. Preferably the A-type CDK further comprises in increasing order of preference at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more overall sequence identity to the amino acid represented in GenBank accession CAA42922 (SEQ ID NO: 8) or to its mutant form represented by SEQ ID NO: 2. The overall sequence identity is determined using a global alignment algorithm, such as the Needleman Wunsch algorithm in the program GAP (GCG Wisconsin Package, Accelrys). Preferably, the A-type CDK belongs to the class 1 A-type CDKs (i.e. CDKA;1).
The term “T161D-type mutation” is defined herein as a mutation in a CDK of the conserved threonine corresponding to threonine 161 in human CDC2 or rice CDKA;1 into aspartic acid or glutamic acid. More particularly, the term “CDK having a T161D-type mutation” encompasses CDK proteins comprising a substitution of the conserved threonine in the T-loop by aspartic acid or glutamic acid; preferably a substitution by aspartic acid. Substitution of threonine by aspartic acid or glutamic acid in a protein results in the introduction of a negative charge, thus mimicking the negative charge of a phosphate group introduced by phosphorylation. Methods for introducing mutations in genes resulting in amino acid substitutions are well known in the art and include site-directed mutagenesis with oligonucleotides or by using PCR.
The various structural domains in a CDKA protein are well known (De Bondt et al., Nature 363, 595-602, 1993) and may be identified using specialised databases e.g. SMART (Schultz et al. (1998) Proc. Natl. Acad. Sci. USA 95, 5857-5864; Letunic et al. (2002) Nucleic Acids Res 30, 242-244; http://smart.embl-heidelberg.de/), InterPro (Mulder et al., (2003) Nucl. Acids. Res. 31, 315-318; http://www.ebi.ac.uk/interpro/), Prosite (Bucher and Bairoch (1994), A generalized profile syntax for biomolecular sequences motifs and its function in automatic sequence interpretation. (In) ISMB-94; Proceedings 2nd International Conference on Intelligent Systems for Molecular Biology. Altman R., Brutlag D., Karp P., Lathrop R., Searls D., Eds., pp 53-61, AAAIPress, Menlo Park; Hulo et al., Nucl. Acids. Res. 32:D134-D137, (2004), http://www.expasy.org/prosite/) or Pfam (Bateman et al., Nucleic Acids Research 30(1):276-280 (2002), http://www.sanger.ac.uk/Software/Pfam/).
The kinase domain of CDK is of an S_TKc-type (SMART accession number SM00220, InterPro accession number IPR002290), and has Ser/Thr kinase activity. The predicted active site (VLHRDLKPQNLLI, wherein D is the predicted catalytic residue) corresponds to the PROSITE signature PS00108. The ATP binding site (IGEGTYGVVYRARDKVTNETIALK) corresponds to the PROSITE signature PS00107.
Methods for the search and identification of A-type CDK homologues would be well within the realm of persons skilled in the art. Such methods comprise comparison of the sequences represented by SEQ ID NO 1 or 2, or by GenBank accession CAA42922, in a computer readable format, with sequences that are available in public databases such as MIPS (http://mips.gsf.de/), GenBank (http://www.ncbi.nim.nih.gov/Genbank/index.html) or EMBL Nucleotide Sequence Database (http://www.ebi.ac.uk/embl/index.html), using algorithms well known in the art for the alignment or comparison of sequences, such as GAP (Needleman and Wunsch, J. Mol. Biol. 48; 443-453 (1970)), BESTFIT (using the local homology algorithm of Smith and Waterman (Advances in Applied Mathematics 2; 482-489 (1981))), BLAST (Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J., J. Mol. Biol. 215:403-410 (1990)), FASTA and TFASTA (W. R. Pearson and D. J. Lipman Proc. Natl. Acad. Sci. USA 85:2444-2448 (1988)). The software for performing BLAST analysis is publicly available through the National Centre for Biotechnology Information (NCBI). The homologues mentioned below were identified using BLAST default parameters (BLOSUM62 matrix, gap opening penalty 11 and gap extension penalty 1) and preferably the full-length sequences are used for analysis. These alignment methods also easily allow the identification of the conserved threonine that corresponds to threonine 161 in human CDC2 or rice CDKA;1 (SEQ ID NO: 8).
Examples of proteins falling under the definition of “A-type CDK or a homologue thereof” include the CDKs with a PSTAIRE motif, such as the proteins listed in Table 1. Persons skilled in the art are aware of the various techniques that may be used for introducing a T161D-type of mutation into these proteins to make them useful in the methods of the present invention.







TABLE 1







examples of plant A-type CDK proteins with their GenBank


or PIR accession numbers (modified from Joubès


et al., Plant Mol. Biol. 43, 607-620, 2000)












Database
SEQ


Gene name
Species
accession
ID NO:













Allce; CDKA; 1
Allium cepa
BAA21673.1
10


Antma; CDKA; 1
Antirrhinum majus
CAA66233.1
12


Antma; CDKA; 2
Antirrhinum majus
CAA66234.1
14


Arath; CDKA; 1
Arabidopsis thaliana
AAA32831.1
16


Betvu; CDKA; 1
Beta vulgaris
CAA96384.1
18


Brana; CDKA; 1
Brassica napus
AAA92823.1
20


Cheru; CDKA; 1
Chenopodium rubrum
CAA71242.1
22


Glyma; CDKA; 1
Glycine max
M93140*
24


Glyma; CDKA; 2
Glycine max
M93139*
26


Lyces; CDKA; 1
Lycopersicon esculentum
CAA76700.1
28


Lyces; CDKA; 2
Lycopersicon esculentum
CAA76701.1
30


Medsa; CDKA; 1
Medicago sativa
AAB41817.1
32


Medsa; CDKA; 2
Medicago sativa
CAA50038.1
34


Nicta; CDKA; 1
Nicotiana tabacum
AAB02567.1
36


Nicta; CDKA; 1
Nicotiana tabacum
AAB02568.1
38


Nicta; CDKA; 3
Nicotiana tabacum
BAA09369.1
40


Orysa; CDKA; 1
Oryza sativa
CAA42922.1
8


Orysa; CDKA; 2
Oryza sativa
CAA42923.1
42


Petcr; CDKA; 1
Petroselinum crispum
AAC41680.1
44


Pethy; CDKA; 1
Petunia hybrida
CAA73997.1
46


Picab; CDKA; 1
Picea abies
CAA54746.1
48


Pinco; CDKA; 1
Pinus contorta
CAA56815.2
50


Pissa; CDKA; 2
Pisum sativum
BAA33152
52


Soltu; CDKA; 2
Solanum tuberosum
AAA98856.1
54


Sesro; CDKA; 1
Sesbania rostrata
CAA99991.1
56


Triae; CDKA; 1
Triticum aestivum
AAD10483.1
58


Triae; CDKA; 2
Triticum aestivum
AAD10484.1
60


Vigac; CDKA; 1
Vigna aconitifolia
AAA34241.1
62


Vigun; CDKA; 1
Vigna unguiculata
CAA61581.1
64


Zeama; CDKA; 1
Zea mays
AAA33479
66





*GenBank accession numbers of the CDS encoding the protein.






It is to be understood that the term “A-type CDK or a homologue thereof” is not to be limited to the sequence represented by SEQ ID NO: 2, but that any polypeptide meeting the criteria of having cyclin dependent kinase activity, having a PSTAIRE domain, and having at least 75% sequence identity to SEQ ID NO: 8, may be suitable for use in the methods of the invention, provided that the CDKA or its homologue comprise a T161D-type mutation. Preferably, the A-type CDK or a homologue thereof is an orthologue of the protein represented by SEQ ID NO: 8.
To determine the kinase activity of A-type CDKs, several assays are available and are well known in the art (for example Current Protocols in Molecular Biology, Volumes 1 and 2, Ausubel et al. (1994), Current Protocols; or online, such as http://www.protocol-online.org).
In brief, the kinase assay generally involves: (1) bringing the kinase protein into contact with a substrate polypeptide containing the target site to be phosphorylated; (2) allowing phosphorylation of the target site in an appropriate kinase buffer under appropriate conditions; (3) separating phosphorylated products from non-phosphorylated substrate after a suitable reaction period. The presence or absence of kinase activity is determined by the presence or absence of the phosphorylated target. In addition, quantitative measurements may be performed. Purified CDK protein, or cell extracts containing or enriched with the CDK protein may be used as a source of the kinase protein. Histone H1 or small peptides are particularly well suited as a substrate. The peptide must comprise one or more serine, threonine, or tyrosine residues in a phosphorylation site motif. A compilation of phosphorylation sites may be found in Biochimica et Biophysica Acta 1314, 191-225, (1996). In addition, the peptide substrates may advantageously have a net positive charge to facilitate binding to phosphocellulose filters, (allowing separation of the phosphorylated from non-phosphorylated peptides and detection of the phosphorylated peptides). If a phosphorylation site motif is not known, a general Ser/Thr kinase substrate may be used. For example, the peptide “ADAQHATPPKKKRKVEDPKDF” (SEQ ID NO: 67) (Marshak et al. J. Cell. Biochem. 45, 391, 1991) is a specific substrate for A-type CDK. To determine the kinetic parameters for phosphorylation of the synthetic peptide, a range of peptide concentrations is required. For initial reactions, a peptide concentration of 0.7-1.5 mM may be used. For each kinase enzyme, it is important to determine the optimal buffer, ionic strength, and pH for activity. A standard 5× Kinase Buffer generally contains 5 mg/ml BSA (Bovine Serum Albumin preventing kinase adsorption to the assay tube), 150 mM Tris-Cl (pH 7.5), 100 mM MgCl2. The optimal concentrations of divalent cations must be determined empirically for each protein kinase. Suitable buffers for CDK assays are known in the art (for example John et al., Protoplasma 161, 70-74, 1991). A commonly used donor of the phosphoryl group is radio-labelled [gamma-32P]ATP (normally at 0.2 mM final concentration). The amount of 32P incorporated in the peptides may be determined by measuring activity on the nitrocellulose dry pads in a scintillation counter.
Furthermore, such “CDKA or homologue or derivative thereof”, when comprising a T161D-type mutation and expressed under control of a shoot specific promoter in Oryza sativa, increases seed yield compared to corresponding wild type plants. This increase in seed yield may be measured in several ways, for example as an increase in the total weight of seeds, as an increase in the number of filled seeds harvested from a plant or as an increased Harvest Index.
The biological and/or functional activity of a CDKA or a homologue thereof according to the present invention includes at least one of having cyclin dependent kinase activity or having yield-increasing activity in plants as described above.
The present invention also provides an isolated mutant A-type cyclin dependent kinase (CDKA), selected from the group consisting of:

    
    
        (a) the amino acid sequence represented by SEQ ID NO: 2;
        (b) a homologue and/or a derivative of a protein as represented by SEQ ID NO: 2, which homologue or derivative is of plant origin and comprises a T161D-type mutation;
        (c) an active fragment of an amino acid sequence as defined in (a) or (b), which active fragment comprises a T161D-type mutation.
    
    


“Active fragments” of an A-type CDK protein encompasses at least 100 amino acid residues of an A-type CDK protein, including a PSTAIRE motif and a T161D-type mutation, which contiguous residues retain similar biological and/or functional activity to the naturally occurring protein that comprises the T161D-type mutation.
A CDKA or a homologue thereof as defined hereinabove is encoded by a CDKA nucleic acid molecule. The nucleic acid encoding a CDKA or a homologue thereof may be any natural or synthetic nucleic acid. Therefore the term “CDKA nucleic acid molecule” or “CDKA gene” as defined herein is any nucleic acid molecule (including those as a result of the degeneration of the genetic code) encoding a CDKA polypeptide or a homologue thereof as defined hereinabove. Examples of CDKA nucleic acid molecules include the one represented by SEQ ID NO: 1, and those encoding the above-mentioned homologues. CDKA nucleic acids and functional variants thereof may be suitable in practicing the methods of the invention, provided that they encode CDKA proteins or homologues thereof comprising a T161D-type mutation. Such functional variant CDKA nucleic acids include portions of a CDKA nucleic acid molecule, allelic variants, splice variants and/or nucleic acids capable of hybridising with a CDKA nucleic acid molecule. The term “functional” in the context of a functional variant refers to a variant (i.e. a portion or a hybridising sequence), which encodes a polypeptide having cyclin-dependent kinase activity and having a T161D-type mutation.
The present invention also provides an isolated nucleic acid molecule selected from the group consisting of:

    
    
        a. a nucleic acid molecule encoding the amino acid sequence represented by SEQ ID NO: 2;
        b. a nucleic acid molecule encoding a homologue, derivative or active fragment of the amino acid molecule represented by SEQ ID NO: 2, which homologue, derivative or fragment is of plant origin and comprises a PSTAIRE motif and a T161D-type mutation;
        c. a nucleic acid molecule capable of hybridising with a nucleic acid of (a) or (b) above, or its complement, wherein the hybridising sequence or the complement thereof encodes a plant CDKA protein that comprises a PSTAIRE motif and a T161D-type mutation;
        d. allelic variants of a nucleic acid according to any of (a) to (c) above, which allelic variants encode a plant CDKA protein comprising a PSTAIRE motif and a T161D-type mutation; and
        e. alternative splice variants of a nucleic acid according to any of (a) to (c), which alternative splice variants encode a plant CDKA protein comprising a PSTAIRE motif and having a T161D-type mutation.
    
    


The term portion as defined herein refers to a piece of a DNA encoding a CDKA, comprising at least 300 nucleotides and which portion encodes a polypeptide having cyclin-dependent kinase activity, having a PSTAIRE motif and having a T161D-type mutation. A portion may be prepared, for example, by making one or more deletions to a CDKA nucleic acid. The portions may be used in isolated form or they may be fused to other coding (or non coding) sequences in order to, for example, produce a protein that combines several activities, one of them being cyclin-dependent kinase activity. When fused to other coding sequences, the resulting polypeptide produced upon translation may be bigger than that predicted for the CDKA fragment. Preferably, the functional portion is a portion of a CDKA nucleic acid, more preferably a portion of the nucleic acid molecule as represented by SEQ ID NO: 1.
Another variant of a CDKA nucleic acid molecule is a nucleic acid molecule capable of hybridising under reduced stringency conditions, preferably under stringent conditions, with a CDKA nucleic acid molecule as hereinbefore defined, which hybridising sequence encodes a CDKA polypeptide comprising a PSTAIRE motif and a T161D-type mutation. Preferably, the hybridising sequence is one that is capable of hybridising to the nucleic acid molecule of SEQ ID NO: 1, or to a nucleic acid encoding one of the above mentioned homologues, or to a portion of any of the aforementioned sequences. Most preferably, the hybridising sequence is capable of hybridising to the nucleic acid molecule of SEQ ID NO: 1.
The term “hybridisation” as defined herein is a process wherein substantially homologous complementary nucleotide sequences anneal to each other. The hybridisation process may occur entirely in solution, i.e. both complementary nucleic acids are in solution. The hybridisation process may also occur with one of the complementary nucleic acids immobilised to a matrix such as magnetic beads, Sepharose beads or any other resin. The hybridisation process can furthermore occur with one of the complementary nucleic acids immobilised to a solid support such as a nitro-cellulose or nylon membrane or immobilised by e.g. photolithography to, for example, a siliceous glass support (the latter known as nucleic acid arrays or microarrays or as nucleic acid chips). In order to allow hybridisation to occur, the nucleic acid molecules are generally thermally or chemically denatured to melt a double strand into two single strands and/or to remove hairpins or other secondary structures from single stranded nucleic acids. The stringency of hybridisation is influenced by conditions such as temperature, salt concentration, ionic strength and hybridisation buffer composition.
“Stringent hybridisation conditions” and “stringent hybridisation wash conditions” in the context of nucleic acid hybridisation experiments such as Southern and Northern hybridisations are sequence dependent and are different under different environmental parameters. The skilled artisan is aware of various parameters which may be altered during hybridisation and washing and which will either maintain or change the stringency conditions.
The Tm is the temperature under defined ionic strength and pH, at which 50% of the target sequence hybridises to a perfectly matched probe. The Tm is dependent upon the solution conditions and the base composition and length of the probe. For example, longer sequences hybridise specifically at higher temperatures. The maximum rate of hybridisation is obtained from about 16° C. up to 32° C. below Tm. The presence of monovalent cations in the hybridisation solution reduce the electrostatic repulsion between the two nucleic acid strands thereby promoting hybrid formation; this effect is visible for sodium concentrations of up to 0.4M. Formamide reduces the melting temperature of DNA-DNA and DNA-RNA duplexes with 0.6 to 0.7° C. for each percent formamide, and addition of 50% formamide allows hybridisation to be performed at 30 to 45° C., though the rate of hybridisation will be lowered. Base pair mismatches reduce the hybridisation rate and the thermal stability of the duplexes. On average and for large probes, the Tm decreases about 1° C. per % base mismatch. The Tm may be calculated using the following equations, depending on the types of hybrids:

    
    
        DNA-DNA hybrids (Meinkoth and Wahl, Anal. Biochem., 138: 267-284, 1984):

Tm=81.5° C.+16.6x log [Na+]a+0.41x%[G/Cb]−500x[Lc]−1−0.61x% formamide

        DNA-RNA or RNA-RNA hybrids:

Tm=79.8+18.5(log10[Na+]a)+0.58 (% G/Cb)+11.8 (% G/Cb)2−820/Lc 

        oligo-DNA or oligo-RNAd hybrids:
    
    


For <20 nucleotides: Tm=2 (ln)
For 20-35 nucleotides: Tm=22+1.46 (ln)
a or for other monovalent cation, but only accurate in the 0.01-0.4 M range.
b only accurate for % GC in the 30% to 75% range.
c L=length of duplex in base pairs.
d Oligo, oligonucleotide; ln, effective length of primer=(no. of G/C)+(no. of A/T).
Note: for each 1% formamide, the Tm is reduced by about 0.6 to 0.7° C., while the presence of 6M urea reduces the Tm by about 30° C.
Specificity of hybridisation is typically the function of post-hybridisation washes. To remove background resulting from non-specific hybridisation, samples are washed with dilute salt solutions. Critical factors of such washes include the ionic strength and temperature of the final wash solution: the lower the salt concentration and the higher the wash temperature, the higher the stringency of the wash. Wash conditions are typically performed at or below hybridisation stringency. Generally, suitable stringent conditions for nucleic acid hybridisation assays or gene amplification detection procedures are as set forth above. More or less stringent conditions may also be selected. Generally, low stringency conditions are selected to be about 50° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. Medium stringency conditions are when the temperature is 20° C. below Tm, and high stringency conditions are when the temperature is 10° C. below Tm. For example, stringent conditions are those that are at least as stringent as, for example, conditions A-L; and reduced stringency conditions are at least as stringent as, for example, conditions M-R. Non-specific binding may be controlled using any one of a number of known techniques such as, for example, blocking the membrane with protein containing solutions, additions of heterologous RNA, DNA, and SDS to the hybridisation buffer, and treatment with Rnase.
Examples of hybridisation and wash conditions are listed in Table 2:











TABLE 2









Wash


Stringency
Polynucleotide
Hybrid Length
Hybridization Temperature
Temperature


Condition
Hybrid ±
(bp)‡
and Buffer†
and Buffer†







A
DNA:DNA
> or equal
65° C. 1xSSC; or 42° C.,
65° C.; 0.3xSSC




to 50
1xSSC and 50% formamide


B
DNA:DNA
<50
Tb*; 1xSSC
Tb*; 1xSSC


C
DNA:RNA
> or equal
67° C. 1xSSC; or 45° C.,
67° C.; 0.3xSSC




to 50
1xSSC and 50% formamide


D
DNA:RNA
<50
Td*; 1xSSC
Td*; 1xSSC


E
RNA:RNA
> or equal
70° C. 1xSSC; or 50° C.,
70° C.; 0.3xSSC




to 50
1xSSC and 50% formamide


F
RNA:RNA
<50
Tf*; 1xSSC
Tf*; 1xSSC


G
DNA:DNA
> or equal
65° C. 4xSSC; or 45° C.,
65° C.; 1xSSC




to 50
4xSSC and 50% formamide


H
DNA:DNA
<50
Th*; 4xSSC
Th*; 4xSSC


I
DNA:RNA
> or equal
67° C. 4xSSC; or 45° C.,
67° C.; 1xSSC




to 50
4xSSC and 50% formamide


J
DNA:RNA
<50
Tj*; 4xSSC
Tj*; 4xSSC


K
RNA:RNA
> or equal
70° C. 4xSSC; or 40° C.,
67° C.; 1xSSC




to 50
6xSSC and 50% formamide


L
RNA:RNA
<50
Tl*; 2xSSC
Tl*; 2xSSC


M
DNA:DNA
> or equal
50° C. 4xSSC; or 40° C.,
50° C.; 2xSSC




to 50
6xSSC and 50% formamide


N
DNA:DNA
<50
Tn*; 6xSSC
Tn*; 6xSSC


O
DNA:RNA
> or equal
55° C. 4xSSC; or 42° C.,
55° C.; 2xSSC




to 50
6xSSC and 50% formamide


P
DNA:RNA
<50
Tp*; 6xSSC
Tp*; 6xSSC


Q
RNA:RNA
> or equal
60° C. 4xSSC; or 45° C.,
60° C.; 2xSSC




to 50
6xSSC and 50% formamide


R
RNA:RNA
<50
Tr*; 4xSSC
Tr*; 4xSSC





‡The “hybrid length” is the anticipated length for the hybridising nucleic acid. When nucleic acids of known sequence are hybridised, the hybrid length may be determined by aligning the sequences and identifying the conserved regions described herein.


†SSPE (1xSSPE is 0.15M NaCl, 10 mM NaH2PO4, and 1.25 mM EDTA, pH 7.4) may be substituted for SSC (1xSSC is 0.15M NaCl and 15 mM sodium citrate) in the hybridisation and wash buffers; washes are performed for 15 minutes after hybridisation is complete. The hybridisations and washes may additionally include 5xDenhardt's reagent, 0.5-1.0% SDS, 100 μg/ml denatured, fragmented salmon sperm DNA, 0.5% sodium pyrophosphate, and up to 50% formamide.


*Tb-Tr: The hybridisation temperature for hybrids anticipated to be less than 50 base pairs in length should be 5-10° C. less than the melting temperature Tm of the hybrids; the Tm is determined according to the above-mentioned equations.


±The present invention also encompasses the substitution of any one, or more DNA or RNA hybrid partners with either a PNA, or a modified nucleic acid.






For the purposes of defining the level of stringency, reference may conveniently be made to Sambrook et al. (2001) Molecular Cloning: a laboratory manual, 3rd Edition Cold Spring Harbor Laboratory Press, CSH, New York or to Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989).
After hybridisation and washing, the duplexes may be detected by autoradiography (when radiolabeled probes were used) or by chemiluminescence, immunodetection, by fluorescent or chromogenic detection, depending on the type of probe labelling. Alternatively, a ribonuclease protection assay may be performed for detection of RNA:RNA hybrids.
The CDKA nucleic acid molecule or variant thereof may be derived from any plant or artificial source. This nucleic acid may be modified from its native form in composition and/or genomic environment through deliberate human manipulation; the CDKA nucleic acids useful in the present invention have at least a mutation causing the T161D substitution. The nucleic acid is preferably of plant origin, whether from the same plant species (for example to the one in which it is to be introduced) or whether from a different plant species. The nucleic acid may be isolated from a monocotyledonous species, preferably from the family Poaceae, further preferably from Oryza sativa. More preferably, the CDKA isolated from Oryza sativa is CDKA;1. Most preferably, the CDKA;1 isolated from Oryza sativa and subsequently mutated is represented by SEQ ID NO: 1 and the CDKA amino acid sequence with the T161D-type mutation is as represented by SEQ ID NO: 2.
The activity of a CDKA polypeptide or a homologue thereof, having a T161D-type mutation, and/or expression of a nucleic acid encoding such a CDKA may be modulated by introducing a genetic modification (preferably in the locus of a CDKA gene). The locus of a gene as defined herein is taken to mean a genomic region which includes the gene of interest and 10 kb up- or downstream of the coding region.
The genetic modification may be introduced, for example, by any one (or more) of the following methods: TILLING, site-directed mutagenesis, directed evolution and homologous recombination or by introducing and expressing in a plant a nucleic acid encoding an A-type CDK polypeptide or a homologue thereof, which CDKA or homologue comprises a PSTAIRE motif and a T161D-type mutation. Following introduction of the genetic modification there follows a step of selecting for increased expression of a nucleic acid encoding a CDK polypeptide with a PSTAIRE motif and a T161D-type mutation and/or selecting for increased activity of a CDK polypeptide with a PSTAIRE motif and a T161D-type mutation, which increase in expression and/or activity gives plants having improved growth characteristics.
A genetic modification may also be introduced in the locus of a CDKA gene using the technique of TILLING (Targeted Induced Local Lesions IN Genomes). This is a mutagenesis technology useful to generate and/or identify, and to eventually isolate mutagenised variants of a nucleic acid molecule encoding an A-type CDK with a T161D-type mutation capable of exhibiting cyclin-dependent kinase activity. TILLING also allows selection of plants carrying such mutant variants. TILLING combines high-density mutagenesis with high-throughput screening methods. The steps typically followed in TILLING are: (a) EMS mutagenesis (Redei and Koncz (1992), In: C Koncz, N-H Chua, J Schell, eds, Methods in Arabidopsis Research. World Scientific, Singapore, pp 16-82; Feldmann et al., (1994) In: E M Meyerowitz, C R Somerville, eds, Arabidopsis. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., pp 137-172; Lightner and Caspar (1998), In: J Martinez-Zapater, J Salinas, eds, Methods on Molecular Biology, Vol. 82. Humana Press, Totowa, N.J., pp 91-104); (b) DNA preparation and pooling of individuals; (c) PCR amplification of a region of interest; (d) denaturation and annealing to allow formation of heteroduplexes; (e) DHPLC, where the presence of a heteroduplex in a pool is detected as an extra peak in the chromatogram; (f) identification of the mutant individual; and (g) sequencing of the mutant PCR product. Methods for TILLING are well known in the art (McCallum Nature Biotechnol. 18, 455-457, 2000, Stemple Nature Rev. Genet. 5, 145-150, 2004).
Site-directed mutagenesis may be used to generate variants of CDKA nucleic acids or portions thereof that retain activity (such as cyclin-dependent kinase activity). Several methods are available to achieve site-directed mutagenesis, the most common being PCR based methods (See for example Ausubel et al., Current Protocols in Molecular Biology. Wiley Eds. http://www.4ulr.com/products/currentprotocols/index.html).
Directed evolution may also be used to generate variants of CDKA nucleic acids. This consists of iterations of DNA shuffling followed by appropriate screening and/or selection to generate variants of CDKA nucleic acids or portions thereof encoding CDKA polypeptides or homologues or portions thereof having a modified biological activity (Castle et al. (2004) Science 304(5674): 1151-4; U.S. Pat. Nos. 5,811,238 and 6,395,547).
TILLING, site-directed mutagenesis and directed evolution are examples of technologies that enable the generation novel alleles and variants of CDKA that retain CDKA function and which are therefore useful in the methods of the invention.
Homologous recombination allows introduction in a genome of a selected nucleic acid at a defined selected position. Homologous recombination is a standard technology used routinely in biological sciences for lower organism such as yeast or the moss Physcomitrella. Methods for performing homologous recombination in plants have been described not only for model plants (Offringa et al. (1990) EMBO J. 9, 3077-3084) but also for crop plants, for example rice (Terada et al., (2002) Nature Biotechnol. 20, 1030-1034; or Iida and Terada (2004) Curr. Opin. Biotechnol. 15, 132-138). The nucleic acid to be targeted (which may be a CDKA nucleic acid molecule or variant thereof as hereinbefore defined) need not be targeted to the locus of a CDKA gene, but may be introduced in, for example, regions of high expression. The nucleic acid to be targeted may be an improved allele used to replace the endogenous gene or may be introduced in addition to the endogenous gene.
A preferred method for introducing a genetic modification (which in this case need not be in the locus of a CDKA gene) is to introduce and express in a plant a nucleic acid encoding a CDKA polypeptide, or a homologue thereof, with a T161D-type mutation. A CDKA polypeptide or a homologue thereof as mentioned above, and suitable for practising the present invention, is one having cyclin-dependent kinase activity and, in increasing order of preference, having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 8, and which CDK polypeptide comprises a PSTAIRE motif and a T161D-type mutation. The nucleic acid to be introduced into a plant may be a portion or a hybridising sequence as hereinbefore defined.
“Homologues” of a protein encompass peptides, oligopeptides, polypeptides, proteins and enzymes having amino acid substitutions, deletions and/or insertions relative to the unmodified protein in question and having similar biological and functional activity as the unmodified protein from which they are derived.
Encompassed by the term “homologues” are orthologous and paralogous sequences, two special forms of homology, which encompass evolutionary concepts used to describe ancestral relationships of genes. Preferably the orthologues and paralogues useful in the present invention have the same structure and activity as an A-type CDK and have the highest similarity to SEQ ID NO: 8 in a reciprocal BLAST search and comprise a T161D-type mutation.
The term “paralogues” relates to homologous genes that result from one or more gene duplications within the genome of a species. Paralogues of a CDKA may easily be identified by performing a BLAST analysis against a set of sequences from the same species as the query sequence.
The term “orthologues” relates to homologous genes in different organisms due to ancestral relationship of these genes. Orthologues in, for example, monocot plant species may easily be found by performing a so-called reciprocal blast search. This may be done by a first blast involving blasting the sequence in question (for example, SEQ ID NO 15 or SEQ ID NO 16, being from Arabidopsis thaliana) against any sequence database, such as the publicly available NCBI database which may be found at: http://www.ncbi.nlm.nih.gov. If orthologues in rice were sought, the sequence in question would be blasted against, for example, the 28,469 full-length cDNA clones from Oryza sativa Nipponbare available at NCBI. BLASTn or tBLASTX may be used when starting from nucleotides or BLASTP or TBLASTN when starting from the protein, with standard default values. The blast results may be filtered. The full-length sequences of either the filtered results or the non-filtered results are then blasted back (second blast) against the sequences of the organism from which the sequence in question is derived, in casu Arabidopsis thaliana. The results of the first and second blasts are then compared. A paralogue is identified if a high-ranking hit from the second blast is from the same species as from which the query sequence is derived; an orthologue is identified if a highest ranking hit is not from the same species as from which the query sequence is derived. Such paralogue or orthologue is also considered a homologue of CDKA, provided that this homologue comprises a serine/threonine kinase domain and comprises a PSTAIRE motif. In the case of large families, ClustalW may be used, followed by the construction of a neighbour joining tree, to help visualize the clustering of related genes and identify orthologues and paralogues.
A homologue may be in the form of a “substitutional variant” of a protein, i.e. where at least one residue in an amino acid sequence has been removed and a different residue inserted in its place. Amino acid substitutions are typically of single residues, but may be clustered depending upon functional constraints placed upon the polypeptide; insertions will usually be of the order of about 1 to 10 amino acid residues. Preferably, amino acid substitutions comprise conservative amino acid substitutions (Table 3). To produce such homologues, amino acids of the protein may be replaced by other amino acids having similar properties (such as similar hydrophobicity, hydrophilicity, antigenicity, propensity to form or break α-helical structures or β-sheet structures). Conservative substitution tables are well known in the art (see for example Creighton (1984) Proteins. W.H. Freeman and Company). The substitutional variant useful in the methods of the present invention is a substitutional variant of a CDKA polypeptide and comprises a PSTAIRE motif and a T161D-type mutation.







TABLE 3







Examples of conserved amino acid substitutions:











Conservative



Residue
Substitutions







Ala
Ser



Arg
Lys



Asn
Gln; His



Asp
Glu



Gln
Asn



Cys
Ser



Glu
Asp



Gly
Pro



His
Asn; Gln



Ile
Leu, Val



Leu
Ile; Val



Lys
Arg; Gln



Met
Leu; Ile



Phe
Met; Leu; Tyr



Ser
Thr; Gly



Thr
Ser; Val



Trp
Tyr



Tyr
Trp; Phe



Val
Ile; Leu










Less conserved substitutions may be made in case the above-mentioned amino acid properties are not so critical.
A homologue may also be in the form of an “insertional variant” of a protein, i.e. where one or more amino acid residues are introduced into a predetermined site in a protein. Insertions may comprise amino-terminal and/or carboxy-terminal fusions as well as intra-sequence insertions of single or multiple amino acids. Generally, insertions within the amino acid sequence will be smaller than amino- or carboxy-terminal fusions, of the order of about 1 to 10 residues. Examples of amino- or carboxy-terminal fusion proteins or peptides include the binding domain or activation domain of a transcriptional activator as used in the yeast two-hybrid system, phage coat proteins, (histidine)6-tag, glutathione S-transferase-tag, protein A, maltose-binding protein, dihydrofolate reductase, Tag 100 epitope, c-myc epitope, FLAG®-epitope, lacZ, CMP (calmodulin-binding peptide), HA epitope, protein C epitope and VSV epitope. The insertional variant useful in the methods of the present invention is a insertional variant of a CDKA polypeptide and comprises a PSTAIRE motif and a T161D-type mutation.
Homologues in the form of “deletion variants” of a protein are characterised by the removal of one or more amino acids from a protein, and encompass active fragments.
Amino acid variants of a protein may readily be made using peptide synthetic techniques well known in the art, such as solid phase peptide synthesis and the like, or by recombinant DNA manipulations. Methods for the manipulation of DNA sequences to produce substitution, insertion or deletion variants of a protein are well known in the art. For example, techniques for making mutations at predetermined sites in DNA are well known to those skilled in the art and include M13 mutagenesis, T7-Gen in vitro mutagenesis (USB, Cleveland, Ohio), QuickChange Site Directed mutagenesis (Stratagene, San Diego, Calif.), PCR-mediated site-directed mutagenesis or other site-directed mutagenesis protocols.
The CDKA polypeptide or homologue thereof with a PSTAIRE motif, may also be a derivative. “Derivatives” include peptides, oligopeptides, polypeptides, proteins and enzymes which may comprise substitutions, deletions or additions of naturally and non-naturally occurring amino acid residues compared to the amino acid sequence of a naturally-occurring form of the protein, for example, as presented in GenBank accession CAA42922 (SEQ ID NO: 8). “Derivatives” of a protein encompass peptides, oligopeptides, polypeptides, proteins and enzymes which may comprise naturally occurring altered, glycosylated, acylated or non-naturally occurring amino acid residues compared to the amino acid sequence of a naturally-occurring form of the polypeptide. A derivative may also comprise one or more non-amino acid substituents compared to the amino acid sequence from which it is derived, for example a reporter molecule or other ligand, covalently or non-covalently bound to the amino acid sequence, such as a reporter molecule which is bound to facilitate its detection, and non-naturally occurring amino acid residues relative to the amino acid sequence of a naturally-occurring protein. The derivative useful in the methods of the present invention is a derivative of a CDKA polypeptide and comprises a PSTAIRE motif and a T161D-type mutation.
The CDK type kinases in plants have a modular structure, consisting of an N-lobe and a C-lobe comprising a catalytic cleft and a T-loop (De Bondt et al. 1993). Therefore, it is envisaged that engineering of the domains of the kinase in such a way that the activity of the CDK protein is retained or modified, may result in the creation of a CDKA mutant that is useful for performing the methods of the invention. A preferred type of variant includes those generated by domain deletion, stacking or shuffling (see for example He et al., Science 288, 2360-2363, 2000; or U.S. Pat. Nos. 5,811,238 and 6,395,547), provided that the resulting CDKA comprises a PSTAIRE motif and a T161D-type mutation.
The CDKA polypeptide or homologue thereof may be encoded by an alternative splice variant of a CDKA nucleic acid molecule or gene. The term “alternative splice variant” as used herein encompasses variants of a nucleic acid sequence in which selected introns and/or exons have been excised, replaced or added. Such variants will be ones that encode polypeptides that comprise a T161D-type mutation and in which the biological activity of the protein is retained, which may be achieved by selectively retaining functional segments of the protein. Such splice variants may be found in nature or may be manmade. Methods for making such splice variants are well known in the art. Preferred splice variants are splice variants derived from the nucleic acid represented by SEQ ID NO 1. Further preferred are splice variants encoding a polypeptide retaining cyclin-dependent kinase activity and having a PSTAIRE motif and a T161D-type mutation.
The homologue may also be encoded by an allelic variant of a nucleic acid encoding a CDKA polypeptide or a homologue thereof, preferably an allelic variant of the nucleic acid represented by SEQ ID NO 1, provided that the polypeptide encoded by the allelic variant has cyclin-dependent kinase activity and comprises a PSTAIRE motif and a T161D-type mutation. Allelic variants exist in nature and encompassed within the methods of the present invention is the use of these natural alleles, provided that these alleles comprise a T161D-type mutation. Allelic variants encompass Single Nucleotide Polymorphisms (SNPs), as well as Small Insertion/Deletion Polymorphisms (INDELs). The size of INDELs is usually less than 100 bp. SNPs and INDELs form the largest set of sequence variants in naturally occurring polymorphic strains of most organisms.
According to a preferred aspect of the present invention, enhanced or increased expression of the CDKA nucleic acid molecule or variant thereof according to the invention is envisaged. Methods for obtaining enhanced or increased expression (overexpression) of genes or gene products are well documented in the art and include, for example, overexpression driven by appropriate promoters, the use of transcription enhancers or translation enhancers. Isolated nucleic acids which serve as promoter or enhancer elements may be introduced in an appropriate position (typically upstream) of a non-heterologous form of a polynucleotide so as to upregulate expression of a CDKA nucleic acid or variant thereof according to the invention. For example, endogenous promoters may be altered in vivo by mutation, deletion, and/or substitution (see, Kmiec, U.S. Pat. No. 5,565,350; Zarling et al., PCT/US93/03868), or isolated promoters may be introduced into a plant cell in the proper orientation and distance from a gene modified according to the present invention so as to control the expression of the gene.
If polypeptide expression is desired, it is generally desirable to include a polyadenylation region at the 3′-end of a polynucleotide coding region. The polyadenylation region may be derived from the natural gene, from a variety of other plant genes, or from T-DNA. The 3′ end sequence to be added may be derived from, for example, the nopaline synthase or octopine synthase genes, or alternatively from another plant gene, or less preferably from any other eukaryotic gene.
An intron sequence may also be added to the 5′ untranslated region or the coding sequence of the partial coding sequence to increase the amount of the mature message that accumulates in the cytosol. Inclusion of a spliceable intron in the transcription unit in both plant and animal expression constructs has been shown to increase gene expression at both the mRNA and protein levels up to 1000-fold (Buchman and Berg, Mol. Cell Biol. 8, 4395-4405 (1988); Callis et al., Genes Dev. 1, 1183-1200 (1987)). Such intron enhancement of gene expression is typically greatest when placed near the 5′ end of the transcription unit. Use of the maize introns Adh1-S intron 1, 2, and 6, the Bronze-1 intron are known in the art. See generally, The Maize Handbook, Chapter 116, Freeling and Walbot, Eds., Springer, N.Y. (1994).
The invention also provides genetic constructs and vectors to facilitate introduction and/or expression of the nucleotide sequences useful in the methods according to the invention.
Therefore, there is provided a gene construct comprising:

    
    
        (i) a CDKA nucleic acid molecule or functional variant thereof, which nucleic acid or variant encodes an A-type CDK comprising a PSTAIRE motif and a T161D-type mutation;
        (ii) one or more control sequence(s) capable of driving expression in a plant of the nucleic acid sequence of (i); and optionally
        (iii) a transcription termination sequence.
    
    


Constructs useful in the methods according to the present invention may be constructed using recombinant DNA technology well known to persons skilled in the art. The gene constructs may be inserted into vectors, which may be commercially available, suitable for transforming into plants and suitable for expression of the gene of interest in the transformed cells.
Plants are transformed with a vector comprising the sequence of interest (i.e., a CDKA nucleic acid or variant thereof according to the present invention). The sequence of interest is operably linked to one or more control sequences (at least to a promoter). The terms “regulatory element”, “control sequence” and “promoter” are all used interchangeably herein and are to be taken in a broad context to refer to regulatory nucleic acid sequences capable of effecting expression of the sequences to which they are ligated. Encompassed by the aforementioned terms are transcriptional regulatory sequences derived from a classical eukaryotic genomic gene (including the TATA box which is required for accurate transcription initiation, with or without a CCAAT box sequence) and additional regulatory elements (i.e. upstream activating sequences, enhancers and silencers) which modulate gene expression in response to developmental and/or external stimuli, or in a tissue-specific manner. Also included within the term is a transcriptional regulatory sequence of a classical prokaryotic gene, in which case it may include a −35 box sequence and/or −10 box transcriptional regulatory sequences. The term “regulatory element” also encompasses a synthetic fusion molecule or derivative which confers, activates or enhances expression of a nucleic acid molecule in a cell, tissue or organ. The term “operably linked” as used herein refers to a functional linkage between the promoter sequence and the gene of interest, such that the promoter sequence is able to initiate transcription of the gene of interest.
Advantageously, any type of promoter may be used to drive expression of the nucleic acid sequence. The promoter may be an inducible promoter, i.e. having induced or increased transcription initiation in response to a developmental, chemical, environmental or physical stimulus. Additionally or alternatively, the promoter may be a constitutive promoter, i.e. a promoter that is expressed predominantly in at least one tissue or organ and predominantly at any life stage of the plant. Additionally or alternatively, the promoter may be a tissue-preferred or cell-preferred promoter, i.e. one that is capable of preferentially initiating transcription in certain tissues, such as the leaves, roots, seed tissue etc, or even in specific cells. Promoters able to initiate transcription only in certain tissues or cells are respectively referred to herein as “tissue-specific”, and “cell-specific”.
Preferably, the CDKA nucleic acid or variant thereof according to the invention is operably linked to a shoot-specific promoter. The term “shoot-specific” as defined herein refers to a promoter that is expressed predominantly in the shoot and at any stage in the life of the plant. The term “shoot” as used in herein encompasses all aerial parts of the plant, including stems and branches, leaves, buds, reproductive organs, including shoot-derived structures such as stolons, corms, rhizomes or tubers. Preferably, the shoot-specific promoter capable of preferentially expressing the nucleic acid throughout the shoot is a weak promoter. Promoter strength and/or expression pattern may be analysed for example by coupling the promoter to a reporter gene and assaying the expression of the reporter gene in various tissues of the plant. One suitable reporter gene well known to persons skilled in the art is beta-glucuronidase. Promoter strength and/or expression pattern can then be compared to that of a well-characterised shoot-specific reference promoter, such as the Cab27 promoter (weak expression, GenBank AP004700), or the putative protochlorophyllid reductase promoter (strong expression, GenBank AL606456). Reference to a “weak promoter” indicates a promoter that drives expression of a coding sequence at a low level, namely at levels of about 1/10,000 transcripts to about 1/100,000 transcripts, to about 1/500,0000 transcripts per cell. Conversely, a “strong promoter” drives expression of a coding sequence at a high level, or at about 1/10 transcripts to about 1/100 transcripts to about 1/1,000 transcripts per cell. Most preferably, the promoter capable of preferentially expressing the nucleic acid throughout the plant is a metallothionein promoter from rice as presented in SEQ ID NO: 6. It should be clear that the applicability of the present invention is not restricted to the CDKA nucleic acid represented by SEQ ID NO: 1, nor is the applicability of the invention restricted to expression of a CDKA nucleic acid when driven by the metallothionein promoter of SEQ ID NO: 6.
Optionally, one or more terminator sequences may also be used in the construct introduced into a plant. The term “terminator” encompasses a control sequence which is a DNA sequence at the end of a transcriptional unit which signals 3′ processing and polyadenylation of a primary transcript and termination of transcription. Additional regulatory elements may include transcriptional as well as translational enhancers. Those skilled in the art will be aware of terminator and enhancer sequences which may be suitable for use in performing the invention. Such sequences would be known or may readily be obtained by a person skilled in the art.
The genetic constructs of the invention may further include an origin of replication sequence, which is required for maintenance and/or replication in a specific cell type. One example is when a genetic construct is required to be maintained in a bacterial cell as an episomal genetic element (e.g. plasmid or cosmid molecule). Preferred origins of replication include, but are not limited to, the f1-ori and colE1.
The genetic construct may optionally comprise a selectable marker gene. As used herein, the term “selectable marker gene” includes any gene which confers a phenotype on a cell in which it is expressed to facilitate the identification and/or selection of cells which are transfected or transformed with a nucleic acid construct of the invention. Suitable markers may be selected from markers that confer antibiotic or herbicide resistance, that introduce a new metabolic trait or that allow visual selection. Examples of selectable marker genes include genes encoding proteins that confer resistance to antibiotics (such as nptII that phosphorylates neomycin and kanamycin, or hpt, phosphorylating hygromycin), to herbicides (for example bar which provides resistance to Basta; aroA or gox providing resistance against glyphosate), or genes that provide a metabolic trait (such as manA that allows plants to use mannose as sole carbon source). Genes encoding visual marker proteins result in the formation of colour (for example β-glucuronidase, GUS), luminescence (such as luciferase) or fluorescence (Green Fluorescent Protein, GFP, and derivatives thereof).
The present invention also encompasses plants or plant cells obtainable by the methods according to the present invention. The present invention therefore provides plants or plant cells obtainable by the method according to the present invention, which plants or plant cells have introduced therein a CDKA nucleic acid or variant thereof, encoding a CDKA comprising a PSTAIRE motif and having a T161D-type mutation.
The invention also provides a method for the production of transgenic plant cells or transgenic plants having improved growth characteristics, comprising introduction and expression in a plant of a CDKA nucleic acid or a variant thereof, encoding a CDKA that comprises a PSTAIRE motif and having a T161D-type mutation.
More specifically, the present invention provides a method for the production of transgenic plants having improved growth characteristics, which method comprises:

    
    
        (i) introducing into a plant or plant cell a nucleic acid encoding an A-type CDK or a homologue thereof comprising a T161D-type mutation; and
        (ii) cultivating the plant cell under conditions promoting plant growth and development.
    
    


The nucleic acid may be introduced directly into a plant cell or into the plant itself (including introduction into a tissue, organ or any other part of a plant). According to a preferred feature of the present invention, the nucleic acid is preferably introduced into a plant by transformation.
The term “transformation” as referred to herein encompasses the transfer of an exogenous polynucleotide into a host cell, irrespective of the method used for transfer. Plant tissue capable of subsequent clonal propagation, whether by organogenesis or embryogenesis, may be transformed with a genetic construct of the present invention and a whole plant regenerated therefrom. The particular tissue chosen will vary depending on the clonal propagation systems available for, and best suited to, the particular species being transformed. Exemplary tissue targets include leaf disks, pollen, embryos, cotyledons, hypocotyls, megagametophytes, callus tissue, existing meristematic tissue (e.g., apical meristem, axillary buds, and root meristems), and induced meristem tissue (e.g., cotyledon meristem and hypocotyl meristem). The polynucleotide may be transiently or stably introduced into a host cell and may be maintained non-integrated, for example, as a plasmid. Alternatively, it may be integrated into the host genome. The resulting transformed plant cell may then be used to regenerate a transformed plant in a manner known to persons skilled in the art.
Transformation of plant species is now a fairly routine technique. Advantageously, any of several transformation methods may be used to introduce the gene of interest into a suitable ancestor cell. Transformation methods include the use of liposomes, electroporation, chemicals that increase free DNA uptake, injection of the DNA directly into the plant, particle gun bombardment, transformation using viruses or pollen and microprojection. Methods may be selected from the calcium/polyethylene glycol method for protoplasts (Krens et al. (1982) Nature 296, 72-74; Negrutiu et al. (1987) Plant Mol. Biol. 8, 363-373); electroporation of protoplasts (Shillito et al. (1985) Bio/Technol 3, 1099-1102); microinjection into plant material (Crossway et al. (1986) Mol. Gen. Genet. 202, 179-185); DNA or RNA-coated particle bombardment (Klein et al. (1987) Nature 327, 70) infection with (non-integrative) viruses and the like. Transgenic rice plants expressing a CDKA according to the present invention are preferably produced via Agrobacterium-mediated transformation using any of the well known methods for rice transformation, such as described in any of the following: published European patent application EP 1198985 A1, Aldemita and Hodges (Planta 199, 612-617, 1996); Chan et al. (Plant Mol. Biol. 22, 491-506, 1993), Hiei et al. (Plant J. 6, 271-282, 1994), which disclosures are incorporated by reference herein as if fully set forth. In the case of corn transformation, the preferred method is as described in either Ishida et al. (Nature Biotechnol. 14, 745-50, 1996) or Frame et al. (Plant Physiol. 129, 13-22, 2002), which disclosures are incorporated by reference herein as if fully set forth.
Generally after transformation, plant cells or cell groupings are selected for the presence of one or more markers which are encoded by plant-expressible genes co-transferred with the gene of interest, following which the transformed material is regenerated into a whole plant.
Following DNA transfer and regeneration, putatively transformed plants may be evaluated, for instance using Southern analysis, for the presence of the gene of interest, copy number and/or genomic organisation. Alternatively or additionally, expression levels of the newly introduced DNA may be monitored using Northern and/or Western analysis, both techniques being well known to persons having ordinary skill in the art.
The generated transformed plants may be propagated by a variety of means, such as by clonal propagation or classical breeding techniques. For example, a first generation (or T1) transformed plant may be selfed to give homozygous second generation (or T2) transformants, and the T2 plants further propagated through classical breeding techniques.
The generated transformed organisms may take a variety of forms. For example, they may be chimeras of transformed cells and non-transformed cells; clonal transformants (e.g., all cells transformed to contain the expression cassette); grafts of transformed and untransformed tissues (e.g., in plants, a transformed rootstock grafted to an untransformed scion).
The present invention clearly extends to any plant cell or plant produced or obtainable by any of the methods described herein, and to all plant parts and propagules thereof. The present invention extends further to encompass the progeny of a primary transformed or transfected cell, tissue, organ or whole plant that has been produced by any of the aforementioned methods, the only requirement being that progeny exhibit the same genotypic and/or phenotypic characteristic(s) as those produced in the parent by the methods according to the invention.
The invention also includes host cells containing an isolated plant CDK nucleic acid or variant thereof, encoding an A-type CDK comprising a T161D-type mutation. Preferred host cells according to the invention are plant cells.
The invention also extends to harvestable parts of a plant according to the invention such as but not limited to seeds, leaves, fruits, flowers, stems, rhizomes, tubers and bulbs. The invention furthermore relates to products derived, preferably directly derived, from a harvestable part of such a plant, such as dry pellets or powders, oil, fat and fatty acids, starch and proteins.
The present invention furthermore encompasses the use of a T161D-type mutation in a CDKA protein for improving the growth characteristics of plants; such improved growth characteristics are as defined herein above.
The present invention also encompasses use of CDKA nucleic acids or variants thereof, and to use of CDKA polypeptides or homologues thereof, which CDKA or homologue comprises a T161D-type mutation, or which CDKA nucleic acid or variant encodes such a protein. One such use relates to improving the growth characteristics of plants, in particular in improving yield, especially seed yield. The seed yield may include any one or more of the following: increased total number of seeds, increased number of filled seeds, increased seed weight, increased harvest index, among others.
CDKA nucleic acids or variants thereof, or CDKA polypeptides or homologues thereof, may find use in breeding programmes in which a DNA marker is identified which may be genetically linked to a CDKA gene or variant thereof. The CDKA or variants thereof, or CDKA or homologues thereof, may be used to define a molecular marker. This DNA or protein marker may then be used in breeding programs to select plants having improved growth characteristics. The CDKA gene or variant thereof may, for example, be a nucleic acid as represented by SEQ ID NO: 1, or a nucleic acid encoding any of the homologues as defined herein.
Allelic variants of a CDKA, which variants comprise a T161D mutation, may also find use in marker-assisted breeding programmes. Such breeding programmes sometimes require introduction of allelic variation by mutagenic treatment of the plants, using for example EMS mutagenesis; alternatively, the programme may start with a collection of allelic variants of so called “natural” origin caused unintentionally. Identification of allelic variants then takes place by, for example, PCR. This is followed by a selection step for selection of superior allelic variants of the sequence in question and which give improved growth characteristics in a plant. Selection is typically carried out by monitoring growth performance of plants containing different allelic variants of the sequence in question, for example, different allelic variants of SEQ ID NO: 1, or of nucleic acids encoding any of the above mentioned homologues. Growth performance may be monitored in a greenhouse or in the field. Further optional steps include crossing plants, in which the superior allelic variant was identified, with another plant. This could be used, for example, to make a combination of interesting phenotypic features.
CDKA nucleic acids or variants thereof according to the invention may also be used as probes for genetically and physically mapping the genes that they are a part of, and as markers for traits linked to those genes. Such information may be useful in plant breeding in order to develop lines with desired phenotypes. Such use of CDKA nucleic acids or variants thereof requires only a nucleic acid sequence of at least 15 nucleotides in length. The CDKA nucleic acids or variants thereof may be used as restriction fragment length polymorphism (RFLP) markers. Southern blots of restriction-digested plant genomic DNA may be probed with the CDKA nucleic acids or variants thereof. The resulting banding patterns may then be subjected to genetic analyses using computer programs such as MapMaker (Lander et al. (1987) Genomics 1, 174-181) in order to construct a genetic map. In addition, the nucleic acids may be used to probe Southern blots containing restriction endonuclease-treated genomic DNAs of a set of individuals representing parent and progeny of a defined genetic cross. Segregation of the DNA polymorphisms is noted and used to calculate the position of the CDKA nucleic acid or variant thereof in the genetic map previously obtained using this population (Botstein et al. (1980) Am. J. Hum. Genet. 32, 314-331).
The production and use of plant gene-derived probes for use in genetic mapping is described in Bernatzky and Tanksley (Genetics 112, 887-898, 1986). Numerous publications describe genetic mapping of specific cDNA clones using the methodology outlined above or variations thereof. For example, F2 intercross populations, backcross populations, randomly mated populations, near isogenic lines, and other sets of individuals may be used for mapping. Such methodologies are well known to those skilled in the art.
The nucleic acid probes may also be used for physical mapping (i.e., placement of sequences on physical maps; see Hoheisel et al. In: Nonmammalian Genomic Analysis: A Practical Guide, Academic press 1996, pp. 319-346, and references cited therein).
In another embodiment, the nucleic acid probes may be used in direct fluorescence in situ hybridization (FISH) mapping (Trask (1991) Trends Genet. 7, 149-154). Although current methods of FISH mapping favour use of large clones (several to several hundred kb; see Laan et al. (1995) Genome Res. 5, 13-20), improvements in sensitivity may allow performance of FISH mapping using shorter probes.
A variety of nucleic acid amplification-based methods of genetic and physical mapping may be carried out using the nucleic acids. Examples include allele-specific amplification (Kazazian (1989) J. Lab. Clin. Med. 11, 95-96), polymorphism of PCR-amplified fragments (CAPS; Sheffield et al. (1993) Genomics 16, 325-332), allele-specific ligation (Landegren et al. (1988) Science 241, 1077-1080), nucleotide extension reactions (Sokolov (1990) Nucleic Acid Res. 18, 3671), Radiation Hybrid Mapping (Walter et al. (1997) Nat. Genet. 7, 22-28) and Happy Mapping (Dear and Cook (1989) Nucleic Acid Res. 17, 6795-6807). For these methods, the sequence of a nucleic acid is used to design and produce primer pairs for use in the amplification reaction or in primer extension reactions. The design of such primers is well known to those skilled in the art. In methods employing PCR-based genetic mapping, it may be necessary to identify DNA sequence differences between the parents of the mapping cross in the region corresponding to the instant nucleic acid sequence. This, however, is generally not necessary for mapping methods.
In this way, generation, identification and/or isolation of improved plants with modulated cyclin-dependent kinase activity displaying improved growth characteristics may be performed.
CDKA nucleic acids or variants thereof or CDKA polypeptides or homologues thereof according to the present invention may also find use as growth regulators. Since these molecules have been shown to be useful in improving the growth characteristics of plants, they would also be useful growth regulators, such as herbicides or growth stimulators. The present invention therefore provides a composition comprising a CDKA or variant thereof or a CDKA polypeptide or homologue thereof, together with a suitable carrier, diluent or excipient, for use as a growth regulator, which CDKA or homologue comprises a T161D mutation, or which CDKA or variant encodes such protein.
The methods according to the present invention result in plants having improved growth characteristics, as described hereinbefore. These advantageous growth characteristics may also be combined with other economically advantageous traits, such as further yield-enhancing traits, tolerance to various stresses, traits modifying various architectural features and/or biochemical and/or physiological features.



DESCRIPTION OF FIGURES
The present invention will now be described with reference to the following figures in which:
FIG. 1 gives a phylogenetic tree of cyclin dependent kinases with a PSTAIRE motif (or A-type CDKs).
FIG. 2 shows the cluster of A-type CDKs of FIG. 1 in more detail.
FIG. 3 details examples of sequences useful in performing the methods according to the present invention. SEQ ID NO 1 and SEQ ID NO 2 represent the nucleotide and protein sequence of the CDKA used in the examples. Start and stop codon are indicated in bold in SEQ ID NO: 1; the mutation is indicated in bold underlined in SEQ ID NO: 1 and 2. SEQ ID NO 3 and SEQ ID NO 4 are primer sequences used for isolating the CDKA;1 nucleic acid. SEQ ID NO: 5 represents the expression cassette used in the present invention, comprising the metallothionein promoter (internal reference PRO0109, nucleotides 1-1208), the coding sequence for the mutated CDKA (internal reference CDS0644—1 (nt 1285-2170) and the terminator (nt 2275-2709). SEQ ID NO: 6 is the sequence of the metallothionein promoter.



EXAMPLES
The present invention will now be described with reference to the following examples, which are by way of illustration alone.
DNA manipulation: unless otherwise stated, recombinant DNA techniques are performed according to standard protocols described in (Sambrook (2001) Molecular Cloning: a laboratory manual, 3rd Edition Cold Spring Harbor Laboratory Press, CSH, New York) or in Volumes 1 and 2 of Ausubel et al. (1994), Current Protocols in Molecular Biology, Current Protocols (http://www.4ulr.com/products/currentprotocols/index.html). Standard materials and methods for plant molecular work are described in Plant Molecular Biology Labfax (1993) by R. D. D. Croy, published by BIOS Scientific Publications Ltd (UK) and Blackwell Scientific Publications (UK).
Example 1
Gene Cloning
The Oryza sativa CDKA;1 was cloned and subsequently mutagenised for introducing the T161D substitution using standard techniques. Next the mutant CDKA;1 (internal code CDS0644-7) was amplified by PCR using Hifi Taq DNA polymerase in standard conditions and primers Prm04553 (SEQ ID NO 3, sense) and Prm04554 (SEQ ID NO 4, reverse complementary), which include the AttB sites for Gateway recombination. The resulting PCR fragment was purified with standard methods. The first step of the Gateway procedure, the BP reaction, was then performed, during which the PCR fragment recombines in vivo with the pDONR201 plasmid to produce, according to the Gateway® terminology, an “entry clone”, p06. Plasmid pDONR201 was purchased from Invitrogen, as part of the Gateway® technology.
Example 2
Vector Construction and Rice Transformation
The entry clone p06 was subsequently used in an LR reaction with p03390, a destination vector used for Oryza sativa transformation. This vector contained as functional elements within the T-DNA borders: a plant selectable marker; a visual marker expression cassette; and a Gateway cassette intended for LR in vivo recombination with the sequence of interest already cloned in the entry clone. A rice metallothionein promoter for shoot specific expression was located upstream of this Gateway cassette.
After the LR recombination step, the resulting expression vector p017, comprising the expression cassette SEQ ID NO: 5, was transformed into the Agrobacterium strain LBA4404 and subsequently to Oryza sativa plants. Transformed rice plants were allowed to grow and were then examined for the parameters described in Example 3.
Example 3
Evaluation of Transformants: Growth Measurements
Approximately 15 to 20 independent TO transformants were generated. The primary transformants were transferred from tissue culture chambers to a greenhouse for growing and harvest of T1 seed. Four events of which the T1 progeny segregated 3:1 for presence/absence of the transgene were retained. For each of these events, 10 T1 seedlings containing the transgene (hetero- and homo-zygotes), and 10 T1 seedlings lacking the transgene (nullizygotes), were selected by visual marker screening. The selected T1 plants were transferred to a greenhouse. Each plant received a unique barcode label to unambiguously link the phenotyping data to the corresponding plant. The selected T1 plants were grown on soil in 10 cm diameter pots under the following environmental settings: photoperiod=11.5 h, daylight intensity=30,000 lux or more, daytime temperature=28° C. or higher, night time temperature=22° C., relative humidity=60-70%. Transgenic plants and the corresponding nullizygotes were grown side-by-side at random positions. From the stage of sowing until the stage of maturity the plants were passed several times through a digital imaging cabinet. At each time point digital images (2048×1536 pixels, 16 million colours) were taken of each plant from at least 6 different angles.
The mature primary panicles were harvested, bagged, barcode-labelled and then dried for three days in the oven at 37° C. The panicles were then threshed and all the seeds collected. The filled husks were separated from the empty ones using an air-blowing device. After separation, both seed lots were then counted using a commercially available counting machine. The empty husks were discarded. The filled husks were weighed on an analytical balance and the cross-sectional area of the seeds was measured using digital imaging. This procedure resulted in the set of seed-related parameters described below.
These parameters were derived in an automated way from the digital images using image analysis software and were analysed statistically. A two factor ANOVA (analyses of variance) corrected for the unbalanced design was used as statistical model for the overall evaluation of plant phenotypic characteristics. An F-test was carried out on all the parameters measured of all the plants of all the events transformed with that gene. The F-test was carried out to check for an effect of the gene over all the transformation events and to verify for an overall effect of the gene, also referred to herein as a “global gene effect”. If the value of the F test showed that the data were significant, than it was concluded that there was a “gene” effect, meaning that not only presence or the position of the gene that was causing the effect. The threshold for significance for a true global gene effect was set at 5% probability level for the F test.
To check for an effect of the genes within an event, i.e., for a line-specific effect, a t-test was performed within each event using data sets from the transgenic plants and the corresponding null plants. “Null plants” or “null segregants” or “nullizygotes” refer to the plants treated in the same way as the transgenic plant, but from which the transgene has segregated. Null plants can also be described as the homozygous negative transformed plants. The threshold for significance for the t-test was set at a 10% probability level. The results for some events can be above or below this threshold. This is based on the hypothesis that a gene might only have an effect in certain positions in the genome, and that the occurrence of this position-dependent effect is not uncommon. This kind of gene effect is also referred to herein as a “line effect of the gene”. The p-value was obtained by comparing the t-value to the t-distribution or alternatively, by comparing the F-value to the F-distribution. The p-value then gives the probability that the null hypothesis (i.e., that there is no effect of the transgene) is correct.
The data obtained in the first experiment were confirmed in a second experiment with T2 plants. Three lines were selected for further analysis. Seed batches from the positive plants (both hetero- and homozygotes) in T1, were screened by monitoring marker expression. For each chosen event, the heterozygote seed batches were then retained for T2 evaluation. Within each seed batch an equal number of positive and negative plants were grown in the greenhouse for evaluation.
A total number of 120 transformed plants were evaluated in the T2 generation, that is 40 plants per event of which 20 were positive for the transgene and 20 negative.
Because two experiments with overlapping events were carried out, a combined analysis was performed. This is useful to check consistency of the effects over the two experiments, and if this is the case, to accumulate evidence from both experiments in order to increase confidence in the conclusion. The method used was a mixed-model approach that takes into account the multilevel structure of the data (i.e. experiment-event-segregants). P-values were obtained by comparing likelihood ratio test to chi square distributions.
Example 4
Evaluation of Transformants: Measurement of Yield-Related Parameters
Upon analysis of the seeds as described above, the inventors found that plants transformed with the CDK gene construct encoding an A-type CDK with a T161D-type mutation had an increased number of filled seeds, an increased total weight of seeds and an increased harvest index compared to plants lacking the CDKA transgene.
Positive results obtained for plants in the T1 generation were again obtained in the T2 generation. In Table 4, data show overall % increases for biomass and TKW, calculated from the data of the individual lines of the T2 generation, and the respective p-values. These T2 data were re-evaluated in a combined analysis with the results for the T1 generation, and the obtained p-values show that the observed effects were significant (Table 4).











TABLE 4









T1 generation
T2 generation
Combined













Overall %
p-value of
Overall %
p-value of
analysis


parameter
increase
F-test
increase
F-test
p-value





Number
62
0.0012
16
0.0230
0.0000


filled seeds


Total weight
60
0.0019
15
0.0392
0.0002


of seeds


Harvest
82
0.0000
14
0.0110
0.0000


Index









Number of Filled Seeds

The number of filled seeds was determined by counting the number of filled husks that remained after the separation step. 3 out of the 4 tested lines showed an increase in filled seed number, amounting to 186%. There was an overall increase of 62% in the number of filled seeds produced by transgenic plants relative to corresponding null segregants, which increase is statistically significant (p-value 0.0012). In the T2 generation, there was increase for 2 of the 3 tested lines. The mean increase for the T2 lines was 14%, this mean increase was also statistically significant (p-value of 0.0230). The combined analysis of T1 and T2 data also confirmed that the global gene effect was highly significant (p-value of 0.0000).
Total Seed Yield
The total seed yield (total weight of seeds) per plant was measured by weighing all filled husks harvested from a plant. 3 of the 4 transgenic T1 lines showed an increase in total seed weight, which varied between 43 and 178%. On average, the increase in seed yield was 60% and this overall effect from the presence of the transgene on seed yield was significant, as evidenced by a P-value from the F test of 0.0019. These results were also observed in the T2 generation. The 3 tested lines had a yield increase between 14 and 48% with an average of 28%. The mean increase (15%) was statistically significant (p-value of 0.0392) and also the combined analysis of the T1 and T2 plants showed there was a global gene effect (p-value of 0.0002).
Harvest Index
The harvest index in the present invention is defined herein as the ratio between the total seed yield and the above ground area (mm2), multiplied by a factor 106. All 4 tested lines showed an increased harvest index, ranging between 9 and 229%. There was a significant overall gene effect (an effect associated with of the presence of the transgene) on harvest index (an overall increase of 82%), with a statistically significant p-value for the F test of 0.0000. Similar results were obtained for T2 plants. The harvest index showed an overall increase of 17% (p-value of 0.0110). Here too, the combined analysis of the T1 and T2 data showed a global gene effect (p-value 0.0000).
Furthermore, there was a tendency for an increased total number of seeds. 3 of the 4 T1 lines showed an increase in the total number of seeds (overall increase 15%), these results were confirmed in the T2 generation (overall increase 9%) and upon a combined analysis these increases were shown to be significant (p-value of 0.0211).

## Claims

1. A method for improving plant growth characteristics relative to a corresponding wild-type plant, comprising introducing and expressing in a plant a nucleic acid encoding an A-type cyclin dependent kinase (CDK) with a T161D-type mutation.

2. The method according to claim 1, wherein said nucleic acid encoding an A-type CDK with a T161D-type mutation is overexpressed in a plant.

3. The method according to claim 1, wherein said nucleic acid encoding an A-type CDK with a T161D-type mutation is derived from a plant.

4. The method according to claim 1, wherein said nucleic acid encoding an A-type CDK having a T161D-type mutation is operably linked to a promoter capable of expressing said nucleic acid predominantly in shoots.

5. The method according to claim 4, wherein said promoter is the rice metallothionein promoter of SEQ ID NO: 6.

6. The method according to claim 1, wherein said improved plant growth characteristic is increased yield relative to corresponding wild type plants.

7. The method according to claim 6, wherein said increased yield is increased seed yield.

8. The method according to claim 7, wherein said increased seed yield is selected from any one or more of: (i) increased seed weight; (ii) increased total number of seeds; (iii) increased number of filled seeds; (iv) increased harvest index.

9. A plant, plant part or plant cell obtained by the method of claim 1.

10. A method for the production of a transgenic plant having improved growth characteristics relative to a corresponding wild-type plant, which method comprises:
(i) introducing into a plant or plant cell a nucleic acid encoding an A-type cyclin dependent kinase (CDK) or a homologue thereof, comprising a T161D-type mutation;
(ii) cultivating the plant or plant cell under conditions promoting plant growth and development.

11. A transgenic plant, plant part or plant cell having improved growth characteristics relative to a corresponding wild-type plant resulting from introducing a nucleic acid encoding an A-type cyclin dependent kinase (CDK) having a T161D-type mutation into said plant.

12. The plant, plant part or plant cell according to claim 11, wherein said plant is a monocotyledonous plant.

13. Harvestable parts of the transgenic plant according to claim 11 and/or products derived from said plants, wherein the harvestable parts and/or products comprise the nucleic acid.

14. The harvestable parts according to claim 13, wherein said harvestable parts are seeds which are true breeding for an isolated nucleic acid encoding an A-type CDK having a T161D-type mutation.

15. The method of claim 1, wherein the nucleic acid is derived from a monocotyledonous plant.

16. The method of claim 15, wherein the monocotyledonous plant is from the family Poaceae.

17. The method of claim 15, wherein the monocotyledonous plant is or sativa.

18. The plant, plant part or plant cell of claim 11, wherein the plant is selected from the group consisting of sugar cane, rice, maize, wheat, barley, millet, rye oats, and sorghum.