Title of Invention

A METHOD FOR INCREASING SEED YIELD OF PLANT

Abstract The present invention concerns a method for modifying the growth characteristics of plants by modulating expression in a plant of a nucleic acid sequence encoding a GRUBX protein and/or modulating activity and/or levels in a plant of a GRUBX protein. The present invention furthermore provides novel GRUBX proteins and nucleic acids encoding such proteins. The invention also relates to constructs comprising GRUBX encoding nucleic acids, and transgenic plants having modified growth characteristics, which plants have modulated expression of a nucleic acid encoding a GRUBX protein.
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1. TITLE OF THE INVENTION
PLANTS HAVING MODIFIED GROWTH CHARACTERISTICS AND METHOD FOR MAKING THE SAME


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The following specification describes the invention



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WO 2005/059147

PCT/EP2004/053594

Plants having modified growth characteristics and method
for making the same
The present invention concerns a method for improving plant growth characteristics. More specifically, the present invention concerns a method for improving plant growth characteristics by modulating expression of a nucleic acid encoding a GRUBX protein and/or by modulating activity and/or levels of a GRUBX protein in a plant. The present invention furthermore provides novel GRUBX proteins and nucleic acids encoding such proteins. The present invention also concerns constructs comprising GRUBX encoding nucleic acids and plants having modulated expression of a nucleic acid encoding a GRUBX protein and/or modulated activity and/or levels of a GRUBX protein, which plants have improved growth characteristics relative to corresponding wild type plants.
Given the ever-increasing world population, and the dwindling area of land available for agriculture, it remains a major goal of agricultural research to improve the efficiency of
15 agriculture and to increase the diversity of plants in horticulture. 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
20 trait being passed on from parent plants. Furthermore, suitable donor species for providing a desired trait may be scarce. Advances in molecular biology have allowed mankind to manipulate 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 led to the
25 development of plants having various improved economic, agronomic or horticultural traits. Traits of particular economic interest are growth characteristics such as high 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 adversely influenced by the typical stresses to which plants or crops are subjected. Such stresses include abiotic stresses, such
30 as temperature stresses caused by atypical high or low temperatures; stresses caused by nutrient deficiency; stresses caused by a lack of or excess water (drought, flooding), stresses caused by chemicals such as fertilisers or insecticides. Typical stresses also include biotic stresses, which may be imposed on plants by other plants (weeds, or the effects of high density planting), by animal pests (including stresses caused by grazing), and by pathogens.
35 Crop yield may not only be increased by combating one or more of the stresses to which the
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crop or plant is subjected, but may also be increased by modifying the inherent growtl mechanisms of a plant. The inherent growth mechanisms of a plant are controlled at several levels and by various metabolic processes. One such process is the control of protein levels in a cell by ubiquitin-mediated protein degradation.
5
Ubiquitination refers to a modification of proteins by conjugation to ubiquitin molecules. The term ubiquitination is often extended to processes that mediate binding of ubiquitin proteins or of proteins that mimic ubiquitin function. Ubiquitination is a versatile tool for eukaryotic cells to control stability, function and the subcelullar localisation of proteins. This mechanism plays a
10 central role in protein degradation, cell cycle control, stress responses, DNA repair, signal transduction, transcriptional regulation and vesicular trafficking. Since ubiquitin mediated protein degradation is at the basis of many cellular processes, it is highly regulated and requires high substrate specificity and ample diversity in downstream effectors. Several ubiquitin-binding proteins are known. These proteins have often a modular domain
15 architecture. For example, ubiquitin-binding proteins typically combine a ubiquitin binding domain with a variable effector domain. Then there are others that do not contain a ubiquitin binding domain, but have a tertiary structure similar to ubiquitin and can therefore mimic certain aspects of ubiquitination (ubiquitin-like domains).
20 The number of ubiquitin-related motifs and domains present in ubiquitin and ubiquitin-like proteins is growing as more information on genome sequences becomes available. Some prototypes of those domains are for example UBA, UBD, UIM and UBX (see for example the Pfam database; Bateman et al., Nucleic Acids Research 30(1):276-280 (2002)). The UBX domain is a sequence approximately 80 amino acid residues long, is of unknown function and
25 is present in proteins of various organisms. Most of these proteins belong to one of five evolutionary conserved families exemplified by the human FAF1, p47, Y33K, REP8, and UBXD1 proteins (Buchberger et al. (2001) J. Mol. Biol. 307, 17-24; Carim-Todd et al. (2001) Biochim. Biophys. Acta 1517, 298-301). Typically, the UBX domain is situated at the C-terminus of a protein.
30
Structural evidence suggests a function of the UBX domain in ubiquitin-related processes; in particular the UBX domain may be involved in protein-protein interactions. Proteins comprising UBX domains are usually predicted to be present mainly in the cytoplasm, but other subcellular localizations have also been reported. For example, phosphorylation which is a specific
35 protein modification used to regulate activity of many proteins, has been shown to also influence transport into the nucleus of FAF-1 (Olsen et al. (2003) FEBS Lett. 546, 218-222.).
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In summary, it has been proposed that animal UBX-containing proteins might be involved in enhanced expression of genes related to apoptosis, cell cycling or targeting of proteins for degradation.
5 In Arabidopsis, the genome of which plant has been fully sequenced, there are at least 15 UBX-containing proteins. They may be classified according to sequence similarity in the FAF1, p47, Y33K and UBXD1 groups, only the group corresponding to REP8 appears not to be present in plants (see Figure 1). As in the animal Kingdom, the UBX domains in plant proteins are present in combination with other domains, like for example SEP, G6PD, PUG, or zinc fingers. UBX-containing proteins and the domain structure of these proteins have been described (see Buchberger (2002) Trends Cell Biol. 12, 216-221) and can be identified by searching using specialised databases such as SMART (Schultz et al. (1998) Proc. Natl. Acad. Sci. USA 95,5857-5864; Letunic et al. (2002) Nucleic Acids Res 30,242-244).
15 PUG domains (in Peptide.N-Glycanases and other putative nuclear UBX-domain-containing proteins; Doerks et al. (2002) Genome Research 12,47-56) co-occur in proteins with domains that are central to ubiquitin-mediated proteolysis, including UBX (in mammals and plants), UBA (in plants) and UBC domains (in Plasmodium). PUG-containing proteins such as PNGases are believed to play a role in the unfolded protein response, an endoplasmatic reticulum (ER)
20 quality control surveillance system that distinguishes aberrant proteins from correctly folded proteins. In some cases, it has been shown that these misfolded and/or unfolded proteins are degraded by a so-called ER-associated degradation mechanism, which involves the ubiquitin-proteasome system (Suzuki et al. (2000) J. Ceil Biol. 149, 1039-1052). Divergent forms of PUG domains are also present in kinases of the IRE1p type which are known to function in the
25 initial stages of the unfolded protein response (Shamu and Walter (1996) EMBO J. 15, 3028-3039).
A recently characterised Arabidopsis protein comprising an UBX domain is PUX1 (Rancour et al. (2004) J. Biol. Chem., online publication 10.1074/jbc.M405498200). PUX1 is a single gene
30 I n Arabidopsis and is probably expressed ubiquitously in planta. The protein was shown to be a non-competitive inhibitor of the AAA-type ATPase CDC48. PUX1 associates through its UBX domain with the non-hexameric form of CDC48, but not with the hexameric CDC48. It is postulated that PUX1 facilitates the disassembly of active hexameric COC48 and that the N-terminal domain of the protein is required for this process, puxl knockout plants showed a
35 faster development to maturity but had no gross morphological abnormalities. Besides PUX1, two other UBX domain comprising proteins, PUX2 and PUX3, were shown to interact with
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CDC48 (Rancour et al., 2004). PUX2 (At2g01650) was previously disclosed in WO 03/08511
(gene and protein sequence described as SEQ ID NO: 1 and SEQ ID NO: 2 respectively).
It has now been found that modulating expression of a nucleic acid encoding a GRUBX protein
(Growth Related UBX domain -comprising protein), and in particular a nucleic acid encoding
the GRUBX protein exemplified by SEQ ID NO: 2, in a plant gives plants having improved
growth characteristics. Therefore, according to a first embodiment of the present invention
there is provided a method for improving the growth characteristics of a plant, comprising
modulating expression in a plant of a nucleic acid encoding a GRUBX protein and/or
modulating activity and/or levels in a plant of a GRUBX protein. According to a preferred
aspect of the invention, the modulated expression is increased expression, the modulated
activity and/or levels are increased activity and/or levels. Optionally, plants having improved
growth characteristics may be selected for.
15 Modulating (enhancing or decreasing) expression of a nucleic acid encoding a GRUBX protein or modulation of the activity and/or levels of the GRUBX protein itself may result from altered expression of a gene and/or altered activity and/or levels of a gene product, namely a polypeptide, in specific cells or tissues. The modulated expression may result from altered expression levels of an endogenous GRUBX gene and/or may result from altered expression
20 of a GRUBX encoding nucleic acid that was previously introduced into a plant. Similarly, modulated levels and/or activity of a GRUBX protein may be the result of altered expression levels of an endogenous GRUBX gene and/or may result from altered expression of a GRUBX encoding nucleic acid that was previously introduced into a plant. Activity may be increased when there is no change in levels of a GRUBX protein, or even when there is a reduction in
25 levels of a GRUBX protein. This may be accomplished by altering the intrinsic properties, for example, by making a mutant that is more active than the wild type. Also encompassed is the inhibition or stimulation of regulatory sequences, or the provision of new regulatory sequences, that drive expression of the native gene encoding a GRUBX or the transgene encoding a GRUBX. Such regulatory sequences may be introduced into a plant. For example, the
30 regulatory sequence introduced into the plant might be a promoter, capable of driving the expression of an endogenous GRUBX gene.
Expression of a gene, and activity and/or levels of a protein may be modulated by introducing a
genetic modification (preferably in the locus of a GRUBX 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.
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The genetic modification may be introduced, for example, by any one (or more) of the following methods: TDNA activation, TILLING, site-directed mutagenesis, homologous recombination or by introducing and expressing in a plant a nucleic acid encoding a GRUBX polypeptide or a homologue thereof. Following introduction of the genetic modification there follows a step of selecting for increased activity of a GRUBX polypeptide, which increase in activity gives plants having improved growth characteristics.
T-DNA activation tagging (Hayashi et al. Science (1992) 1350-1353) involves insertion of T-DNA usually containing a promoter (may also be a translation enhancer or an intron), in the
10 genomic region of the gene of interest or 10 kB up- or downstream of the coding region of a gene in a configuration such that such promoter directs expression of the targeted gene. Typically, regulation of expression of the targeted gene by its natural promoter is disrupted and the gene falls under the control of the newly introduced promoter. The promoter is typically embedded in a T-DNA. This T-DNA is randomly inserted into the plant genome, for example,
15 through Agrobacterium infection and leads to overexpression of genes near to the inserted T-DNA. The resulting transgenic plants show dominant phenotypes due to overexpression of genes close to the introduced promoter. The promoter to be introduced may be any promoter capable of directing expression of a gene in the desired organism, in this case a plant. For example, constitutive, tissue-specific, cell type-specific and inducible promoters are all suitable
20 for use in T-DNA activation.
A genetic modification may also be introduced in the locus of a GRUBX 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
25 a GRUBX nucleic acid capable of exhibiting GRUBX activity. TILLING also allows selection of plants carrying such mutant variants. These mutant variants may even exhibit higher GRUBX activity than that exhibited by the gene in its natural form. 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,
30 Methods in Arabidopsis Research. World Scientific, Singapore, pp 16-82; Feldmann et al., (1994) In: EM Meyerowitz, CR Somerville, eds, Arabidopsis. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp 137-172; Lightner and Caspar (1998), In: J Martinez-Zapater, J Salinas, eds, Methods on Molecular Biology, Vol. 82. Humana Press, Totowa, NJ, pp 91-104); (b) DNA preparation and pooling of individuals; (c) PCR amplification of a region of
35 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
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product. Methods for TILLING are well known in the art (McCallum, Nat Biotechnol. 2000 Ap 18(4):455-7, Stemple, Nature Rev. Genet. 5,145-150,2004).
Site-directed mutagenesis may be used to generate variants of GRUBX nucleic acids or portions thereof that retain GRUBX activity, for example cation transporter 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. http7/www.4ulr.com/products/currentprotocols/index.html).
10 TDNA activation, TILLING and site-directed mutagenesis are examples of technologies that enable the generation of novel alleles and variants of GRUBX that retain GRUBX 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
15 defined selected position. Homologous recombination is a standard technology used routinely in biological sciences for lower organism such as yeast and the moss Physcomitmlla. 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 lida and Terada
20 (2004) Curr. Opin. Biotechnol. 15, 132-138). The nucleic acid to be targeted (which may be a GRUBX nucleic acid molecule or variant thereof as hereinbefore defined) need not be targeted to the locus of a GRUBX 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.
25
A preferred approach for modulating expression of a GRUBX gene, or modulating the activity and/or levels of a GRUBX protein, comprises introducing into a plant an isolated nucleic acid sequence encoding a GRUBX protein or a homologue, derivative or active fragment thereof. The nucleic acid may be introduced into a plant by, for example, transformation. Therefore,
30 according to a preferred aspect of the present invention, there is provided a method for improving the growth characteristics of a plant comprising introducing and expressing a GRUBX encoding nucleic acid into a plant.
The term GRUBX protein, as defined herein, refers to a protein comprising at least an UBX
35 domain, preferably an UBX and a PUG domain, and optionally also a Zinc finger domain.
Preferably, the GRUBX protein is structurally related to the human UBXD1 protein (SPTrEMBL
AAH07414). Preferably, the GRUBX protein is from a plant. Further preferably, the GRUBX
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protein is from the family of Solanaceae, more preferably the GRUBX is a protein from Nicotiana tabacum, most preferably the GRUBX is a protein as represented by SEQ ID NO: 2 or a homologue, derivative or active fragment thereof, which homofogues, derivatives or active fragments have similar biological activity to that of SEQ ID NO: 2. However, it should be understood that GRUBX proteins from monocotyledonous plants could equally well be used in the methods of the present invention, including GRUBX proteins from Zea mays, Saccharum officinarum (SEQ ID NO 4), Oryza sativa (SEQ ID NO 7), Triticum sp., Hordeum sp., and Sorghum sp, since these sequences are related to SEQ ID NO 2 (see Figure 1b).
10 One of the activities of a GRUBX protein is increasing seed yield, in particular increasing harvest index, when a nucleic acid encoding such GRUBX protein is expressed in rice under control of a prolamin promoter as used in the present invention. Advantageously, a GRUBX protein is able to interact with plant CDC48 proteins under conditions described in Rancour et al. (2004).
15
The GRUBX proteins of Nicotiana tabacum were analysed with the SMART tool and were used to screen the Pfam (Version 11.0, November 2003; Bateman et al. (2002) Nucl. Acids Res. 30, 276-280) and InterPro database (Release 7.0, 22 July 2003; Mulder et al. (2003) Nucl. Acids. Res. 31, 315-318). GRUBX proteins comprise an UBX domain (PF00789,
20 SM00166, IPR001012) and a PUG domain (SM00580, IPR006567). The UBX domain, as defined in InterPro, is found in ubiquitin-regulatory proteins, which are members of the ubiquitination pathway, as well as a number of other proteins including FAF-1 (FAS-associated factor 1), the human Rep-8 reproduction protein and several hypothetical proteins from yeast. In Arabidopsis, there are approximately twenty proteins predicted to comprise this domain.
25 The PUG domain is found in protein kinases, N-glycanases and other nuclear proteins in eukaryotes and is postulated to be involved in protein-protein interactions (for a review see Suzuki & Lennarz (2003) Biochem Biophys Res Commun. 302,1-5 and Biochem Biophys Res Commun. 303, 732) and in RNA binding (Doerks et al., 2002). PUG domains are often found together with UBA or UBX domains in Arabidopsis proteins (Doerks et al, 2002). A consensus
30 sequence for the UBX and PUG domains, as defined in the SMART database (Software Version 4.0, sequence database update of 15 September 2003) is given in Figure 2a; Figure 2b shows the UBX and PUG domains of respectively SEQ ID NO 2 and SPTrEMBL Q9ZU93; Figure 2c shows a BLAST alignment of these 2 proteins; and Figures 2d and 2e display an alignment between SEQ ID NO 2 and SEQ ID NO 4, and SEQ ID NO 4 and SEQ ID NO 7,
35 respectively. The PUG and UBX domains are indicated.
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Optionally, a zinc finger domain may be present in the GRUBX protein. Zinc finger domains as defined in InterPro, are nudeic acid-binding protein structures that were first identified in the Xenopus laevis transcription factor TFIIIA. These domains have since been found in numerous nucleic acid-binding proteins. A zinc finger domain is composed of 25 to 30 amino- add residues induding 2 conserved Cys and 2 conserved His residues in a C-2-C-12-H-3-H type motif. The 12 residues separating the second Cys and the first His are mainly polar and basic, indicating that this region is involved in nudeic acid binding. The zinc finger motif is an unusually small, self-folding domain in which Zn is a crucial component of its tertiary structure. All Zinc finger domains bind an atom of Zn in a tetrahedral array resulting in the formation of a
10 finger-like projection which may interad with nudeotides in the major groove of the nudeic add. The Zn binds to the conserved Cys and His residues. Fingers have been found to bind to about 5 base pairs of nudeic add-containing short runs of guanine residues, and have the ability to bind to both RNA and DNA. The zinc finger may thus represent the original nudeic acid binding protein. It has also been suggested that a Zn-centred domain could be used in a
15 protein interaction, for example in protein kinase C. Many dasses of zinc fingers are characterized according to the number and positions of the histidine and cysteine residues involved in the spatial positioning of the zinc atom. In the first dass to be characterized, called C2H2 (IPR007087), the first pair of zinc coordinating residues consists of cysteines, while the second pair are histidines. Another Zinc finger domain (IPR006642) may be of the type found
20 in the Saccharvmyces ceravisiae protein Rad18. Here too, the zinc finger domain is a putative nudeic add binding sequence. The optional Zinc finger domain in the GRUBX protein as defined herein is however not restricted to the C2H2 or Rad18 type, but can be any type of Zinc finger domain.
25 The term GRUBX nudeic acid/gene, as defined herein, refers to any nudeic acid encoding a GRUBX protein, or the complement thereof. The nudeic acid may be derived (either directly or indirectly (if subsequently modified)) from any natural or artificial source provided that the nudeic acid, when expressed in a plant, leads to modulated expression of a GRUBX nudeic acid/gene or modulated activity and/or levels of a GRUBX protein. The nudeic acid may be
30 isolated from a microbial source, such as bacteria, yeast or fungi, or from a plant, algal or animal (induding human) source. Preferably the nudeic add is derived from a eukaryotic organism. Preferably the GRUBX nudeic acid is of plant origin, further preferably of monocotyledonous or dicotyledonous plant origin, more preferably the GRUBX nudeic acid encodes a GRUBX protein from the family of Solanaceae, furthermore preferably the GRUBX
35 nudeic acid is a nudeic acid sequence from Nicotiana tabacum, most preferably the GRUBX nudeic add is a nudeic acid sequence as represented by SEQ ID NO: 1 or a functional portion thereof, or is a nudeic acid sequence capable of hybridising therewith, which hybridising
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sequence encodes a protein having GRUBX protein activity, i.e. similar biological activity to that of SEQ ID NO: 1, and also encompasses nucleic acids encoding an amino acid sequence represented by SEQ ID NO: 2 or homologues, derivatives or active fragments thereof. Alternatively, the nucleic acid encoding a GRUBX protein may be derived from the family of the
5 Poaceae, preferably from Oryza sativa. This nucleic acid may be substantially modified from its native form in composition and/or genomic environment through deliberate human manipulation. The nucleic acid sequence is preferably a homologous nucleic acid sequence, i.e. a structurally and/or functionally related nucleic acid sequence, preferably obtained from a plant, whether from the same plant species or different.
10
The term "functional portion" refers to a portion of a Gf?L/BXgene which encodes a polypeptide that retains the same biological activity of a GRUBX protein and that has an UBX domain, and preferably additionally a PUG domain, and optionally a Zinc finger domain. The term "GRUBX nucleic acid/gene" also encompasses a variant of the nucleic acid encoding a
15 GRUBX protein due to the degeneracy of the genetic code, an allelic variant of the nucleic acid encoding a GRUBX, different splice variant of the nucleic acid encoding a GRUBX and variants that are interrupted by one or more intervening sequences.
Advantageously, the method according to the present invention may also be practised using
20 portions of a nucleic acid sequence encoding a GRUBX protein (such as the sequence represented by SEQ ID NO: 1), or by using sequences that hybridise preferably under stringent conditions to a nucleic acid sequence encoding a GRUBX protein (which hybridising sequences encode proteins having GRUBX activity), or by using homologues, derivatives or active fragments of a GRUBX protein, such as the sequence according to SEQ ID NO: 2, or by
25 using the nucleic acids encoding these homologues, derivatives or active fragments.
Homologues of GRUBX proteins such as the one represented in SEQ ID NO 2 may be found in various eukaryotic organisms. The closest homologues are generally found in the plant kingdom. The Arabidopsis thaliana genome seems to have at least 15 GRUBX proteins, of
30 which the homologue with a sequence submitted in SPTrEMBL Q9ZU93 and Q8LGE5 (MIPS No. At2G01650, or GenBank AY084317 and AAM60904) is the closest homologue to SEQ ID NO: 2, other suitable homologues of SEQ ID NO: 2 include SEQ ID NO 4 from Sacchamm officinamm, encoded by a nucleic acid represented in SEQ ID N03, SEQ ID NO 7 (encoded by the nucleic acid sequence presented in SEQ ID NO 6) from Oryza sativa, and GenBank
35 Accession Nos. BQ198347 and BF778922 from Pinus taeda.


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Methods for the search and identification of GRUBX homologues would be well within In realm of persons skilled in the art. Such methods comprise comparison of the sequences represented by SEQ ID NO 1 or 2, in a computer readable format, with sequences that are available in public databases such as MIPS (Munich Information Center for Protein
5 Sequences, http://mips.gsf.de/), GenBank (http://www.ncbi.nlm.nih.QOv/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
10 (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. The abovementioned homologues were identified using blast default parameters (for example
15 BLASTN Program Advanced Options: G-Cost (to open a gap)=5; E-Cost (to extend a gap)=2; q-Penalty (for a mismatch)=-3; r-Reward (for a match)=1; e-Expectation value (E)=10.0; W-Word size=11; TBLASTN Program Advanced Options: G-Cost (to open a gap)=11; E-Cost (to extend a gap)=1; e-Expectation value (E)=10.0; W-Word size=3). As more genomes are being sequenced, it is expected that many more GRUBX homologues will be identifiable.
20
The sequence represented by SEQ ID NO: 6 was hitherto unknown. There is therefore provided in a second embodiment of the invention an isolated nucleic acid sequence comprising:
(a) a nucleic acid sequence represented by SEQ ID NO: 6, or the complement strand
25 thereof;
(b) a nucleic acid sequence encoding an amino acid sequence represented by SEQ ID NO: 7, or homologues, derivatives or active fragments thereof;
(c) a nucleic acid sequence capable of hybridising (preferably under stringent conditions) with a nucleic acid sequence of (i) or (ii) above, which hybridising
30 sequence preferably encodes a protein having GRUBX activity;
(d) a nucleic acid sequence according to (i) to (iii) above which is degenerate as a result of the genetic code;
(e) a nucleic acid which is an allelic variant of the nucleic acid sequences according to (a) to (d);
35 (0 a nucleic acid which is an alternative splice variant of the nucleic acid sequences
according to (a) to (e);
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(g) a nucleic acid sequence which has 75.00%, 80.00%, 85.00%, 90.00%, 95.00%,
96.00%, 97.00%, 98.00% or 99.00% sequence identity to any one or more of the
sequence defined in (a) to (f);
(h) a portion of a nucleic acid sequence according to any one of (a) to (g) above, which
5 portion preferably encodes a protein having GRUBX activity.
The sequence represented by SEQ ID NO: 4 was assembled from 4 EST sequences
(CA154270, CA144028, BQ535511 & CA184742) and was hitherto unknown. There is
therefore provided an isolated GRUBX protein selected from the group consisting of:
10 (i) a polypeptide as given in SEQ ID NO 4;
(ii) a polypeptide as given in SEQ ID NO 7;
(iii) a polypeptide with an amino acid sequence which has at least 40.00% sequence
identity, preferably 50.00%, 60.00%, 70.00% sequence identity, more preferably
80% or 90% sequence identity, most preferably 95.00%, 96.00%, 97.00%, 98.00%
15 or 99.00% sequence identity to the amino acid sequence as given in SEQ ID NO 4
or 7;
(iv) a polypeptide comprising at least an UBX domain, preferably an UBX and a PUG
domain, and optionally a Zinc finger domain;
(v) a homologue, a derivative, an immunologically active and/or functional fragment of
20 a protein as defined in any of (i) to (iv),
with the proviso that the polypeptide sequence is not a sequence as represented by SEQ ID NO 2, or database entries Q9ZU93, AAR01744, Q9D7L9, Q9BZV1, Q99PL6, ENSANGP00000020442, Q7SXA8, Q9V8K8, Q96IK9, ENSRNOP00000037228, or AAH07414.
25
The term GRUBX includes proteins homologous to the GRUBX as presented in SEQ ID NO 2. Accordingly, preferred homologues to be used in the methods of the present invention comprise at least an UBX domain, preferably they comprise an UBX and a PUG domain. "Homologues" of a GRUBX protein encompass peptides, oligopeptides, polypeptides, proteins
30 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. 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 a-helicai structures or B-35 sheet structures). Conservative substitution tables are well known in the art (see for example Creighton (1984) Proteins. W.H. Freeman and Company).
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The homologues useful in the methods according to the invention have at least 40.00% sequence identity or similarity (functional identity) to the unmodified protein, alternatively at least 50.00% sequence identity or similarity to an unmodified protein, alternatively at least 60.00% sequence identity or similarity to an unmodified protein, alternatively at least 70.00%
5 sequence identity or similarity to an unmodified protein. Typically, the homologues have at least 80% sequence identity or similarity to an unmodified protein, preferably at least 85.00% sequence identity or similarity, further preferably at least 90.00% sequence identity or similarity to an unmodified protein, most preferably at least 95.00%, 96.00%, 97.00%, 98.00% or 99.00% sequence identity or similarity to an unmodified protein. The percentage of identity
10 can be calculated using alignment programs such as GAP. Despite what may appear to be a relatively low sequence homology (as low as approximately 40.00%), GRUBX proteins are highly conserved in structure, with all full-length proteins having at least an UBX domain, preferably an UBX domain and a PUG domain, and further optionally a Zinc finger domain. GRUBX proteins in other plant species may therefore easily be found (as evidenced by the
15 above-mentioned novel sequences of rice and sugar cane).
Homologous proteins can be grouped in "protein families". A protein family can be defined by functional and sequence similarity analysis, such as, for example, Clustal W. A neighbour-joining tree of the proteins homologous to GRUBX can be generated by the Clustal W program
20 and gives a good overview of its structural and ancestral relationship (see for example Figure 1a and b, constructed with Vector NTI Suite 5.5, Informax). In a particular embodiment of the present invention, the GRUBX homologue(s) belong(s) to the same protein family as the protein corresponding to SEQ ID NO 2. In the Arabidopsis genome a preferred family member of the GRUBX protein was identified
25 (Q9ZU93, GenBank Refseq NM_126226). Also in other plants such as rice, sugarcane or other monocotyledonous plants, family members of the GRUBX protein were identified as shown above. Advantageously also these family members are useful in the methods of the present invention.
30 Two special forms of homology, orthologous and paralogous, are evolutionary concepts used to describe ancestral relationships of genes. The term "paralogous" relates to homologous genes that result from one or more gene duplications within the genome of a species. The term "orthologous" relates to homologous genes in different organisms due to ancestral relationship of these genes. The term "homologues" as used herein also encompasses
35 paralogues and orthologues of the proteins useful in the methods according to the invention. Orthologous genes can be identified by querying one or more gene databases with a query gene of interest, using for example the BLAST program. The highest-ranking subject genes
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that result from the search are then again subjected to a BLAST analysis, and only those subject genes that match again with the query gene are retained as true orthologous genes. If for example orthologues in rice were sought, the sequence in question would be blasted against the 28,469 full-length cDNA clones from Oryza sativa Nipponbare available at NCBI.
5 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 Nicotiana tabacum. The results of the first and second blasts
10 are then compared. An orthologue is found when the results of the second blast give as hits with the highest similarity a query GRUBX nucleic acid or GRUBX polypeptide. If for a specific query sequence the highest hit is found with a paralogue of GRUBX then such query sequence is also considered a homologue of GRUBX, provided that this homologue has GRUBX activity and comprises at least an UBX domain, preferably an UBX domain and a PUG domain, and
15 optionally also a Zinc finger domain. The results may be further refined when the resulting sequences are analysed with ClustalW and visualised in a neighbour joining tree. The method can be used in identifying orthologues in many different species.
A further way to identify a functional orthologue within a group of related proteins is to
20 determine the expression pattern and tissue distribution of the members of this protein family, whereby sequences present in the same tissues and with a similar expression pattern are expected to perform related functions. A further way to identify functional homologues of a protein is by identifying sequences with a similar conserved domain structure. Proteins carrying the same domains and particularly when the distribution of the domains is conserved,
25 are expected to perform similar functions. Thus, similarities in chemical structure and in regulation (expression pattern, tissue specificity) could be useful to identify functional homologues of GRUBX.
"Homologues" of GRUBX encompass proteins having amino acid substitutions, insertions
30 and/or deletions relative to the unmodified protein.
"Substitutional variants" of a protein are those in which 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
35 constraints placed upon the polypeptide; insertions will usually be of the order of about 1 to 10 amino acid residues, and deletions will range from about 1 to 20 residues. Preferably, amino acid substitutions comprise conservative amino acid substitutions.
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"Insertional variants" of a protein are those in which one or more amino acid residues are introduced into a predetermined site in a protein. Insertions can comprise amino-tenminal and/or carboxy-terminal fusions as well as intra-sequence insertions of single or multiple amino
5 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, (histidineytag, glutathione S-transferase-tag, protein A, maltose-binding protein, dihydrofolate
10 reductase, Tag-100 epitope, c-myc epitope, FLAG®-epitope, lacZ, CMP (calmodulin-binding peptide), HA epitope, protein C epitope and VSV epitope.
"Deletion variants" of a protein are characterised by the removal of one or more amino acids from the protein. Amino acid variants of a protein may readily be made using peptide synthetic
15 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 substitution 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,
20 Cleveland, OH), QuickChange Site Directed mutagenesis (Stratagene, San Diego, CA), PCR-mediated site-directed mutagenesis or other site-directed mutagenesis protocols.
The term "derivatives" refers to peptides, oligopeptides, polypeptides, proteins and enzymes which may comprise substitutions, deletions or additions of naturally and non-naturally
25 occurring amino acid residues compared to the amino acid sequence of a naturally-occurring form of the protein, for example, as presented in SEQ ID NO: 2 or 4. "Derivatives" of GRUBX 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
30 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.
35
"Active fragments" of a GRUBX protein encompasses at least 80 contiguous amino acid residues of a protein, which residues retain similar biological and/or functional activity to the
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naturally occurring protein. The active fragment at least comprises an UBX domain, preferably the active fragment comprise an UBX and a PUG domain.
Advantageously, the method according to the present invention may also be practised using portions of a DNA or nucleic acid sequence, which portions encode a polypeptide retaining GRUBX activity. Portions of a DNA sequence refer to a piece of DNA derived or prepared from an original (larger) DNA molecule, which DNA portion, when expressed in a plant, gives rise to plants having improved growth characteristics. The portion comprises at least 200 nucleotides, and comprises at least a sequence encoding an UBX domain, preferably an UBX
10 domain and a PUG domain, and optionally a Zinc finger domain. A portion may be prepared, for example, by making one 57 moreTdeletionsi'fd a GRUBX nucleic acid molecule. The portion may comprise many genes, with or without additional control elements, or may contain just spacer sequences etc. The portion may be in isolated form or it may be fused to other coding (or non-coding) sequences in order to, for example, produce a protein that combines several
15 activities, one of them being increasing seed yield when expressed in plants under the control of a prolamin promoter. Preferably, the portion is of any one of SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 6.
The present invention also encompasses nucleic acid sequences capable of hybridising with a
20 nucleic acid sequence encoding a GRUBX protein, which nucleic acid sequences may also be useful in practising the methods according to the invention. The term "hybridisation" as defined herein is a process wherein substantially homologous complementary nucleotide sequences anneal to each other. The hybridisation process can occur entirely in solution, i.e. both complementary nucleic acids are in solution. Tools in molecular biology relying on such a
25 process include the polymerase chain reaction (PCR; and all methods based thereon), subtractive hybridisation, random primer extension, nuclease S1 mapping, primer extension, reverse transcription, cDNA synthesis, differential display of RNAs, and DNA sequence determination. The hybridisation process can also occur with one of the complementary nucleic acids immobilised to a matrix such as magnetic beads, Sepharose beads or any other
30 resin. Tools in molecular biology relying on such a process include the isolation of poly (A+) mRNA. 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, for example, photolithography to, for example, a siliceous glass support (the latter known as nucleic acid arrays or microarrays or as nucleic acid chips). Tools in molecular
35 biology relying on such a process include RNA and DNA gel blot analysis, colony hybridisation, plaque hybridisation, in situ hybridisation and micro array hybridisation. In order to allow hybridisation to occur, the nucleic acid molecules are generally thermally or chemically
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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 and hybridisation buffer composition.
5
For applications requiring high selectivity, one skilled in the art will typically desire to employ relatively stringent conditions to form the hybrids, for example, one will select relatively low salt and/or high temperature conditions, such as provided by about 0.02 M to about 0.15 M NaCI at temperatures of about 50°C to about 70°C. High stringency conditions for hybridisation thus
10 include high temperature and/or low salt concentration (salts include NaCI and Na3-citrate) but may also be influenced by the inclusion of formamide in the hybridisation buffer and/or lowering the concentration of compounds such as SDS (sodium dodecyl sulphate) in the hybridisation buffer and/or exclusion of compounds such as dextran sulphate or polyethylene glycol (promoting molecular crowding) from the hybridisation buffer. Sufficiently low stringency
15 hybridisation conditions are particularly preferred for the isolation of nucleic acids homologous to the DNA sequences of the invention defined supra. Elements contributing to homology include allelism, degeneration of the genetic code and differences in preferred codon usage.
"Stringent hybridisation conditions" and "stringent hybridisation wash conditions" in the context
20 of nucleic acid hybridisation experiments such as Southern and Northern hybridisations are sequence dependent and are different under different environmental parameters. For example, longer sequences hybridise specifically at higher temperatures. The Tm is the temperature under defined ionic strength and pH, at which 50% of the target sequence hybridises to a perfectly matched probe. Specificity is typically the function of post-
25 hybridisation washes. Critical factors of such washes include the ionic strength and temperature of the final wash solution.
Generally, stringent 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. The Tm is the 30 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 of the probe, and may be calculated using the following equation:
Tm= 79.8°C + (18-Sxlogpsla4]) + (58.4°Cx%[G+C]) - (820x(#bp in duplex)"1) - (0.5x% formamide)
35
More preferred stringent conditions are when the temperature is 20°C below Tm, and the most preferred stringent conditions are when the temperature is 10°C below Tm. Non-specific
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binding may also be controlled using any one of a number of known techniques such as blocking the membrane with protein-containing solutions, additions of heterologous RNA, DNA, and SDS to the hybridisation buffer, and treatment with Rnase.
5 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.
10 For the purposes of defining the level of stringency, reference can 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). An example of low stringency conditions is 4-6x SSC / 0.1-0.5% w/v SDS at 37-45°C for 2-3 hours. Depending on the source and concentration of the nucleic acid
15 involved in the hybridisation, alternative conditions of stringency may be employed such as medium stringent conditions. Examples of medium stringent conditions include 1-4x SSC / 0.25% w/v SDS at £ 45°C for 2-3 hours. An example of high stringency conditions includes 0.1-1x SSC / 0.1% w/v SDS at 60°C for 1-3 hours. The skilled artisan is aware of various parameters which may be altered during hybridisation and washing and which will either
20 maintain or change the stringency conditions. For example, another stringent hybridisation condition is hybridisation at 4x SSC at 65°C, followed by a washing in 0.1 x SSC, at 65°C for about one hour. Alternatively, another stringent hybridisation condition is 50% formamide, 4x SSC, at 42°C. Still another example of stringent conditions include hybridisation at 62°C in 6x SSC, 0.05x BLOTTO and washing at 2x SSC, 0.1 % w/v SDS at 62°C.
25
The methods according to the present invention may also be practiced using an alternative splice variant of a nucleic acid sequence encoding a GRUBX protein. 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
30 ones in which the biological activity of the protein remains unaffected, which can be achieved by selectively retaining functional segments of the protein. Such splice variants may be found in nature or can be manmade. Methods for making such splice variants are well known in the art. Therefore according to another aspect of the present invention, there is provided, a method for improving the growth characteristics of plants, comprising modulating expression in
35 a plant of an alternative splice variant of a nucleic acid sequence encoding a GRUBX protein and/or by modulating activity and/or levels of a GRUBX protein encoded by the alternative
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splice variant. Preferably, the splice variant is a splice variant of the sequence represented be SEQ ID NO: 1.
Advantageously, the methods according to the present invention may also be practiced using
5 allelic variants of a nucleic acid sequence encoding a GRUBX protein, preferably an allelic variant of a sequence represented by SEQ ID NO: 1. Allelic variants exist in nature and encompassed within the methods of the present invention is the use of these natural alleles. 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).
10 SNPs and INDELs form the largest set of sequence variants in naturally occurring polymorphic strains of most organisms.
The use of these allelic variants in particular conventional breeding programmes, such as in marker-assisted breeding is also encompassed by the present invention; this may be in
15 addition to their use in the methods according to the present invention. Such breeding programmes sometimes require the introduction of allelic variations in the plants by mutagenic treatment of a plant. One suitable mutagenic method is EMS mutagenesis. Identification of allelic variants then may take place by, for example, PCR. This is followed by a selection step for selection of superior allelic variants of the GRUBX sequence in question and which give
20 rise to 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. Monitoring growth performance can be done 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
25 could be used, for example, to make a combination of interesting phenotypic features.
Therefore, as mutations in the GRUBX gene may occur naturally, they may form the basis for selection of plants showing higher yield. Accordingly, as another aspect of the invention, there is provided a method for the selection of plants having improved growth characteristics, which method is based on the selection of superior allelic variants of the GRUBX sequence and
30 which give rise to improved growth characteristics in a plant.
The methods according to the present invention may also be practiced by introducing into a plant at least a part of a (natural or artificial) chromosome (such as a Bacterial Artificial Chromosome (BAC)), which chromosome contains at least a gene/nucleic acid sequence
35 encoding a GRUBX protein (such as SEQ ID NO: 1 or SEQ ID NO 3), preferably together with one or more related gene family members and/or nucleic acid sequence(s) encoding regulatory proteins for GRUBX expression and/or activity. Therefore, according to a further
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aspect of the present invention, there is provided a method for improving the growth characteristics of plants by introducing into a plant at least a part of a chromosome comprising at least a gene/nucleic acid encoding a GRUBX protein.
5 According to another aspect of the present invention, advantage may be taken of the nucleic acid encoding a GRUBX protein in breeding programmes. The nucleic acid sequence may be on a chromosome, or a part thereof, comprising at least the nucleic acid sequence encoding the GRUBX protein and preferably also one or more related family members. In an example of such a breeding programme, a DNA marker is identified which may be genetically linked to a
10 gene capable of modulating expression of a nucleic acid encoding a GRUBX protein in a plant, which gene may be a gene encoding the GRUBX protein itself or any other gene which may directly or indirectly influence expression of the gene encoding a GRUBX protein and/or activity of the GRUBX protein itself. This DNA marker may then be used in breeding programs to select plants having improved growth characteristics.
15
The present invention therefore extends to the use of a nucleic acid sequence encoding a GRUBX protein in breeding programs.
GRUBX nucleic acids or variants thereof or GRUBX polypeptides or homologues thereof may 20 find use in breeding programmes in which a DNA marker, a desired trait or a Quantitative Trait
Locus (QTL), is identified which may be genetically linked to a GRUBX gene or variant thereof.
This desirable trait or QTL may comprise a single gene or a cluster of linked genes that affect
the desirable trait The GRUBX or variants thereof or GRUBX or homologues thereof may be
used to define a molecular marker. This DNA or protein marker may then be used in breeding 25 programmes to select plants having improved growth characteristics. The GRUBX 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 above mentioned homologues.
Allelic variants of a GRUBX may also find use in marker-assisted breeding programmes. Such
30 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
35 and which give rise to improved growth characteristics in a plant, such as increased harvest index. 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
WO 2005/059147 PCT/EP2004/053594
SEQ ID NO: 1, or of nucleic acids encoding any of the above mentioned plant homologuesi Growth performance may be monitored in a greenhouse or in the field. Further optional steps include crossing plants, in which the superior allelic variant resulting in increased GRUBX activity was identified, with another plant. This could be used, for example, to make a
5 combination of interesting phenotypic features.
A GRUBX nucleic acid or variant thereof 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
10 phenotypes. Such use of GRUBX nucleic acids or variants thereof requires only a nucleic acid sequence of at least 10 nucleotides in length. The GRUBX 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 GRUBX nucleic acids or variants thereof. The resulting banding patterns may then be subjected to genetic analyses
15 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 GRUBX nucleic acid or
20 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 Bematzky and Tanksley (Plant Mol. Biol. Reporter 4, 37-41, 1986). Numerous publications
25 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.
30 The nucleic acid probes may also be used for physical mapping (i.e., placement of sequences on physical maps; see Hoheisel et al. In: Non-mammalian 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
35 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
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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
5 carried out using the nudeic adds. Examples indude 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), nudeotide extension reactions (Sokolov (1990) Nudeic Acid Res. 18, 3671), Radiation Hybrid Mapping (Walter et al. (1997) Nat. Genet. 7, 22-28) and Happy
10 Mapping (Dear and Cook (1989) Nudeic Add Res. 17, 6795-6807). For these methods, the sequence of a nudeic 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
15 in the region corresponding to the instant nudeic acid sequence. This, however, is generally not necessary for mapping methods.
In this way, generation, identification and/or isolation of improved plants with altered GRUBX activity displaying improved growth characteristics can be performed.
20
According to another feature of the present invention, there is provided a method for improving
plant growth characteristics, comprising modulating expression in a plant of a nudeic add
sequence encoding a GRUBX protein and/or modulating levels and/or activity of a GRUBX
protein, wherein said nudeic acid sequence and said protein indudes variants chosen from:
25 (i) an alternative splice variant of a nudeic add sequence encoding a GRUBX protein
or wherein said GRUBX protein is encoded by a splice variant;
(ii) an allelic variant of a nudeic add sequence encoding a GRUBX protein or wherein
said GRUBX protein is encoded by an allelic variant;
(iii) a nudeic add sequence encoding a GRUBX protein and that is comprised on at
30 least a part of an artificial chromosome, which artificial chromosome preferably also
comprises one or more related gene family members; (iv) a functional portion of a GRUBX encoding nudeic acid; (v) sequence capable of hybridising to a GRUBX encoding nudeic add; (vi) homologues, derivatives and active fragments of a GRUBX protein.
35
According to a preferred aspect of the present invention, enhanced or increased expression of a nudeic acid is envisaged. Methods for obtaining enhanced or increased expression of
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genes or gene products are well documented in the art and include, for example* overexpression driven by a (strong) promoter, 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
5 polynucleotide so as to upregulate expression of a GRUBX nucleic acid or variant thereof. 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 of the present invention so as to control the expression of the gene. Preferably, the
10 nucleic acids useful in the present invention are overexpressed in a plant or plant cell. The term overexpression as used herein means any form of expression that is additional to the original wild-type expression level. Preferably the nucleic acid to be introduced into the plant and/or the nucleic acid that is to be overexpressed in the plants is in a sense direction with respect to the promoter to which it is operably linked. Preferably, the nucleic acid to be
15 overexpressed encodes a GRUBX protein, further preferably the nucleic acid sequence encoding the GRUBX protein is isolated from a dicotyledonous plant, preferably of the family Solanaceae, further preferably wherein the sequence is isolated from Nicotiana tabacum, most preferably the nucleic acid sequence is as represented by SEQ ID NO: 1 or a portion thereof, or encodes an amino acid sequence as represented by SEQ ID NO: 2 or a homologue,
20 derivative or active fragment thereof. Alternatively, the nucleic acid sequence encoding the GRUBX protein is as represented by MIPS No. At2g01650, SEQ ID NO: 3 or 6, or is a portion thereof, or encodes an amino acid sequence as represented by Q9ZU93, SEQ ID NO: 4 or 7, or encodes a homologue, derivative or active fragment thereof. It should be noted that the applicability of the invention does not rest upon the use of the nucleic acid represented by SEQ
25 ID NO: 1, nor upon the nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 2, but that other nucleic acid sequences encoding homologues, derivatives or active fragments of SEQ ID NO: 2, or portions of SEQ ID NO: 1, or sequences hybridising with SEQ ID NO: 1 may be used in the methods of the present invention. In particular, the nucleic acids useful in the methods of the present invention encode proteins comprising at least an UBX
30 domain, preferably an UBX domain and a PUG domain, and optionally also a Zinc finger domain.
According to a further embodiment of the present invention, genetic constructs and vectors to facilitate introduction and/or expression of the nucleotide sequences useful in the methods
35 according to the invention are provided. Therefore, according to a third embodiment of the present invention, there is provided a gene construct comprising: (i) a nucleic acid encoding a GRUBX protein;
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(ii) one or more control sequences capable of regulating expression of the nucleic acid
sequence of (i); and optionally (iii) a transcription termination sequence, provided that said nucleic acid encoding a GRUBX protein is not the nucleic acid represented
5 in GenBank Accession number AX927140.
Constructs useful in the methods according to the present invention may be created 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
10 plants and suitable for expression of the gene of interest in the transformed cells. The genetic construct can be an expression vector wherein the nucleic acid sequence is operably linked to one or more control sequences allowing expression in prokaryotic and/or eukaryotic host cells.
According to a preferred embodiment of the invention, the genetic construct is an expression
vector designed to overexpress the nucleic acid sequence. The nucleic acid sequence may be a nucleic acid sequence encoding a GRUBX protein or a homologue, derivative or active fragment thereof, such as any of the nucleic acid sequences described hereinbefore. A preferred nucleic acid sequence is the sequence represented by SEQ ID NO: 1 or a portion thereof or sequences capable of hybridising therewith or a nucleic acid sequence encoding a
20 sequence represented by SEQ ID NO: 2 or a homologue, derivative or active fragment thereof. Preferably, this nucleic acid is cloned in the sense orientation relative to the control sequence to which it is operably linked.
Plants are transformed with a vector comprising the sequence of interest (i.e., the nucleic acid
25 sequence capable of modulating expression of nucleic acid encoding a GRUBX protein), which
sequence is operably linked to one or more control sequences (at least a promoter). The
terms "regulatory element", "control sequence" and "promoter" are all used herein
interchangeably 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.
30 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 alter
gene expression in response to developmental and/or external stimuli, or in a tissue-specific
35 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
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synthetic fusion molecule or derivative which confers, activates or enhances expression of 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.
5
Advantageously, any type of promoter may be used to drive expression of the nucleic acid sequence depending on the desired outcome. Suitable promoters include promoters that are active in monocotyledonous plants such as rice or maize.
10 Preferably, the nucleic acid sequence capable of modulating expression of a gene encoding a GRUBX protein is operably linked to a seed-preferred promoter. The term "seed-preferred" as defined herein refers to a promoter that is expressed predominantly in seed tissue, but not necessarily exclusively in this tissue. The term "seed-preferred" encompasses all promoters that are active in seeds. Seed tissue encompasses any part of the seed including the
15 endosperm, aleurone or embryo. Preferably, the seed-preferred promoter is a prolamin promoter, or a promoter of similar strength and/or a promoter with a similar expression pattern. Most preferably, the prolamin promoter is as represented by nucleotides 1-654 in the expression cassette of SEQ ID NO: 5. Promoter strength and/or expression pattern can be analysed for example by coupling the promoter to a reporter gene and assay the expression of
20 the reporter gene in various tissues of the plant. One suitable reporter gene well known to a person skilled in the art is bacterial beta-glucuronidase. Examples of other seed-preferred promoters are presented in Table 1, and these promoters are useful for the methods of the present invention.
25 TABLE 1: Examples of seed-preferred promoters for use in the performance of the present
invention:

GENE SOURCE EXPRESSION PATTERN REFERENCE
seed-specific genes seed Simon, et al., Plant Mol. Biol. 5:191, 1985; Scofield, ef al., J. Biol. Chem. 262: 12202, 1987.; Baszczynski, et al., Plant Mol. Biol. 14:633,1990.
Brazil Nut albumin seed Pearson, etal., Plant Mol. Biol. 18: 235-245.1992.
legumin seed Ellis, et al., Plant Mol. Biol. 10: 203-214, 1988.
glutelin (rice) seed Takaiwa, et al., Mol. Gen. Genet. 208:
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15-22, 1986; Takaiwa, et al., FEBS Letts. 221: 43-47,1987.
zein seed Matzke et al Plant Mol Biol, 14(3):323-321990
napA seed Stalberg, et al, Planta 199:515 -519, 1996.
wheat LMW and HMW glutenin-1 endosperm Mol Gen Genet 216:81-90, 1989; NAR 17:461-2,1989
wheat SPA seed Albani etal, Plant Cell, 9:171-184,1997
wheat a, p, y-gliadins endosperm EMBO J. 3:1409-15, 1984
barley ttrl promoter endosperm
barley B1, C, D, hordein endosperm Theor Appl Gen 98:1253-62, 1999; Plant J 4:343-55, 1993; Mol Gen Genet 250:750-60,1996
barley DOF endosperm Mena et al, The Rant Journal, 116(1): 53-62,1998
btz2 endosperm EP99106056.7
synthetic promoter endosperm Vicente-Carbajosa et al., Plant J. 13: 629-640,1998.
rice prolamin NRP33 endosperm Wu et al, Plant Cell Physiology 39(8) 885-889,1998
rice a-globulin Glb-1 endosperm Wu et al, Plant Cell Physiology 39(8) 885-889,1998
rice OSH1 embryo Sato et al, Proc. Natl. Acad. Sci. USA, 93:8117-8122,1996
rice a-globulin REB/OHP-1 endosperm Nakase et al. Plant Mol. Biol. 33: 513-522, 1997
rice ADP-glucose PP endosperm Trans Res 6:157-68,1997
maize ESR gene family endosperm Plant J 12:235-46,1997
sorgum y-kafirin endosperm PMB 32:1029-35,1996
KNOX embryo Postma-Haarsma et al, Plant Mol. Biol. 39:257-71,1999
rice oleosin embryo and aleuron Wu et at, J. Biochem., 123:386,1998
sunflower oleosin seed (embryo and dry seed) Cummins, et al., Plant Mol. Biol. 19: 873-876,1992
PRO0117, putative rice 40S ribosomal protein weak in endosperm WO2004/070039
PRO0135, rice alpha-globulin strong in endosperm
PRO0136, rice alanine weak in endosperm
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aminotransferase 4
PRO0147, trypsin inhibitor ITR1 (barley) weak in endosperm
PRO0151,riceWSI18 embryo + stress WO2004/070039
PRO0175, rice RAB21 embryo + stress WO2004/070039
PRO0218, rice oleosin 18kd aleurone + embryo
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); Call is 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
10 Maize Handbook, Chapter 116, Freeling and Walbot, Eds., Springer, N.Y. (1994).
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 translations 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 (for example plasmid or cosmid molecule). Preferred origins of replication include, but
are not limited to, the f 1 -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
26

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or that allow visual selection. Examples of selectable marker genes include genes conferring resistance to antibiotics (such as npttt 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, allowing plants to use mannose as sole carbon source). Visual marker genes result in the formation of colour (for example p-glucuronidase, GUS), luminescence (such as luciferase) or fluorescence (Green Fluorescent Protein, GFP, and derivatives thereof).
10 In a preferred embodiment, the genetic construct as mentioned above, comprises a GRUBX in sense orientation coupled to a promoter that is preferably a seed-preferred promoter, such as for example the rice prolamin promoter. Therefore, another aspect of the present invention is a vector construct comprising an expression cassette essentially similar to SEQ ID NO 5, comprising a prolamin promoter, the Nicotiana tabacum GRUBX gene and the T-zein + T-
15 rubisco deltaGA transcription terminator sequence. A sequence essentially similar to SEQ ID NO 5 encompasses a first nucleic acid sequence encoding a protein homologous to SEQ ID NO 2 or hybridising to SEQ ID NO 1, which first nucleic acid is operably linked to a prolamin promoter or a promoter with a similar expression pattern, additionally or alternatively the first nucleic acid is linked to a transcription termination sequence.
20
Therefore according to another aspect of the invention, there is provided a nucleic acid construct, comprising an expression cassette in which is located a nucleic acid sequence encoding a GRUBX protein, chosen from the group comprising:
(i) a nucleic acid sequence represented by SEQ ID NO: 1 or the complement strand
thereof;
(ii) a nucleic acid sequence encoding an amino acid sequence represented by SEQ ID
NO: 2 or homologues, derivatives or active fragments thereof;
(iii) a nucleic acid sequence capable of hybridising (preferably under stringent
conditions) with a nucleic acid sequence of (i) or (ii) above, which hybridising

30 sequence preferably encodes a protein having GRUBX protein activity;
(iv) a nucleic acid sequence according to (i) to (iii) above which is degenerate as a
results of the genetic code;
( v) nucleic acid sequence which is an allelic variant of the nucleic acid sequences
according to (i) to (iv);
35 (vi) nucleic acid sequence which is an alternative splice variant of the nucleic acid
sequences according to (i) to (v).
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The present invention also encompasses plants obtainable by the methods according to tnepresent invention. The present invention therefore provides plants obtainable by the method according to the present invention, which plants have improved growth characteristics and which plants have altered GRUBX protein activity and/or levels and/or altered expression of a
5 nucleic acid encoding a GRUBX protein, with the proviso that said GRUBX protein is not encoded by the nucleic acid sequence represented by the GenBank accession AX927140.
Thus, according to a fourth embodiment of the present invention, there is provided a method for the production of transgenic plants having improved growth characteristics, comprising
10 introduction and expression in a plant of a nucleic acid molecule of the invention.
More specifically, the present invention provides a method for the production of transgenic plants having improved growth characteristics, which method comprises:
(a) introducing into a plant or plant cell a nucleic acid sequence, a nucleic acid
15 sequence capable of hybridising therewith or a portion thereof, encoding a GRUBX
protein or a homologue, derivative or active fragment thereof;
(b) cultivating the plant cell under conditions promoting plant growth.
The GRUBX protein itself and/or the GRUBX nucleic acid itself may be introduced directly into a plant cell or into the plant itself (including introduction into a tissue, organ or any other part of
20 the plant). According to a preferred feature of the present invention, the nucleic acid is preferably introduced into a plant by transformation. The nucleic acid is preferably as represented by SEQ ID NO: 1 or a portion thereof or sequences capable of hybridising therewith, or is a nucleic acid encoding an amino acid sequence represented by SEQ ID NO: 2 or a homologue, derivative or active fragment thereof. Alternatively, the nucleic acid sequence
25 is as represented by any of MIPS No. At2g01650, SEQ ID NO: 3, SEQ ID NO 6, or by a portion thereof or by sequences capable of hybridising with any of the aforementioned sequences. The amino acid sequence may alternatively be a sequence as represented by any of SPTrEMBL Q9ZU93, GenBank Ace. Nr. AAR01744, SEQ ID NO: 4, SEQ ID NO 7, or by homologues, derivatives or active fragments thereof.
30
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
35 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
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tissue, existing meristematic tissue (for example, apical meristem, axillary buds, and root meristems), and induced meristem tissue (for example, 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
5 into the host genome. The resulting transformed plant cell can then be used to regenerate a transformed plant in a manner known to persons skilled in the art.
Transformation of a 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
10 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, F.A. et al., 1882, Nature 296, 72-74; Negrutiu I. et al., June 1987, Plant Mol. Biol. 8, 363-373);
15 electroporation of protoplasts (Shillito R.D. et al., 1985 Bio/Technol 3, 1099-1102); microinjection into plant material (Crossway A. et al., 1986, Mol. Gen Genet 202, 179-185); DNA or RNA-coated particle bombardment (Klein T.M. et al., 1987, Nature 327, 70) infection with (non-integrative) viruses and the like. Transgenic rice plants expressing a GRUBX gene are preferably produced via Agrobacterium-mediated transformation using any of the well
20 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 (3) 491-506, 1993), Hiei et al. (Plant J. 6 (2) 271-282, 1994), which disclosures are incorporated by reference herein as if fully set forth. In the case of com transformation, the preferred method is as described in either Ishida et al. (Nat.
25 Biotechnol. 1996 Jun; 14(6): 745-50) or Frame et al. (Plant Physiol. 2002 May; 129(1): 13-22), 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
30 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
35 DNA may be monitored using Northern and/or Western analysis, both techniques being well known to persons having ordinary skill in the art.
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The generated transformed plants may be propagated by a variety of means, such as by clon 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.
5
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 (for example, all cells transformed to contain the expression cassette); grafts of transformed and untransformed tissues (for example, in plants, a transformed rootstock grafted to an untransformed scion).
10
The present invention clearly extends to any plant cell or plant produced by any of the methods described herein, and to all plant parts, propagules and progeny 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
15 methods, the only requirement being that progeny exhibit the same genotypic and/or phenotypic characteristics) as those produced in the parent by the methods according to the invention. The invention also includes host cells containing an isolated nucleic acid molecule encoding a GRUBX protein. Preferred host cells according to the invention are plant cells. Therefore, the invention also encompasses host cells, transgenic plant cells or transgenic
20 plants having improved growth characteristics, characterized in that said host cell, transgenic plant or plant cell has increased expression of a nucleic acid sequence encoding a GRUBX protein and/or increased activity and/or levels of a GRUBX protein.
The invention also extends to harvestable parts of a plant such as but not limited to seeds, 25 leaves, fruits, flowers, stems or stem cultures, rhizomes, roots, tubers and bulbs, and to products directly derived thereof, such as dry pellets or powders, oil, fat and fatty acids, starch or proteins.
The term "plant" as used herein encompasses whole plants, ancestors and progeny of the
30 plants, plant parts, plant cells, tissues and organs. The term "plant" also therefore encompasses suspension cultures, embryos, meristematic regions, callus tissue, leaves, flowers, fruits, seeds, roots (including rhizomes and tubers), shoots, bulbs, stems, gametophytes, sporophytes, pollen, and microspores. Plants that are particularly useful in the methods of the invention include algae, ferns, and all plants which belong to the superfamiiy
35 Viridiplantae, in particular monocotyiedonous 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.,
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Amaranthus spp., Ananas comosus, Annona spp., Apium graveolens, Arabidopsis thaliana, Arachis spp, Artocarpus spp., Asparagus officinalis, Avena sativa, Avenfioa carambola, Benincasa hispida, Bertholletia excelsea, Beta vulgaris, Brassica spp., Cadaba farinosa, Camellia sinensis, Canna indica, Capsicum spp., Carica papaya, Carissa macrocarpa, 5 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., Cucurbits spp., Cynara spp., Daucus carota, Desmodium spp., Dimocarpus longan, Dioscor&a spp., Diospyros spp., Echinochloa spp., Eleusine coracana, Eriobotrya japonica, Eugenia uniflora, Fagopyrum spp., Fag/t/s spp., F/a/s ca/fca,
10 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., Lufi^ acutangula, Lupinus spp., Macrotyloma spp., Malpighia emarginata, Malus spp., Mammea americana, Mangifera indica, Manihot spp., Manilkara zapota, Medicago sativa, Melilotus spp.,
15 Mentha spp., Momordica spp., Mon/s n/gra, Musa spp., Nicotiana spp., O/ea spp., Opuntia spp., Omithopus spp., Oryza spp., Panicum miliaceum, Passiflora edulis, Pastinaca sativa, Persea spp., Petroselinum crispum, Phaseolus spp., Phoenix spp., Physalis spp., P/nus spp., Pistacia vera, Pisum spp., F*oa spp., Populus spp., Prosopis spp., Prunus spp., Psidium spp., Punica granatum, Pyrus communis, Quercus spp., Raphanus sativus, Rheum rhabarbanim,
20 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., Mrc/a spp., v7gna spp., Wfts spp., Zea mays, Zizania palustris, Ziziphus spp., amongst others.
25 According to a preferred feature of the present invention, the plant is a crop plant comprising soybean, sunflower, canola, alfalfa, rapeseed or cotton. Further preferably, the plant according to the present invention is a monocotyledonous plant such as sugarcane, most preferably a cereal, such as rice, maize, wheat, millet, barley, rye, sorghum or oats. However, it is envisaged that the methods of the present invention can be applied to a wide
30 variety of plants, since the domain conservation among the known eukaryotic GRUBX homologues suggests an equally conserved function in cellular metabolism.
Advantageously, performance of the methods according to the present invention results in plants having a variety of improved growth characteristics, such improved growth 35 characteristics including improved growth, increased yield and/or increased biomass, modified architecture and a modified cell division, each relative to corresponding wild type plants.
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The present invention relates to methods to improve growth characteristics of a plant or tl methods to produce plants with improved growth characteristics, wherein the growth characteristics comprise any one or more selected from: increased yield, increased biomass, increased total above ground area, increased plant height, increased number of tillers, 5 increased number of first panicles, increased number of second panicles, increased total number of seeds, increased number of filled seeds, increased total seed yield per plant, increased seed biomass, increased seed size, increased seed volume, increased harvest index, increased Thousand Kernel Weight (TKW), altered cycling time and/or an altered growth curve. The present invention also provides methods to alter one of the above mentioned 10 growth characteristics, without causing a penalty on one of the other growth characteristics, for example increase of the above-ground green tissue area while retaining the same number of filled seeds and the same seed yield.
The term "increased yield" encompasses an increase in biomass in one or more parts of a
15 plant relative to the biomass of corresponding wild-type plants. The term also encompasses
an increase in seed yield, which includes an increase in the biomass of the seed (seed weight)
and/or an increase in the number of (filled) seeds and/or in the size of the seeds and/or an
increase in seed volume, each relative to corresponding wild-type plants. For maize, the
increase of seed yield may be reflected in an increase of rows (of seeds) per ear and/or an
20 increased number of kernels per row. 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, among others. An increase in seed size and/or
volume may also influence the composition of seeds. An increase in seed yield could be due
25 to an increase in the number and/or size of flowers. An increase in yield might also increase
the harvest index, which is expressed as a ratio of the total biomass over the yield of
harvestable parts, such as seeds; or Thousand Kernel Weight. Increased yield also
encompasses the capacity for planting at higher density (number of plants per hectare or
acre).
30
The term "modified cell division" encompasses an increase or decrease in cell division or an abnormal cell division/cytokinesis, altered plane of division, altered cell polarity, altered cell differentiation. The term also comprises phenomena such as endomitosis, acytokinesis, polyploidy, polyteny and endoreduplication.
35
It can be envisaged that plants having increased biomass and height exhibit a modified growth rate when compared to corresponding wild-type plants. The term "modified growth rate" as
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used herein encompasses, but is not limited to, a faster rate of growth in one or more parts of a plant (including green biomass and including seeds), at one or more stages in the life cycle of a plant. The term "modified growth" encompasses enhanced vigour, earlier flowering, modified cycling time. If the growth rate is sufficiently increased, the resulting shorter cycling
5 time may allow for an additional harvest within one conventional growing period. Harvesting additional times from the same root stock in the case of some plants may also be possible. Improving 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
10 modified 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. Plants with modified growth may show a modified growth curve and may have modified values for
15 their Tmia or Tgo (respectively the time needed to reach half of their maximal area or 90% of their area, each relative to corresponding wild-type plants).
According to a preferred feature of the present invention, performance of the methods according to the present invention result in plants having increased yield. Preferably, the
20 increased yield includes at least an increase in harvest index, relative to control plants. Therefore, according to the present invention, there is provided a method for increasing yield of plants, in particular harvest index, which method comprises increasing expression of a nucleic acid sequence encoding a GRUBX protein and/or increasing activity of a GRUBX protein itself in a plant, preferably wherein the GRUBX protein is encoded by a nucleic acid
25 sequence represented by SEQ ID NO: 1 or a portion thereof or by sequences capable of hybridising therewith or wherein the GRUBX protein is represented by SEQ ID NO: 2 or a homologue, derivative or active fragment thereof. Alternatively, the GRUBX may be encoded by a nucleic acid sequence represented by any of MIPS No. At2g01650, SEQ ID NO: 3, or by a portion thereof or by sequences capable of hybridising therewith, or wherein the GRUBX is
30 represented by any of SPTrEMBL Q9ZU93, SEQ ID NO: 4, or a homologue, derivative or active fragment of any thereof.
The methods of the present invention are favourable to apply to crop plants because the methods of the present invention are used to increase the harvest index of a plant. Therefore,
35 the methods of the present invention are particularly useful for crop plants cultivated for their seeds, such as cereals. Accordingly, a particular embodiment of the present invention relates to a method to increase the harvest index of a cereal.
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An increase in yield and/or growth 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 5 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, fertilisation, pesticide treatments) severe stresses are not often encountered in cultivated crop plants. As a consequence, the compromised growth induced by mild stress is
10 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
15 chemicals. Biotic stresses are typically those stresses caused by pathogens, such as bacteria, viruses, fungi or insects.
"Modified architecture" may be due to change in cell division. The term "architecture" as used herein encompasses the appearance or morphology of a plant, including any one or more
20 structural features or combination of structural features thereof. Such structural features include the shape, size, number, position, texture, arrangement, and pattern of any cell, tissue or organ or groups of cells, tissues or organs of a plant, including the root, leaf, shoot, stem or tiller, petiole, trichome, flower, inflorescence (for monocotyledonous and dicotyledonous plants), panicles, petal, stigma, style, stamen, pollen, ovule, seed, embryo, endosperm, seed
25 coat, aleurone, fibre, cambium, wood, heartwood, parenchyma, aerenchyma, sieve elements, phloem or vascular tissue, amongst others. Modified architecture therefore includes all aspects of modified growth of the plant.
The present invention also relates to the use of a nucleic acid encoding a GRUBX protein and 30 to the use of portions thereof or nucleic acids hybridising therewith in improving the growth characteristics of plants, preferably in increasing the yield and/or biomass of a plant. The present invention also relates to the use of a GRUBX protein and to the use of homologues, derivatives and active fragments thereof in improving the growth characteristics of plants. The nucleic acid sequence is preferably as represented by SEQ ID NO: 1, 6, or a portion thereof or sequences capable of hybridising therewith or encodes an amino acid sequence represented by SEQ ID NO: 2,4,7, or a homologue, derivative or active fragment thereof.
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The present invention also relates to the use of a nucleic acid sequence encoding a GRUBX protein and variants thereof, and to the use of the GRUBX protein itself and of homologues, derivatives and active fragments thereof as growth regulators. The nucleic acid sequences hereinbefore described (and portions of the same and sequences capable of hybridising with
5 the same) and the amino acid sequences hereinbefore described (and homologues, derivatives and active fragments of the same) are useful in improving the growth characteristics of plants, as hereinbefore described. The sequences would therefore find use as growth regulators, to stimulate or inhibit plant growth. Therefore, the present invention provides a composition comprising a GRUBX protein or a protein represented by SEQ ID NO 2
10 or a homologue, derivative or active fragment thereof for use in improving the growth characteristics of plants. The present invention furthermore provides a composition comprising a nucleic add encoding a GRUBX protein, or a nucleic acid as represented by SEQ ID NO 1 or a portion thereof or a sequence hybridising therewith for use in improving the growth characteristics of plants. The present invention also provides a composition comprising a
15 protein represented by any of the aforementioned amino acid sequences or homologues, derivatives or active fragments thereof for the use as a growth regulator.
The present invention will now be described with reference to the following figures in which: Figure 1a. Phylogenetic tree representing Arabidopsis thaliana proteins and animal reference
20 proteins comprising an UBX domain, as recognised by the SMART tool. The human proteins are represented by their GenBank Accession numbers NP_079517 (Homo sapiens UBX domain containing 1 (UBXD1)), AAP97263 (Homo sapiens Fas-associated protein factor FAF1 mRNA), NP_005662 (Homo sapiens reproduction 8 (D8S2298E), REP8) and a rat protein by NP_114187 (Rattus norvegicus p47 protein). The other identifiers (except for SEQ ID NO 2,
25 SEQ ID NO 4 and SEQ ID NO 7) are GenBank or SPTrEMBL accession numbers for Arabidopsis thaliana proteins.
Figure 1b. Phylogenetic tree representing plant proteins comprising a PUG domain, as recognised by the SMART tool. SEQ ID NO 2 and SEQ ID NO 4 are compared with Arabidopsis thaliana proteins (SPTrEMBL accessions Q9ZU93 (Expressed protein), Q9FKI1
30 (Similarity to zinc metalloproteinase), Q9MAT3 (F13M7.16 protein), Q9FKC7 (Genomic DNA, chromosome 5, TAC done:K24G6), Q9SF12 (Hypothetical protein), Q9C5S2 (Endoribonudease/protein kinase IRE1), Q8RX75 (AT5g24360/K16H17_7), Q94IG5 (Ire1 homolog-1)), and with the rice protein SPTrEMBL Q7XIT1 (Oslrelp).
35 Figure 2a. Definition of UBX1 and PUG domains by their consensus sequences (SMART database). CONSENSUS/50%, respectively /65% and /80% are the consensus sequences for the top 50, 65 and 80% of the reference sequences comprising the UBX1 or PUG domain.
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The capital letters are the standard single letter IUPAC codes for the various amino acids, that
other letters symbolise the nature of the amino acids as outlined below:
Class Key Residues
Alcohol o S,T
5 Aliphatic I I.L.V
Any A,C,D,E,F,G,H,I,K,LIM,N,P,Q>R,S,T,V1W,Y
Aromatic a F.H.W.Y
Charged c D,E,H,K,R
Hydrophobic h AIC,FIG,H,I,K,L1M,R)T,VIW,Y
10 Negative - D,E
Polar p C.D.E.H.K.N.Q.R.S.T
Positive + H.K.R
Small s A,C,D,G1N>PIS,T,V
Tiny u A,G,S
15 Turnpike t A.C.D.E.G.H.K.N.Q.R.S.T
Figure 2b. UBX and PUG domain sequences present in SEQ ID NO 2 and in Q9ZU93. Figure 2c. Alignment of Q9ZU93 and SEQ ID NO 2, PUG domains underlined, UBX domains in bold. 20 Figure 2d. Alignment of SEQ ID NO 2 and SEQ ID NO 4, PUG domains underlined, UBX domains in bold.
Figure 2e. Alignment of SEQ ID NO 4 and SEQ ID NO 7, PUG domains underlined, UBX domains in bold.
25 Figure 3. Schematic presentation of the entry clone p77, containing CDS0669 within the AttL1 and AttL2 sites for Gateway® cloning in the pDONR201 backbone. CDS0669 is the internal code for the tobacco GRUBX coding sequence. This vector contains also a bacterial kanamycin-resistance cassette and a bacterial origin of replication.
30 Figure 4. Binary vector for the expression in Oryza sativa of the tobacco GRUBX gene (CDS0669) under the control of the prolamin promoter (PRO0090). This vector contains a T-DNA derived from the Ti Plasmid, limited by a left border (LB repeat, LB Ti C58) and a right border (RB repeat, RB Ti C58)). From the left border to the right border, this T-DNA contains: a cassette for antibiotic selection of transformed plants; a cassette for visual screening of
35 transformed plants; the PRO0090 -CDS0669 -zein and rbcS-deltaGA double terminator cassette for expression of the tobacco GRUBX gene. This vector also contains an origin of
36

WO 2005/059147 PCT/EP2004/053594
replication from pBR322 for bacterial replication and a selectable marker (Spe/SmeR) for bacterial selection with spectinomycin and streptomycin.
Figure 5. Examples of sequences useful in the present invention. SEQ ID NO: 1 and SEQ ID 5 NO: 2 are the sequences of the GRUBX nucleic acid and GRUBX protein respectively that were used in the examples. SEQ ID NO: 3 and SEQ ID NO: 4 represent the coding sequence and the protein sequence of the sugarcane GRUBX orthologue, SEQ ID NO:
5 is the sequence of the expression cassette that was used in the transformed rice plants, SEQ ID NO: 6 and SEQ ID NO: 7 represent the encoding sequence respectively protein sequence of the rice
10 GRUBX orthologue.
Examples
The present invention will now be described with reference to the following examples, which are by way of illustration alone.
15 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. (Current Protocols in Molecular Biology. New York: John Wiley and Sons, 1998). Standard materials and methods for plant molecular work are
20 described in Plant Molecular Biology Lab fax (1993) by R.D.D. Croy, published by BIOS Scientific Publications Ltd (UK) and Blackwell Scientific Publications (UK).
Example 1: Cloning of the CDS0669 sequence Cloning of the GRUBX gene fragment from tobacco
25 A cDNA-AFLP experiment was performed on a synchronized tobacco BY2 cell culture (Nicotiana tabacum L. cv. Bright Yellow-2), and BY2 expressed sequence tags that were cell cycle modulated were elected for further cloning. The expressed sequence tags were used to screen a tobacco cDNA library and to isolate the full-length cDNA of interest, namely one coding for GRUBX gene (CDS0669).
30
Synchronization of BY2 cells.
A tobacco BY2 (Nicotiana tabacum L. cv. Bright Yellow-2) cultured cell suspension was synchronized by blocking cells in early S-phase with aphidicolin as follows. The cell suspension of Nicotiana tabacum L. cv. Bright Yellow 2 was maintained as described (Nagata et
35 al. Int. Rev. Cytol. 132, 1-30, 1992). For synchronization, a 7-day-old stationary culture was diluted 10-fold in fresh medium supplemented with aphidicolin (Sigma-Aldrich, St. Louis, MO;
37

WO 2005/059147 PCT/EP2004/053594
5 mg/l), a DNA-polymerase a inhibiting drug. After 24 h, cells were released from the block by
several washings with fresh medium after which their cell cycle progression resumed.
RNA extraction and cDNA synthesis.
5 Total RNA was prepared using LiCI precipitation (Sambrook et al, 2001) and poly(A+) RNA was extracted from 500 ug of total RNA using Oligotex columns (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Starting from 1 ug of poly(A+) RNA, first-strand cDNA was synthesized by reverse transcription with a biotinylated oligo-dT2s primer (Genset, Paris, France) and Superscript II (Life Technologies, Gaithersburg, MD). Second-strand
10 synthesis was done by strand displacement with Escherichia coli ligase (Life Technologies), DNA polymerase I (USB, Cleveland, OH) and RNAse-H (USB).
cDNA-AFLP analysis.
Five hundred ng of double-stranded cDNA was used for AFLP analysis as described (Vos et al.,
15 Nucleic Acids Res. 23 (21) 4407-4414, 1995; Bachem et al., Plant J. 9 (5) 745-53, 1996) with
modifications. The restriction enzymes used were BsfYl and Mse\ (Biolabs) and the digestion
was done in two separate steps. After the first restriction digest with one of the enzymes, the 3'
end fragments were trapped on Dyna beads (Dynal, Oslo, Norway) by means of their
biotinylated tafl, while the other fragments were washed away. After digestion with the second
20 enzyme, the released restriction fragments were collected and used as templates in the
subsequent AFLP steps. For pre-amplificattons, a Mse\ primer without selective nucleotides was
combined with a BsfYl primer containing either a T or a C as 3" most nucleotide. PCR conditions
were as described (Vos et al., 1995). The obtained amplification mixtures were diluted 600-fold
and 5|ii was used for selective amplifications using a P^-labeled BsfYl primer and the
25 Amplitaq-Gold polymerase (Roche Diagnostics, Brussels, Belgium). Amplification products were
separated on 5% polyacrylamide gels using the Sequigel system (Biorad). Dried gels were
exposed to Kodak Btomax films as well as scanned in a Phosphorlmager (Amersham Pharmacia
Biotech, Little Chalfont, UK).
30 Characterization of AFLP fragments.
Bands corresponding to differentially expressed transcripts, among which the (partial)
transcript corresponding to SEQ ID NO 1 (or CDS0669), were isolated from the gel and eluted
DNA was re-amplified under the same conditions as for selective amplification. Sequence
information was obtained either by direct sequencing of the re-amplified polymerase chain
35 reaction product with the selective BsfYl primer or after cloning the fragments in pGEM-T easy
(Promega, Madison, Wl) and sequencing of individual clones. The obtained sequences were
compared against nucleotide and protein sequences present in the publicly available
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WO 2005/059147 PCT/EP2004/053594
databases by BLAST sequence alignments (Altschul ef a/., Nucleic Acids Res. 25 (17) 3389-3402 1997). When available, tag sequences were replaced with longer EST or isolated cDNA sequences to increase the chance of finding significant homology. The physical cDNA clone corresponding to SEQ ID NO 1 (COS0669) was subsequently amplified from a commercial
5 tobacco cDNA library as follows:
Cloning of the GRUBX gene (CDS0669)
A c-DNA library with an average size of inserts of 1,400 bp was prepared from poly (A+) RNA isolated from actively dividing, non-synchronized BY2 tobacco cells. These library-inserts
10 were cloned in the vector pCMVSPORT6.0, comprising an attB Gateway cassette (Life Technologies). From this library, 46,000 clones were selected, arrayed in 384-well microtiter plates, and subsequently spotted in duplicate on nylon filters. The arrayed clones were screened using pools of several hundreds of radioactively labelled tags as probes (including the BY2-tag corresponding to the sequence CDS0669, SEQ IDNO 1). Positive clones were
15 isolated (among which the clone corresponding to COS0669, SEQ I NO 1), sequenced, and
aligned with the tag sequence. Where the hybridisation with the tag failed, the full-length
cDNA corresponding to the tag was selected by PCR amplification: tag-specific primers were
designed using primer3 program mttp://www-
genome.wi.mit.edu/Qenome software/other/primer3.html) and used in combination with a
20 common vector primer to amplify partial cDNA inserts. Pools of DNA from 50,000, 100,000, 150,000, and 300,000 cDNA clones were used as templates in the PCR amplifications. Amplification products were then isolated from agarose gels, cloned, sequenced and their sequence aligned with those of the tags. Next, the full-length cDNA corresponding to the nucleotide sequence of SEQ ID NO 1 was cloned from the pCMVsport6.0 library vector into
25 pDONR201, a Gateway® donor vector (Invitrogen, Paisley, UK) via a LR reaction, resulting in the entry clone p77 (Figure 3).
Example 2: Vector construction
The entry clone p77 was subsequently used in an LR reaction with p0830, a destination vector
30 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 prolamin promoter for seed-preferred expression (PRO0090) was upstream of this Gateway cassette. After the LR recombination step, the resulting expression
35 vector p72 (Figure 4) was transformed into Agmbacterium strain LBA4404 and subsequently into Oryza sativa plants.
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WO 2005/059147 PCT/EP2004/053594
Example 3: Transformation of rice with the PRO0090-CDS0669 construct
Mature dry seeds of Oryza sativa japonica cultivar Nipponbare were dehusked. Sterilization was done by incubating the seeds for one minute in 70% ethanol, followed by 30 minutes in 0.2% HgC!2 and by 6 washes of 15 minutes with sterile distilled water. The sterile seeds were
5 then germinated on a medium containing 2,4-D (callus induction medium). After a 4-week incubation in the dark, embryogenic, scutellum-derived calli were excised and propagated on the same medium. Two weeks later, the calli were multiplied or propagated by subculture on the same medium for another 2 weeks. 3 days before co-cultivation, embryogenic callus pieces were sub-cultured on fresh medium to boost cell division activity. The Agrobacterium
10 strain LBA4414 harbouring binary vector p72 was used for co-cultivation. The Agrobacterium strain was cultured for 3 days at 28°C on AB medium with the appropriate antibiotics. The bacteria were then collected and suspended in liquid co-cultivation medium at an ODeoo of about 1. The suspension was transferred to a petri dish and the calli were immersed in the suspension for 15 minutes. Next, the callus tissues were blotted dry on a filter paper,
15 transferred to solidified co-cultivation medium and incubated for 3 days in the dark at 25°C. Thereafter, co-cultivated callus was grown on 2,4-D-containing medium for 4 weeks in the dark at 28°C in the presence of a selective agent at a suitable concentration. During this period, rapidly growing resistant callus islands developed. Upon transfer of this material to a regeneration medium and incubation in the light, the embryogenic potential was released and
20 shoots developed in the next four to five weeks. Shoots were excised from the callus and incubated for 2 to 3 weeks on an auxin-containing medium from which they were transferred to soil. Hardened shoots were grown under high humidity and short days in a greenhouse. Finally seeds were harvested three to five months after transplanting. The method yielded single locus transformants at a rate of over 50 % (Aldemita and Hodges, Planta 199, 612-617,
25 1996; Chan et al.. Plant Mol. Biol. 22(3), 491-506, 1993; Hiei et al., Rant J. 6(2), 271-282, 1994).
Example 4: Evaluation of transgenic rice transformed with the PRO0090-CDS0669 construct
30 Approximately 15 to 20 independent TO rice transformants were generated. The primary transformants were transferred from tissue culture chambers to a greenhouse for growing and harvest of T1 seed. 6 events, of which the T1 progeny segregated 3:1 for presence/absence of the transgene, were retained. For each of these events, approximately 10 T1 seedlings containing the transgene (hetero- and homo-zygotes), and approximately 10 T1 seedlings
35 lacking the transgene (nullizygotes), were selected by monitoring visual marker expression. A
number of parameters related to vegetative growth and seed production were evaluated and
all data were statistically analysed as outlined below:
40

WO 2005/059147

PCT/EP2004/053594

Statistical analysis: t-test and F-test:
A two factor ANOVA (analysis of variants) was used as statistical model for the overall evaluation of plant phenotypic characteristics. An F-test was carried out on all the parameters
5 measured of all the plants of all the events transformed with the gene of the present invention. The F-test is 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 named herein "global gene effect". If the value of the F-test shows that the data are significant, than it is concluded that there is a "gene" effect, meaning that not only presence or the position of the gene is causing the
10 differences in phenotype. The threshold for significance for a true global gene effect is set at 5% probability level for the F-test.
4.1 Vegetative growth measurements:
The selected T1 plants (approximately 10 with the transgene and approximately 10 without the
15 transgene) were transferred to a greenhouse. Each plant received a unique barcode label to
link unambiguously 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
20 corresponding nullizygotes were grown side-by-side at random positions. From the stage of
sowing until the stage of maturity each plant was passed several times through a digital
imaging cabinet and imaged. At each time point digital images (2048x1536 pixels, 16 million
colours) were taken of each plant from at least 6 different angles. Several parameters can be
derived in an automated way from all the digital images of all the plants, using image analysis
25 software.
4.2 Seed-related parameter measurements:
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 were
30 collected and counted. The filled husks were separated from the empty ones using an air-blowing device. The empty husks were discarded and the remaining fraction was counted again. The filled husks were weighed on an analytical balance. This procedure allows to derive a set of seed-related parameters.
35 Harvest index of plants
The harvest index in the present invention is defined as the ratio between the total seed yield and the above ground area (mm2), multiplied by a factor 106. The total seed yield per plant
41

WO 2005/059147 PCT/EP2004/053594
was measured by weighing all filled husks harvested from a plant as described above. Plani aboveground area was determined by counting the total number of pixels of the digital images from aboveground plant parts discriminated from the background. This value was averaged for the pictures taken on the same time point from the different angles and was converted to a
5 physical surface value expressed in square mm by calibration. Experiments showed that the aboveground plant area measured this way correlates with the biomass of plant parts above ground.
The data obtained in the first experiment were confirmed in a second experiment with T2
10 plants. Three lines that had the correct expression pattern 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.
15
A total number of 120 GRUBX transformed plants were evaluated in the T2 generation, that is 40 plants per event of which 20 positives for the transgene, and 20 negatives.
Because two experiments with overlapping events have been carried out, a combined analysis
20 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 are obtained by comparing likelihood ratio test to chi square distributions.
25
In a first experiment, six lines in T1 generation were evaluated. There was an average increase of the harvest index and two lines had a significant increase of 50% or more compared to the nullizygote lines (Table 2).
30 Table 2: Evaluation of the two best performing T1 events

Harvest index:
Line TR null dif %dif p-value
10 74.9 49.9 24.97 50 0.039
4 35 21.7 13.28 61 0.0656
Mean absolute values of the measurements of harvest index for the transgenic lines (TR) and
control plants (null) in the T1 generation are given in columns 2 and 3, the absolute difference
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WO 2005/059147 PCT/EP2004/053594
in column 4 and the difference in % in column 5, significance, expressed as a p-value obtained in a t-test, is given in column 6.
The results obtained for the T1 generation were confirmed in the T2 generation; the average
5 increase for harvest index was 13% and an F-test showed this increase was significant (p-value of 0.0447). Furthermore, these T2 data were re-evaluated in a combined analysis with the results for the T1 generation, and the p-value obtained from an F-test showed again that the observed effects were significant (p-value 0.0181).
10


43
WO 2005/059147 PCT/EP2004/053594
Claims
1. Method for improving plant growth characteristics, comprising increasing expression in
a plant of a nudeic add sequence encoding a GRUBX protein and/or comprising
increasing activity and/or increasing levels in a plant of a GRUBX protein, and
5 optionally selecting for plants having improved growth characteristics.
2. Method of claim 1, wherein said increase is effected by introducing a genetic
modification, preferably in the locus of a gene encoding a GRUBX protein.
10 3. Method according to claim 2, wherein said genetic modification is effected by one of
site-directed mutagenesis, homologous recombination, TILLING and T-DNA activation.
4. Method for improving plant growth characteristics, comprising introducing and
expressing in a plant an isolated nudeic add sequence encoding a GRUBX protein.
15
5. Method according to claim 4, wherein said nudeic acid encoding a GRUBX protein is
overexpressed in a plant.
6. Method according to claim 4 or 5, wherein said nudeic add is derived from a eukaryotic
20 organism, preferably from a plant.
7. Method according to claim 6, wherein said nudeic add is derived from a
dicotyledonous plant, preferably from the family Solanaceae, more preferably from
Nicotiana tabacum.
25
8. Method according to claim 6, wherein said nudeic acid is derived from a
monocotyledonous plant, preferably from the family Poaceae, more preferably from
Oryza sativa.
30 9. Method according to claim 7, wherein said nudeic add is as represented by SEQ ID
NO: 1 or is a portion thereof or is a sequence capable of hybridising therewith or encodes a GRUBX protein, wherein said GRUBX protein is represented by SEQ ID NO: 2 or a homologue, derivative or active fragment thereof.
35 10. Method according to any of claims 4 to 9, wherein said nudeic acid sequence and said
proteins indude variants chosen from:
44

WO 2005/059147 PCT/EP2004/053594
(i) an alternative splice variant of a nucleic acid sequence encoding a GRUBX
protein or wherein said GRUBX protein is encoded by a splice variant; (ii) an allelic variant of a nucleic acid sequence encoding a GRUBX protein or
wherein said GRUBX protein is encoded by an allelic variant;
5 (iii) a nucleic acid sequence that is comprised on at least a part of an artificial
chromosome, which artificial chromosome preferably also comprises one or more
related gene family members;
(iv) a functional portion of a GRUBX encoding nucleic acid;
(v) sequence capable of hybridising to a GRUBX encoding nucleic acid;
10 (vi) homologues, derivatives and active fragments of a GRUBX protein.
11. Method according to any of claims 4 to 10, wherein expression of said nucleic acid
encoding a GRUBX protein is driven by a seed -preferred promoter, preferably a
prolamin promoter.
15
12. Method according to any of claims 1 to 11, wherein said improved growth characteristic
is increased yield and/or modified plant architecture, each relative to corresponding
wild type plants.
20 13. Method according to any of claims 1 to 12, wherein said increased yield is increased
seed yield.
14. Method according to any of claims 1 to 13, wherein said increased yield and said
modified plant architecture comprise one or more of (i) increased seed biomass, (ii)
25 increased total number of seeds, (iii) increased number of filled seeds, (iv) increased
seed size, (v) increased seed volume, (vi) increased harvest index, and (vii) increased Thousand Kernel Weight, all relative to corresponding wild type plants.
15. Method for increasing the yield of a plant, which method comprises increasing
30 expression in a plant of a GRUBX encoding nucleic acid and/or increasing activity
and/or levels in a plant of a GRUBX protein.
16. Method for the production of a transgenic plant having improved growth characteristics,
which method comprises:
35 a. introducing into a plant or plant cell a nucleic acid sequence, a nucleic acid
sequence capable of hybridising therewith or a portion thereof, encoding a GRUBX protein or a homologue, derivative or active fragment thereof;
45

WO 2005/059147 PCT/EP2004/053594
b. cultivating the plant cell under conditions promoting plant growth.
17. Method for the selection of plants having improved growth characteristics, which method is based on the selection of superior allelic variants of a GRUBX encoding sequence and which alleles give rise to improved growth characteristics in a plant.
18. Plants obtainable by a method according to any of claims 1 to 17, with the proviso that said GRUBX protein is not encoded by the nucleic acid sequence represented by the GenBank accession AX927140.
10
19. An isolated nucleic acid sequence comprising:
(i) a nucleic acid sequence represented by SEQ ID NO: 6, or the complement
strand thereof;
(ii) a nucleic acid sequence encoding an amino acid sequence represented by
15 SEQ ID NO: 7, or homologues, derivatives or active fragments thereof;
(iii) a nucleic acid sequence capable of hybridising (preferably under stringent
conditions) with a nucleic acid sequence of (i) or (ii) above, which
hybridising sequence preferably encodes a protein having GRUBX activity;
(iv) a nucleic acid sequence according to (i) to (iii) above which is degenerate as
20 a result of the genetic code;
(v) a nucleic acid which is an allelic variant of the nucleic acid sequences
according to (i) to (iv);
(vi) a nucleic acid which is an alternative splice variant of the nucleic acid
sequences according to (i) to (v);
25 (vii) a nucleic acid sequence which has 75.00%, 80.00%, 85.00%, 90.00%,
95.00%, 96.00%, 97.00%, 98.00% or 99.00% sequence identity to any one or more of the sequence defined in (i) to (vi); (viii) a portion of a nucleic acid sequence according to any of (i) to (vii) above, which portion preferably encodes a protein having GRUBX activity.
30
20. An isolated protein comprising at least part of one of the polypeptides selected from the group consisting of:
(i) a polypeptide as given in SEQ ID NO 4;
(ii) a polypeptide as given in SEQ ID NO 7;
35 (iii) a polypeptide with an amino acid sequence which has at least 40.00%
sequence identity, preferably 50.00%, 60.00%, 70.00% sequence identity, more preferably 80% or 90% sequence identity, most preferably 95.00%, 96.00%,

46

CD-107-PCT
97.00%, 98.00% or 99.00% sequence identity to the amino acid sequence as
given in SEQ ID NO 4 or SEQ ID NO 7; (iv) a polypeptide comprising at least an UBX domain, preferably an UBX domain
and a PUG domain, and optionally a Zinc finger domain;
5 (v) a homologue, a derivative, an immunologically active and/or functional fragment
of a protein as defined in any of (i) to (iv),
with the proviso that the protein sequence is not a sequence represented by SEQ ID
NO 2, or database entries Q9ZU93, AAR01744, Q9D7L9, Q9BZV1, Q99PL6,
ENSANGP00000020442, Q7SXA8, Q9V8K8, Q96IK9, ENSRNOP00000037228, or
10 AAH07414.
21. Construct comprising:
(i) a nucleic acid sequence encoding a GRUBX protein;
(ii) one or more control sequences capable of driving expression in a plant of the
15 nucleic acid sequence of (i); and optionally,
(iii) a transcription termination sequence,
provided that said nucleic acid encoding a GRUBX protein is not the nucleic acid represented in GenBank Accession number AX927140.
20 22. Construct according to claim 21, wherein said nucleic acid encoding a GRUBX protein
is a nucleic acid sequence encoding a protein represented by SEQ ID NO 2 or a protein according to any of (i) to (v) in claim 20.
23. Construct according to claims 21 or 22, wherein said control sequences comprise at 25 least a seed-preferred promoter, preferably a prolamin promoter.
24. Construct comprising an expression cassette essentially similar to SEQ ID NO 5.
25. Transgenic plant or plant cell, characterized in that said plant or plant cell has 30 increased expression of a nucleic acid sequence encoding a GRUBX protein and/or
increased activity and/or levels of a GRUBX protein.
26. Transgenic plant or plant cell of claim 25 having improved growth characteristics.
35 27. Transgenic plant according to claim 25 or 26, wherein said plant is a crop plant
comprising soybean, sunflower, canola, alfalfa, rapeseed or cotton, preferably a
47

CD-107-PCT
monocotyledonous plant such as sugarcane, most preferably a cereal, such as rice maize, wheat, millet, barley, rye, sorghum or oats.
28. Plant cells, plant parts, including harvestable parts and/or products directly derived
5 thereof, propagules or progeny of a plant according to any of claims 18, 25, 26 or 27.
29. Use of a nucleic acid sequence encoding a GRUBX protein, portions thereof or nucleic
acids hybridising therewith, in improving the growth characteristics of a plant.
10 30. Use of a GRUBX protein, homologues, derivatives and active fragments thereof, in
improving the growth characteristics of a plant.
31. Use of a GRUBX protein according to claim 20 in improving the growth characteristics
of a plant.
15
32. A composition comprising a GRUBX protein for use in improving the growth
characteristics of plants.
33. A composition comprising a nucleic acid encoding a GRUBX protein for use in 20 improving the growth characteristics of plants.
34. Use of a nucleic acid sequence encoding a GRUBX protein in breeding programs.
Mumbai, this 14th day of June, 2006
25
FOR CROPDESIGN N. V. (By their Agent)
VIPUL N BHUTA ADITYA & ASSOCIATES
48

CD-107-PCT
Abstract
Plants having modified growth characteristics and method for
making the same
5
The present invention concerns a method for modifying the growth characteristics of plants by modulating expression in a plant of a nucleic acid sequence encoding a GRUBX protein and/or modulating activity and/or levels in a plant of a GRUBX protein. The present invention furthermore provides novel GRUBX proteins and nucleic acids encoding such proteins. The invention also relates to constructs comprising GRUBX encoding nucleic acids, and transgenic plants having modified growth characteristics, which plants have modulated expression of a nucleic acid encoding a GRUBX protein.
49

Documents:

698-MUMNP-2006-ABSTRACT(14-6-2006).pdf

698-MUMNP-2006-ABSTRACT(17-9-2010).pdf

698-MUMNP-2006-ABSTRACT(GRANTED)-(18-10-2011).pdf

698-mumnp-2006-abstract.doc

698-mumnp-2006-abstract.pdf

698-MUMNP-2006-ASSIGNMENT(15-9-2011).pdf

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698-MUMNP-2006-CLAIMS(AMENDED)-(15-9-2011).pdf

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698-mumnp-2006-claims.pdf

698-mumnp-2006-correspondance-received.pdf

698-MUMNP-2006-CORRESPONDENCE(01-12-2010).pdf

698-MUMNP-2006-CORRESPONDENCE(25-5-2011).pdf

698-MUMNP-2006-CORRESPONDENCE(27-3-2012).pdf

698-MUMNP-2006-CORRESPONDENCE(27-4-2012).pdf

698-mumnp-2006-correspondence(6-8-2007).pdf

698-MUMNP-2006-CORRESPONDENCE(IPO)-(18-10-2011).pdf

698-mumnp-2006-correspondence(ipo)-(29-10-2009).pdf

698-mumnp-2006-description (complete).pdf

698-MUMNP-2006-DESCRIPTION(GRANTED)-(18-10-2011).pdf

698-MUMNP-2006-DRAWING(14-6-2006).pdf

698-MUMNP-2006-DRAWING(17-9-2010).pdf

698-MUMNP-2006-DRAWING(GRANTED)-(18-10-2011).pdf

698-MUMNP-2006-FORM 1(14-6-2006).pdf

698-MUMNP-2006-FORM 1(15-9-2011).pdf

698-MUMNP-2006-FORM 13(27-3-2012).pdf

698-mumnp-2006-form 18(6-8-2007).pdf

698-MUMNP-2006-FORM 2(GRANTED)-(18-10-2011).pdf

698-MUMNP-2006-FORM 2(TITLE PAGE)-(14-6-2006).pdf

698-MUMNP-2006-FORM 2(TITLE PAGE)-(15-9-2011).pdf

698-MUMNP-2006-FORM 2(TITLE PAGE)-(17-9-2010).pdf

698-MUMNP-2006-FORM 2(TITLE PAGE)-(GRANTED)-(18-10-2011).pdf

698-MUMNP-2006-FORM 26(15-9-2011).pdf

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698-mumnp-2006-form 3(5-12-2006).pdf

698-mumnp-2006-form-1.pdf

698-mumnp-2006-form-2.doc

698-mumnp-2006-form-2.pdf

698-mumnp-2006-form-26.pdf

698-mumnp-2006-form-5.pdf

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698-MUMNP-2006-SEQUENCE LISTING(18-10-2011).pdf

698-mumnp-2006-sequence listing-cd107pct.prj

698-mumnp-2006-sequence listing-cd107pct.st25.txt

698-MUMNP-2006-SPECIFICATION(AMENDED)-(17-9-2010).pdf

698-MUMNP-2006-WO INTERNATIONAL PUBLICATION REPORT(14-6-2006).pdf

abstract1.jpg


Patent Number 249371
Indian Patent Application Number 698/MUMNP/2006
PG Journal Number 42/2011
Publication Date 21-Oct-2011
Grant Date 18-Oct-2011
Date of Filing 14-Jun-2006
Name of Patentee CORPDESIGN N.V.
Applicant Address Technologiepark 3 B-9052 Zwijnaarde
Inventors:
# Inventor's Name Inventor's Address
1 FRANKARD VALERIE Bierenberg 47 B-1640 Sint-Genesius-Rode
2 MIRONOV VLADIMIR Zebrastraat 17 B-9000 Gent
3 SANZ MOLINERO ANA ISABEL Bernheimlaan 38 B-9050 Gentbrugge
PCT International Classification Number C12N15/82;A01H 5/00
PCT International Application Number PCT/EP2004/053594
PCT International Filing date 2004-12-17
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 03104764.0 2003-12-17 EUROPEAN UNION
2 60/531.866 2003-12-22 EUROPEAN UNION