Title of Invention

A METHOD FOR INCREASING ACTIVITY OF A YIPPEE-LIKE POLYPEPTIDE IN A PLANT

Abstract The present invention concerns a method for improving the growth characteristics of plants by increasing activity in a plant of a YIPPEE-like polypeptide or a homologue thereof. One such method comprises introducing into a plant a YIPPEE-like nucleic acid or variant thereof. The invention also relates to transgenic plants having introduced therein a YIPPEE-like nucleic acid or variant thereof, which plants have improved growth characteristics relative to corresponding wild type plants. The present invention also concerns constructs useful in the methods of the invention.
Full Text

Plants having improved growth characteristics and method for
making the same
The present invention relates generally to the field of molecular biology and concerns a method for improving plant growth characteristics. More specifically, the present invention concerns a method for improving plant growth characteristics, in particular yield, by increasing activity in a plant of a YIPPEE-like polypeptide or a homologue thereof. The present invention also concerns plants having increased activity of a YIPPEE-like polypeptide or a homologue thereof, which plants have improved growth characteristics relative to corresponding wild type plants. The invention also provides constructs useful in the methods of the invention.
The ever-increasing world population and the dwindling supply of arable land available for agriculture fuel agricultural research towards improving the efficiency of agriculture. Conventional means for crop and horticultural improvements utilise selective breeding techniques to identify plants having desirable characteristics. However, such selective breeding techniques have several drawbacks, namely that these techniques are typically labour intensive and result in plants that often contain heterogeneous genetic components that may not always result in the desirable trait being passed on from parent plants. Advances in molecular biology have allowed mankind to modify the germplasm of animals and plants. Genetic engineering of plants entails the isolation and manipulation of genetic material (typically in the form of DNA or RNA) and the subsequent introduction of that genetic material into a plant. Such technology has the capacity to deliver crops or plants having various improved economic, agronomic or horticultural traits. A trait of particular economic interest is yield. Yield is normally defined as the measurable produce of economic value from a crop. This may be defined in terms of quantity and/or quality. Yield is directly dependent on several factors, for example, the number and size of the organs, plant architecture (for example, the number of branches), seed production and more. Root development, nutrient uptake and stress tolerance may also be important factors in determining yield. Crop yield may therefore be increased by optimizing one of the abovementioned factors.
The ability to improve various growth characteristics of a plant would have many applications in areas such as crop enhancement, plant breeding, in the production of ornamental plants, aboriculture, horticulture and forestry. Improving growth characteristics, such as yield may also find use in the production of algae for use in bioreactors (for the biotechnological production of substances such as Pharmaceuticals, antibodies, or vaccines, or for the byconversion of organic waste) and other such areas.

It has now been found that increasing activity in a plant of a YIPPEE-iike polypeptide gives plants having improved growth characteristics relative to corresponding wild type plants.
The YIPPEE gene was first identified in Drosophita and revealed a novel family of putative zinc binding proteins highly conserved among eukaryotes (Roxstrom-Lindquist K. and Faye I. (2001) Insect Mol Biol. 10(1): 77-86). The YIPPEE protein was characterized and was found to contain a putative zinc-finger-like metal binding domain. It was the first characterized member of a conserved gene family of proteins present in diverse eukaryotic organisms, ranging from cellular slime mould to humans. The YIPPEE gene is ubiquitously expressed in different developmental stages of Drosophila. The high degree of YIPPEE-like sequence conservation between a wide range of species is an indication that the YIPPEE protein is of general importance in eukaryotes.
According to the present invention, there is provided a method for improving the growth characteristics of a plant, comprising increasing activity in a plant of a YIPPEE-like polypeptide or a homologue thereof.
Advantageously, performance of the methods according to the present invention results in plants having a variety of improved growth characteristics, especially increased yield, particularly seed yield.
The term "increased yield" as defined herein is taken to mean an increase in any one or more of the following, each relative to corresponding wild type planlS: (i) increased biomass (weight) of one or more parts of a plant, particularly aboveground (harvestable) parts, increased root biomass or increased biomass of any other harvestable part; (ii) increased seed yield, which includes an increase in seed biomass (seed weight) and which may be an increase in the seed weight per plant or on an individual seed basis; (iii) increased number of (filled) seeds; (iv) increased seed size, which may also influence the composition of seeds; (v) increased seed volume, which may also influence the composition of seeds; (vi) increased harvest index, which is expressed as a ratio of the yield of harvestable parts, such as seeds, over the total bbmass; and (vii) increased thousand kernel weight (TKW), which is extrapolated from the number of filled seeds counted and their total weight. An increased TKW may result from an increased seed size and/or seed weight.
Taking corn as an example, a yield increase may be manifested as one or more of the following: increase in the number of plants per hectare or acre, an increase in the number of ears per plant, an increase in the number of rows, number of kernels per row, kernel weight,

thousand kernel weight, ear length/diameter, among others. Taking rice as an example, a yield increase may be manifested by an increase in one or more of the following: number of plants per hectare or acre, number of panicles per plant, number of spikelets per panicle, number of flowers per panicle, increase in the seed filling rate, increase in thousand kernel weight, among others. An increase in yield may also result in modified architecture, or may occur as a result of modified architecture.
According to a preferred feature, performance of the methods of the invention result in plants having increased yield, particularly seed yield. Therefore, according to the present invention, there is provided a method for increasing plant yield, particularly seed yield, which method comprises increasing activity in a plant of a YIPPEE-like polypeptide or a homologue thereof.
Since the transgenic plants according to the present invention have increased yield, it is likely that these plants exhibit an increased growth rate (during at least part of their life cycle), relative to the growth rate of corresponding wild type plants at a corresponding stage in their life cycle. The increased growth rate may be specific to one or more parts of a plant (including seeds), or may be throughout substantially the whole plant. A plant having an increased growth rate may even exhibit early flowering. The increase in growth rate may take place at one or more stages in the life cycle of a plant or during substantially the whole plant life cycle. Increased growth rate during the early stages in the life cycle of a plant may reflect enhanced vigour. The increase in growth rate may alter the harvest cycle of a plant allowing plants to be sown later and/or harvested sooner than would otherwise be possible. If the growth rate is sufficiently increased, it may allow for the sowing of further seeds of the same plant species (for example sowing and harvesting of rice plants followed by sowing and harvesting of further rice plants all within one conventional growing period). Similarly, if the growth rate is sufficiently increased, it may allow for the sowing of further seeds of different plants species (for example the sowing and harvesting of rice plants followed by, for example, the sowing and optional harvesting of soy bean, potato or any other suitable plant). Harvesting additional times from the same rootstock in the case of some plants may also be possible. Altering the harvest cycle of a plant may lead to an increase in annual biomass production per acre (due to an increase in the number of times (say in a year) that any particular plant may be grown and harvested). An increase in growth rate may also allow for the cultivation of transgenic plants in a wider geographical area than their wild-type counterparts, since the territorial limitations for growing a crop are often determined by adverse environmental conditions either at the time of planting (early season) or at the time of harvesting (late season). Such adverse conditions may be avoided if the harvest cycle is shortened. The growth rate may be determined by deriving various parameters from growth curves, such parameters may be: T-Mid (the time

taken for plants to reach 50% of their maximal size) and T-90 (time taken for plants to reach 90% of their maximal size), amongst others.
Performance of the methods of the invention gives plants having an increased growth rate. Therefore, according to the present invention, there is provided a method for increasing the growth rate of plants, which method comprises increasing activity in a plant of a YIPPEE-like polypeptide or a homologue thereof.
An increase in yield and/or growth rate occurs whether the plant is under non-stress conditions or whether the plant is exposed to various stresses compared to control plants. Plants typically respond to exposure to stress by growing more slowly. In conditions of severe stress, the plant may even stop growing altogether. Mild stress on the other hand is defined herein as being any stress to which a plant is exposed which does not result in the plant ceasing to grow altogether without the capacity to resume growth. Due to advances in agricultural practices (irrigation, fertilization, pesticide treatments) severe stresses are not often encountered in cultivated crop plants. As a consequence, the compromised growth induced by mild stress is often an undesirable feature for agriculture. Mild stresses are the typical stresses to which a plant may be exposed. These stresses may be the everyday biotic and/or abiotic (environmental) stresses to which a plant is exposed. Typical abiotic or environmental stresses include temperature stresses caused by atypical hot or cold/freezing temperatures; salt stress; water stress (drought or excess water). Abiotic stresses may also be caused by chemicals. Biotic stresses are typically those stresses caused by pathogens, such as bacteria, viruses, fungi and insects.
The abovementioned growth characteristics may advantageously be modified in any plant
The term "plant" as used herein encompasses whole plants, ancestors and progeny of the plants and plant parts, including seeds, shoots, stems, leaves, roots, flowers (including tubers), and tissues and organs, wherein each of the aforementioned comprise the gene/nudeic acid of interest and/or a genetic modification, preferably in the locus of a YIPPEE-like gene. The term "plant" also encompasses suspension cultures, callus tissue, embryos, meristematic regions, gametophytes, sporophytes, pollen, and microspores, again wherein each of the aforementioned comprise the gene/nucleic acid of interest and/or a genetic modification, preferably in the locus of a YIPPEE-like gene.
Plants that are particularly useful in the methods of the invention include all plants which belong to the superfamiiy Viridiplantae, in particular monocotyledonous and dicotyledonous

plants including fodder or forage legumes, ornamental plants, food crops, trees or shrubs selected from the list comprising Acacia spp.,-Acer spp., Actinidia spp., Aesculus spp., Agathis australis, Albizia amara, Alsophila tricolor, Andropogon spp., Arachis spp, Areca catechu, Astelia fragrans, Astragalus cicer, Baikiaea plurijuga, Betula spp., Brassica spp., Bruguiera gymnorrhiza, Burkea africana, Butea frondosa, Cadaba farinosa, Calliandra spp, Camellia sinensis, Canna indica, Capsicum spp., Cassia spp., Centroema pubescens, Chaenomeles spp., Cinnamomum cassia, Coffea arabica, Colophospermum mopane, Coronil/ia varia, Cotoneaster semtina, Crataegus spp., Cucumis spp., Cupressus spp., Cyathea dealbata, Cydonia oblongaf Cryptorneria japonica, Cymbopogon spp., Cynthea dealbata, Cydonia oblonga, Dalbergia monetaria, DavalUa divaricate, Desmodium spp., Dicksonia squarosa, Diheteropogon ampledens, Dioclea spp, Dolichos spp., Dorycnium rectum, Echinochloa pyramidalis, Ehrartia spp., Eleusine coracana, Eragrestis spp., Erythrina spp., Eucalyptus spp., Euclea schimperi, Eulalia vittosa, Fagopyrum spp., Feijoa sellowiana, Fragaria spp., Flewingia spp, Freycinetia banksii, Geranium thunbergii, Ginkgo biloba, Glycine javanica, Gliricidia spp, Gossypium hirsutum, Grevillea spp., Guibourtia coleosperma, Hedysarum spp., Hemarthia altissima, Heteropogon contortus, Hordeum vulgare, Hyparrhenia rufa, Hypericum erectum, Hypertheiia dissoluta, Indigo incarnate, Iris spp., Leptarrhena pyrolrfblia, Lespediza spp., Lettuce spp., Leucaena leucocephala, LoudeUa simplex, Lotonus bainesii, Lotus spp., Macrotyloma axillare, Malus spp., Manihot esculenta, Medicago sativa, Metasequoia glyptostroboides, Musa sapientum, Nicotianum spp., Onobrychis spp., Orn'rthopus spp., Oryza spp., Peltophorum africanum, Pennisetum spp., Persea gratissima, Petunia spp., Phaseolus spp., Phoenix canariensis, Phormium cookianum, Photinia spp., Picea glauca, Pinus spp., Pisum sativum, Podocarpus totara, Pogonarthria fieckii, Pogonarihria squarrosa, Populus spp., Prosopis cineraria, Pseudotsuga menziesii, Pterolobium stellatum, Pyms communis, Quercus spp., Rhaphiolepsis umbellate, Rhopalostylis sapida, Rhus natalensis, Ribes grossularia, Ribes spp., Robinia pseudoacacia, Rosa spp., Rubus spp., Salix spp., Schyzachyrium sanguineum, Sciadopitys verticillata, Sequoia sempervirens, Sequoiadendrvn giganteum, Sorghum bicolor, Spinacia spp., Sporobolus fimbriatus, Stiburus alopecuroides, Stylosanthos humilis, Tadehagi spp, Taxodium distichum, Themeda triandra, Trifolium sppM Triticum spp., Tsuga heterophylla, Vaccinium spp., Vicia spp., Vitis vinifera, Watsonia pyramidata, Zantedeschia aethiopica, Zea mays, amaranth, artichoke, asparagus, broccoli, Brussel sprouts, cabbage, canola, carrot, cauliflower, celery, collard greens, flax, kale, lentil, oilseed rape, okra, onion, potato, rice, soybean, strawberry, sugar beet, sugar cane, sunflower, tomato, squash, tea and algae, amongst others. According to a preferred embodiment of the present invention, the plant is a crop plant such as soybean, sunflower, canola, alfalfa, rapeseed, cotton, tomato, potato or tobacco. Further preferably, the plant is a

monocotyiedonous plant, such as sugar cane. More preferably the plant is a cereal, such as rice, maize, wheat, barley, millet, rye, sorghum or oats.
The activity of a YIPPEE-like polypeptide may be increased by increasing levels in a plant of the polypeptide. Alternatively, activity may also be increased when there is no change in levels of a YIPPEE-like polypeptide, or even when there is a reduction in levels of a YIPPEE-like polypeptide. This may occur when the intrinsic properties of the polypeptide are altered, for example, by making mutant versions that are more active that the wild type polypeptide.
The term "YIPPEE-like polypeptide or a homologue thereof as defined herein refers to a polypeptide comprising: (i) a putative zinc-binding motif. 2xCXXC, where X is any amino acid residue; and (ii) the motif KYKEGK, allowing for one amino acid substitution at any position and any conservative amino acid substitution; and (iii) the motif GRAYLF, allowing for one amino acid substitution at any position and any conservative amino acid substitution. The putative zinc-binding motif: 2xCXXC is typically found with a gap of about 52 amino adds residues between the first and second CXXC, i.e. CXXC{52 amino acids}CXXC. The term "any conservative amino acid substitution* means that any one or more of the amino acid residues may be replaced with a conservative substitution. Conservative substitution tables are readily available in the art. The table below gives examples of conserved amino acid substitutions.
Table 1: Examples of conserved amino acid substitutions

Residue Conservative Substitutions Residue Conservative Substitutions
Ala Ser Leu He; Vai
Arg Lys Lys Arg; Gin
Asn Gin; His Met Leu; He
Asp Glu Phe Met; Leu; Tyr
Gin Asn Ser Thn Gly
Cys Ser Thr Ser; Val
Glu Asp Trp Tyr
Gly Pro Tyr Trp; Phe
His Asn; Gin Val He; Leu
He Leu, Val
A "YIPPEE-like polypeptide or a homologue thereof may readily be identified using routine techniques well known in the art. The motifs defined above are highly conserved, thereby

allowing a person skilled in the art to readily identify other YIPPEE-like sequences based on the presence of these motifs.
The plant YIPPEE-like polypeptide sequence represented by SEQ ID NO: 2, encoded by the nucleic acid of SEQ ID NO: 1, was found on the basis of homology to a transcription factor in Drosophila. Examples of plant-derived polypeptides falling under the definition of a "YIPPEE-like polypeptide or a homologue thereof include: /H3g08990 (SEQ ID NO: 4), Ai3g11230 (SEQ ID NO: 6), Af2g40110 (SEQ ID NO: 8), /U4g27740 (SEQ ID NO: 10) and Af5g53940 (SEQ ID NO: 12), all from Arabidopsis thaliana; AB061267 (SEQ ID NO: 14), from potato; AY109711.1 (SEQ ID NO: 16) and AY104347.1 (SEQ ID NO: 18) protein predictions in maize; NMJI96100.1 (SEQ ID NO: 20), AK121352.1 (SEQ ID NO: 22) and AK109500.1 (SEQ ID NO: 24), protein predictions in rice. The table below shows the percentage homology of the aforementioned YIPPEE-like polypeptide sequences with SEQ ID NO: 2 based on overall global sequence alignment. Accession numbers 1 to 7 in the table refer to the protein and the remaining accession numbers refer to the mRNA with the corresponding SEQ ID NO giving the protein prediction. The percentage identity was calculated using an NCBI Align program with default parameters.

It is to be understood that sequences felling under the definition of "YIPPEE-like polypeptide or homologue thereof are not to be limited to the sequences represented by SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22 and SEQ ID NO: 24, but that any polypeptide meeting the criteria of comprising: (i) a putative zinc-binding motif: 2xCXXC, where X is any amino acid residue; and (ii) the motif KYKEGK, allowing for one amino acid

substitution at any position and any conservative amino acid substitution; and (in) the motif GRAYLF, allowing for one amino acid substitution at any position and any conservative amino acid substitution may be suitable for use in the methods of the invention. The methods of the invention may also be performed when the polypeptide has at least one of the aforementioned motifs (i) to (iii).
The nucleic acid encoding a YIPPEE-like polypeptide or a homologue thereof may be any natural or synthetic nucleic acid. A YIPPEE-like polypeptide or a homologue thereof as defined hereinabove is one that is encoded by a YIPPEE-like nucleic acid/gene. Therefore the term *YIPPEE~like nucleic acid/gene" as defined herein is any nucleic acid/gene encoding a YIPPEE-like polypeptide or a homologue thereof as defined hereinabove. Examples of Yippee-like nucleic acids include those represented by any one of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO; 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21 and SEQ ID NO: 23. YIPPEE-like nucleic acids/genes and variants thereof may be suitable in practising the methods of the invention. Variant YIPPEE-like nucleic acid/genes include portions of a YIPPEE-like nucleic acid/gene and/or nucleic acids capable of hybridising with a YIPPEE-like nucleic acid/gene.
The term portion as defined herein refers to a piece of DNA comprising at least 249 nucleotides and which portion encodes a polypeptide comprising any one or more of, and preferably all of. (i) a putative zino-binding motif: 2xCXXC, where X is any amino acid residue; (ii) the motif KYKEGK, allowing for one amino acid substitution at any position and any conservative amino acid substitution; (iii) the motif GRAYLF, allowing for one amino acid substitution at any position and any conservative amino acid substitution. A portion may be prepared, for example, by making one or more deletions to a YIPPEE-like nucleic acid. The portions may be used in isdated form or they may be fused to other coding (or non coding) sequences in order to, for example, produce a protein that combines several activities. When fused to other coding sequences, the resulting polypeptide produced upon translation could be bigger than that predicted for the YJPPEE-like fragment. Preferably, the functional portion is a portion of a nucleic acid as represented by any one of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21 and SEQ ID NO: 23.
Another variant YIPPEE-like nucleic acid/gene is a nucleic acid capable of hybridising under reduced stringency conditions, preferaWy under stringent conditions, with a YIPPEE-like nucleic acid/gene as hereinbefore defined, which hybridising sequence encodes a polypeptide comprising any one or more of, and preferably all of. (i) a putative zinc-binding motif: 2xCXXC,

where X is any amino acid residue; (ii) the motif KYKEGK, allowing for one amino acid substitution at any position and any conservative amino acid substitution; (iii) the motif GRAYLF, allowing for one amino acid substitution at any pos'rtion and any conservative amino acid substitution. Preferably, the hybridising sequence is one that is capable of hybridising to a nucleic acid as represented by any one of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO; 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21 and SEQ ID NO: 23 or to a portion of any of the aforementioned sequences as defined hereinabove.
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. 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 resin. The hybridisation process can furthermore occur with one of the complementary nucleic acids immobilised to a solid support such as a nitro-cellulose or nylon membrane or immobilised by e.g. photolithography to, for example, a siliceous glass support (the latter known as nucleic acid arrays or microarrays or as nucleic acid chips). In order to allow hybridisation to occur, the nucleic acid molecules are generally thermally or chemically denatured to melt a double strand into two single strands and/or to remove hairpins or other secondary structures from single stranded nucleic acids. The stringency of hybridisation is influenced by conditions such as temperature, salt concentration, ionic strength and hybridisation buffer composition.
"Stringent hybridisation conditions" and "stringent hybridisation wash conditions" in the context of nucleic acid hybridisation experiments such as Southern and Northern hybridisations are sequence dependent and are different under different environmental parameters. The skilled artisan is aware of various parameters which may be altered during hybridisation and washing and which will either maintain or change the stringency conditions.
The Tm is the temperature under defined ionic strength and pH, at which 50% of the target sequence hybridises to a perfectly matched probe. The Tm is dependent upon the solution conditions and the base composition and length of the probe. For example, longer sequences hybridise specifically at higher temperatures. The maximum rate of hybridisation is obtained from about 16°C up to 32°C below Tm. The presence of monovalent cations in the hybridisation solution reduce the electrostatic repulsion between the two nucleic acid strands thereby promoting hybrid formation; this effect is visible for sodium concentrations of up to CK4M. Formamide reduces the melting temperature of DNA-DNA and DNA-RNA duplexes with

0.6 to 0.7°C for each percent formamide, and addition of 50% formamide allows hybridisaton to be performed at 30 to 45°C, though the rate of hybridisation will be lowered. Base pair mismatches reduce the hybridisation rate and the thermal stability of the duplexes. On average and for large probes, the Tm decreases about 1°C per % base mismatch. The Tm may be calculated using the following equations, depending on the types of hybrids:
1. DNA-DNA hybrids (Meinkoth and Wahl, Anal. Biochem., 138:267-284,1984):
2. Tm= 81.5°C + 16.6xlog[Na*]a + 0A1x%[GIC*\ - 500x[LT1 - 0.61x% formamide
3. DNA-RNA or RNA-RNA hybrids:
Tm= 79.8 + 18.5 (logiofNaT) + 0.58 (%G/tf>) + 11.8 (%G/Cb)2 - 820/Lc
3. oligo-DNA or oligo-RNAd hybrids:
For For 20-35 nucleotides: Tm= 22 + 1.46 (/„)
8 or for other monovalent cation, but only accurate in the 0.01-0.4 M range.
b only accurate for %GC in the 30% to 75% range.
c L = length of duplex in base pairs.
d Oligo, oligonudeotide; /n, effective length of primer = 2x(no. of G/C)+(no. of A/T).
Note: for each 1% formamide, the Tm is reduced by about 0.6 to 0.7°C, white the presence of 6 M urea reduces the Tm by about 30°C
Specificity of hybridisation is typically the function of post-hybridisation washes. To remove background resulting from non-specific hybridisation, samples are washed with dilute salt solutions. Critical factors of such washes include the ionic strength and temperature of the final wash solution: the lower the salt concentration and the higher the wash temperature, the higher the stringency of the wash. Wash conditions are typically performed at or below hybridisation stringency. Generally, suitable stringent conditions for nucleic acid hybridisation assays or gene ampiificatfon detection procedures are as set forth above- Conditions of greater or less stringency may also be selected. Generally, low stringency conditions are selected to be about 50°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. Medium stringency conditions are when the temperature is 20°C below Tm, and high stringency conditions are when the temperature fs 10°C below Tm. For example, stringent conditions are those that are at least as stringent as, for example, conditions A-L; and reduced stringency conditions are at least as stringent as, for example, conditions M-R. Non-specific binding may be controlled using any one of a number of known techniques such as, for example, blocking the membrane with protein containing solutions,



dicotyledonous species, preferably from the family Brassicaceae, further preferably from Arabidopsis thaliana. More preferably, the Yippee-iike nucleic add isolated from Arabidopsis thaliana is represented by SEQ ID NO: 1 and the YIPPEE-like amino acid sequence is as represented by SEQ ID NO: 2.
The activity of a YIPPEE-like polypeptide or a homologue thereof may be increased by introducing a genetic modification (preferably in the locus of a YIPPEE-like gene). The locus of a gene as defined herein is taken to mean a genomic region, which includes the gene of interest and 10KB up- or down stream of the coding region.
The genetic modification may be introduced, for example, by any one (or more) of the following methods: TDNA activation, TILLING, site-directed mutagenesis, directed evolution, homologous recombination or by introducing and expressing in a plant a nucleic acid encoding a YIPPEE-like polypeptide or a homologue thereof. Following introduction of the genetic modification, there follows a step of selecting for increased activity of a YIPPEE-like polypeptide, which increase in activity gives plants having improved growth characteristics.
T-DNA activation tagging (Hayashi et a/. Science (1992) 1350-1353) involves insertion of T-DNA usually containing a promoter (may also be a translation enhancer or an intron), in the genomic region of the gene of interest or 10KB up- or down stream of the coding region of a gene in a configuration such that the promoter directs expression of the targeted gene. Typically, regulation of expression of the targeted gene by its natural promoter is disrupted and the gene fells 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, 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-preferred, cell type-preferred and inducible promoters are all suitable for use in T-DNA activation.
A genetic modification may also be introduced in the locus of a YIPPEE-like gene using the technique of TILLING (Targeted Induced Local Lesions IN Genomes). This is a mutagenesis technology useful to generate and/or identify, and to eventually isolate mutagenised variants of a YIPPEE-like nucleic acid capable of exhibiting YIPPEE-like activity. TILLING also allows selection of plants carrying such mutant variants. These mutant variants may even exhibit higher YIPPEE-like activity than that exhibited by the gene in its natural form. TILLNG


* The "hybrid length" is the anticipated length for the hybridising nucleic acid. When nucleic
acids of known sequence are hybridised, the hybrid length may be determined by aligning the
sequences and identifying the conserved regions described herein.
* SSPE (1xSSPE is 0.15M NaCI, 10mM NaH2PO^, and 1.25mM EDTA, pH7.4) may be
substituted for SSC (1*SSC is 0.15M NaCI and 15mM sodium citrate) in the hybridisation and
wash buffers; washes are performed for 15 minutes after hybridisation is complete. The
hybridisations and washes may additionally include 5 * Denhardfs reagent, .5-1.0% SDS, 100
pg/ml denatured, fragmented salmon sperm DNA, 0.5% sodium pyrophosphate, and up to
50% formamide.
* Tb-Tr The hybridisation temperature for hybrids anticipated to be less than 50 base pairs in
* length should be 5-10°C less than the melting temperature Tm of the hybrids; the Tm is
* determined according to the above-mentioned equations.
* The present invention also encompasses the substitution of any one, or more DNA or RNA
* hybrid partners with either a PNA, or a modified nucleic acid.
For the purposes of defining the level of stringency, reference can be made to Sambrook etaL (2001) Molecular Cloning: a laboratory manual, 3rd Edition Cold Spring Harbor Laboratory Press, CSH, New Yorfc or to Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989).
The YIPPEE-like nucleic acid or variant thereof may be derived from any natural or artificial source. The nucleic acid/gene or variant thereof may be isolated from a microbial source, such as bacteria, yeast or fungi, or from a plant, algae or animal (including human) source. This nucleic acid may be modified from its native form in composition and/or genomic environment through deliberate human manipulation. The nucleic acid is preferably of plant origin, whether from the same plant species (for example to the one in which it is to be introduced) or whether from a different plant species. The nucleic acid may be isolated from a

combines high-density mutagenesis with high-throughput screening methods. The steps typically followed in TILLING are: (a) EMS mutagenesis (Redei and Koncz, 1992; Feldmann et a/., 1994; Lightner and Caspar, 1998); (b) DNA preparation and pooling of individuals; (c) PCR amplification of a region of interest; (d) denaturation and annealing to allow formation of heteroduplexes; (e) DHPLC, where the presence of a heteroduplex in a pool is detected as an extra peak in the chromatogram; (f) identification of the mutant individual; and (g) sequencing of the mutant PCR product. Methods for TILLING are well known in the art (McCallum Nat Biotechnol. 2000 Apr; 18(4):455-7, reviewed by Stemple 2004 (TILLING-a high-throughput harvest for functional genomics. Nat Rev Genet. 2004 Feb;5(2):145-50.)).
Site directed mutagenesis may be used to generated variants of YIPPEE-like nucleic acids or portions thereof. Several methods are available to achieve site directed mutagenesis, the most common being PCR based methods (current protocols in molecular biology. Wiley Eds. http://www.4ulr.a3m/products/currentprotocols/index.htrnl).
Directed evolution may also be used to generate variants of YIPPEE-like nucleic acids. This consists of iterations of DNA shuffling followed by appropriate screening and/or selection to generate variants of YIPPEE-like nucleic acids or portions thereof encoding polypeptides having a modified biological activity (Castle ef a/., (2004) Science 304(5674): 1151-4; US patents 5,811,238 and 6,395,547).
TDNA activation, TILLING, directed evolution and site-directed mutagenesis are examples of technologies that enable the generation of novel alleles and YIPPEE-like variants.
Homologous recombination allows introduction in a genome of a selected nucleic acid at a defined selected position. Homologous recombination is a standard technology used routinely in biological sciences for lower organisms such as yeast or the moss physcom'rtrella. Methods for performing homologous recombination in plants have been described not only for model plants (Offringa ef a/. Extrachromosomal homologous recombination and gene targeting in plant cells after /Ignobactem/m-mediated transformation. 1990 EMBO J. 1990 Oct; 9(10):3077-S4) but also for crop plants, for example rice (Terada R, Urawa H, Inagaki Y, Tsugane K, lida S. Efficient gene targeting by homologous recombination in rice. Nat Biotechnol. 2002. lida and Terada: A tale of two integrations, transgene and T-DNA: gene targeting by homologous recombination in rioe. Curr Opin Biotechnol. 2004 Apr; 15(2):132-8). The nucleic acid to be targeted (which may be a YIPPEE-like nucleic acid or variant thereof as hereinbefore defined) need not be targeted to the locus of a YIPPEE-like 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.
According to a preferred embodiment of the invention, plant growth characteristics may be improved by introducing and expressing in a plant a nucleic acid encoding a YIPPEE-like polypeptide or a homologue thereof.
A preferred method for introducing a genetic modification (which in this case need not be in the locus of an YiPPEE-like gene) is to introduce and express in a plant a nudeic acid encoding a YIPPEE-like polypeptide or a homologue thereof. An YIPPEE-like polypeptide or a homologue thereof as mentioned above is one comprising (i) a putative zinc-binding motif. 2xCXXC, where X is any amino acid residue; and (ii) the motif KYKEGK, allowing for one amino acid substitution at any position and any conservative amino acid substitution; and (iii) the motif GRAYLF, allowing for one amino acid substitution at any position and any conservative amino acid substitution. The nudeic acid to be introduced into a plant may be a full-length nudeic acid or may be a portion or a hybridizing sequence as hereinbefore defined.
"Homologues" of a protein encompass peptides, oligopeptides, polypeptides, proteins and enzymes having amino acid substitutions, deletions and/or insertions relative to the unmodified protein in question and having similar biological and functional activity as the unmodified protein from which they are derived. 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-helical structures or 13-sheet structures). Conservative substitution tables are well known in the art (see for example Creighton (1984) Proteins. W.H. Freeman and Company and Table 1 above).
According to a preferred feature of the invention, the homologue has at least 45% sequence identity to the amino acid sequence represented by SEQ ID NO: 2. Whether a polypeptide has at least 45% identity to the amino acid represented by SEQ ID NO: 2 may readily be established by sequence alignment. Methods for the alignment of sequences for comparison are well known in the art, such methods include GAP, BESTFIT, BLAST, FASTA and TFASTA. GAP uses the algorithm of Needleman and Wunsch (J. Mol. Biol. 48: 443-453, 1970) to find the alignment of two complete sequences that maximises the number of matches and minimises the number of gaps. The BLAST algorithm calculates percent sequence identity and performs a statistical analysis of the similarity between the two sequences. The software for performing BLAST analysis is publicly available through the National Centre for

Biotechnology Information. A YIPPEE-like polypeptide or a homologue thereof having at least 45% identity to the amino acid represented by SEQ ID NO: 2 may readily be identified by aligning a query sequence (preferably a protein sequence) with known YIPPEE-like protein sequences (see for example the alignment shown in Figure 1). The query sequence may be aligned (with known YIPPEE-like sequences) using, for example, the VNTI AlignX multiple alignment program, based on a modified clustal W algorithm (InforMax, Bethesda, MD, http://www.informaxinc.com), with default settings for gap opening penalty of 10 and a gap extension of 0.05.
Also encompassed by the term "homologues" are two special forms of homology, which include orthologous sequences and paralogous sequences, which encompass evolutionary concepts used to describe ancestral relationships of genes. The term "paralogous" relates to gene-duplications within the genome of a species leading to paralogous genes. The term "orthologous" relates to homologous genes in different organisms due to speciation.
Othologues in, for example, monocot plant species may easily be found by performing a so-called reciprocal blast search. This may be done by a first blast involving blasting the sequence in question (for example, SEQ ID NO: 1 or SEQ ID NO: 2) against any sequence database, such as the publicly available NCBI database which may be found at: http://www.ncbi.nlm.nih.gov. If orthoiogues in rice were sought, the sequence in question would be blasted against, for example, the 28,469 full-length cDNA clones from Oryza sativa Nipponbare available at NCBI. BLASTn or tBLASTX may be used when starting from nucleotides or BLASTP or TBLASTN when starting from the protein, with standard default values. The blast results may be filtered. The full-length sequences of either the filtered results or the non-filtered results are then blasted back (second blast) against the sequences of the organism from which the sequence in question is derived. The results of the first and second blasts are then compared. An orthologue is found when the results of the second blast give as hits with the highest similarity an YIPPEE-like nucleic acid or YIPPEE-like polypeptide, for example, if one of the organisms is Arabidopsis thaliana then a paralogue is found. In the case of large families, ClustalW may be used, followed by a neighbour joining tree, to help visualize the clustering.
A homologue may be in the form of a "substitutional variant" of a protein, i.e. where at least one residue in an amino acid sequence has been removed and a different residue inserted in its place. Amino acid substitutions are typically of single residues, but may be clustered depending upon functional constraints placed upon the polypeptide; insertions will usually be

of the order of about 1 to 10 amino acid residues. Preferably, amino acid substitutions comprise conservative amino acid substitutions.
A homologue may also be in the form of an "insertional variant" of a protein, i.e. where one or more amino acid residues are introduced into a predetermined site in a protein. Insertions may comprise amino-terminal and/or carboxy-terminal fusions as well as intra-sequence insertions of single or multiple amino acids. Generally, insertions within the amino acid sequence will be smaller than amino- or carboxy-terminal fusions, of the order of about 1 to 10 residues. Examples of amino- or carboxy-terminal fusion proteins or peptides include the binding domain or activation domain of a transcriptional activator as used in the yeast two-hybrid system, phage coat proteins, (histidine^-tag, glutathione S-transferase-tag, protein A, maltose-binding protein, dihydrofolate reductase, Tag-100 epitope, c-myc epitope, FLAG®-epitope, lacZ, CMP (calmodulin-binding peptide), HA epitope, protein C epitope and VSV epitope.
Homologues in the form of "deletion variants" of a protein are characterised by the removal of one or more amino acids from a protein.
Amino acid variants of a protein may readily be made using peptide synthetic techniques well known in the art, such as solid phase peptide synthesis and the like, or by recombinant DNA manipulations. Methods for the manipulation of DNA sequences to produce substitution, insertion or deletion variants of a protein are well known in the art. For example, techniques for making 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, Cleveland, OH), QuickChange Site Directed mutagenesis (Stratagene, San Diego, CA), PCR-mediated site-directed mutagenesis or other site-directed mutagenesis protocols.
The YIPPEE-like polypeptide or homologue thereof may be a derivative. "Derivatives" include peptides, oligopeptides, polypeptides, proteins and enzymes which may comprise substitutions, deletions or additions of naturally and non-naturally occurring amino acid residues compared to the amino acid sequence of a naturally-occurring form of the protein, for example, as presented in SEQ ID NO: 2. "Derivatives" of a protein encompass peptides, oligopeptides, polypeptides, proteins and enzymes which may comprise naturally occurring altered, glycosyiated, acylated or non-naturally occurring amino acid residues compared to the amino acid sequence of a naturally-occurring form of the polypeptide. A derivative may also comprise one or more non-amino acid substituents compared to the amino acid sequence from which it is derived, for example a reporter molecule or other ligand, covalently or non-covalently bound to the amino acid sequence, such as a reporter molecule which is bound to

facilitate its detection, and non-naturally occurring amino acid residues relative to the amino acid sequence of a naturally-occurring protein.
The YIPPEE-like polypeptide or homologue thereof may be encoded by an alternative splice variant of a YIPPEE-like nucleic acid/gene. The term "alternative splice variant" as used herein encompasses variants of a nucleic acid sequence in which selected introns and/or exons have been excised, replaced or added. Such variants will be ones in which the biological activity of the protein is retained, which may be achieved by selectively retaining functional segments of the protein. Such splice variants may be found in nature or may be manmade. Methods for making such splice variants are well known in the art. Preferred splice variants are splice variants of the nucleic acid represented by SEQ ID NO: 1. Further preferred are splice variants encoding a polypeptide comprising any one or more of, and preferably all of: (i) a putative zinc-binding motif: 2xCXXC, where X is any amino acid residue; (ii) the motif KYKEGK, allowing for one amino acid substitution and any conservative amino acid substitution; (iii) the motif GRAYLF, allowing for one amino acid substitution and any conservative amino acid substitution.
The homologue may also be encoded by an allelic variant of a nucleic acid encoding a YIPPEE-like polypeptide or a homologue thereof, preferably an allelic variant of the nucleic acid represented by SEQ ID NO: 1. Further preferably, the polypeptide encoded by the allelic variant comprises any one or more of, and preferably all of: (i) a putative zinc-binding motif: 2xCXXC, where X is any amino acid residue; (ii) the motif KYKEGK, allowing for one amino acid substitution at any position and any conservative amino acid substitution; (iii) the motif GRAYLF, allowing for one amino acid substitution at any position and any conservative amino acid substitution. 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. SNPs and INDELs form the largest set of sequence variants in naturally occurring polymorphic strains of most organisms/
According to a preferred aspect of the present invention, enhanced or increased expression of the YIPPEE-like nucleic acid or variant thereof is envisaged. Methods for obtaining enhanced or increased expression of genes or gene products are well documented in the art and include, for example, overexpression driven by appropriate promoters, the use of transcription enhancers or translation enhancers. Isolated nucleic acids which serve as promoter or enhancer elements may be introduced in an appropriate position (typically upstream) of a non-heterologous form of a polynucleotide so as to upregulate expression of a YIPPEE-like 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; Zariing et a/., PCT7US93/03868), 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.
If polypeptide expression is desired, it is generally desirable to include a polyadenylation region at the 3'-end of a polynucleotide coding region. The polyadenylation region can be derived from the natural gene, from a variety of other plant genes, or from T-DNA. The 31 end sequence to be added may be derived from, for example, the nopaline synthase or octopine synthase genes, or alternatively from another plant gene, or less preferably from any other eukaryotic gene-
An intron sequence may also be added to the 5' untranslated region or the coding sequence of the partial coding sequence to increase the amount of the mature message that accumulates in the cytosol. Inclusion of a spliceable intron in the transcription unit in both plant and animal expression constructs has been shown to increase gene expression at both the mRNA and protein levels up to 1000-fold, Buchman and Berg, Mol. Cell bid. 8:4395-4405 (1988); Cadis et a/., 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 AdM-S intron 1, 2, and 6, the Bronze-1 intron are known in the art. See generally, The Maize Handbook, Chapter.116, Freeling and Walbot, Eds., Springer, N.Y. (1994).
The invention also provides genetic constructs and vectors to facilitate introduction and/or expression of the nucleotide sequences useful in the methods according to the invention.
Therefore, there is provided a gene construct comprising: (i) A YIPPEE-like nucleic acid or variant thereof; (ii) One or more control sequences capable of driving expression of the nucleic acid
sequence of (i); and optionally (iii) A transcription termination sequence.
Constructs useful in the methods according to the present invention may be constructed using recombinant DNA technology well known to persons skilled in the art. The gene constructs may be inserted into vectors, which may be commercially available, suitable for transforming into plants and suitable for expression of the gene of interest in the transformed cells.

Plants are transformed with a vector comprising the sequence of interest (i.e., a YIPPEE-like nucleic acid or variant thereof). The sequence of interest is operabiy linked to one or more control sequences (at least to a promoter). The terms "regulatory element", "control sequence" and "promoter" are all used interchangeably herein and are to be taken in a broad context to refer to regulatory nucleic acid sequences capable of effecting expression of the sequences to which they are ligated. Encompassed by the aforementioned terms are transcriptional regulatory sequences derived from a classical eukaryotic genomic gene (including the TATA box which is required for accurate transcription initiation, with or without a CCAAT box sequence) and additional regulatory elements (i.e. upstream activating sequences, enhancers and silencers) which alter gene expression in response to developmental and/or external stimuli, or in a tissue-specific manner. Also included within the term is a transcriptional regulatory sequence of a classical prokaryotic gene, in which case it may include a -35 box sequence and/or-10 box transcriptional regulatory sequences. The term "regulatory element* also encompasses a synthetic fusion molecule or derivative which confers, activates or enhances expression of a nucleic acid molecule in a cell, tissue or organ. The term "operabiy linked" as used herein refers to a functional linkage between the promoter sequence and the gene of interest, such that the promoter sequence is able to initiate transcription of the gene of interest.
Advantageously, any type of promoter may be used to drive expression of the nucleic acid sequence. The promoter may be an inducible promoter, he. having induced or increased transcription initiation in response to a developmental, chemical, environmental or physical stimulus. An example of an inducible promoter being a stress-indudble promoter, i.e. a promoter activated when a plant is exposed to various stress conditions. Additionally or alternatively, the promoter may be a tissue-preferred promoter, i.e. one that is capable of preferentially initiating transcription in certain tissues, such as the leaves, roots, seed tissue etc. Promoters able to initiate transcription in certain tissues only are referred to herein as "tissue-specific11.
Preferably, the YIPPEE-like nucleic acid or variant thereof is operabiy linked to a constitutive promoter. A constitutive promoter is transcriptionally active during most, but not necessarily all, phases of its growth and development and is substantially ubiquitously expressed. Preferably, the constitutive promoter is a GOS2 promoter (from rice) (SEQ ID NO: 25). It should be clear that the applicability of the present invention is not restricted to the YIPPEE-like nucleic add represented by SEQ ID NO: 1, nor is the applicability of the invention restricted to expression of an YIPPEE-like nucleic acid when driven by a G0S2 promoter. Examples of other constitutive promoters are shown in Table 4 below.


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 31 processing and polyadenylation of a primary transcript and termination of transcription. Additional regulatory elements may include transcriptional as well as translational enhancers. Those skilled in the art will be aware of terminator and enhancer sequences which may be suitable for use in performing the invention. Such sequences would be known or may readily be obtained by a person skilled in the art.
The genetic constructs of the invention may further include an origin of replication sequence which is required for maintenance and/or replication in a specific cell type. One example is when a genetic construct is required to be maintained in a bacterial cell as an episomal genetic element (e.g. plasmid or cosmid molecule). Preferred origins of replication include, but are not limited to, the fl-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. Cells containing the recombinant DNA will thus be able to survive in the presence of antibiotic or herbicide concentrations that kill untransformed cells. Examples of selectable marker genes include the bar gene which provides resistance to the herbicide Basta; the npt gene which confers resistance to the antibiotic kanamycin; the hpt gene which confers hygromycin resistance. Visual markers, such as the Green Fluorescent Protein (GFP, Haseloff et a/., 1997), p-glucuronidase (GUS) or luciferase may also be used as selectable markers. Further examples of suitable selectable marker genes include the ampicillin resistance (Ampr), tetracydine resistance gene (Tcr), phosphinothricin resistance gene, , hygromycin resistance geneand the chloramphenicol acetyltransferase (CAT) gene, amongst others.
The present invention also encompasses plants obtainable by the methods according to the present invention. The present invention therefore provides plants obtainable by the method according to the present invention, which plants have introduced therein a YIPPEE-like nucleic acid or variant thereof and/or which plants have a genetic modification preferably in the locus of a YIPPEE-like gene.
The invention also provides a method for the production of transgenic plants having improved growth characteristics, comprising introduction and expression in 8 plant of a YIPPEE-like nucleic acid or a variant thereof and/or comprising introduction of a genetic modification preferably in the locus of a YIPPEE-like gene.
More specifically, the present invention provides a method for the production of transgenic plants having improved growth characteristics, which method comprises:
(i) introducing into a plant or plant cell a YIPPEE-like nucleic acid or variant thereof and/or introducing a genetic modification preferably in the locus of a YIPPEE-like gene; and (ii) cultivating the plant cell under conditions promoting plant growth and development.
The nucleic acid may be introduced directly into a plant cell or into the plant itself (induding introduction into a tissue, organ or any other part of a plant). According to a preferred feature of the present invention, the nucleic acid is preferably introduced into a plant by transformation.

The term "transformation* as referred to herein encompasses the transfer of an exogenous polynucleoticle into a host cell, irrespective of the method used for transfer. Rant tissue capable of subsequent clonal propagation, whether by organogenesis or embryogenesis, may be transformed with a genetic construct of the present invention and a whole plant regenerated therefrom. The particular tissue chosen will vary depending on the clonal propagation systems available for, and best suited to, the particular species being transformed. Exemplary tissue targets include leaf disks, pollen, embryos, cotyledons, hypocotyls, megagametophytes, callus tissue, existing meristematic tissue (e.g., apical meristem, axillary buds, and root meristems), and induced meristem tissue (e.g., cotyledon meristem and hypocotyl meristem). The polynucleotide may be transiently or stably introduced into a host cell and may be maintained non-integrated, for example, as a plasmid. Alternatively, it may be integrated into the host genome. The resulting transformed plant cell may then be used to regenerate a transformed plant in a manner known to persons skilled in the art.
Transformation of plant species is now a fairly routine technique. Advantageously, any of several transformation methods may be used to introduce the gene of interest into a suitable ancestor cell. Transformation methods include the use of liposomes, electroporation, chemicals that increase free DNA uptake, injection of the DNA directly into the plant, particle gun bombardment, transformation using viruses or pollen and microprojection. Methods may be selected from the calcium/polyethylene glycol method for protoplasts (Krens, F.A. et a/.f 1882, Nature 296, 72-74; Negrutiu I. et a/., June 1987, Plant Mol. Biol. 8, 363-373); electroporation of protoplasts (Shillito R.D. et a/., 1985 Bio/Technol 3, 1099-1102); microinjection into plant material (Crossway A. et a/., 1986, Mol. Gen Genet 202, 179-185); DNA or RNA-coated particle bombardment (Klein T.M. et a/., 1987, Nature 327, 70) infection with (non-integrative) viruses and the like, Transgenic rice plants expressing a YIPPEE-Iike nucleic acid/gene are preferably produced via Agrvbacterium~rr\e6iated transformation using any of the well known methods for rice transformation, such as described in any of the following: published European patent application EP 1198985 A1, Aidemita and Hodges (Planta, 199, 612-617, 1996); Chan et aL (Plant Mol. Biol. 22 (3) 491-506, 1993), Hiei et a/. (Plant J. 6 (2) 271-282, 1994), which disclosures are incorporated by reference herein as if fully set forth. In the case of corn transformation, the preferred method is as described in either Ishida et aL (Nat. 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 gene of interest, following which the transformed material is regenerated into a whole plant
Following DNA transfer and regeneration, putatively transformed plants may be evaluated, for instance using Southern analysis, for the presence of the gene of interest, copy number and/or genomic organisation. Alternatively or additionally, expression levels of the newly introduced DNA may be monitored using Northern and/or Western analysis, both techniques being well known to persons having ordinary skill in the art.
The generated transformed plants may be propagated by a variety of means, such as by clonal propagation or classical breeding techniques. For example, a first generation (or T1) transformed plant may be selfed to give homozygous second generation (or T2) transformants, and the T2 plants further propagated through classical breeding techniques.
The generated transformed organisms may take a variety of forms. For example, they may be chimeras of transformed cells and non-transformed cells; clonal transformants (e.g., all cells transformed to contain the expression cassette); grafts of transformed and untransformed tissues (e.g., in plants, a transformed rootstock grafted to an untransformed scion).
The present invention dearly extends to any plant cell or plant produced by any of the methods described herein, and to all plant parts and propagules thereof. The present invention extends further to encompass the progeny of a primary transformed or transfected ceil, tissue, organ or whole plant that has been produced by any of the aforementioned methods, the only requirement being that progeny exhibit the same genotypic and/or phenotypic characteristics) as those produced in the parent by the methods according to the invention. The invention also includes host cells containing an isolated YIPPEE-like nucleic acid or variant thereof. Preferred host cells according to the invention are plant cells. The invention also extends to harvestable parts of a plant such as but not limited to seeds, leaves, fruits, flowers, stem cultures, rhizomes, tubers and bulbs.
The present invention also encompasses the use of YIPPEE-like nucleic acids or variants thereof and to the use of YIPPEE-like polypeptides or homologues thereof.
One such use relates to improving the growth characteristics of plants, in particular in improving yield/biomass, especially seed yield. The seed yield may include one or more of the

following: increased number of (filled) seeds, increased seed weight, increased harvest index among others.
YIPPEE-like nucleic acids or variants thereof, or YIPPEE-like polypeptides or homologues thereof may find use in breeding programmes in which a DNA marker is identified which may be genetically linked to a YIPPEE-like gene or variant thereof. The YIPPEE-like nucleic acids/ genes or variants thereof, or YIPPEE-like polypeptides or homologues thereof may be used to define a molecular marker. This DNA or protein marker may then be used in breeding programs to select plants having improved growth characteristics. The YIPPEE-like gene or variant thereof may, for example, be a nucleic acid as represented by any one of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21 and SEQ ID NO: 23.
Allelic variants of a YIPPEE-like nucleic acid/gene may also find use in marker-assisted breeding programmes. Such breeding programmes sometimes require introduction of allelic variation by mutagenic treatment of the plants, using for example EMS mutagenesis; alternatively, the programme may start with a collection of allelic variants of so called "natural" origin caused unintentionally. Identification of allelic variants then takes place by, for example, PCR. This is followed by a selection step for selection of superior allelic variants of the sequence in question and which give improved growth characteristics in a plant. Selection is typically carried out by monitoring growth performance of plants containing different allelic variants of the sequence in question, for example, different allelic variants of any one of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21 and SEQ ID NO: 23. Growth performance may be monitored in a greenhouse or in the field. Further optional steps include crossing plants, in which the superior allelic variant was identified, with another plant. This could be used, for example, to make a combination of interesting phenotypic features.
A YIPPEE-like 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 phenotypes. Such use of YIPPEE-like nucleic acids or variants thereof requires only a nucleic acid sequence of at least 15 nucleotides in length. The YIPPEE-like nucleic acids or variants thereof may be used as restriction fragment length polymorphism (RFLP) markers. Southern blots (Maniatis) of restriction-digested plant genomic DNA may be probed with the YIPPEE-like nucleic acids or variants thereof. The resulting banding patterns may then be subjected to

genetic analyses using computer programs such as MapMaker (Lander et a/. (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 YIPPEE-like nucleic acid or variant thereof in the genetic map previously obtained using this population (Botstein et ai (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 (1986) Plant Mol. Biol. Reporter 4:37-41. Numerous publications describe genetic mapping of specific cDNA clones using the methodology outlined above or variations thereof. For example, F2 intercross populations, backcross populations, randomly mated populations, near isogenic lines, and other sets of individuals may be used for mapping. Such methodologies are well known to those skilled in the art
The nucleic acid probes may also be used for physical mapping (i.e., placement of sequences on physical maps; see Hoheisel et ai 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 hybridization (FISH) mapping (Trask (1991) Trends Genet. 7:149-154). Although current methods of FISH mapping favor use of large clones (several to several hundred KB; see Laan et al. (1995) *©enorne Res. 5:13-20), improvements in sensitivity may allow performance of FISH mapping using shorter probes.
A variety of nucleic add amplification-based methods for genetic and physical mapping may be carried out using the nucleic acids. Examples include allele-specffic amplification (Kazazian (1989) J. Lab. CHn. Med 11:95-96), polymorphism of PCR-ampfified fragments (CAPS; Sheffield et al. (1993) Genomics 16:325-332), allele-specific ligation (Landegren ef a/. (1988) Science 241:1077-1080), nucteotide extension reactions (Sokolov (1990) Nucleic Acid Res. 18:3671), Radiation Hybrid Mapping (Walter et al. (1997) Nat. Gen&t 7:22-28) and Happy Mapping (Dear and Cook (1989) Nucleic Acid Res. 17:6795-6807). For these methods, the sequence of a nucleic add is used to design and produce primer pairs for use in the amplification reaction or in primer extension reactions. The design of such primers is well known to those skilled in the art. In methods employing PCR-based genetic mapping, it may be necessary to identify DNA sequence differences between the parents of the mapping cross

in the region corresponding to the instant nucleic acid sequence. This, however, is generally not necessary for mapping methods.
YIPPEE-like nucleic acids or variants thereof or YIPPEE-like pofypeptides or homologues thereof may also find use as growth regulators. Since these molecules have been shown to be useful in improving the growth characteristics of plants, they would also be useful growth regulators, such as herbicides or growth stimulators. The present invention therefore provides a composition, for use as a growth regulator, comprising a YIPPEE-like nucleic add/gene or variant thereof, or a YIPPEE-like polypeptide or homologue thereof, together with a suitable carrier, diluent or excipient.
The methods according to the present invention result in plants having improved growth characteristics, as described hereinbefore. These advantageous growth characteristics may also be combined with other economically advantageous traits, such as further yield-enhancing traits, tolerance to various stresses, traits modifying various architectural features and/or biochemical and/or physiological features.
Description of figures
The present invention will now be described with reference to the following figures in which:
Fig- 1 shows a multiple alignment of several plant YIPPEE-like polypeptides. The cysteine residues of the putative zinc-binding motif 2xCXXCs where X may be any amino acid, are in bold. The motif KYKEGK and the motif GRAYLF are boxed. At denotes Arabidopsis thaliana.
Fig. 2 shows a binary vector for expression in Oryza sativa of an Arabidopsis thaliana YIPPEE-like (internal reference CDS1522) under the control of a GOS2 promoter (internal reference PRO0129).
Fig. 3 details examples of sequences useful in performing the methods according to the present invention.
Examples
The present invention will now be described with reference to the following examples, which are by way of illustration alone.
DNA manipulation: unless otherwise stated, recombinant DNA techniques are performed according to standard protocols described in (Sambrook (2001) Molecular Cloning: a

laboratory manual, 3rd Edition Cold Spring Harbor Laboratory Press, CSH, New York) or in Volumes 1 and 2 of Ausubel et ai (1994), Current Protocols in Molecular Biology, Current Protocols. Standard materials and methods for plant molecular work are described in Plant Molecular Biology Labfase (1993) by R.D.D. Croy( published by BIOS Scientific Publications Ltd (UK) and Blackwell Scientific Publications (UK).
Example 1: Gene Cloning
The Arabidopsis thaliana YIPPEE-like gene (CDS1522) was amplified by PCR using as template an Arabidopsis thaliana seedling cDNA library (Invitrogen, Paisley, UK). After reverse transcription of RNA extracted from seedlings, the cDNAs were cloned into pCMV Sport 6.0. Average insert size of the bank was 1.5 kb and the original number of clones was of the order of 1.59x107 cfu. Original titer was determined to be 9.6x105 cfu/ml after first amplification of 6x10" cfu/ml. After plasmid extraction, 200 ng of template was used in a 50 pi PCR mix. Primers prmO3196 (sense, start codon in bold, AttB1 site in italic: 5'-GGGGACAAGTrTGTACAAAAAAGCAGGCTTCfiCAATGGCTGTCGGAG/KTGAT 31) and prmO3199 (reverse, complementary, stop codon in bold, AttB2 site in italic: 5' GGGGACCAC77TG7/\CAAGAMGCrGGG7"AATCAAGCATCATCTCCATCACTAAC 3'), which include the AttB sites for Gateway recombination, were used for PCR amplification. PCR was performed using Hifi Taq DNA polymerase in standard conditions. A PCR fragment of 366 bp was amplified and purified also using standard methods. The first step of the Gateway procedure, the BP reaction, was then performed, during which the PCR fragment recombines in vivo with the pDONR201 plasmid to produce, according to the Gateway terminology, an "entry clone", p3956, Plasmid pDOMR201 was purchased from Invitrogen, as part of the Gateway® technology.
Example 2: Vector Construction
The entry clone p3956 was subsequently used in an LR reaction with p0640, a destination vector used for Oryza sativa transformation. This vector contains as functional elements within the T-DNA borders: a plant selectable marker; a screenable marker expression cassette; and a Gateway cassette intended for LR in vivo recombination with the sequence of interest already cloned in the entry clone. A rice GOS2 promoter for constitutive expression (PRO0129) was upstream of this Gateway cassette (De Pater et a/., Plant J. 1992 Nov; 2(6):837-44).
After the LR recombination step, the resulting expression vector (Figure 2) was transformed into Agrobacterium strain LBA4044 and subsequently to Oryza sativa plants. Transformed rice plants were allowed to grow and were then examined for the parameters described in Example 3.

Example 3: Evaluation and Results
Approximately 15 to 20 independent TO rice transformants were generated. The primary transformants were transferred from a tissue culture chamber to a greenhouse for growing and harvest of T1 seed. 5 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 lacking the transgene (nullizygotes) were selected by monitoring visual marker expression.
Statistical analysis: F-test
A two factor ANOVA (analysis of variants) was used as a statistical model for the overall evaluation of plant phenotypic characteristics. An F4est was carried out on all the parameters measured of all the plants of all the events transformed with the gene of the present invention. The F-test was carried out to check for an effect of the gene over all the transformation events and to verify for an overall effect of the gene, also known as a global gene effect. The threshold for significance for a true global gene effect was set at a 5% probability level for the F-test. A significant F-test value points to a gene effect, meaning that it is not only the presence or position of the gene that is causing the differences in phenotype.
3.1 Seed-related parameter measurements
The mature primary panicles were harvested, bagged, barcode-labeled and then dried for three days in an oven at 37°C. The panicles were then threshed and all the seeds were 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. The number of filled seeds was determined by counting the number of filled husks that remained after the separation step. The total seed yield was measured by weighing all filled husks harvested from a plant. Total seed number per plant was measured by counting the number of husks harvested from a plant. The harvest index in the present invention is defined as the ratio of total seed yield and the above ground area (mm2) multiplied by a factor 106.
3.2 Aboveground Area
Plant aboveground area was determined by counting the total number of pixels 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 physical surface value expressed in square mm by calibration. Experiments show that the

aboveground plant area measured this way correlates with the biomass of plant parts above ground.
The Table of results below show the p values from the F test for the T1 evaluations and for extra T1 events generated. The percentage difference between the transgenics and the corresponding nullizygotes is also shown. For example, for total seed weight, 3 out of 6 lines were positive for total seed weight (i.e., showed an increase in total seed weight (of greater than 32%) compared to the seed weight of corresponding nullizygote plants). 1 out of 6 of these lines showed a significant increase in total seed weight with a p value from the F test of 0.18.


Claims
1. Method for improving plant growth characteristics, comprising the steps of (A)
2. increasing activity in a plant of a YIPPEE-like poiypeptide or a homologue thereof
3. comprising any one or more of, and preferably all of: (i) a putative zinc-binding motif
4. 2xCXXC, where X is any amino acid residue; (ii) the motif KYKEGK, allowing for one
5. amino acid substitution at any position and any conservative amino acid substitution;
6. (ifi) the motif GRAYLF, allowing for one amino acid substitution at any position and any
7. conservative amino acid substitution; and (B) optionally selecting for plants having
8. improved growth characteristics.
9. Method according to claim 1, wherein said increased activity is effected by introducing a
10. genetic modification preferably in the locus of a gene encoding a YIPPEE-like
11. poiypeptide or a homologue thereof.
12. Method according to claim 2, wherein said genetic modification is effected by any one
13. or more: site-directed mutagenesis, directed evolution, homologous recombination,
14. TILLING, T-DNA activation and by introducing and expressing in a plant a nucleic acid
15. encoding a YIPPEE-like poiypeptide.
16. Method for improving plant growth characteristics, comprising introducing and
17. expressing in a plant a YIPPEE-like nucleic acid or a variant thereof, which YIPPEE-like
18. nucleic acid or variant thereof encodes a YIPPEE-like poiypeptide or a homologue
19. thereof comprising any one or more of, and preferably all of: (i) a putative zinc-binding
20. motif: 2xCXXC, where X is any amino acid residue; (ii) the motif KYKEGK, allowing for
21. one amino acid substitution at any position and any conservative amino acid
22. substitution; (Hi) the motif GRAYLF, allowing for one amino acid substitution at any
23. position and any conservative amino acid substitution.
24. Method according to claim 4, wherein said variant is a portion of a YIPPEE-like nucleic
25. acid or a sequence capable of hybridising to a YIPPEE-like nucleic acid.
26. Method according to claim 4 or 5, wherein said YIPPEE-Hke nucleic acid or variant
27. thereof is overexpressed in a plant.
28. Method according to any one of claims 4 to 6, wherein said YIPPEE-like nucleic acid or
29. variant thereof is of plant origin, preferably from a dicotyledonous plant, further

preferably from the family Brassicaceae, more preferably the nudeic add is from Arabidopsis thaliana.
8. Method according to any one of daims 4 to 7, wherein said YIPPEE-like nudeic acid or
9. variant thereof is operably linked to a constitutive promoter.
10. Method according to daim 8, wherein said constitutive promoter is a GOS2 promoter.
11. Method according to any one of daims 1 to 9, wherein said improved plant growth
12. characteristic is increased yield relative to corresponding wild type plants.
13. Method according to any one of daims 1 to 10, wherein said improved plant growth
14. characteristic is increased plant biomass.
15. Method according to daim 10, wherein said increased yield is increased seed yield.
16. Method according to daim 12, wherein said increased seed yield is selected from any
17. one or more of (i) increased seed biomass; (ii) increased number of (filled) seeds; (iii)
18. increased seed size; (iv) increased seed volume; (v) increased harvest index; and (vi)
19. increased thousand kernel weight (TKW).
20. Method according to any of one of claims 1 to 13, wherein said improved plant growth
21. characteristic is increased growth rate.
22. Plants obtainable by a method according to any of daims 1 to 14.
23. Construct comprising:
(i) A YIPPEE-like nudeic acid or variant thereof;
(ii) One or more control sequence capable of driving expression of the nudeic acid
sequence of (i); and optionally (iii) A transcription termination sequence.
17. Construct according to claim 16, wherein said promoter is a constitutive promoter.
18. Construct according to daim 17, wherein said promoter is a GOS2 promoter.
19. Plant transformed with a construct according to any on one of claims 16 to 18.

20. Method for the production of a transgenic plant having improved growth characteristics,
which method comprises:
(i) introducing into a plant a YIPPEE-like nucleic acid or variant thereof and/or introducing a genetic modification in the locus of a YIPPEE-like gene;
(ii) cultivating the plant cell under conditions promoting plant growth and development
21. Transgenic plant having improved growth characteristics resulting from a YIPPEE-like
nucleic acid or variant thereof introduced into said plant and/or resulting from a genetic
modification introduced in the locus of a YIPPEE-like gene.
22. Transgenic plant according to claim 15, 19 or 21, wherein said plant is a
monocotyledonous plant, such as sugar cane or wherein the plant is a cereal, such as
rice, maize, wheat, barley, millet, rye oats or sorghum.
23. Harvestable parts of a plant according to any one of claims 15,19,21 or 22.
24. Harvestable parts according to claim 23, wherein said harvestable parts are seeds.
25. Products directly derived from a plant according to any one of claims 15, 19, 21 or 22
and/or products derived directly from harvestable parts according to claims 23 or 24.
26. Use of a YIPPEE-like nucleic acidfgene or variant thereof or use of a YIPPEE-like
polypeptide or homologue thereof in improving the growth characteristics of plants, in
particular in improving yield/biomass, especially seed yield.
27. Use according to claim 25, wherein said seed yield includes one or more of the
following: increased number of (filled) seeds, increased seed weight, increased harvest
index and increased TKW.
28. Use of a YIPPEE-like nucleic acid/gene or variant thereof or use of a YIPPEE-like
polypeptide or homologue thereof as a molecular marker.



Documents:

0118-chenp-2007-abstract.pdf

0118-chenp-2007-claims.pdf

0118-chenp-2007-correspondnece-others.pdf

0118-chenp-2007-description(complete).pdf

0118-chenp-2007-drawings.pdf

0118-chenp-2007-form 1.pdf

0118-chenp-2007-form 3.pdf

0118-chenp-2007-form 5.pdf

0118-chenp-2007-pct.pdf

118-chenp-2007 amended pages of specification 10-12-2010.pdf

118-chenp-2007 amended claims 10-12-2010.pdf

118-chenp-2007 power of attorney 10-12-2010.pdf

118-CHENP-2007 AMENDED CLAIMS 25-10-2010.pdf

118-CHENP-2007 AMENDED PAGES OF SPECIFICATION 25-10-2010.pdf

118-chenp-2007 form-1 10-12-2010.pdf

118-CHENP-2007 POWER OF ATTORNEY 25-10-2010.pdf

118-chenp-2007 correspondence others 10-12-2010.pdf

118-CHENP-2007 CORRESPONDENCE OTHERS 05-02-2010.pdf

118-CHENP-2007 CORRESPONDENCE OTHERS 19-04-2007.pdf

118-CHENP-2007 CORRESPONDENCE OTHERS 29-05-2007.pdf

118-CHENP-2007 EXAMINATION REPORT REPLY RECIEVED 25-10-2010.pdf

118-CHENP-2007 FORM-1 19-04-2007.pdf

118-chenp-2007 form-3 25-10-2010.pdf

118-CHENP-2007 OTHER PATENT DOCUMENT 25-10-2010.pdf

118-CHENP-2007 OTHERS 22-11-2007.pdf

118-CHENP-2007 OTHERS 29-05-2007.pdf


Patent Number 245184
Indian Patent Application Number 118/CHENP/2007
PG Journal Number 2/2011
Publication Date 14-Jan-2011
Grant Date 06-Jan-2011
Date of Filing 11-Jan-2007
Name of Patentee CROPDSESIGN N.V.
Applicant Address TECHNOLOGIEPARK 3, B-9052 ZWIJNAARDE,
Inventors:
# Inventor's Name Inventor's Address
1 REUZEAU, CHRISTOPHE LA CHAPELLE GONAGUET, F-24350 TOCANE,
PCT International Classification Number C07K 14/415
PCT International Application Number PCT/EP05/53324
PCT International Filing date 2005-07-12
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 04103303.6 2004-07-12 EUROPEAN UNION
2 60/588,917 2004-07-16 EUROPEAN UNION