WO2006079655A2 - Plants having increased yield and a method for making the same - Google Patents

Plants having increased yield and a method for making the same Download PDF

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Publication number
WO2006079655A2
WO2006079655A2 PCT/EP2006/050489 EP2006050489W WO2006079655A2 WO 2006079655 A2 WO2006079655 A2 WO 2006079655A2 EP 2006050489 W EP2006050489 W EP 2006050489W WO 2006079655 A2 WO2006079655 A2 WO 2006079655A2
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Prior art keywords
seq
plant
syt
nucleic acid
plants
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PCT/EP2006/050489
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French (fr)
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WO2006079655A3 (en
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Valerie Frankard
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Cropdesign N.V.
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Priority to BRPI0607211-9A priority Critical patent/BRPI0607211A2/en
Priority to AU2006208779A priority patent/AU2006208779B2/en
Priority to JP2007552650A priority patent/JP2008528014A/en
Priority to KR1020137028798A priority patent/KR20130122989A/en
Application filed by Cropdesign N.V. filed Critical Cropdesign N.V.
Priority to EP06701687A priority patent/EP1844151A2/en
Priority to US11/795,976 priority patent/US8426683B2/en
Priority to CA002595672A priority patent/CA2595672A1/en
Priority to KR1020077017270A priority patent/KR101356311B1/en
Priority to NZ556605A priority patent/NZ556605A/en
Priority to MX2007008962A priority patent/MX2007008962A/en
Publication of WO2006079655A2 publication Critical patent/WO2006079655A2/en
Publication of WO2006079655A3 publication Critical patent/WO2006079655A3/en
Priority to IL184655A priority patent/IL184655A0/en
Priority to US13/847,251 priority patent/US20130269049A1/en
Priority to PH12013501782A priority patent/PH12013501782A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

  • the present invention relates generally to the field of molecular biology and concerns a method for increasing plant yield relative to corresponding wild type plants. More specifically, the present invention concerns a method for increasing plant yield comprising modulating expression in a plant of a nucleic acid encoding a synovial sarcoma translocation (SYT) polypeptide or a homologue thereof. The present invention also concerns plants having modulated expression of a nucleic acid encoding a SYT polypeptide or a homologue thereof, which plants have increased yield relative to corresponding wild type plants. The invention also provides constructs useful in the methods of the invention.
  • SYT synovial sarcoma translocation
  • Plant seeds are an important source of human and animal nutrition. Crops such as, corn, rice, wheat, canola and soybean account for over half of total human caloric intake, whether through direct consumption of the seeds themselves or through consumption of meat products raised on processed seeds. They are also a source of sugars, oils and many kinds of metabolites used in industrial processes. Seeds contain an embryo, the source of new shoots and roots after germination, and an endosperm, the source of nutrients for embryo growth, during germination and early growth of seedlings.
  • the development of a seed involves many genes, and requires the transfer of metabolites from roots, leaves and stems into the growing seed.
  • the endosperm in particular, assimilates the metabolic precursors of carbohydrate polymers, oil and proteins and synthesizes them into storage macromolecules to fill out the grain.
  • the ability to increase plant seed yield, whether through seed number, seed biomass, seed development, seed filling or any other seed- related trait would have many applications in agriculture, and even many non-agricultural uses such as in the biotechnological production of substances such as pharmaceuticals, antibodies or vaccines.
  • Yield may also depend on factors, such as the number and size of 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. Optimizing these factors may therefore also contribute to increasing crop yield.
  • SYT is a transcriptional co-activator which, in plants, forms a functional complex with transcription activators of the GRF (growth-regulating factor) family of proteins (Kim HJ, Kende H (2004) Proc Nat Acad Sc 101 : 13374-9). SYT is also called GIF for GRF- interacting factor.
  • the GRF transcription activators share structural domains (in the N- terminal region) with the SWI/SNF proteins of the chromatin-remodelling complexes in yeast (van der Knaap E et al., (2000) Plant Phys 122: 695-704).
  • Transcriptional co-activators of these complexes are proposed to be involved in recruiting SWI/SNF complexes to enhancer and promoter regions to effect local chromatin remodelling (review Naar AM et al., (2001) Annu Rev Biochem 70: 475-501).
  • the alteration in local chromatin structure modulates transcriptional activation.
  • SYT is proposed to interact with plant SWI/SNF complex to affect transcriptional activation of GRF target gene(s) (Kim HJ, Kende H (2004) Proc Nat Acad Sc 101 : 13374-9).
  • SYT belongs to a gene family of three members in Arabidopsis.
  • the SYT polypeptide shares homology with the human SYT.
  • the human SYT polypeptide was shown to be a transcriptional co-activator (Thaete et al. (1999) Hum Molec Genet 8: 585-591).
  • Three domains characterize the mammalian SYT polypeptide:
  • the SNH domain is well conserved.
  • the C-terminal domain is rich in glycine and glutamine, but not in proline or tyrosine. It has therefore been named the QG-rich domain in contrast to the QPGY domain of mammals.
  • a Met-rich domain may be identified N-terminally of the QG domain.
  • the QG-rich domain may be taken to be substantially the C-terminal remainder of the protein (minus the SHN domain); the Met-rich domain is typically comprised within the first half of the QG-rich (from the N-terminus to the C-terminus).
  • a second Met-rich domain may precede the SNH domain in plant SYT polypeptides (see Fig 1 ).
  • a method for increasing plant yield comprising modulating expression in a plant of a nucleic acid encoding a SYT polypeptide or a homologue thereof.
  • control plant refers not only to whole plants, but also to plant parts, including seeds and seed parts.
  • performance of the methods according to the present invention results in plants having increased yield, particularly seed yield, relative to corresponding wild type plants.
  • the term "increased yield” as defined herein is taken to mean an increase in any one or more of the following, each relative to corresponding wild type plants: (i) increased biomass (weight) of one or more parts of a plant, particularly aboveground (harvestable) parts, increased root biomass or increased biomass of any other harvestable part (such as fruits, nuts and pulses); (ii) increased total seed yield, which includes an increase in seed biomass (seed weight) and which may be an increase in the seed weight per plant or on an individual seed basis; (iii) increased number of (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 (including oil, protein and carbohydrate total content and composition); (vi) increased individual seed area; (vii) increased individual seed length or width; (viii) increased harvest index, which is expressed as a ratio of the yield of harvestable parts, such as seeds, over the total biomass; and (ix) increased thousand kernel weight (TKW), which is extrapolated from the
  • An increased TKW may result from an increased seed size and/or seed weight.
  • An increased TKW may result from an increase in embryo size and/or endosperm size.
  • An increase in seed size, seed volume, seed area, seed perimeter, seed width and seed length may be due to an increase in specific parts of a seed, for example due to an increase in the size of the embryo and/or endosperm and/or aleurone and/or scutellum, or other parts of a seed.
  • 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, increase in the seed filling rate (which is the number of filled seeds divided by the total number of seeds and multiplied by 100), among others.
  • 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 (florets) per panicle (which is expressed as a ratio of the number of filled seeds over the number of primary panicles), increase in the seed filling rate (which is the number of filled seeds divided by the total number of seeds and multiplied by 100), 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.
  • a method for increasing seed yield in a plant which method comprises modulating expression in a plant of a nucleic acid encoding a SYT polypeptide or a homologue thereof.
  • 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.
  • the growth rate is sufficiently increased, it may allow for the further sowing of seeds of the same plant species (for example sowing and harvesting of rice plants followed by sowing and harvesting of further rice plants all within one conventional growing period). Similarly, if the growth rate is sufficiently increased, it may allow for the further sowing of seeds of different plants species (for example the sowing and harvesting of rice plants followed by, for example, the sowing and optional harvesting of soybean, potato or any other suitable plant). Harvesting additional times from the same rootstock in the case of some crop 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 relative to corresponding wild type plants. Therefore, according to the present invention, there is provided a method for increasing growth rate in plants, which method comprises modulating expression in a plant of a nucleic acid encoding a SYT polypeptide or a homologue thereof.
  • Mild stress 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). Chemicals may also cause abiotic stresses.
  • Biotic stresses are typically those stresses caused by pathogens, such as bacteria, viruses, fungi and insects.
  • yield may be modified in any plant.
  • plant as used herein encompasses whole plants, ancestors and progeny of the plants and plant parts, including seeds, shoots, stems, leaves, roots (including tubers), flowers, and tissues and organs, wherein each of the aforementioned comprise the transgene of interest.
  • plant also encompasses plant cells, suspension cultures, callus tissue, embryos, meristematic regions, gametophytes, sporophytes, pollen and microspores, again wherein each of the aforementioned comprise the transgene.
  • Plants that are particularly useful in the methods of the invention include all plants which belong to the superfamily Viridiplantae, in particular monocotyledonous and dicotyledonous plants including fodder or forage legumes, ornamental plants, food crops, trees or shrubs selected from the list comprising 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., Centroem
  • the plant is a crop plant.
  • crop plants include amongst others soybean, sunflower, canola, alfalfa, rapeseed, cotton, tomato, potato or tobacco.
  • Arabidopsis thaliana is generally not considered as a crop plant.
  • the plant is a monocotyledonous plant, such as sugarcane. More preferably the plant is a cereal, such as rice, maize, wheat, barley, millet, rye, sorghum or oats.
  • SYT polypeptide or homologue thereof as defined herein refers to a polypeptide comprising from N-terminal to C-terminal: (i) an SNH domain having in increasing order of preference at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the SNH domain of SEQ ID NO: 2; and (ii) a Met-rich domain; and (iii) a QG-rich domain.
  • SNH domain having at least 40% identity to the SNH domain of SEQ ID NO: 2 comprises the residues shown in black in Figure 2. Further preferably, the SNH domain is represented by SEQ ID NO: 1.
  • the SYT polypeptide or a homologue thereof may comprise one or more of the following: (a) SEQ ID NO: 90; (b) SEQ ID NO: 91; and (c) a Met-rich domain at the N- terminal preceding the SNH domain.
  • a SYT polypeptide or a homologue thereof typically interacts with GRF (growth-regulating factor) polypeptides in yeast two-hybrid systems. Yeast two-hybrid interaction assays are well known in the art (see Field et al. (1989) Nature 340(6230): 245-246).
  • the SYT polypeptide as represented by SEQ ID NO: 4 is capable of interacting with AtGRF5 and with AtGRF9.
  • SYT polypeptide and homologues thereof have been demonstrated by the inventors to increase yield, particularly seed yield, in plants.
  • SYT nucleic acid/gene is any nucleic acid/gene encoding a SYT polypeptide or a homologue thereof as defined hereinabove.
  • SYT polypeptides or homologues thereof may readily be identified using routine techniques well known in the art, such as 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 ((1970) J MoI Biol 48: 443-453) to find the alignment of two complete sequences that maximizes the number of matches and minimizes the number of gaps. The BLAST algorithm (Altschul et al. (1990) J MoI Biol 215: 403-10) 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.
  • Homologues of SYT comprising an SNH domain having at least 40% sequence identity to the SNH domain of SEQ ID NO: 2 and/or comprising SEQ ID NO: 90 and/or SEQ ID NO: 91 , may readily be identified using, for example, the ClustalW multiple sequence alignment algorithm (version 1.83) available at http://clustalw.genome.jp/sit-bin/nph-clustalw, with the default pairwise alignment parameters, and a scoring method in percentage.
  • a sequence having a 40% identity to the SNH domain of SEQ ID NO: 2 is sufficient to identify a sequence as being a SYT.
  • the presence of a Met-rich domain or a QG-rich domain may also readily be identified.
  • the Met-rich domain and QG-rich domain follows the SNH domain.
  • the QG-rich domain may be taken to be substantially the C-terminal remainder of the protein (minus the SHN domain); the Met-rich domain is typically comprised within the first half of the QG-rich (from the N-term to the C-term).
  • Primary amino acid composition (in %) to determine if a polypeptide domain is rich in specific amino acids may be calculated using software programs from the ExPASy server (Gasteiger E et al. (2003) ExPASy: the proteomics server for in-depth protein knowledge and analysis.
  • the composition of the protein of interest may then be compared to the average amino acid composition (in %) in the Swiss-Prot Protein Sequence data bank.
  • the average Met (M) content is of 2.37%
  • the average GIn (Q) content is of 3.93%
  • the average GIy (G) content is of 6.93%.
  • a Met-rich domain or a QG-rich domain has Met content (in %) or a GIn and GIy content (in %) above the average amino acid composition (in %) in the Swiss-Prot Protein Sequence data bank.
  • SYT polypeptide or homologues thereof include (encoded by polynucleotide sequence accession number in parenthesis; see also Table 1): Arabidopsis thaliana Arath_SYT1 (AY102639.1) SEQ ID NO: 4, Arabidopsis thaliana Arath_SYT2 (AY102640.1) SEQ ID NO: 6, Arabidopsis thaliana Arath_SYT3 (AY102641.1) SEQ ID NO: 8, Aspergillus officinalis Aspof_SYT (CV287542) SEQ ID NO: 10, Brassica napus Brana_SYT (CD823592) SEQ ID NO: 12, Citrus sinensis Citsi_SYT (CB290588) SEQ ID NO: 14, Gossypium arboreum Gosar_SYT (BM359324) SEQ ID NO: 16, Medicago trunculata Medtr_SYT (CA858507.1) SEQ ID NO:
  • Sacof_SYT2 (CA110367) SEQ ID NO: 74, Saccharum officinarum Sacof_SYT3 (CA161933, CA265085) SEQ ID NO: 76, Solanum tuberosum Soltu_SYT1 (CK265597) SEQ ID NO: 78, Sorghum bicolor Sorbi_SYT3 (CX611128) SEQ ID NO: 80, Triticum aestivum Triae_SYT2 (CD901951) SEQ ID NO: 82, Triticum aestivum Triae_SYT3 (BJ246754, BJ252709) SEQ ID NO: 84, Vitis vinifera Vitvi_SYT1 (DV219834) SEQ ID NO: 86, Tea mays Zeama_SYT3 (CO468901) SEQ ID NO: 88.
  • sequences falling under the definition of "SYT polypeptide or homologue thereof are not to be limited to the sequences represented by 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, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68
  • SYT nucleic acids include but are not limited to those represented by any one of 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 , SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31 , SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41 , SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51 , SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61 , SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO:
  • SYT nucleic acids/genes and variants thereof may be suitable in practising the methods of the invention.
  • Variant SYT nucleic acid/genes typically are those having the same function as a naturally occurring SYT nucleic acid/genes, which can be the same biological function or the function of increasing yield when expression of the nucleic acids/genes is modulated in a plant.
  • Such variants include portions of a SYT nucleic acid/gene and/or nucleic acids capable of hybridising with a SYT nucleic acid/gene as defined below.
  • portion refers to a piece of DNA encoding a polypeptide comprising from N-terminal to C-terminal: (i) an SNH domain having in increasing order of preference at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the SNH domain of SEQ ID NO: 2 and (ii) a Met-rich domain; and (iii) a QG-rich domain.
  • a portion may be prepared, for example, by making one or more deletions to a SYT nucleic acid.
  • the portions may be used in isolated form or they may be fused to other coding (or non coding) sequences in order to, for example, produce a protein that combines several activities. When fused to other coding sequences, the resulting polypeptide produced upon translation may be bigger than that predicted for the SYT fragment.
  • the portion is a portion of a nucleic acid as represented by any one of 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 , SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31 , SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51 , SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61 , SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO:
  • SYT nucleic acid/gene is a nucleic acid capable of hybridising under reduced stringency conditions, preferably under stringent conditions, with a SYT nucleic acid/gene as hereinbefore defined, which hybridising sequence encodes a polypeptide comprising from N-terminal to C-terminal: (i) an SNH domain having in increasing order of preference at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the SNH domain of SEQ ID NO: 2 and (ii) a Met-rich domain; and (iii) a QG-rich domain.
  • the hybridising sequence is one that is capable of hybridising to a nucleic acid as represented by any one of 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 , SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51 , SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61 , SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, S
  • hybridisation 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).
  • 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 T m is the temperature under defined ionic strength and pH, at which 50% of the target sequence hybridises to a perfectly matched probe. The T m 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 T m .
  • the presence of monovalent cations in the hybridisation solution reduce the electrostatic repulsion between the two nucleic acid strands thereby promoting hybrid formation; this effect is visible for sodium concentrations of up to 0.4M.
  • Formamide reduces the melting temperature of DNA- DNA and DNA-RNA duplexes with 0.6 to 0.7 0 C for each percent formamide, and addition of 50% formamide allows hybridisation to be performed at 30 to 45°C, though the rate of hybridisation will be lowered.
  • Base pair mismatches reduce the hybridisation rate and the thermal stability of the duplexes.
  • the T m decreases about 1°C per % base mismatch.
  • the T m may be calculated using the following equations, depending on the types of hybrids:
  • T m 79.8 + 18.5 (logio[Na + ] a ) + 0.58 (%G/C b ) + 11.8 (%G/C b ) 2 - 820/L c
  • T m 2 (/ n )
  • T m 22 + 1.46 (/ n )
  • 0 L length of duplex in base pairs.
  • d Oligo, oligonucleotide; / n , effective length of primer 2 ⁇ (no. of G/C)+(no. of AfT).
  • the T m is reduced by about 0.6 to 0.7 0 C, while the presence of 6 M urea reduces the T m by about 30°C
  • Specificity of hybridisation is typically the function of post-hybridisation washes. To remove background resulting from non-specific hybridisation, samples are washed with dilute salt solutions. Critical factors of such washes include the ionic strength and temperature of the final wash solution: the lower the salt concentration and the higher the wash temperature, the higher the stringency of the wash. Wash conditions are typically performed at or below hybridisation stringency. Generally, suitable stringent conditions for nucleic acid hybridisation assays or gene amplification detection procedures are as set forth above. Conditions of greater or less stringency may also be selected. Generally, low stringency conditions are selected to be about 50 0 C lower than the thermal melting point (T m ) for the specific sequence at a defined ionic strength and pH.
  • Medium stringency conditions are when the temperature is 20 0 C below T m
  • high stringency conditions are when the temperature is 10°C below T m
  • stringent conditions are those that are at least as stringent as, for example, conditions A-L
  • reduced stringency conditions are at least as stringent as, for example, conditions M-R.
  • Non-specific binding may be controlled using any one of a number of known techniques such as, for example, blocking the membrane with protein containing solutions, additions of heterologous RNA, DNA, and SDS to the hybridisation buffer, and treatment with RNase. Examples of hybridisation and wash conditions are listed in Table 2 below.
  • a DNA DNA equal to 50 and 50% formamide 0.3 ⁇ SSC
  • E RNA RNA equal to 50 and 50% formamide 0.3 ⁇ SSC
  • RNA RNA ⁇ 50 Tl* ; 2 xSSC Tl* ; 2 ⁇ SSC
  • RNA RNA equal to 50 and 50% formamide 2 ⁇ SSC
  • hybrid length is the anticipated length for the hybridising nucleic acid.
  • the hybrid length may be determined by aligning the sequences and identifying the conserved regions described herein.
  • f SSPE (I xSSPE is 0.15M NaCI, 1OmM NaH 2 PO 4 , 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 x Denhardfs reagent, 0.5-1.0% SDS, 100 ⁇ g/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 T m of the hybrids; the T m 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.
  • the SYT nucleic acid or variant thereof may be derived from any artificial source or natural source, such as plant, algae or animal. 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 of plant origin encodes a SYT1.
  • the nucleic acid may encode a SYT2 or SYT3, which are closely related to one another on a polypeptide level.
  • the nucleic acid may be isolated from a dicotyledonous species, preferably from the family Brassicaceae, further preferably from Arabidopsis thaliana. More preferably, the three SYT nucleic acids isolated from Arabidopsis thaliana are represented by SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 7, and the three SYT amino acid sequences are as represented by SEQ ID NO: 4, SEQ ID NO: 6 and SEQ ID NO: 8.
  • nucleic acid encoding a SYT polypeptide or a homologue thereof may be modulated by introducing a genetic modification (preferably in the locus of a SYT gene).
  • a genetic modification preferably in the locus of a SYT gene.
  • the locus of a gene as defined herein is taken to mean a genomic region, which includes the gene of interest and 10 kb up- or downstream of the coding region.
  • the genetic modification may be introduced, for example, by any one (or more) of the following methods: T-DNA activation, TILLING, site-directed mutagenesis, directed evolution and homologous recombination, or by introducing and expressing in a plant a nucleic acid encoding a SYT polypeptide or a homologue thereof.
  • T-DNA activation TILLING
  • site-directed mutagenesis site-directed mutagenesis
  • directed evolution and homologous recombination or by introducing and expressing in a plant a nucleic acid encoding a SYT polypeptide or a homologue thereof.
  • a step of selecting for modulated expression of a nucleic acid encoding a SYT polypeptide or a homologue thereof which modulated expression gives plants having increased yield, particularly increased seed yield.
  • T-DNA activation tagging 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 10 kb up- or downstream of the coding region of a gene in a configuration such that the promoter directs expression of the targeted gene.
  • a promoter may also be a translation enhancer or an intron
  • 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, through Agrobacterium infection and leads to overexpression of genes near 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.
  • 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 SYT gene using the technique of TILLING (Targeted Induced Local Lesions In Genomes).
  • TILLING Tueted Induced Local Lesions In Genomes. This is a mutagenesis technology useful to generate and/or identify, and to eventually isolate mutagenised variants of a SYT nucleic acid encoding a protein with enhanced SYT activity.
  • TILLING also allows selection of plants carrying such mutant variants. These mutant variants may even exhibit higher SYT activity than that exhibited by the gene in its natural form.
  • TILLNG combines high-density mutagenesis with high-throughput screening methods.
  • Site-directed mutagenesis may be used to generate variants of SYT nucleic acids.
  • 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.com/products/currentprotocols/index.html).
  • Directed evolution may also be used to generate variants of SYT nucleic acids. This consists of iterations of DNA shuffling followed by appropriate screening and/or selection to generate variants of SYT nucleic acids or portions thereof encoding SYT polypeptides or homologues or portions thereof having an modified biological activity (Castle et al., (2004) Science 304(5674): 1151-4; US patents 5,811,238 and 6,395,547). T-DNA activation, TILLING, site-directed mutagenesis and directed evolution are examples of technologies that enable the generation of novel SYT alleles and 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 Physcomitrella. Methods for performing homologous recombination in plants have been described not only for model plants (Offringa et al. (1990) EMBO J 9(10): 3077-84) but also for crop plants, for example rice (Terada et al. (2002) Nat Biotech 20(10): 1030-4; lida and Terada (2004) Curr Opin Biotech 15(2): 132-8).
  • the nucleic acid to be targeted (which may be a SYT nucleic acid or variant thereof as hereinbefore defined) is targeted to the locus of a SYT gene.
  • the nucleic acid to be targeted may be an improved allele used to replace the endogenous gene or may be introduced in addition to the endogenous gene.
  • a preferred method for introducing a genetic modification is to introduce and express in a plant a nucleic acid encoding a SYT polypeptide or a homologue thereof.
  • a SYT polypeptide or a homologue thereof is defined as a polypeptide comprising from N-terminal to C-terminal: (i) an SNH domain having in increasing order of preference at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the SNH domain of SEQ ID NO: 2; and (ii) a Met-rich domain; and (iii) a QG-rich domain.
  • SNH domain having at least 40% identity to the SNH domain of SEQ ID NO: 2 comprises the residues shown in black in Figure 2. Further preferably, the SNH domain is represented by SEQ ID NO: 1.
  • the nucleic acid to be introduced into a plant may be a full-length nucleic 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.
  • 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 ⁇ -helical structures or ⁇ -sheet structures).
  • Conservative substitution tables are well known in the art (see for example Creighton (1984) Proteins. W.H. Freeman and Company and Table 3 below).
  • Homologues include orthologues and paralogues, which encompass evolutionary concepts used to describe ancestral relationships of genes.
  • Paralogues are genes within the same species that have originated through duplication of an ancestral gene and orthologues are genes from different organisms that have originated through speciation.
  • Orthologues in, for example, monocot plant species may easily be found by performing a so-called reciprocal blast search. This may be done by a first blast involving blasting a query sequence (for example, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8) against any sequence database, such as the publicly available NCBI database which may be found at: http://www.ncbi.nlm.nih.gov.
  • BLASTN or TBLASTX (using standard default values) may be used when starting from a nucleotide sequence and BLASTP or TBLASTN (using standard default values) may be used when starting from a protein sequence.
  • the BLAST results may optionally be filtered.
  • the full- length sequences of either the filtered results or non-filtered results are then BLASTed back (second BLAST) against sequences from the organism from which the query sequence is derived (where the query sequence is SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8 the second blast would therefore be against Arabidopsis sequences).
  • the results of the first and second BLASTs are then compared.
  • a paralogue is identified if a high-ranking hit from the second blast is from the same species as from which the query sequence is derived; an orthologue is identified if a high-ranking hit is not from the same species as from which the query sequence is derived.
  • High-ranking hits are those having a low E-value.
  • Computation of the E-value is well known in the art. In the case of large families, ClustalW may be used, followed by a neighbour joining tree, to help visualize clustering of related genes and to identify orthologues and paralogues.
  • a homologue may be in the form of a "substitutional variant" of a protein, i.e. where at least one residue in an amino acid sequence has been removed and a different residue inserted in its place.
  • Amino acid substitutions are typically of single residues, but may be clustered depending upon functional constraints placed upon the polypeptide; insertions will usually be of the order of about 1 to 10 amino acid residues.
  • amino acid substitutions comprise conservative amino acid substitutions.
  • Conservative substitution tables are readily available in the art. The table below gives examples of conserved amino acid substitutions. Table 3: Examples of conserved 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 N-terminal and/or C-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 N- or C-terminal fusions, of the order of about 1 to 10 residues.
  • N- or C-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.
  • a transcriptional activator as used in the yeast two-hybrid system
  • phage coat proteins phage coat proteins
  • (histidine)- ⁇ -tag glutathione S-transferase-tag
  • protein A maltose-binding protein
  • dihydrofolate reductase Tag-100 epitope
  • c-myc epitope FLAG®-
  • 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 M 13 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 SYT polypeptide or homologue thereof may be a derivative.
  • “Derivatives” include peptides, oligopeptides, polypeptides, proteins and enzymes which may comprise substitutions, deletions or additions of 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: 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, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52
  • “Derivatives” of a protein encompass peptides, oligopeptides, polypeptides, proteins and enzymes which may comprise naturally occurring altered, glycosylated, acylated, prenylated 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 SYT polypeptide or homologue thereof may be encoded by an alternative splice variant of a SYT 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, or in which introns have been shortened or lengthened. 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 encoding a polypeptide comprising from N-terminal to C-terminal: (i) an SNH domain having in increasing order of preference at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the SNH domain of SEQ ID NO: 2; and (ii) a Met-rich domain; and (iii) a QG-rich domain.
  • SNH domain having at least 40% identity to the SNH domain of SEQ ID NO: 2 comprises the residues shown in black in Figure 2.
  • the SNH domain is represented by SEQ ID NO: 1.
  • the SYT polypeptide or a homologue thereof may comprise one or more of the following: (i) SEQ ID NO: 90; and/or (ii) SEQ ID NO: 91; and/or (iii) a Met-rich domain at the N-terminal preceding the SNH domain.
  • the homologue may also be encoded by an allelic variant of a nucleic acid encoding a SYT polypeptide or a homologue thereof, preferably an allelic variant of the nucleic acid encoding a polypeptide comprising from N-terminal to C-terminal: (i) an SNH domain having in increasing order of preference at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the SNH domain of SEQ ID NO: 2; and (ii) a Met-rich domain; and (iii) a QG-rich domain.
  • SNH domain having at least 40% identity to the SNH domain of SEQ ID NO: 2 comprises the residues shown in black in Figure 2.
  • the SNH domain is represented by SEQ ID NO: 1.
  • the SYT polypeptide or a homologue thereof may comprise one or more of the following: (i) SEQ ID NO: 90; and/or (ii) SEQ ID NO: 91; and/or (iii) a Met-rich domain at the N-terminal preceding the SNH domain.
  • the allelic variant is an allelic variant of a nucleic acid as represented by any one of 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 , SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31 , SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41 , SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61 , SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67,
  • 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.
  • the modulated expression of a SYT nucleic acid or variant thereof is increased expression.
  • the increase in expression may lead to raised SYT mRNA or polypeptide levels, which could equate to raised activity of the SYT polypeptide; or the activity may also be raised when there is no change in polypeptide levels, or even when there is a reduction in polypeptide levels. 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.
  • Methods for increasing 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 SYT nucleic acid or variant thereof.
  • endogenous promoters may be altered in vivo by mutation, deletion, and/or substitution (see, Kmiec, US 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. Methods for reducing the expression of genes or gene products are well documented in the art.
  • polypeptide expression 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 3 1 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, MoI. Cell biol. 8:4395-4405 (1988); Callis et a/., Genes Dev. 1 :1183-1200 (1987).
  • Such intron enhancement of gene expression is typically greatest when placed near the 5 1 end of the transcription unit.
  • Use of the maize introns Adh1-S intron 1 , 2, and 6, the Bronze-1 intron are known in the art. See generally, The Maize Handbook, Chapter 116, Freeling and Walbot, Eds., Springer, N.Y. (1994).
  • the invention also provides genetic constructs and vectors to facilitate introduction and/or expression of the nucleotide sequences useful in the methods according to the invention.
  • control sequences capable of driving expression of the nucleic acid sequence of (i); and optionally (iii) A transcription termination sequence.
  • a preferred construct is one whether the control sequence is a promoter derived from a plant, preferably from a monocotyledonous plant.
  • 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 nucleic acid encoding a SYT polypeptide or homologue thereof).
  • the sequence of interest is operably linked to one or more control sequences (at least to a promoter).
  • control sequence at least to a promoter.
  • 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.
  • 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 which alter gene expression in response to developmental and/or external stimuli, or in a tissue-specific manner.
  • 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.
  • regulatory element also encompasses a synthetic fusion molecule or derivative that confers, activates or enhances expression of a nucleic acid molecule in a cell, tissue or organ.
  • operably linked 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.
  • any type of promoter may be used to drive expression of the nucleic acid sequence.
  • the promoter may be an inducible promoter, i.e. having induced or increased transcription initiation in response to a developmental, chemical, environmental or physical stimulus.
  • An example of an inducible promoter being a stress-inducible promoter, i.e. a promoter activated when a plant is exposed to various stress conditions.
  • 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-specific".
  • the SYT nucleic acid or variant thereof is operably 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.
  • the promoter is derived from a plant, further preferably a monocotyledonous plant. Most preferred is use of a GOS2 promoter (from rice) (SEQ ID NO: 89). It should be clear that the applicability of the present invention is not restricted to the SYT nucleic acid represented by SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7, nor is the applicability of the invention restricted to expression of a SYT nucleic acid when driven by a GOS2 promoter. Examples of other constitutive promoters which may also be used to drive expression of a SYT nucleic acid are shown in Table 4 below. Table 4: Examples of constitutive promoters
  • terminator sequences may also be used in the construct introduced into a plant.
  • the term "terminator” encompasses a control sequence which is a DNA sequence at the end of a transcriptional unit which signals 3' processing and polyadenylation of a primary transcript and termination of transcription. Additional regulatory elements may include transcriptional as well as translational enhancers. Those skilled in the art will be aware of terminator and enhancer sequences that 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 that is required for maintenance and/or replication in a specific cell type.
  • an origin of replication sequence that is required for maintenance and/or replication in a specific cell type.
  • Preferred origins of replication include, but are not limited to, the f1-ori and colE1.
  • the genetic construct may optionally comprise a selectable marker gene.
  • selectable marker gene includes any gene that confers a phenotype on a cell in which it is expressed to facilitate the identification and/or selection of cells that are transfected or transformed with a nucleic acid construct of the invention.
  • Suitable markers may be selected from markers that confer antibiotic or herbicide resistance, that introduce a new metabolic trait or that allow visual selection.
  • selectable marker genes include genes conferring resistance to antibiotics (such as nptll that phosphorylates neomycin and kanamycin, or hpt, phosphorylating hygromycin), to herbicides (for example bar which provides resistance to Basta; aroA or gox providing resistance against glyphosate), or genes that provide a metabolic trait (such as manA that allows plants to use mannose as sole carbon source).
  • Visual marker genes result in the formation of colour (for example ⁇ -glucuronidase, GUS), luminescence (such as luciferase) or fluorescence (Green Fluorescent Protein, GFP, and derivatives thereof).
  • the present invention also encompasses plants obtainable by the methods according to the present invention.
  • the present invention therefore provides plants, plant parts and plant cells obtainable by the methods according to the present invention, which plants have introduced therein a SYT nucleic acid or variant thereof and which plants, plant parts and plant cells are preferably from a crop plant, further preferably from a monocotyledonous plant.
  • the invention also provides a method for the production of transgenic plants having increased yield, comprising introduction and expression in a plant of a SYT nucleic acid or a variant thereof.
  • the present invention provides a method for the production of transgenic plants, preferably monocotyledonous plants, having increased yield, which method comprises:
  • Subsequent generations of the plants obtained from cultivating step (ii) may be propagated by a variety of means, such as by clonal propagation or classical breeding techniques.
  • 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 nucleic acid may be introduced directly into a plant cell or into the plant itself (including introduction into a tissue, organ or any other part of a plant). According to a preferred feature of the present invention, the nucleic acid is introduced into a plant by transformation.
  • transformation 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 may be transformed with a genetic construct of the present invention and a whole plant regenerated from there.
  • the particular tissue chosen will vary depending on the clonal propagation systems available for, and best suited to, the particular species being transformed.
  • 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.
  • 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 al., (1982) Nature 296, 72-74; Negrutiu I et al. (1987) Plant MoI Biol 8: 363-373); electroporation of protoplasts (Shillito R.D.
  • Transgenic rice plants expressing a SYT nucleic acid/gene are preferably produced via Agrobacterium-med ⁇ ated transformation using any of the well known methods for rice transformation, such as described in any of the following: published European patent application EP 1198985 A1, Aldemita and Hodges (Planta 199: 612-617, 1996); Chan et al.
  • plant 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.
  • 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.
  • expression levels of the newly introduced DNA may be monitored using Northern and/or Western analysis, quantitative PCR, such 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.
  • 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).
  • clonal transformants e.g., all cells transformed to contain the expression cassette
  • grafts of transformed and untransformed tissues e.g., in plants, a transformed rootstock grafted to an untransformed scion.
  • the present invention clearly extends to any plant cell or plant produced by any of the methods described herein, and to all plant parts and propagules thereof.
  • the present invention extends further to encompass the progeny of a primary transformed or transfected cell, tissue, organ or whole plant that has been produced by any of the aforementioned methods, the only requirement being that progeny exhibit the same genotypic and/or phenotypic characteristic(s) as those produced by the parent in the methods according to the invention.
  • the invention also includes host cells containing an isolated SYT 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 invention furthermore relates to products derived, preferably directly derived, from a harvestable part of such a plant, such as dry pellets or powders, meal, oil, fat and fatty acids, starch or proteins.
  • the present invention also encompasses use of SYT nucleic acids or variants thereof and use of SYT polypeptides or homologues thereof and to use of a construct as defined hereinabove in increasing plant yield, especially seed yield.
  • the seed yield is as defined above and preferably includes increased total seed yield or increased TKW.
  • SYT nucleic acids or variants thereof, or SYT polypeptides or homologues thereof may find use in breeding programmes in which a DNA marker is identified which may be genetically linked to a SYT gene or variant thereof.
  • the SYT nucleic acids/ genes or variants thereof, or SYT polypeptides or homologues thereof may be used to define a molecular marker. This DNA or protein marker may then be used in breeding programmes to select plants having increased yield.
  • the SYT gene or variant thereof may, for example, be a nucleic acid as represented by any one of 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, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41 , SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51 , SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61 , SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ
  • Allelic variants of a SYT 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, for example, by PCR. This is followed by a step for selection of superior allelic variants of the sequence in question and which give increased yield.
  • 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: 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 , SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31 , SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41 , SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51 , SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO
  • a SYT 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 SYT nucleic acids or variants thereof requires only a nucleic acid sequence of at least 15 nucleotides in length. The SYT nucleic acids or variants thereof may be used as restriction fragment length polymorphism (RFLP) markers. Southern blots (Sambrook J, Fritsch EF and Maniatis T (1989) Molecular Cloning, A Laboratory Manual) of restriction-digested plant genomic DNA may be probed with the SYT nucleic acids or variants thereof.
  • RFLP restriction fragment length polymorphism
  • the resulting banding patterns may then be subjected to genetic analyses using computer programs such as MapMaker (Lander et al. (1987) Genomics 1 : 174-181) in order to construct a genetic map.
  • 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 SYT nucleic acid or variant thereof in the genetic map previously obtained using this population (Botstein et al. (1980) Am. J. Hum. Genet. 32:314-331).
  • the 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).
  • the nucleic acid probes may be used in direct fluorescence in situ hybridization (FISH) mapping (Trask (1991) Trends Genet. 7:149-154).
  • FISH direct fluorescence in situ hybridization
  • nucleic acid amplification-based methods for genetic and physical mapping may be carried out using the nucleic acids. Examples include allele-specific amplification (Kazazian (1989) J. Lab. Clin. Med 11 :95-96), polymorphism of PCR-amplified fragments (CAPS; Sheffield et al. (1993) Genomics 16:325-332), allele-specific ligation (Landegren et al. (1988) Science 241 :1077-1080), nucleotide extension reactions (Sokolov (1990) Nucleic Acid Res. 18:3671), Radiation Hybrid Mapping (Walter et al. (1997) Nat. Genet.
  • yield-enhancing traits 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.
  • Fig. 1 shows the typical domain structure of SYT polypeptides from plants and mammals.
  • the conserved SNH domain is located at the N-terminal end of the protein.
  • the C-terminal remainder of the protein domain consists of a QG-rich domain in plant SYT polypeptides, and of a QPGY-rich domain in mammalian SYT polypeptides.
  • a Met-rich domain is typically comprised within the first half of the QG-rich (from the N-term to the C-term) in plants or QPGY-rich in mammals.
  • a second Met-rich domain may precede the SNH domain in plant SYT polypeptides Fig.
  • FIG. 2 shows a multiple alignment of the N-terminal end of several SYT polypeptides, using VNTI AlignX multiple alignment program, based on a modified ClustalW algorithm (InforMax, Bethesda, MD, http://www.informaxinc.com), with default settings for gap opening penalty of 10 and a gap extension of 0.05).
  • the SNH domain is boxed across the plant and human SYT polypeptides.
  • the last line in the alignment consists of a consensus sequence derived from the aligned sequences.
  • Fig.3 shows a multiple alignment of several plant SYT polypeptides, using VNTI AlignX multiple alignment program, based on a modified ClustalW algorithm (InforMax, Bethesda, MD, http://www.informaxinc.com), with default settings for gap opening penalty of 10 and a gap extension of 0.05).
  • the two main domains, from N-terminal to C-terminal, are boxed and identified as SNH domain and the Met-rich/QG-rich domain. Additionally, the N-terminal Met-rich domain is also boxed, and the positions of SEQ ID NO: 90 and SEQ ID NO 91 are underlined in bold.
  • Fig. 4 shows a Neighbour joining tree resulting from the alignment of multiple SYT polypeptides using CLUSTALW 1.83 (http://align.genome.jp/sit-bin/clustalw).
  • CLUSTALW 1.83 http://align.genome.jp/sit-bin/clustalw.
  • the SYT1 and SYT2/SYT3 clades are identified with brackets.
  • Fig. 5 shows a binary vector pO523, for expression in Oryza sativa of an Arabidopsis thaliana AtSYTI under the control of a GOS2 promoter (internal reference PRO0129).
  • Fig. 6 shows a binary vector pO524, for expression in Oryza sativa of an Arabidopsis thaliana AtSYT2 under the control of a GOS2 promoter (internal reference PRO0129).
  • Fig. 7 shows a binary vector pO767, for expression in Oryza sativa of an Arabidopsis thaliana AtSYT3 under the control of a GOS2 promoter (internal reference PRO0129).
  • Fig. 8 details examples of sequences useful in performing the methods according to the present invention.
  • SYT nucleic acid sequences are presented from start to stop. The majority of these sequences are derived from EST sequencing, which is of lower quality. Therefore, nucleic acid substitutions may be encountered.
  • the Arabidopsis thaliana AtSYTI gene 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 1.59 x 10 7 cfu. Original titer was determined to be 9.6 x 10 5 cfu/ml after first amplification of 6x10 11 cfu/ml. After plasmid extraction, 200 ng of template was used in a 50 ⁇ l PCR mix.
  • Primers prmO6681 (SEQ ID NO: 92; sense, start codon in bold, AttB1 site in italic: 5'-GGGGyACyAyAGrTTGLACyAAAAAyAGCyAGGCTTAAACAATGCAACAGCACCTGATG -3') and prmO6682 (SEQ ID NO: 93; reverse, complementary, AttB2 site in italic: 5'- GGGGACCyACTTTGTyACAAGAAAGCTGGGTCATCATTAAGATTCCTTGTGC-S'), 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 727 bp was amplified and purified also using standard methods.
  • Plasmid pDONR201 was purchased from Invitrogen, as part of the Gateway® technology.
  • the Arabidopsis thaliana AtSYT2 gene was amplified by PCR using the same method as the Arabidopsis thaliana AtSYTI gene.
  • Primers prmO6685 SEQ ID NO: 94; sense, start codon in bold, AttB1 site in italic: 5'-GGGGACAAGTTTGTACAAAAAAGCAGG CTTAAACAATGCAGCAGCAGCAGTCT 3'
  • prmO6686 SEQ ID NO: 95
  • reverse, stop codon in bold, complementary, AttB2 site in italic: 5' GGGGACCACTTTGTACAAGAAAG CTGGGTTCTTTGGATCCTTTTCACTTG 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 666 bp (including attB sites) was amplified and purified as above. The entry clone was numbered pO7467.
  • the Arabidopsis thaliana AtSYT3 gene was amplified by PCR using the same method as the Arabidopsis thaliana AtSYTI and AtSYT2 genes.
  • Primers prmO6683 (SEQ ID NO: 96; sense, start codon in bold, AttB1 site in italic: 5' GGGGACAAGTTTGTACAAAAAAG CyAGGCTTAAACAATGCAGCAATCTCCACAGAT 3') and prmO6684 (SEQ ID NO: 97; reverse, stop codon in bold, complementary, AttB2 site in italic: 5' GGGGACCACTTTGTAC A4G>4A4GC7GGG7TCCTCTATTTCATTTTCCTTCAG 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 745 bp (including attB sites) was amplified and purified as above. The entry clone was numbered p07604.
  • the entry clones pO7466, pO7467 and p07604 were subsequently used in an LR reaction with p00640, 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 (SEQ ID NO: 89) for constitutive expression (PRO0129) was located upstream of this Gateway cassette.
  • the resulting expression vectors were 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 of AtSYTI, AtSYT2 and AtSYT3 under the control of the rice GOS2 promoter
  • a two factor ANOVA analysis of variants was used as a statistical model for the overall evaluation of plant phenotypic characteristics.
  • An F-test was carried out on all the parameters measured of all the plants of all the events transformed with 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 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.
  • 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.
  • Thousand Kernel Weight (TKW) is extrapolated from the number of filled seeds counted and their total weight.
  • the total seed yield and TKW measurement results for AtSYTI , AtSYT2 and AtSYT3 transgenic plants for the T1 generation are shown in Tables 5 to 7, respectively.
  • the number of lines with an increase in either parameter is indicated.
  • the percentage difference between the transgenics and the corresponding nullizygotes is also shown, as well as the P values from the F test.
  • Both the total seed yield and TKW are significantly increased in the T1 generation for AtSYTI, AtSYT2 and ATSYT3 transgenic plants (Tables 5 to 7, respectively).
  • Table 5 Results of total seed yield and TKW measurements in the T1 generation of AtSYTI transgenic plants.
  • Table 6 Results of total seed yield and TKW measurements in the T1 generation of AtSYT2 transgenic plants.
  • Table 7 Results of total seed yield and TKW measurements in the T1 generation of AtSYT3 transgenic plants.
  • the average individual seed area, length and width measurement results of the T3 seeds (harvested from the T2 plants) for the Oryza sativa AtSYTI transgenic plants are shown in Table 8.
  • the percentage difference between the transgenics and the corresponding nullizygotes is shown, as well as the number of events with an increase in a given parameter and the p values from the F test.
  • the average individual seed area, length and width of the T3 husked and dehusked seeds were all significantly increased compared to their null counterparts (Table 8).
  • Table 8 Individual seed area, length and width measurements of the T3 husked and dehusked seeds (harvested from the T2 plants) of the Oryza sativa AtSYTI transgenic plants compared to their null counterparts.
  • Embryo and endosperm size were also measured by longitudinally cutting in half dehusked seeds and staining the seed halves for 2 to 3 hours at 35 0 C with colouring agent, 2,3,5- triphenyltetrazolium chloride. Following staining, the two halves were placed on agarose gel in a Petri dish ready for imaging. Three independent events were taken, and from each event 120 seeds homozygous for the transgene and 120 seeds without the transgene were analysed. Digital photographs of the seeds were taken and the images analysed with ImagePro software. The results for the three events are given below.
  • AtSYTI homozygous transgenic plants and their corresponding controls were transplanted into the field in September and harvested in December. Four repetitions were planted for each entry (four events) with 104 plants per repeat. The spacing between plants was of 20 by 20 cm. The field was flooded and irrigated. After seed harvest, the seeds were measured for TKW as described above. Results of these measurements are presented in Table 9.
  • Table 9 Results of TKW measurements in the T3 generation of AtSYTI transgenic plants grown in the field.
  • the TKW is increased in all the transgenic events evaluated in the field.

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Abstract

The present invention concerns a method for increasing plant yield by modulating expression in a plant of a nucleic acid encoding a synovial sarcoma translocation (SYT) polypeptide or a homologue thereof. One such method comprises introducing into a plant a SYT nucleic acid or variant thereof. The invention also relates to transgenic plants having introduced therein a SYT nucleic acid or variant thereof, which plants have increased yield relative to corresponding wild type plants. The present invention also concerns constructs useful in the methods of the invention.

Description

Plants having increased yield and a method for making the same
The present invention relates generally to the field of molecular biology and concerns a method for increasing plant yield relative to corresponding wild type plants. More specifically, the present invention concerns a method for increasing plant yield comprising modulating expression in a plant of a nucleic acid encoding a synovial sarcoma translocation (SYT) polypeptide or a homologue thereof. The present invention also concerns plants having modulated expression of a nucleic acid encoding a SYT polypeptide or a homologue thereof, which plants have increased yield 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 fuels 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, and in the case of many plants seed 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. Plant seeds are an important source of human and animal nutrition. Crops such as, corn, rice, wheat, canola and soybean account for over half of total human caloric intake, whether through direct consumption of the seeds themselves or through consumption of meat products raised on processed seeds. They are also a source of sugars, oils and many kinds of metabolites used in industrial processes. Seeds contain an embryo, the source of new shoots and roots after germination, and an endosperm, the source of nutrients for embryo growth, during germination and early growth of seedlings. The development of a seed involves many genes, and requires the transfer of metabolites from roots, leaves and stems into the growing seed. The endosperm, in particular, assimilates the metabolic precursors of carbohydrate polymers, oil and proteins and synthesizes them into storage macromolecules to fill out the grain. The ability to increase plant seed yield, whether through seed number, seed biomass, seed development, seed filling or any other seed- related trait would have many applications in agriculture, and even many non-agricultural uses such as in the biotechnological production of substances such as pharmaceuticals, antibodies or vaccines.
Yield may also depend on factors, such as the number and size of 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. Optimizing these factors may therefore also contribute to increasing crop yield.
It has now been found that modulating expression in a plant of a nucleic acid encoding a SYT polypeptide or a homologue thereof gives plants having increased yield relative to corresponding wild type plants.
SYT is a transcriptional co-activator which, in plants, forms a functional complex with transcription activators of the GRF (growth-regulating factor) family of proteins (Kim HJ, Kende H (2004) Proc Nat Acad Sc 101 : 13374-9). SYT is also called GIF for GRF- interacting factor. The GRF transcription activators share structural domains (in the N- terminal region) with the SWI/SNF proteins of the chromatin-remodelling complexes in yeast (van der Knaap E et al., (2000) Plant Phys 122: 695-704). Transcriptional co-activators of these complexes are proposed to be involved in recruiting SWI/SNF complexes to enhancer and promoter regions to effect local chromatin remodelling (review Naar AM et al., (2001) Annu Rev Biochem 70: 475-501). The alteration in local chromatin structure modulates transcriptional activation. More precisely, SYT is proposed to interact with plant SWI/SNF complex to affect transcriptional activation of GRF target gene(s) (Kim HJ, Kende H (2004) Proc Nat Acad Sc 101 : 13374-9).
SYT belongs to a gene family of three members in Arabidopsis. The SYT polypeptide shares homology with the human SYT. The human SYT polypeptide was shown to be a transcriptional co-activator (Thaete et al. (1999) Hum Molec Genet 8: 585-591). Three domains characterize the mammalian SYT polypeptide:
(i) the N-terminal SNH (SYT N[-terminal homology) domain, conserved in mammals, plants, nematodes and fish;
(ii) the C-terminal QPGY-rich domain, composed predominantly of glycine, proline, glutamine and tyrosine, occurring at variable intervals; (iii) a methionine-rich (Met-rich) domain located between the two previous domains.
In plant SYT polypeptides, the SNH domain is well conserved. The C-terminal domain is rich in glycine and glutamine, but not in proline or tyrosine. It has therefore been named the QG-rich domain in contrast to the QPGY domain of mammals. As with mammalian SYT, a Met-rich domain may be identified N-terminally of the QG domain. The QG-rich domain may be taken to be substantially the C-terminal remainder of the protein (minus the SHN domain); the Met-rich domain is typically comprised within the first half of the QG-rich (from the N-terminus to the C-terminus). A second Met-rich domain may precede the SNH domain in plant SYT polypeptides (see Fig 1 ).
A SYT loss-of function mutant and transgenic plants with reduced expression of SYT was reported to develop small and narrow leaves and petals, which have fewer cells (Kim HJ, Kende H (2004) Proc Nat Acad Sc 101 : 13374-9).
According to the present invention, there is provided a method for increasing plant yield, comprising modulating expression in a plant of a nucleic acid encoding a SYT polypeptide or a homologue thereof.
Reference herein to "corresponding wild type plants" is taken to mean any suitable control plant or plants, the choice of which would be well within the capabilities of a person skilled in the art and may include, for example, corresponding wild type plants or corresponding plants without the gene of interest. A "control plant" as used herein refers not only to whole plants, but also to plant parts, including seeds and seed parts.
Advantageously, performance of the methods according to the present invention results in plants having increased yield, particularly seed yield, relative to corresponding wild type plants.
The term "increased yield" as defined herein is taken to mean an increase in any one or more of the following, each relative to corresponding wild type plants: (i) increased biomass (weight) of one or more parts of a plant, particularly aboveground (harvestable) parts, increased root biomass or increased biomass of any other harvestable part (such as fruits, nuts and pulses); (ii) increased total seed yield, which includes an increase in seed biomass (seed weight) and which may be an increase in the seed weight per plant or on an individual seed basis; (iii) increased number of (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 (including oil, protein and carbohydrate total content and composition); (vi) increased individual seed area; (vii) increased individual seed length or width; (viii) increased harvest index, which is expressed as a ratio of the yield of harvestable parts, such as seeds, over the total biomass; and (ix) increased thousand kernel weight (TKW), which is extrapolated from the number of filled seeds counted and their total weight. An increased TKW may result from an increased seed size and/or seed weight. An increased TKW may result from an increase in embryo size and/or endosperm size. An increase in seed size, seed volume, seed area, seed perimeter, seed width and seed length may be due to an increase in specific parts of a seed, for example due to an increase in the size of the embryo and/or endosperm and/or aleurone and/or scutellum, or other parts of a seed.
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, increase in the seed filling rate (which is the number of filled seeds divided by the total number of seeds and multiplied by 100), 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 (florets) per panicle (which is expressed as a ratio of the number of filled seeds over the number of primary panicles), increase in the seed filling rate (which is the number of filled seeds divided by the total number of seeds and multiplied by 100), 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 seed yield. Therefore, according to the present invention, there is provided a method for increasing seed yield in a plant, which method comprises modulating expression in a plant of a nucleic acid encoding a SYT 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 further sowing of seeds of the same plant species (for example sowing and harvesting of rice plants followed by sowing and harvesting of further rice plants all within one conventional growing period). Similarly, if the growth rate is sufficiently increased, it may allow for the further sowing of seeds of different plants species (for example the sowing and harvesting of rice plants followed by, for example, the sowing and optional harvesting of soybean, potato or any other suitable plant). Harvesting additional times from the same rootstock in the case of some crop 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 relative to corresponding wild type plants. Therefore, according to the present invention, there is provided a method for increasing growth rate in plants, which method comprises modulating expression in a plant of a nucleic acid encoding a SYT polypeptide or a homologue thereof.
An increase in (seed) 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 suitable 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). Chemicals may also cause abiotic stresses. Biotic stresses are typically those stresses caused by pathogens, such as bacteria, viruses, fungi and insects.
Advantageously, yield may be modified in any plant.
The term "plant" as used herein encompasses whole plants, ancestors and progeny of the plants and plant parts, including seeds, shoots, stems, leaves, roots (including tubers), flowers, and tissues and organs, wherein each of the aforementioned comprise the transgene of interest. The term "plant" also encompasses plant cells, suspension cultures, callus tissue, embryos, meristematic regions, gametophytes, sporophytes, pollen and microspores, again wherein each of the aforementioned comprise the transgene.
Plants that are particularly useful in the methods of the invention include all plants which belong to the superfamily Viridiplantae, in particular monocotyledonous and dicotyledonous plants including fodder or forage legumes, ornamental plants, food crops, trees or shrubs selected from the list comprising 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, Coronillia varia, Cotoneaster serotina, Crataegus spp., Cucumis spp., Cupressus spp., Cyathea dealbata, Cydonia oblonga, Cryptomeria japonica, Cymbopogon spp., Cynthea dealbata, Cydonia oblonga, Dalbergia monetaria, Davallia divaricata, Desmodium spp., Dicksonia squarosa, Diheteropogon amplectens, Dioclea spp, Dolichos spp., Dorycnium rectum, Echinochloa pyramidalis, Ehrartia spp., Eleusine coracana, Eragrestis spp., Erythrina spp., Eucalyptus spp., Euclea schimperi, Eulalia villosa, Fagopyrum spp., Feijoa sellowiana, Fragaria spp., Flemingia 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, Hyperthelia dissoluta, Indigo incarnata, Iris spp., Leptarrhena pyrolifolia, Lespediza spp., Letiuca spp., Leucaena leucocephala, Loudetia simplex, Lotonus bainesii, Lotus spp., Macrotyloma axillare, Malus spp., Manihot esculenta, Medicago sativa, Metasequoia glyptostroboides, Musa sapientum, Nicotianum spp., Onobrychis spp., Ornithopus 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 fleckii, Pogonarthria squarrosa, Populus spp., Prosopis cineraria, Pseudotsuga menziesii, Pterolobium stellatum, Pyrus communis, Quercus spp., Rhaphiolepsis umbellata, Rhopalostylis sapida, Rhus natalensis, Ribes grossularia, Ribes spp., Robinia pseudoacacia, Rosa spp., Rubus spp., Salix spp., Schyzachyrium sanguineum, Sciadopitys verticillata, Sequoia sempervirens, Sequoiadendron giganteum, Sorghum bicolor, Spinacia spp., Sporobolus fimbriatus, Stiburus alopecuroides, Stylosanthos humilis, Tadehagi spp, Taxodium distichum, Themeda triandra, Trifolium spp., Triticum spp., Tsuga heterophylla, Vaccinium spp., Vicia spp., Vitis vinifera, Watsonia pyramidata, Zantedeschia aethiopica, Zea mays, amaranth, artichoke, asparagus, broccoli, Brussels 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. Examples of crop plants include amongst others soybean, sunflower, canola, alfalfa, rapeseed, cotton, tomato, potato or tobacco. Arabidopsis thaliana is generally not considered as a crop plant. Further preferably, the plant is a monocotyledonous plant, such as sugarcane. More preferably the plant is a cereal, such as rice, maize, wheat, barley, millet, rye, sorghum or oats.
The term "SYT polypeptide or homologue thereof as defined herein refers to a polypeptide comprising from N-terminal to C-terminal: (i) an SNH domain having in increasing order of preference at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the SNH domain of SEQ ID NO: 2; and (ii) a Met-rich domain; and (iii) a QG-rich domain.
Preferably, SNH domain having at least 40% identity to the SNH domain of SEQ ID NO: 2 comprises the residues shown in black in Figure 2. Further preferably, the SNH domain is represented by SEQ ID NO: 1.
Additionally, the SYT polypeptide or a homologue thereof may comprise one or more of the following: (a) SEQ ID NO: 90; (b) SEQ ID NO: 91; and (c) a Met-rich domain at the N- terminal preceding the SNH domain. A SYT polypeptide or a homologue thereof typically interacts with GRF (growth-regulating factor) polypeptides in yeast two-hybrid systems. Yeast two-hybrid interaction assays are well known in the art (see Field et al. (1989) Nature 340(6230): 245-246). For example, the SYT polypeptide as represented by SEQ ID NO: 4 is capable of interacting with AtGRF5 and with AtGRF9. SYT polypeptide and homologues thereof have been demonstrated by the inventors to increase yield, particularly seed yield, in plants.
A SYT polypeptide or homologue thereof is encoded by a SYT nucleic acid/gene. Therefore the term "SYT nucleic acid/gene" as defined herein is any nucleic acid/gene encoding a SYT polypeptide or a homologue thereof as defined hereinabove.
SYT polypeptides or homologues thereof may readily be identified using routine techniques well known in the art, such as 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 ((1970) J MoI Biol 48: 443-453) to find the alignment of two complete sequences that maximizes the number of matches and minimizes the number of gaps. The BLAST algorithm (Altschul et al. (1990) J MoI Biol 215: 403-10) 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. Homologues of SYT comprising an SNH domain having at least 40% sequence identity to the SNH domain of SEQ ID NO: 2 and/or comprising SEQ ID NO: 90 and/or SEQ ID NO: 91 , may readily be identified using, for example, the ClustalW multiple sequence alignment algorithm (version 1.83) available at http://clustalw.genome.jp/sit-bin/nph-clustalw, with the default pairwise alignment parameters, and a scoring method in percentage. A sequence having a 40% identity to the SNH domain of SEQ ID NO: 2 is sufficient to identify a sequence as being a SYT.
Furthermore, the presence of a Met-rich domain or a QG-rich domain may also readily be identified. As shown in Figure 3, the Met-rich domain and QG-rich domain follows the SNH domain. The QG-rich domain may be taken to be substantially the C-terminal remainder of the protein (minus the SHN domain); the Met-rich domain is typically comprised within the first half of the QG-rich (from the N-term to the C-term). Primary amino acid composition (in %) to determine if a polypeptide domain is rich in specific amino acids may be calculated using software programs from the ExPASy server (Gasteiger E et al. (2003) ExPASy: the proteomics server for in-depth protein knowledge and analysis. Nucleic Acids Res 31 :3784- 3788), in particular the ProtParam tool. The composition of the protein of interest may then be compared to the average amino acid composition (in %) in the Swiss-Prot Protein Sequence data bank. Within this databank, the average Met (M) content is of 2.37%, the average GIn (Q) content is of 3.93% and the average GIy (G) content is of 6.93%. As defined herein, a Met-rich domain or a QG-rich domain has Met content (in %) or a GIn and GIy content (in %) above the average amino acid composition (in %) in the Swiss-Prot Protein Sequence data bank.
Examples of SYT polypeptide or homologues thereof include (encoded by polynucleotide sequence accession number in parenthesis; see also Table 1): Arabidopsis thaliana Arath_SYT1 (AY102639.1) SEQ ID NO: 4, Arabidopsis thaliana Arath_SYT2 (AY102640.1) SEQ ID NO: 6, Arabidopsis thaliana Arath_SYT3 (AY102641.1) SEQ ID NO: 8, Aspergillus officinalis Aspof_SYT (CV287542) SEQ ID NO: 10, Brassica napus Brana_SYT (CD823592) SEQ ID NO: 12, Citrus sinensis Citsi_SYT (CB290588) SEQ ID NO: 14, Gossypium arboreum Gosar_SYT (BM359324) SEQ ID NO: 16, Medicago trunculata Medtr_SYT (CA858507.1) SEQ ID NO: 18, Oryza sativa Orysa_SYT1 (AK058575) SEQ ID NO: 20, Oryza sativa Orysa_SYT2 (AK105366) SEQ ID NO: 22, Oryza sativa Orysa_SYT3 (BP185008) SEQ ID NO: 24, Solanum tuberosum Soltu_SYT (BG590990) SEQ ID NO: 26, Tea mays Zeama_SYT1 (BG874129.1 , CA409022.1) SEQ ID NO: 28, Tea mays Zeama_SYT2 (AY106697) SEQ ID NO: 30, Homo sapiens Homsa_SYT (CAG46900) SEQ ID NO: 32, Allium cepa Allce_SYT2 (CF437485) SEQ ID NO: 34, Aquilegia formosa x Aquilegia pubescens Aqufo_SYT1 (DT758802) SEQ ID NO: 36, Brachypodium distachyon Bradi_SYT3 (DV480064) SEQ ID NO: 38, Brassica napus Brana_SYT2 (CN732814) SEQ ID NO: 40, Citrus sinensis Citsi_SYT2 (CV717501) SEQ ID NO: 42, Euphorbia esula Eupes_SYT2 (DV144834) SEQ ID NO: 44, Glycine max Glyma_SYT2 (BQ612648) SEQ ID NO: 46, Glycine soya Glyso_SYT2 (CA799921) SEQ ID NO: 48, Gossypium hirsutum Goshi_SYT1 (DT558852) SEQ ID NO: 50, Gossypium hirsutum Goshi_SYT2 (DT563805) SEQ ID NO: 52, Hordeum vulgare Horvu_SYT2 (CA032350) SEQ ID NO: 54, Lactuca serriola Lacse_SYT2 (DW110765) SEQ ID NO: 56, Lycopersicon esculentum Lyces_SYT1 (AW934450, BP893155) SEQ ID NO: 58, Malus domestica Maldo_SYT2 (CV084230, DR997566) SEQ ID NO: 60, Medicago trunculata Medtr_SYT2 (CA858743, BI310799, AL382135) SEQ ID NO: 62, Panicum virgatum Panvi_SYT3 (DN152517) SEQ ID NO: 64, Picea sitchensis Picsi_SYT1 (DR484100, DR478464) SEQ ID NO: 66, Pinus taeda Pinta_SYT1 (DT625916) SEQ ID NO: 68, Populus tremula Poptr_SYT1 (DT476906) SEQ ID NO: 70, Saccharum officinarum Sacof_SYT1 (CA078249, CA078630, CA082679, CA234526, CA239244, CA083312) SEQ ID NO: 72, Saccharum officinarum. Sacof_SYT2 (CA110367) SEQ ID NO: 74, Saccharum officinarum Sacof_SYT3 (CA161933, CA265085) SEQ ID NO: 76, Solanum tuberosum Soltu_SYT1 (CK265597) SEQ ID NO: 78, Sorghum bicolor Sorbi_SYT3 (CX611128) SEQ ID NO: 80, Triticum aestivum Triae_SYT2 (CD901951) SEQ ID NO: 82, Triticum aestivum Triae_SYT3 (BJ246754, BJ252709) SEQ ID NO: 84, Vitis vinifera Vitvi_SYT1 (DV219834) SEQ ID NO: 86, Tea mays Zeama_SYT3 (CO468901) SEQ ID NO: 88.
Figure imgf000011_0001
Figure imgf000012_0001
"Compiled from cited accessions
It is to be understood that sequences falling under the definition of "SYT polypeptide or homologue thereof are not to be limited to the sequences represented by 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, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, but that any polypeptide comprising from N-terminal to C-terminal: (i) an SNH domain having at least 40% sequence identity to the SNH domain of SEQ ID NO: 2; and (ii) a Met-rich domain; and (iii) a QG-rich domain may be suitable in performing the methods of the invention.
Examples of SYT nucleic acids include but are not limited to those represented by any one of 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 , SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31 , SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41 , SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51 , SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61 , SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87. SYT nucleic acids/genes and variants thereof may be suitable in practising the methods of the invention. Variant SYT nucleic acid/genes typically are those having the same function as a naturally occurring SYT nucleic acid/genes, which can be the same biological function or the function of increasing yield when expression of the nucleic acids/genes is modulated in a plant. Such variants include portions of a SYT nucleic acid/gene and/or nucleic acids capable of hybridising with a SYT nucleic acid/gene as defined below.
The term portion as defined herein refers to a piece of DNA encoding a polypeptide comprising from N-terminal to C-terminal: (i) an SNH domain having in increasing order of preference at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the SNH domain of SEQ ID NO: 2 and (ii) a Met-rich domain; and (iii) a QG-rich domain. A portion may be prepared, for example, by making one or more deletions to a SYT nucleic acid. The portions may be used in isolated form or they may be fused to other coding (or non coding) sequences in order to, for example, produce a protein that combines several activities. When fused to other coding sequences, the resulting polypeptide produced upon translation may be bigger than that predicted for the SYT fragment. Preferably, the portion is a portion of a nucleic acid as represented by any one of 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 , SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31 , SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51 , SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61 , SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71 , SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87. Most preferably the portion of a nucleic acid is as represented by SEQ ID NO: 3 SEQ ID NO: 5 or SED IQ NO: 7.
Another variant of a SYT nucleic acid/gene is a nucleic acid capable of hybridising under reduced stringency conditions, preferably under stringent conditions, with a SYT nucleic acid/gene as hereinbefore defined, which hybridising sequence encodes a polypeptide comprising from N-terminal to C-terminal: (i) an SNH domain having in increasing order of preference at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the SNH domain of SEQ ID NO: 2 and (ii) a Met-rich domain; and (iii) a QG-rich domain. Preferably, the hybridising sequence is one that is capable of hybridising to a nucleic acid as represented by any one of 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 , SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51 , SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61 , SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71 , SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81 , SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87 or to a portion of any of the aforementioned sequences as defined hereinabove. Most preferably the hybridizing sequence of a nucleic acid is as represented by SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7.
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 0.4M. Formamide reduces the melting temperature of DNA- DNA and DNA-RNA duplexes with 0.6 to 0.70C for each percent formamide, and addition of 50% formamide allows hybridisation to be performed at 30 to 45°C, though the rate of hybridisation will be lowered. Base pair mismatches reduce the hybridisation rate and the thermal stability of the duplexes. On average and for large probes, the Tm decreases about 1°C per % base mismatch. The Tm may be calculated using the following equations, depending on the types of hybrids:
1. DNA-DNA hybrids (Meinkoth and Wahl, Anal. Biochem., 138: 267-284, 1984): Tm= 81.5°C + 16.6xlog[Na+]a + 0.41x%[G/Cb] - 500x[Lc]"1 - 0.61x% formamide
2. DNA-RNA or RNA-RNA hybrids:
Tm= 79.8 + 18.5 (logio[Na+]a) + 0.58 (%G/Cb) + 11.8 (%G/Cb)2 - 820/Lc
3. oligo-DNA or oligo-RNAd hybrids:
For <20 nucleotides: Tm= 2 (/n)
For 20-35 nucleotides: Tm= 22 + 1.46 (/n )
a or for other monovalent cation, but only accurate in the 0.01-0.4 M range.
6 only accurate for %GC in the 30% to 75% range.
0 L = length of duplex in base pairs. d Oligo, oligonucleotide; /n, effective length of primer = 2χ(no. of G/C)+(no. of AfT).
Note: for each 1% formamide, the Tm is reduced by about 0.6 to 0.70C, while 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 amplification 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 500C 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 200C below Tm, and high stringency conditions are when the temperature is 10°C below Tm. For example, stringent conditions are those that are at least as stringent as, for example, conditions A-L; and reduced stringency conditions are at least as stringent as, for example, conditions M-R. Non-specific binding may be controlled using any one of a number of known techniques such as, for example, blocking the membrane with protein containing solutions, additions of heterologous RNA, DNA, and SDS to the hybridisation buffer, and treatment with RNase. Examples of hybridisation and wash conditions are listed in Table 2 below.
Table 2: Examples of hybridisation and wash conditions
Wash
Stringency Polynucleotide Hybrid Hybridization Temperature Temperature Condition Hybrid ± Length (bp) * and Buffer f and Buffer*
> or 65° C IxSSC; or 42 °C, IxSSC 650C;
A DNA: DNA equal to 50 and 50% formamide 0.3χSSC
B DNAr DNA <50 Tb* ; I xSSC Tb* ; I xSSC
> or 67 ° C I xSSC; or 45°C, I xSSC 670C;
C DNA: RNA equal to 50 and 50% formamide 0.3χSSC
D DNAr RNA <50 Td* ; I xSSC Td* ; I xSSC
> or 70 ° C I xSSC; or 500C, I xSSC 700C;
E RNA: RNA equal to 50 and 50% formamide 0.3χSSC
F RNAr RNA <50 Tf* ; I xSSC Tf* ; I xSSC
> or 65° C 4 χSSC; or 45°C, 4 χSSC
G DNAr DNA 65°C; IxSSC equal to 50 and 50% formamide
H DNAr DNA <50 Th* ; 4 xSSC Th* ; 4 xSSC
> or 67 ° C 4 χSSC; or 45°C, 4 χSSC
I DNAr RNA 67 °C; IxSSC equal to 50 and 50% formamide
J DNAr RNA <50 Tj * ; 4 xSSC Tj * ; 4 xSSC > or 70 °C 4 χSSC; or 40°c, 6χSSC
K RNAr RNA 670C; IxSSC equal to 50 and 50% formamide
L RNA: RNA <50 Tl* ; 2 xSSC Tl* ; 2 χSSC
> or 50 ° C 4 χSSC; or 40 °c, 6χSSC
M DNA: DNA 500C; 2χSSC equal to 50 and 50% formamide
N DNAr DNA <50 Tn* ; 6 xSSC Tn* ; 6χSSC
> or 55° C 4 χSSC; or 42 °c, 6χSSC
O DNA: RNA 55°C; 2χSSC equal to 50 and 50% formamide
P DNAr RNA <50 Tp* ; 6 xSSC Tp* ; 6χSSC
> or 60 ° C 4 χSSC; or 45 °c, 6χSSC 600C . ;
Q RNA: RNA equal to 50 and 50% formamide 2 χSSC
R RNAr RNA <50 Tr* ; 4 xSSC Tr* ; 4 χSSC
* 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.
f SSPE (I xSSPE is 0.15M NaCI, 1OmM NaH2PO4, 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 x Denhardfs reagent, 0.5-1.0% SDS, 100 μg/ml denatured, fragmented salmon sperm DNA, 0.5% sodium pyrophosphate, and up to 50% formamide.
* Tb-Tr: The hybridisation temperature for hybrids anticipated to be less than 50 base pairs in length should be 5-10°C less than the melting temperature Tm of the hybrids; the Tm is determined according to the above-mentioned equations.
* The present invention also encompasses the substitution of any one, or more DNA or RNA hybrid partners with either a PNA, or a modified nucleic acid.
For the purposes of defining the level of stringency, reference can 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).
The SYT nucleic acid or variant thereof may be derived from any artificial source or natural source, such as plant, algae or animal. 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. Preferably the nucleic acid of plant origin encodes a SYT1. Alternatively, the nucleic acid may encode a SYT2 or SYT3, which are closely related to one another on a polypeptide level. The nucleic acid may be isolated from a dicotyledonous species, preferably from the family Brassicaceae, further preferably from Arabidopsis thaliana. More preferably, the three SYT nucleic acids isolated from Arabidopsis thaliana are represented by SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 7, and the three SYT amino acid sequences are as represented by SEQ ID NO: 4, SEQ ID NO: 6 and SEQ ID NO: 8.
The expression of a nucleic acid encoding a SYT polypeptide or a homologue thereof may be modulated by introducing a genetic modification (preferably in the locus of a SYT gene). The locus of a gene as defined herein is taken to mean a genomic region, which includes the gene of interest and 10 kb up- or downstream of the coding region.
The genetic modification may be introduced, for example, by any one (or more) of the following methods: T-DNA activation, TILLING, site-directed mutagenesis, directed evolution and homologous recombination, or by introducing and expressing in a plant a nucleic acid encoding a SYT polypeptide or a homologue thereof. Following introduction of the genetic modification, there follows a step of selecting for modulated expression of a nucleic acid encoding a SYT polypeptide or a homologue thereof, which modulated expression gives plants having increased yield, particularly increased seed yield.
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 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 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 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, through Agrobacterium infection and leads to overexpression of genes near 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 SYT 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 SYT nucleic acid encoding a protein with enhanced SYT activity. TILLING also allows selection of plants carrying such mutant variants. These mutant variants may even exhibit higher SYT activity than that exhibited by the gene in its natural form. TILLNG combines high-density mutagenesis with high-throughput screening methods. The steps typically followed in TILLING are: (a) EMS mutagenesis (Redei GP and Koncz C (1992) In Methods in Arabidopsis Research, Koncz C, Chua NH, Schell J, eds. Singapore, World Scientific Publishing Co, pp. 16-82; Feldmann et al., (1994) In Meyerowitz EM, Somerville CR, eds, Arabidopsis. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp 137-172; Lightner J and Caspar T (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 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 et al., (2000) Nat Biotechnol 18: 455-457; reviewed by Stemple (2004) Nat Rev Genet 5(2): 145-50).
Site-directed mutagenesis may be used to generate variants of SYT nucleic acids. 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.com/products/currentprotocols/index.html).
Directed evolution may also be used to generate variants of SYT nucleic acids. This consists of iterations of DNA shuffling followed by appropriate screening and/or selection to generate variants of SYT nucleic acids or portions thereof encoding SYT polypeptides or homologues or portions thereof having an modified biological activity (Castle et al., (2004) Science 304(5674): 1151-4; US patents 5,811,238 and 6,395,547). T-DNA activation, TILLING, site-directed mutagenesis and directed evolution are examples of technologies that enable the generation of novel SYT alleles and 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 Physcomitrella. Methods for performing homologous recombination in plants have been described not only for model plants (Offringa et al. (1990) EMBO J 9(10): 3077-84) but also for crop plants, for example rice (Terada et al. (2002) Nat Biotech 20(10): 1030-4; lida and Terada (2004) Curr Opin Biotech 15(2): 132-8). The nucleic acid to be targeted (which may be a SYT nucleic acid or variant thereof as hereinbefore defined) is targeted to the locus of a SYT gene. The nucleic acid to be targeted may be an improved allele used to replace the endogenous gene or may be introduced in addition to the endogenous gene.
A preferred method for introducing a genetic modification (which in this case need not be in the locus of a SYT gene) is to introduce and express in a plant a nucleic acid encoding a SYT polypeptide or a homologue thereof. A SYT polypeptide or a homologue thereof is defined as a polypeptide comprising from N-terminal to C-terminal: (i) an SNH domain having in increasing order of preference at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the SNH domain of SEQ ID NO: 2; and (ii) a Met-rich domain; and (iii) a QG-rich domain.
Preferably, SNH domain having at least 40% identity to the SNH domain of SEQ ID NO: 2 comprises the residues shown in black in Figure 2. Further preferably, the SNH domain is represented by SEQ ID NO: 1.
The nucleic acid to be introduced into a plant may be a full-length nucleic 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 α-helical structures or β-sheet structures). Conservative substitution tables are well known in the art (see for example Creighton (1984) Proteins. W.H. Freeman and Company and Table 3 below).
Homologues include orthologues and paralogues, which encompass evolutionary concepts used to describe ancestral relationships of genes. Paralogues are genes within the same species that have originated through duplication of an ancestral gene and orthologues are genes from different organisms that have originated through speciation.
Orthologues in, for example, monocot plant species may easily be found by performing a so-called reciprocal blast search. This may be done by a first blast involving blasting a query sequence (for example, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8) against any sequence database, such as the publicly available NCBI database which may be found at: http://www.ncbi.nlm.nih.gov. BLASTN or TBLASTX (using standard default values) may be used when starting from a nucleotide sequence and BLASTP or TBLASTN (using standard default values) may be used when starting from a protein sequence. The BLAST results may optionally be filtered. The full- length sequences of either the filtered results or non-filtered results are then BLASTed back (second BLAST) against sequences from the organism from which the query sequence is derived (where the query sequence is SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8 the second blast would therefore be against Arabidopsis sequences). The results of the first and second BLASTs are then compared. A paralogue is identified if a high-ranking hit from the second blast is from the same species as from which the query sequence is derived; an orthologue is identified if a high-ranking hit is not from the same species as from which the query sequence is derived. High-ranking hits are those having a low E-value. The lower the E-value, the more significant the score (or in other words the lower the chance that the hit was found by chance). Computation of the E-value is well known in the art. In the case of large families, ClustalW may be used, followed by a neighbour joining tree, to help visualize clustering of related genes and to identify orthologues and paralogues.
A homologue may be in the form of a "substitutional variant" of a protein, i.e. where at least one residue in an amino acid sequence has been removed and a different residue inserted in its place. Amino acid substitutions are typically of single residues, but may be clustered depending upon functional constraints placed upon the polypeptide; insertions will usually be of the order of about 1 to 10 amino acid residues. Preferably, amino acid substitutions comprise conservative amino acid substitutions. Conservative substitution tables are readily available in the art. The table below gives examples of conserved amino acid substitutions. Table 3: Examples of conserved amino acid substitutions
Figure imgf000022_0001
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 N-terminal and/or C-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 N- or C-terminal fusions, of the order of about 1 to 10 residues. Examples of N- or C-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)-β-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 M 13 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 SYT polypeptide or homologue thereof may be a derivative. "Derivatives" include peptides, oligopeptides, polypeptides, proteins and enzymes which may comprise substitutions, deletions or additions of 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: 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, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86 and SEQ ID NO: 88.
"Derivatives" of a protein encompass peptides, oligopeptides, polypeptides, proteins and enzymes which may comprise naturally occurring altered, glycosylated, acylated, prenylated 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 SYT polypeptide or homologue thereof may be encoded by an alternative splice variant of a SYT 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, or in which introns have been shortened or lengthened. 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 encoding a polypeptide comprising from N-terminal to C-terminal: (i) an SNH domain having in increasing order of preference at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the SNH domain of SEQ ID NO: 2; and (ii) a Met-rich domain; and (iii) a QG-rich domain. Preferably, SNH domain having at least 40% identity to the SNH domain of SEQ ID NO: 2 comprises the residues shown in black in Figure 2. Further preferably, the SNH domain is represented by SEQ ID NO: 1.
Additionally, the SYT polypeptide or a homologue thereof may comprise one or more of the following: (i) SEQ ID NO: 90; and/or (ii) SEQ ID NO: 91; and/or (iii) a Met-rich domain at the N-terminal preceding the SNH domain.
Further preferred are splice variants of nucleic acids represented by 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 , SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31 , SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51 , SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61 , SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81 , SEQ ID NO: 83, SEQ ID NO: 85 and SEQ ID NO: 87 . Most preferred are splice variants of a SYT nucleic acid/gene represented by SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 7.
The homologue may also be encoded by an allelic variant of a nucleic acid encoding a SYT polypeptide or a homologue thereof, preferably an allelic variant of the nucleic acid encoding a polypeptide comprising from N-terminal to C-terminal: (i) an SNH domain having in increasing order of preference at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the SNH domain of SEQ ID NO: 2; and (ii) a Met-rich domain; and (iii) a QG-rich domain. Preferably, SNH domain having at least 40% identity to the SNH domain of SEQ ID NO: 2 comprises the residues shown in black in Figure 2. Further preferably, the SNH domain is represented by SEQ ID NO: 1. Additionally, the SYT polypeptide or a homologue thereof may comprise one or more of the following: (i) SEQ ID NO: 90; and/or (ii) SEQ ID NO: 91; and/or (iii) a Met-rich domain at the N-terminal preceding the SNH domain.
Further preferably, the allelic variant is an allelic variant of a nucleic acid as represented by any one of 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 , SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31 , SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41 , SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61 , SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71 , SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85 and SEQ ID NO: 87. Most preferably, the allelic variant is an allelic variant of a nucleic acid as represented by any one of SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 7.
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, the modulated expression of a SYT nucleic acid or variant thereof is increased expression. The increase in expression may lead to raised SYT mRNA or polypeptide levels, which could equate to raised activity of the SYT polypeptide; or the activity may also be raised when there is no change in polypeptide levels, or even when there is a reduction in polypeptide levels. 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. Methods for increasing 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 SYT nucleic acid or variant thereof. For example, endogenous promoters may be altered in vivo by mutation, deletion, and/or substitution (see, Kmiec, US 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. Methods for reducing the expression of genes or gene products are well documented in the art.
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, MoI. Cell biol. 8:4395-4405 (1988); Callis et a/., Genes Dev. 1 :1183-1200 (1987). Such intron enhancement of gene expression is typically greatest when placed near the 51 end of the transcription unit. Use of the maize introns Adh1-S intron 1 , 2, and 6, the Bronze-1 intron are known in the art. See generally, The Maize Handbook, Chapter 116, Freeling and Walbot, Eds., Springer, N.Y. (1994).
The invention also provides genetic constructs and vectors to facilitate introduction and/or expression of the nucleotide sequences useful in the methods according to the invention.
Therefore, there is provided a gene construct comprising:
(i) Any SYT nucleic acid or variant thereof, as defined hereinabove;
(ii) One or more control sequences capable of driving expression of the nucleic acid sequence of (i); and optionally (iii) A transcription termination sequence.
A preferred construct is one whether the control sequence is a promoter derived from a plant, preferably from a monocotyledonous plant.
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 nucleic acid encoding a SYT polypeptide or homologue thereof). The sequence of interest is operably linked to one or more control sequences (at least to a promoter). The terms "regulatory element", "control sequence" and "promoter" are all used interchangeably herein and are to be taken in a broad context to refer to regulatory nucleic acid sequences capable of effecting expression of the sequences to which they are ligated. Encompassed by the aforementioned terms are transcriptional regulatory sequences derived from a classical eukaryotic genomic gene (including the TATA box which is required for accurate transcription initiation, with or without a CCAAT box sequence) and additional regulatory elements (i.e. upstream activating sequences, enhancers and silencers) which 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 that confers, activates or enhances expression of a nucleic acid molecule in a cell, tissue or organ. The term "operably linked" as used herein refers to a functional linkage between the promoter sequence and the gene of interest, such that the promoter sequence is able to initiate transcription of the gene of interest.
Advantageously, any type of promoter may be used to drive expression of the nucleic acid sequence. The promoter may be an inducible promoter, i.e. having induced or increased transcription initiation in response to a developmental, chemical, environmental or physical stimulus. An example of an inducible promoter being a stress-inducible 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-specific".
Preferably, the SYT nucleic acid or variant thereof is operably 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 promoter is derived from a plant, further preferably a monocotyledonous plant. Most preferred is use of a GOS2 promoter (from rice) (SEQ ID NO: 89). It should be clear that the applicability of the present invention is not restricted to the SYT nucleic acid represented by SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7, nor is the applicability of the invention restricted to expression of a SYT nucleic acid when driven by a GOS2 promoter. Examples of other constitutive promoters which may also be used to drive expression of a SYT nucleic acid are shown in Table 4 below. Table 4: Examples of constitutive promoters
Figure imgf000028_0001
Optionally, one or more terminator sequences may also be used in the construct introduced into a plant. The term "terminator" encompasses a control sequence which is a DNA sequence at the end of a transcriptional unit which signals 3' processing and polyadenylation of a primary transcript and termination of transcription. Additional regulatory elements may include transcriptional as well as translational enhancers. Those skilled in the art will be aware of terminator and enhancer sequences that 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 that is required for maintenance and/or replication in a specific cell type. One example is when a genetic construct is required to be maintained in a bacterial cell as an episomal genetic element (e.g. plasmid or cosmid molecule). Preferred origins of replication include, but are not limited to, the f1-ori and colE1. The genetic construct may optionally comprise a selectable marker gene. As used herein, the term "selectable marker gene" includes any gene that confers a phenotype on a cell in which it is expressed to facilitate the identification and/or selection of cells that are transfected or transformed with a nucleic acid construct of the invention. Suitable markers may be selected from markers that confer antibiotic or herbicide resistance, that introduce a new metabolic trait or that allow visual selection. Examples of selectable marker genes include genes conferring resistance to antibiotics (such as nptll that phosphorylates neomycin and kanamycin, or hpt, phosphorylating hygromycin), to herbicides (for example bar which provides resistance to Basta; aroA or gox providing resistance against glyphosate), or genes that provide a metabolic trait (such as manA that allows plants to use mannose as sole carbon source). Visual marker genes result in the formation of colour (for example β-glucuronidase, GUS), luminescence (such as luciferase) or fluorescence (Green Fluorescent Protein, GFP, and derivatives thereof).
The present invention also encompasses plants obtainable by the methods according to the present invention. The present invention therefore provides plants, plant parts and plant cells obtainable by the methods according to the present invention, which plants have introduced therein a SYT nucleic acid or variant thereof and which plants, plant parts and plant cells are preferably from a crop plant, further preferably from a monocotyledonous plant.
The invention also provides a method for the production of transgenic plants having increased yield, comprising introduction and expression in a plant of a SYT nucleic acid or a variant thereof.
More specifically, the present invention provides a method for the production of transgenic plants, preferably monocotyledonous plants, having increased yield, which method comprises:
(i) introducing and expressing in a plant or plant cell a SYT nucleic acid or variant thereof; and
(ii) cultivating the plant cell under conditions promoting plant growth and development.
Subsequent generations of the plants obtained from cultivating step (ii) 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 nucleic acid may be introduced directly into a plant cell or into the plant itself (including introduction into a tissue, organ or any other part of a plant). According to a preferred feature of the present invention, the nucleic acid is introduced into a plant by transformation.
The term "transformation" as referred to herein encompasses the transfer of an exogenous polynucleotide into a host cell, irrespective of the method used for transfer. Plant tissue capable of subsequent clonal propagation, whether by organogenesis or embryogenesis, may be transformed with a genetic construct of the present invention and a whole plant regenerated from there. 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 al., (1982) Nature 296, 72-74; Negrutiu I et al. (1987) Plant MoI Biol 8: 363-373); electroporation of protoplasts (Shillito R.D. et al., 1985 Bio/Technol 3, 1099-1102); microinjection into plant material (Crossway A et al., (1986) MoI. Gen Genet 202: 179-185); DNA or RNA-coated particle bombardment (Klein TM et al., (1987) Nature 327: 70) infection with (non-integrative) viruses and the like. Transgenic rice plants expressing a SYT nucleic acid/gene are preferably produced via Agrobacterium-med\ated transformation using any of the well known methods for rice transformation, such as described in any of the following: published European patent application EP 1198985 A1, Aldemita and Hodges (Planta 199: 612-617, 1996); Chan et al. (Plant MoI 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 corn transformation, the preferred method is as described in either lshida et al. (Nat. Biotechnol 14(6): 745-50, 1996) or Frame et al. (Plant Physiol 129(1): 13-22, 2002), which disclosures are incorporated by reference herein as if fully set forth.
Generally after transformation, plant cells or cell groupings are selected for the presence of one or more markers which are encoded by plant-expressible genes co-transferred with the gene of interest, following which the transformed material is regenerated into a whole plant.
Following DNA transfer and regeneration, putatively transformed plants may be evaluated, for instance using Southern analysis, for the presence of the gene of interest, copy number and/or genomic organisation. Alternatively or additionally, expression levels of the newly introduced DNA may be monitored using Northern and/or Western analysis, quantitative PCR, such techniques being well known to persons having ordinary skill in the art.
The generated transformed plants may be propagated by a variety of means, such as by clonal propagation or classical breeding techniques. For example, a first generation (or T1) transformed plant may be selfed to give homozygous second generation (or T2) transformants, and the T2 plants further propagated through classical breeding techniques.
The generated transformed organisms may take a variety of forms. For example, they may be chimeras of transformed cells and non-transformed cells; clonal transformants (e.g., all cells transformed to contain the expression cassette); grafts of transformed and untransformed tissues (e.g., in plants, a transformed rootstock grafted to an untransformed scion).
The present invention clearly extends to any plant cell or plant produced by any of the methods described herein, and to all plant parts and propagules thereof. The present invention extends further to encompass the progeny of a primary transformed or transfected cell, tissue, organ or whole plant that has been produced by any of the aforementioned methods, the only requirement being that progeny exhibit the same genotypic and/or phenotypic characteristic(s) as those produced by the parent in the methods according to the invention. The invention also includes host cells containing an isolated SYT 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 invention furthermore relates to products derived, preferably directly derived, from a harvestable part of such a plant, such as dry pellets or powders, meal, oil, fat and fatty acids, starch or proteins.
The present invention also encompasses use of SYT nucleic acids or variants thereof and use of SYT polypeptides or homologues thereof and to use of a construct as defined hereinabove in increasing plant yield, especially seed yield. The seed yield is as defined above and preferably includes increased total seed yield or increased TKW.
SYT nucleic acids or variants thereof, or SYT polypeptides or homologues thereof may find use in breeding programmes in which a DNA marker is identified which may be genetically linked to a SYT gene or variant thereof. The SYT nucleic acids/ genes or variants thereof, or SYT polypeptides or homologues thereof may be used to define a molecular marker. This DNA or protein marker may then be used in breeding programmes to select plants having increased yield. The SYT gene or variant thereof may, for example, be a nucleic acid as represented by any one of 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, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41 , SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51 , SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61 , SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85 and SEQ ID NO: 87.
Allelic variants of a SYT 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, for example, by PCR. This is followed by a step for selection of superior allelic variants of the sequence in question and which give increased yield. 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: 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 , SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31 , SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41 , SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51 , SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81 , SEQ ID NO: 83, SEQ ID NO: 85 and SEQ ID NO: 87. 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 SYT 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 SYT nucleic acids or variants thereof requires only a nucleic acid sequence of at least 15 nucleotides in length. The SYT nucleic acids or variants thereof may be used as restriction fragment length polymorphism (RFLP) markers. Southern blots (Sambrook J, Fritsch EF and Maniatis T (1989) Molecular Cloning, A Laboratory Manual) of restriction-digested plant genomic DNA may be probed with the SYT nucleic acids or variants thereof. The resulting banding patterns may then be subjected to genetic analyses using computer programs such as MapMaker (Lander et al. (1987) Genomics 1 : 174-181) in order to construct a genetic map. In addition, the nucleic acids may be used to probe Southern blots containing restriction endonuclease-treated genomic DNAs of a set of individuals representing parent and progeny of a defined genetic cross. Segregation of the DNA polymorphisms is noted and used to calculate the position of the SYT nucleic acid or variant thereof in the genetic map previously obtained using this population (Botstein et al. (1980) Am. J. Hum. Genet. 32:314-331).
The production and use of plant gene-derived probes for use in genetic mapping is described in Bematzky and Tanksley (1986) Plant MoI. 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 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 hybridization (FISH) mapping (Trask (1991) Trends Genet. 7:149-154). Although current methods of FISH mapping favor use of large clones (several kb to several hundred kb; see Laan et al. (1995) Genome Res. 5:13-20), improvements in sensitivity may allow performance of FISH mapping using shorter probes.
A variety of nucleic acid amplification-based methods for genetic and physical mapping may be carried out using the nucleic acids. Examples include allele-specific amplification (Kazazian (1989) J. Lab. Clin. Med 11 :95-96), polymorphism of PCR-amplified fragments (CAPS; Sheffield et al. (1993) Genomics 16:325-332), allele-specific ligation (Landegren et al. (1988) Science 241 :1077-1080), nucleotide extension reactions (Sokolov (1990) Nucleic Acid Res. 18:3671), Radiation Hybrid Mapping (Walter et al. (1997) Nat. Genet. 7:22-28) and Happy Mapping (Dear and Cook (1989) Nucleic Acid Res. 17:6795-6807). For these methods, the sequence of a nucleic acid is used to design and produce primer pairs for use in the amplification reaction or in primer extension reactions. The design of such primers is well known to those skilled in the art. In methods employing PCR-based genetic mapping, it may be necessary to identify DNA sequence differences between the parents of the mapping cross in the region corresponding to the instant nucleic acid sequence. This, however, is generally not necessary for mapping methods.
The methods according to the present invention result in plants having increased yield, as described hereinbefore. These yield-enhancing traits 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 the typical domain structure of SYT polypeptides from plants and mammals. The conserved SNH domain is located at the N-terminal end of the protein. The C-terminal remainder of the protein domain consists of a QG-rich domain in plant SYT polypeptides, and of a QPGY-rich domain in mammalian SYT polypeptides. A Met-rich domain is typically comprised within the first half of the QG-rich (from the N-term to the C-term) in plants or QPGY-rich in mammals. A second Met-rich domain may precede the SNH domain in plant SYT polypeptides Fig. 2 shows a multiple alignment of the N-terminal end of several SYT polypeptides, using VNTI AlignX multiple alignment program, based on a modified ClustalW algorithm (InforMax, Bethesda, MD, http://www.informaxinc.com), with default settings for gap opening penalty of 10 and a gap extension of 0.05). The SNH domain is boxed across the plant and human SYT polypeptides. The last line in the alignment consists of a consensus sequence derived from the aligned sequences.
Fig.3 shows a multiple alignment of several plant SYT polypeptides, using VNTI AlignX multiple alignment program, based on a modified ClustalW algorithm (InforMax, Bethesda, MD, http://www.informaxinc.com), with default settings for gap opening penalty of 10 and a gap extension of 0.05). The two main domains, from N-terminal to C-terminal, are boxed and identified as SNH domain and the Met-rich/QG-rich domain. Additionally, the N-terminal Met-rich domain is also boxed, and the positions of SEQ ID NO: 90 and SEQ ID NO 91 are underlined in bold.
Fig. 4 shows a Neighbour joining tree resulting from the alignment of multiple SYT polypeptides using CLUSTALW 1.83 (http://align.genome.jp/sit-bin/clustalw). The SYT1 and SYT2/SYT3 clades are identified with brackets.
Fig. 5 shows a binary vector pO523, for expression in Oryza sativa of an Arabidopsis thaliana AtSYTI under the control of a GOS2 promoter (internal reference PRO0129).
Fig. 6 shows a binary vector pO524, for expression in Oryza sativa of an Arabidopsis thaliana AtSYT2 under the control of a GOS2 promoter (internal reference PRO0129).
Fig. 7 shows a binary vector pO767, for expression in Oryza sativa of an Arabidopsis thaliana AtSYT3 under the control of a GOS2 promoter (internal reference PRO0129).
Fig. 8 details examples of sequences useful in performing the methods according to the present invention. SYT nucleic acid sequences are presented from start to stop. The majority of these sequences are derived from EST sequencing, which is of lower quality. Therefore, nucleic acid substitutions may be encountered.
Examples
The present invention will now be described with reference to the following examples, which are by way of illustration alone. DNA manipulation: unless otherwise stated, recombinant DNA techniques are performed according to standard protocols described in (Sambrook (2001) Molecular Cloning: a laboratory manual, 3rd Edition Cold Spring Harbor Laboratory Press, CSH, New York) or in Volumes 1 and 2 of Ausubel et al. (1994), Current Protocols in Molecular Biology, Current Protocols. Standard materials and methods for plant molecular work are described in Plant Molecular Biology Labfase (1993) by R.D.D. Cray, published by BIOS Scientific Publications Ltd (UK) and Blackwell Scientific Publications (UK).
Example 1: Gene Cloning of AtSYTI, AtSYT2 and AtSYT3
The Arabidopsis thaliana AtSYTI gene 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 1.59 x 107 cfu. Original titer was determined to be 9.6 x 105 cfu/ml after first amplification of 6x1011 cfu/ml. After plasmid extraction, 200 ng of template was used in a 50 μl PCR mix. Primers prmO6681 (SEQ ID NO: 92; sense, start codon in bold, AttB1 site in italic: 5'-GGGGyACyAyAGrTTGLACyAAAAAyAGCyAGGCTTAAACAATGCAACAGCACCTGATG -3') and prmO6682 (SEQ ID NO: 93; reverse, complementary, AttB2 site in italic: 5'- GGGGACCyACTTTGTyACAAGAAAGCTGGGTCATCATTAAGATTCCTTGTGC-S'), 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 727 bp (including attB sites) 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", pO7466. Plasmid pDONR201 was purchased from Invitrogen, as part of the Gateway® technology.
The Arabidopsis thaliana AtSYT2 gene was amplified by PCR using the same method as the Arabidopsis thaliana AtSYTI gene. Primers prmO6685 (SEQ ID NO: 94; sense, start codon in bold, AttB1 site in italic: 5'-GGGGACAAGTTTGTACAAAAAAGCAGG CTTAAACAATGCAGCAGCAGCAGTCT 3') and prmO6686 (SEQ ID NO: 95); reverse, stop codon in bold, complementary, AttB2 site in italic: 5' GGGGACCACTTTGTACAAGAAAG CTGGGTTCTTTGGATCCTTTTCACTTG 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 666 bp (including attB sites) was amplified and purified as above. The entry clone was numbered pO7467. The Arabidopsis thaliana AtSYT3 gene was amplified by PCR using the same method as the Arabidopsis thaliana AtSYTI and AtSYT2 genes. Primers prmO6683 (SEQ ID NO: 96; sense, start codon in bold, AttB1 site in italic: 5' GGGGACAAGTTTGTACAAAAAAG CyAGGCTTAAACAATGCAGCAATCTCCACAGAT 3') and prmO6684 (SEQ ID NO: 97; reverse, stop codon in bold, complementary, AttB2 site in italic: 5' GGGGACCACTTTGTAC A4G>4A4GC7GGG7TCCTCTATTTCATTTTCCTTCAG 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 745 bp (including attB sites) was amplified and purified as above. The entry clone was numbered p07604.
Example 2: Vector Construction
The entry clones pO7466, pO7467 and p07604 were subsequently used in an LR reaction with p00640, 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 (SEQ ID NO: 89) for constitutive expression (PRO0129) was located upstream of this Gateway cassette.
After the LR recombination step, the resulting expression vectors, respectively pO523 for AtSYTI , pO524 for AtSYT2 and pO767 for AtSYT3 (Figures 5 to 7) were 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 of AtSYTI, AtSYT2 and AtSYT3 under the control of the rice GOS2 promoter
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. Six 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 F-test 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 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.
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. Thousand Kernel Weight (TKW) is extrapolated from the number of filled seeds counted and their total weight.
Individual seed parameters (including width, length, area, weight) were measured using a custom-made device consisting of two main components, a weighing and imaging device, coupled to software for image analysis.
3.1 Total seed yield and TKW measurement results for transgenic plants grown in the greenhouse
The total seed yield and TKW measurement results for AtSYTI , AtSYT2 and AtSYT3 transgenic plants for the T1 generation are shown in Tables 5 to 7, respectively. The number of lines with an increase in either parameter is indicated. The percentage difference between the transgenics and the corresponding nullizygotes is also shown, as well as the P values from the F test. Both the total seed yield and TKW are significantly increased in the T1 generation for AtSYTI, AtSYT2 and ATSYT3 transgenic plants (Tables 5 to 7, respectively).
Table 5: Results of total seed yield and TKW measurements in the T1 generation of AtSYTI transgenic plants.
Figure imgf000039_0001
Table 6: Results of total seed yield and TKW measurements in the T1 generation of AtSYT2 transgenic plants.
Figure imgf000039_0002
Table 7: Results of total seed yield and TKW measurements in the T1 generation of AtSYT3 transgenic plants.
Figure imgf000039_0003
3.2 Seed size measurements results of seeds from 12 generation AtSYTI transgenic plants
Individual seed parameters (width, length and area) were measured on the seeds from the T2 plants, using a custom-made device consisting of two main components, a weighing and an imaging device, coupled to software for image analysis. Measurements were performed on both husked and dehusked seeds.
The average individual seed area, length and width measurement results of the T3 seeds (harvested from the T2 plants) for the Oryza sativa AtSYTI transgenic plants are shown in Table 8. The percentage difference between the transgenics and the corresponding nullizygotes is shown, as well as the number of events with an increase in a given parameter and the p values from the F test. The average individual seed area, length and width of the T3 husked and dehusked seeds (harvested from the T2 transgenic Oryza sativa AtSYTI plants) were all significantly increased compared to their null counterparts (Table 8).
Table 8: Individual seed area, length and width measurements of the T3 husked and dehusked seeds (harvested from the T2 plants) of the Oryza sativa AtSYTI transgenic plants compared to their null counterparts.
Figure imgf000040_0001
3.3 Embryo and endosperm size measurement results of seeds from T2 generation AtSYTI transgenic plants
Embryo and endosperm size were also measured by longitudinally cutting in half dehusked seeds and staining the seed halves for 2 to 3 hours at 350C with colouring agent, 2,3,5- triphenyltetrazolium chloride. Following staining, the two halves were placed on agarose gel in a Petri dish ready for imaging. Three independent events were taken, and from each event 120 seeds homozygous for the transgene and 120 seeds without the transgene were analysed. Digital photographs of the seeds were taken and the images analysed with ImagePro software. The results for the three events are given below.
For all three events, embryos of seeds homozygous for the transgene were bigger than the embryos of seeds without the transgene. There was a significant increase in the average area of the embryo for the seeds of each of the three events, with p values from the t-test of 0.0325, <0.0001 and <0.0001. Similarly, there was a significant increase in the average perimeter of the embryo for the seeds of each of the three events, with p values from the t- test of 0.0176, <0.0001 and <0.0001. Furthermore, there was a significant increase in the average area and perimeter of the endosperm for the seeds of each of the three events, all giving p values of <0.0001. 3.4 TKW measurement results for AtSYTI transgenic plants grown in the field
The AtSYTI homozygous transgenic plants and their corresponding controls were transplanted into the field in September and harvested in December. Four repetitions were planted for each entry (four events) with 104 plants per repeat. The spacing between plants was of 20 by 20 cm. The field was flooded and irrigated. After seed harvest, the seeds were measured for TKW as described above. Results of these measurements are presented in Table 9.
Table 9: Results of TKW measurements in the T3 generation of AtSYTI transgenic plants grown in the field.
Figure imgf000041_0001
The TKW is increased in all the transgenic events evaluated in the field.

Claims

Claims
1. Method for increasing plant yield relative to corresponding wild type plants, comprising modulating expression in a plant of a nucleic acid encoding a synovial sarcoma translocation (SYT) polypeptide or homologue thereof, and optionally selecting for plants having increased yield, wherein said SYT polypeptide or homologue comprises from N-terminal to C-terminal: (i) an SNH domain having at least 40% sequence identity to the SNH domain of SEQ ID NO: 2; and (ii) a Met-rich domain; and (iii) a QG-rich domain.
2. Method according to claim 1, wherein said SNH domain comprises the residues shown in black in Figure 2.
3. Method according to claim 1, wherein said SNH domain is represented by SEQ ID NO: 1.
4. Method according to any one of claims 1 to 3, wherein said SYT polypeptide or homologue thereof further comprises one or more of the following: (i) SEQ ID NO: 90; (ii) SEQ ID NO: 91 ; (iii) a Met-rich domain at the N-terminus preceding the SNH domain.
5. Method according to any one of claims 1 to 4, wherein said modulated expression is effected by introducing a genetic modification, preferably in the locus of a gene encoding a SYT polypeptide or a homologue thereof.
6. Method according to claim 5, wherein said genetic modification is effected by one of: T-DNA activation, TILLING, site-directed mutagenesis or directed evolution.
7. Method for increasing yield, particularly seed yield, relative to that of corresponding wild type plants, comprising introducing and expressing in a plant, plant part or plant cell a SYT nucleic acid or a variant thereof.
8. Method according to claim 7, wherein said variant is a portion of a SYT nucleic acid or a sequence capable of hybridising to a SYT nucleic acid, which portion or hybridising sequence encodes a polypeptide comprising from N-terminal to C- terminal: (i) an SNH domain having at least 40% sequence identity to the SNH domain of SEQ ID NO: 2; and (ii) a Met-rich domain; and (iii) a QG-rich domain.
9. Method according to claim 7, wherein said SNH domain comprises the residues shown in black in Figure 2.
10. Method according to claim 7, wherein said SNH domain is represented by SEQ ID NO: 1.
11. Method according to any one of claims 7 to 10, wherein said SYT polypeptide or homologue thereof further comprises one or more of the following: (i) SEQ ID NO: 90; (ii) SEQ ID NO: 91 ; (iii) a Met-rich domain at the N-terminus preceding the SNH domain.
12. Method according to any one of claims 7 to 11, wherein said SYT nucleic acid or variant thereof is overexpressed in a plant.
13. Method according to any one of claims 7 to 12, wherein said SYT nucleic acid or variant thereof is of plant origin, preferably from a dicotyledonous plant, further preferably from the family Brassicaceae, more preferably the nucleic acid is from Arabidopsis thaliana.
14. Method according to any one of claims 7 to 13, wherein said variant encodes an orthologue or paralogue of the SYT protein of SEQ ID NO: 4, SEQ ID NO: 6 and SEQ ID NO: 8.
15. Method according to any one of claims 7 to 14, wherein said SYT nucleic acid or variant thereof is operably linked to a constitutive promoter.
16. Method according to claim 15, wherein said constitutive promoter is plant-derived, preferably from a monocotyledonous plant.
17. Method according to claim 15 or 16, wherein said constitutive promoter is a GOS2 promoter.
18. Method according to any one of claims 1 to 17, wherein said increased yield is increased seed yield.
19. Method according to any one of claims 1 to 18, wherein said increased yield is increased total seed yield and/or increased TKW.
20. Plant, plant part or plant cell obtainable by a method according to any one of claims 1 to 19.
21. Construct comprising:
(i) a SYT nucleic acid or variant thereof;
(ii) one or more control sequences capable of driving expression of the nucleic acid sequence of (a); and optionally
(iii) a transcription termination sequence.
22. Construct according to claim 21 , wherein said control sequence is a constitutive promoter derived from a monocot plant.
23. Construct according to claim 22, wherein said constitutive promoter is a GOS2 promoter.
24. Construct according to claim 23, wherein said GOS2 promoter is as represented by SEQ ID NO: 89.
25. Plant, plant part or plant cell transformed with a construct according to any one of claims 21 to 24.
26. Method for the production of a transgenic plant, preferably a monocotyledonous plant, having increased yield, particularly increased seed yield, which method comprises:
(i) introducing and expressing in a plant or plant cell a SYT nucleic acid or variant thereof; (ii) cultivating the plant cell under conditions promoting plant growth and development.
27. Method according to claim 26, comprising generating one or more subsequent generations of plants and parts thereof including seeds by crossing plants obtained by said cultivating step (ii).
28. Transgenic plant or part thereof having increased yield, particularly increased seed yield, resulting from a SYT nucleic acid or a variant thereof introduced into said plant or plant part, said increased yield being relative to corresponding wild type plants.
29. Transgenic plant according to claim 20, 25 or 28, 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.
30. Harvestable parts of a plant according to any one of claims 20, 25, 28 or 29.
31. Harvestable parts of a plant according to claim 30 wherein said harvestable parts are seeds.
32. Products derived, preferably directly derived, from a plant according to claim 29 and/or from harvestable parts of a plant according to claims 30 or 31.
33. Use of a SYT nucleic acid/gene or variant thereof, or use of a SYT polypeptide or homologue thereof, or use of a construct according to any one of claims 21 to 24, in improving yield, especially seed yield, relative to corresponding wild type plants.
34. Use according to claim 33, wherein said seed yield is increased total seed yield and increased TKW.
35. Use of a SYT nucleic acid/gene or variant thereof, or use of a SYT polypeptide or homologue thereof, as a molecular marker.
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009018018A2 (en) * 2007-07-31 2009-02-05 University Of Utah Research Foundation Animal model of synovial sarcoma
WO2009037338A1 (en) * 2007-09-21 2009-03-26 Basf Plant Science Gmbh Plants having increased yield-related traits and a method for making the same
WO2010023320A2 (en) * 2008-08-29 2010-03-04 Basf Plant Science Company Gmbh Plants having enhanced yield-related traits and a method for making the same
EP2199395A1 (en) * 2006-08-02 2010-06-23 CropDesign N.V. Plants transformed with SYT-polypeptide having increased yield under abiotic stress and a method for making the same
WO2012153267A1 (en) * 2011-05-09 2012-11-15 Basf Plant Science Company Gmbh Plants having enhanced yield-related traits and method for making the same
US8426683B2 (en) 2005-01-27 2013-04-23 Cropdesign N.V. Plants having increased yield and a method for making the same
CN104099368A (en) * 2006-08-02 2014-10-15 克罗普迪塞恩股份有限公司 Plants having improved characteristics and a method for making the same
WO2022152660A1 (en) * 2021-01-12 2022-07-21 Vib Vzw Means and methods for producing drought tolerant cereals

Families Citing this family (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
BRPI0818076A2 (en) * 2007-10-19 2014-10-07 Pioneer Hi Bred Int ISOLATED POLYPEPTIDE WITH TRANSCRIPTION FACTOR ACTIVITY, ISOLATED POLYNUCLEOTIDES, TRANSGENIC PLANT, TRANSFORMED SEED, PLANT, METHOD FOR MODULATING PLANT TRANSCRIPTION FACTOR ACTIVITY, NULLECULA EXCELLULA MOLLECULES,
KR101315068B1 (en) * 2011-02-07 2013-10-08 한국생명공학연구원 SbtA gene from Synechocystis sp. PCC6803 and uses thereof
KR101273279B1 (en) * 2011-08-25 2013-06-11 경북대학교 산학협력단 Use of mdhar gene from Oryza sativa as regulator of yield and environmental stresses
KR101493978B1 (en) * 2013-10-08 2015-02-17 대한민국 Indel marker for discrimination of soybean cultivar
EP3107373A4 (en) * 2014-02-21 2017-11-08 Syngenta Participations AG Genetic loci associated with increased fertility in maize
KR101635497B1 (en) * 2014-10-06 2016-07-01 김용재 Novel Citrullus vulgaris reduced seed number and breeding method thereof
AU2015344482B2 (en) * 2014-11-04 2021-04-01 Agresearch Limited Methods for monocot plant improvement
KR102665987B1 (en) * 2023-09-08 2024-05-24 주식회사눌라바이오 Method for increasing regeneration efficiency using hemp immature embryo

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2004350553A (en) * 2003-05-28 2004-12-16 Japan Science & Technology Agency An3 gene of arabidopsis thaliana

Family Cites Families (21)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU691550B2 (en) 1993-12-09 1998-05-21 Thomas Jefferson University Compounds and methods for site-directed mutations in eukaryotic cells
US6395547B1 (en) 1994-02-17 2002-05-28 Maxygen, Inc. Methods for generating polynucleotides having desired characteristics by iterative selection and recombination
US5605793A (en) 1994-02-17 1997-02-25 Affymax Technologies N.V. Methods for in vitro recombination
EP1036184B1 (en) * 1997-12-11 2007-03-21 Syngenta Limited Genetic method
US20050086718A1 (en) * 1999-03-23 2005-04-21 Mendel Biotechnology, Inc. Plant transcriptional regulators of abiotic stress
US7345217B2 (en) * 1998-09-22 2008-03-18 Mendel Biotechnology, Inc. Polynucleotides and polypeptides in plants
EP1033405A3 (en) 1999-02-25 2001-08-01 Ceres Incorporated Sequence-determined DNA fragments and corresponding polypeptides encoded thereby
CZ20013859A3 (en) 1999-04-29 2002-04-17 Syngenta Ltd. Plants resistant to herbicides
US20100293669A2 (en) * 1999-05-06 2010-11-18 Jingdong Liu Nucleic Acid Molecules and Other Molecules Associated with Plants and Uses Thereof for Plant Improvement
CA2366104C (en) 1999-07-22 2010-07-06 Japan As Represented By Director General Of National Institute Of Agrobiological Resources, Ministry Of Agriculture, Forestry And Fisheries Ultra-fast transformation technique for monocotyledons
KR100350215B1 (en) * 2000-12-02 2002-08-28 (주)제노마인 Novel transcriptional factor enhancing the resistance of plants to osmotic stress
JP2002356498A (en) 2001-04-24 2002-12-13 Hokkaido Technology Licence Office Co Ltd Antigen peptide for synovial sarcoma
CN1678748B (en) 2002-09-05 2011-11-16 作物培植股份有限公司 Plants having changed development and a method for making the same
BR0317754A (en) 2002-12-24 2005-11-22 Cropdesign Nv Plants having modified growth characteristics and method for producing them
WO2004106528A1 (en) 2003-06-03 2004-12-09 Cropdesign N.V. Transgenic monocotyledonous plants overexpressing a nhx protein and having improved growth charcteristics and a method for making the same
AU2005261646B2 (en) * 2004-07-12 2012-02-02 Cropdesign N.V. Plants having improved growth characteristics and method for making the same
BRPI0513393A (en) 2004-07-16 2008-05-06 Cropdesign Nv methods for improving the growth characteristics of a plant, and for producing a transgenic plant, plant or plant cell, building, transgenic plant or plant cell, harvestable parts and / or products derived directly from them, use of an acid molecule snrk2 nucleic acid or functional variant thereof or use of a snrk2 polypeptide or homologue thereof, and, composition
US8426683B2 (en) 2005-01-27 2013-04-23 Cropdesign N.V. Plants having increased yield and a method for making the same
EP2540832A1 (en) * 2006-08-02 2013-01-02 CropDesign N.V. Plants transformed with a small inducible kinase having improved yield related traits and a method for making the same
NL1033850C2 (en) 2007-05-15 2008-11-18 3Force B V Burner system with premixed burners and flame transfer agents.
MX2010002463A (en) * 2007-09-21 2010-03-29 Basf Plant Science Gmbh Plants having increased yield-related traits and a method for making the same.

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2004350553A (en) * 2003-05-28 2004-12-16 Japan Science & Technology Agency An3 gene of arabidopsis thaliana

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
JEONG HOE KIM ET AL: "THE ATGRF FAMILY OF PUTATIVE TRANSCRIPTION FACTORS US INVOLVED IN LEAF AND COTYLEDON GROWTH IN ARABIDOPSIS" PLANT JOURNAL, BLACKWELL SCIENTIFIC PUBLICATIONS, OXFORD, GB, vol. 36, no. 1, October 2003 (2003-10), pages 94-104, XP008058318 ISSN: 0960-7412 *
KIM JEONG HOE ET AL: "A transcriptional coactivator, AtGIF1, is involved in regulating leaf growth and morphology in Arabidopsis" PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA, NATIONAL ACADEMY OF SCIENCE, WASHINGTON, DC, US, vol. 101, no. 36, 7 September 2004 (2004-09-07), pages 13374-13379, XP002362467 ISSN: 0027-8424 cited in the application *
KIM J-H ET AL: "STUDIES ON THE FUNCTION OF OSGRF1-LIKE (ATGRL) GENES IN ARABIDOPSIS" PLANT BIOLOGY, NEW YORK, NY, US, August 2002 (2002-08), page 56, XP008058260 *
PATENT ABSTRACTS OF JAPAN vol. 2003, no. 12, 5 December 2003 (2003-12-05) & JP 2004 350553 A (JAPAN SCIENCE & TECHNOLOGY AGENCY), 16 December 2004 (2004-12-16) *

Cited By (22)

* Cited by examiner, † Cited by third party
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US20100199382A1 (en) * 2007-09-21 2010-08-05 Basf Plant Science Gmbh Plants Having Increased Yield-Related Traits And A Method For Making The Same
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