# Patent

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

## Abstract

The present invention relates generally to the field of molecular biology and concerns a method for increasing plant yield relative to suitable control plants. More specifically, the present invention concerns a method for increasing plant yield comprising increasing expression in a plant of a nucleic acid encoding a Dof (DNA-binding with one finger) domain transcription factor polypeptide. The present invention also concerns plants having increased expression of a nucleic acid encoding a Dof domain transcription factor polypeptide, which plants have increased yield relative to suitable control plants. The invention also provides constructs useful in the methods of the invention.

## Background
RELATED APPLICATIONS
This application is a national stage application (under 35 U.S.C. 371) of PCT/US2006/045721 filed Nov. 29, 2006, which claims benefit of European application EP 05111597.0 filed Dec. 1, 2005, European application EP 05111691.1 filed Dec. 5, 2005, U.S. Provisional application U.S. 60/742,352 filed Dec. 5, 2005, European application EP 05111786.9 filed Dec. 7, 2005, U.S. Provisional application U.S. 60/748,903 filed Dec. 8, 2005, U.S. Provisional application U.S. 60/749,219 filed Dec. 9, 2005, European application EP 05111996.4 filed Dec. 12, 2005, U.S. Provisional application U.S. 60/750,143 filed Dec. 14, 2005, European application EP 05112562.3 filed Dec. 21, 2005, U.S. Provisional application U.S. 60/753,650 filed Dec. 23, 2005, European application EP 05113110.0 filed Dec. 30, 2005, European application EP 05113111.8 filed Dec. 30, 2005, U.S. Provisional application U.S. 60/756,086 filed Jan. 4, 2006, and U.S. Provisional application U.S. 60/756,042 filed Jan. 4, 2006.
SEQUENCE LISTING SUBMISSION
The Sequence Listing associated with this application is filed in electronic format via EFS-Web and hereby incorporated by reference into the specification in its entirety. The name of the text file containing the Sequence Listing is Revised_Sequence_Listing—14546—00031_US. The size of the text file is 614 KB, and the text file was created on Jun. 3, 2011.


The present invention relates generally to the field of molecular biology and concerns a method for improving various plant growth characteristics by modulating expression in a plant of a nucleic acid encoding a GRP (Growth-Related Protein). The present invention also concerns plants having modulated expression of a nucleic acid encoding a GRP, which plants have improved growth characteristics relative to corresponding wild type plants or other control plants. The invention also provides constructs useful in the methods of the invention.
Given the ever-increasing world population, and the dwindling area of land available for agriculture, it remains a major goal of research to improve the efficiency of agriculture and to increase the diversity of plants in horticulture. Conventional means for crop and horticultural improvements utilise selective breeding techniques to identify plants having desirable characteristics. However, such selective breeding techniques have several drawbacks, namely that these techniques are typically labour intensive and result in plants that often contain heterogeneous genetic complements that may not always result in the desirable trait being passed on from parent plants. Advances in molecular biology have allowed mankind to manipulate the germplasm of animals and plants. Genetic engineering of plants entails the isolation and manipulation of genetic material (typically in the form of DNA or RNA) and the subsequent introduction of that genetic material into a plant. Such technology has led to the development of plants having various improved economic, agronomic or horticultural traits. Traits of particular economic interest are growth characteristics such as high yield. Yield is normally defined as the measurable produce of economic value from a crop. This may be defined in terms of quantity and/or quality. Yield is directly dependent on several factors, for example, the number and size of the organs, plant architecture (for example, the number of branches), seed production and more. Root development, nutrient uptake and stress tolerance may also be important factors in determining yield.
Seed yield is a particularly important trait, since the seeds of many plants are important for human and animal nutrition. Crops such as, corm, rice, wheat, canola and soybean account for over half the 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) and an endosperm (the source of nutrients for embryo growth during germination and during early growth of seedlings). The development of a seed involves many genes, and requires the transfer of metabolites from the roots, leaves and stems into the growing seed. The endosperm, in particular, assimilates the metabolic precursors of carbohydrates, oils and proteins and synthesizes them into storage macromolecules to fill out the grain.
Another important trait for many crops is early vigour. Improving early vigour is an important objective of modern rice breeding programs in both temperate and tropical rice cultivars. Long roots are important for proper soil anchorage in water-seeded rice. Where rice is sown directly into flooded fields, and where plants must emerge rapidly through water, longer shoots are associated with vigour. Where drill-seeding is practiced, longer mesocotyls and coleoptiles are important for good seedling emergence. Early vigour may also result from increased plant fitness due to, for example, the plants being better adapted to their environment (i.e. being more able to cope with various abiotic or biotic stress factors). Plants having early vigour also show better establishment of the crop (with the crop growing in a more uniform manner, i.e. with the majority of plants reaching the various stages of development at substantially the same time), and show better growth and often better yield.
A further important trait is that of improved abiotic stress tolerance. Abiotic stress is a primary cause of crop loss worldwide, reducing average yields for most major crop plants by more than 50% (Wang et al., Planta (2003) 218: 1-14). Abiotic stresses may be caused by drought, salinity, extremes of temperature, chemical toxicity and oxidative stress. The ability to improve plant tolerance to abiotic stress would be of great economic advantage to farmers worldwide and would allow for the cultivation of crops during adverse conditions and in territories where cultivation of crops may not otherwise be possible.
Crop yield may therefore be increased by optimising one of the above-mentioned factors.
Depending on the end use, the modification of certain yield traits may be favoured over others. For example for applications such as forage or wood production, or bio-fuel resource, an increase in the leafy parts of a plant may be desirable, and for applications such as flour, starch or oil production, an increase in seed parameters may be particularly desirable. Even amongst the seed parameters, some may be favoured over others, depending on the application. Various mechanisms may contribute to increasing seed yield, whether that is in the form of increased seed size or increased seed number.
One approach to increasing (seed) yield in plants may be through modification of the inherent growth mechanisms of a plant. One such mechanism is the cell cycle.
It has now been found that various growth characteristics may be improved in plants by modulating expression in a plant of a nucleic acid encoding a GRP (Growth-Related Protein) in a plant. The GRP may be one of the following: Seed Yield Regulator (SYR), FG-GAP, CYP90B, CDC27, AT-hook transcription factors, DOF transcription factors and Cyclin Dependent Kinase Inhibitors (CKIs).
BACKGROUND
Seed Yield Regulator (SYR)
There is a continuous need to find new seed yield enhancement genes and several approaches have been used so far, for example through manipulation of plant hormone levels (WO 03/050287), through manipulation of the cell cycle (WO 2005/061702), through manipulation of genes involved in salt stress response (WO 2004/058980) amongst other strategies.
SYR is a new protein that has hitherto not been characterised. SYR shows some homology (around 48% sequence identity on the DNA level, around 45% on the protein level) to an Arabidopsis protein named ARGOS (Hu et al., Plant Cell 15, 1951-1961, 2003; US 2005/0108793). Hu et al. postulated that ARGOS is a protein of unique function and is encoded by a single gene. The major phenotypes of ARGOS overexpression in Arabidopsis are increased leafy biomass and delayed flowering.
FG-GAP
FG-GAP proteins are putative transmembrane proteins. They are characterised by the presence of one or more FG-GAP domains (Pfam accession number PF01839) and by the presence of an N-terminal signal peptide and a transmembrane domain in the C-terminal half of the protein.
One such protein, DEX1, was isolated from Arabidopsis and was reported to play a role during pollen development (Paxson-Sowders et al. Plant Physiol. 127, 1739-1749, 2001). Dex1 mutant plants were shown to be defective in pollen wall pattern formation. The DEX1 gene encodes an 896-amino acid protein that is predicted to localize to the plasma membrane, with residues 1 through to 860 being located outside of the cell, residues 880 through to 895 on the cytoplasmic side of the membrane, and amino acids 861 through to 879 representing a potential membrane-spanning domain. Twelve potential N-glycosylation sites are present in DEX1. Therefore, the protein has the potential to be heavily modified and interact with various components of the cell wall. DEX1 shows the greatest sequence similarity to a hemolysin-like protein from V. cholerae, whereas an approximately 200-amino acid segment of DEX1 (amino acids 439643) also shows limited similarity to the calcium-binding domain of alpha-integrins. In this region are at least two sets of putative calcium-binding ligands that are also present in a predicted Arabidopsis calmodulin protein (AC009853). Therefore, it appears that DEX1 may be a calcium-binding protein. DEX1 appears to be a unique plant protein; homologs are not present in bacteria, fungi, or animals.
The alterations observed in dex1 plants, as well as the predicted structure of DEX1, raise several possibilities for the role of the protein in pollen wall formation (Paxson-Sowders et al., 2001):

    
    
        (a) DEX1 could be a linker protein. It may associate with the microspore membrane and participate in attaching either the primexine or sporopollenin to the plasma membrane. Absence of the protein from the microspore surface could result in structural alterations in the primexine. The numerous potential N-glycosylation sites are consistent with attachment of DEX1 to the callose wall, the intine, or both.
        (b) DEX1 may be a component of the primexine matrix and play a role in the initial polymerization of the primexine. Changes in Ca+2 ion concentrations appear to be important for pollen wall synthesis; beta-glucan synthase is activated by micromolar concentrations of Ca+2 during callose wall formation.
    
    


(c) DEX1 could be part of the rough ER and be involved in processing and/or transport of primexine precursors to the membrane. The delayed appearance and general alterations in the primexine are consistent with a general absence of primexine precursors. The primexine matrix is initially composed of polysaccharides, proteins, and cellulose, followed by the incorporation of more resistant materials. Therefore, DEX1 may participate in the formation or transport of any number of different components.
CYP90B
Brassinosteroids (BRs) are a class of plant hormones that are important for promoting plant growth, division and development. The term BR collectively refers to more than forty naturally occurring poly-hydroxylated sterol derivatives, with structural similarity to animal steroid hormones. Among these, brassinolide has been shown to be the most biologically active (for review, Clouse (2002) Brassinosteroids. The Arabidopsis Book: 1-23).
The BR biosynthetic pathway has been elucidated using biochemical and mutational analyses. BRs are synthesized via at least two branched biochemical pathways starting from the same initial precursor, campesterol (Fujioka et al. (1997) Physiol Plant 100:710-715). The discovered BR biosynthesis genes have been found to encode mostly cytochrome P450 monooxygenases (CYP) (Bishop and Yokota (2001) Plant Cell Physiol 42:114-120). CYP superfamily of enzymes catalyses the oxidation of many chemicals, and in the present case more specifically catalyse essential oxidative reactions in the biosynthesis of BRs. One of the important steps identified consists in the hydroxylation of the steroid side chain of BR intermediates campestanol and 6-oxocampestanol to form 6-deoxocathasterone and cathasterone respectively. These two parallel oxidative steps are also collectively called the early steroid C-22 alpha-hydroxylation step (Choe et al. (1998) Plant Cell 10: 231-243). In Arabidopsis, a specific CYP enzyme, CYP90B1 or DWF4, performs this step (for general reference on plant CYP nomenclature, Nelson et al. (2004) Plant Phys 135: 756-772).
Arabidopsis mutant plants lacking steroid 22 alpha hydroxylase activity due insertion of a T-DNA in the DWF4 locus displayed a dwarfed phenotype due to lack of cell elongation (Choe et al. (1998) Plant Cell 10: 231-243). Biochemical feeding studies with BR biosynthesis intermediates showed that all of the downstream compounds rescued the phenotype, whereas the known precursors failed to do so.
Transgenic Arabidopsis and tobacco plants, both dicotyledonous, were generated that ectopically overexpressed an Arabidopsis DWF4 genomic fragment, using the cauliflower mosaic virus 35S promoter (Choe et al. (2001) Plant J 26(6): 573-582). Phenotypic characterisation of the plants showed that the hypocotyl length, plant height at maturity, total number of branches and total number of seeds were increased in the transgenics compared to control plants. Choe et al. found that the increased seed production was due to a greater number of seeds per plant, seed size increase being within the range of standard deviation. These experiments are further described in WO00/47715.
U.S. Pat. No. 6,545,200 relates to isolated nucleic acid fragments encoding sterol biosynthetic genes, and more specifically claims a nucleotide sequence encoding a polypeptide having C-8,7 sterol isomerase activity. Partial nucleotides sequences encoding DWF4 are disclosed.
US 2004/0060079 relates to a method of producing a modified monocotyledonous plant having a desired trait. An example is provided in which the rice DWF4-encoding nucleotide sequence (referred to either OsDWF4 or CYP90B2) is placed under the control of a constitutive promoter, the rice actin promoter. Fourteen of the thirty-six transgenic rice plants expressing the chimeric construct show an increased number of grains per spike as compared to non-transformed control plants. According to the inventors, the yield increase in the transgenics compared to the wild types is due to an increase in total number of seeds, as no significant difference is found in the “weight of 10 grains”. CDC27
Depending on the end use, the modification of certain yield traits may be favoured over others. For example for applications such as forage or wood production, or bio-fuel resource, an increase in the leafy parts of a plant may be desirable, and for applications such as flour, starch or oil production, an increase in seed parameters may be particularly desirable. Even within the seed parameters, some may be favoured over others, depending on the application. Various mechanisms may contribute to increasing seed yield, whether that is in the form of increased seed size or increased seed number. One such mechanism is the cell cycle.
Progression through the cell cycle is fundamental to the growth and development of all multicellular organisms and is crucial to cell proliferation. The major components of the cell cycle are highly conserved in yeast, mammals, and plants. The cell cycle is typically divided into the following sequential phases: G0-G1-S-G2-M. DNA replication or synthesis generally takes place during the 8 phase (“S” is for DNA synthesis) and mitotic segregation of the chromosomes occurs during the M phase (the “M” is for mitosis), with intervening gap phases, G1 (during which cells grow before DNA replication) and G2 (a period after DNA replication during which the cell prepares for division). Cell division is completed after cytokinesis, the last step of the M phase. Cells that have exited the cell cycle and that have become quiescent are said to be in the G0 phase. Cells in this phase can be stimulated to renter the cell cycle at the G1 phase. The “G” in G1, G2 and G0 stands for “gap” Completion of the cell cycle process allows each daughter cell during cell division to receive a full copy of the parental genome.
Cell division is controlled by two principal cell cycle events, namely initiation of DNA synthesis and initiation of mitosis. Each transition to each of these key events is controlled by a checkpoint represented by specific protein complexes (involved in DNA replication and division). The expression of genes necessary for DNA synthesis at the G1/S boundary is regulated by the E2F family of transcription factors in mammals and plant cells (La Thangue, 1994; Muller et al., 2001; De Veylder et al., 2002). Entry into the cell cycle is regulated/triggered by an E2F/Rb complex that integrates signals and allows activation of transcription of cell cycle genes. The transition between the different phases of the cell cycle, and therefore progression through the cell cycle, is driven by the formation and activation of different heterodimeric serine/threonine protein kinases, generally referred to as cyclin-dependent kinases (CDKs). A prerequisite for activity of these kinases is the physical association with a specific cyclin, the timing of activation being largely dependent upon cyclin expression. Cyclin-binding induces conformational changes in the N-terminal lobe of the associating CDK and contributes to the localisation and substrate specificity of the complex. Monomeric CDKs are activated when they are associated with cyclins and thus have a kinase activity. Cyclin protein levels fluctuate in the cell cycle and therefore represent a major factor in determining timing of CDK activation. The periodic activation of these complexes containing cyclins and CDK during cell cycle mediates the temporal regulation of cell cycle transitions (checkpoints).
Mechanisms exist to ensure that DNA replication occurs only once during the cell cycle. For example, CDC16, CDC23 and CDC27 proteins are part of a high molecular weight complex known as the anaphase promoting complex (APC) or cyclosome, (see Romanowski and Madine, Trends in Cell Biology 6, 184-188, 1996, and Wuarin and Nurse, Cell 85, 785787 (1996). The complex in yeast is composed of at least eight proteins, the TPR-(tetratrico peptide repeat) containing proteins CDC16, CDC23 and CDC27, and five other subunits named APC1, APC2, APC4, APC5 and APC7 (Peters et al. 1996, Science 274, 1199-1201). The APC targets its substrates for proteolytic degradation by catalyzing the ligation of ubiquitin molecules to these substrates. APC-dependent proteolysis is required for the separation of the sister chromatids at meta- to anaphase transition and for the final exit from mitosis. Among the APC-substrates are the anaphase inhibitor protein Pds1p and mitotic cyclins such as cyclin B, respectively (Ciosk et al. 1998, Cell 93, 1067-1076; Cohen-Fix et al. 1996, Genes Dev 10, 3081-3093; Sudakin et al. 1995, Mol Biol Cell 6, 185-198; Jorgensen et al. 1998, Mol Cell Biol 18, 468-476; Townsley and Ruderman 1998, Trends Cell Biol 8, 238-244). To become active as an ubiquitin-ligase, at least CDC16, CDC23 and CDC27 need to be phosphorylated in the M-phase (Ollendorf and Donoghue 1997, J Biol Chem 272, 32011-32018). Activated APC persists throughout G1 of the subsequent cell cycle to prevent premature appearance of B-type cyclins, which would result in an uncontrolled entry into the S-phase (Imiger and Nasmyth 1997, J Cell Sci 110, 1523-1531). It has been demonstrated in yeast that mutations in either of at least two of the APC components, CDC16 and CDC27, can result in DNA overreplication without intervening passages through M-phases (Heichman and Roberts 1996, Cell 85, 39-48). This process of replication of nuclear DNA without subsequent mitosis and cell division is called DNA endoreduplication, and leads to increased cell size.
CDC16, CDC23 and CDC27 all are tetratrico peptide repeat (TPR; 34 amino acids long) containing proteins. A suggested minimal consensus sequence of the TPR motif is as follows: X3-W-X2-L-G-X2-Y-X8-A-X3-F-X2-A-X4-P-X2, (SEQ ID NO: 286) where X is any amino acid (Lamb et al. 1994, EMBO J. 13, 4321-4328). The consensus residues can exhibit significant degeneracy and little or no homology is present in non-consensus residues. It is the hydrophobicity and size of the consensus residues, rather than their identity, that seems to be of importance. TPR motifs are present in a wide variety of proteins functional in yeast and higher eukaryotes in mitosis (including the APC protein components CDC16, CDC23 and CDC27), transcription, splicing, protein import and neurogenesis (Goebl and Yanagida 1991, Trends Biochem Sci 16, 173-177). The TPR forms an α-helical structure; tandem repeats organize into a superhelical structure ideally suited as interfaces for protein recognition (Groves and Barford 1999, Curr Opin Struct Biol 9, 383-389). Within the α-helix, two amphipathic domains are usually present, one at the NH2 terminal region and the other near the COOH terminal region (Sikorski et al. 1990, Cell 60, 307-317).
CDC27 (also known as Hobbit; others names include CDC27, BimA, Nuc2 or makos) has been isolated from various organisms, including Aspergillus nidulans, yeast, drosophila, human and various plants (such as Arabidopsis thaliana and Otyza sativa). The gene encoding CDC27 is present as a single copy in most genomes, but two copies may exceptionally be found within the same genome, for example in Arabidopsis thaliana. The two genes encoding CDC27 proteins have been named CDC27A and CDC27B (MIPS references At3g16320 and At2g20000 respectively).
Published International Patent Application, WO01/02430 describes CDC27A (CDC27A1 and CDC27A2) and CDC27B sequences. Also described in this document is a truncated CDC27B amino add sequence in which 161 amino acids are missing from the NH2 terminal region. Reference is made in this document to GenBank accession number AC006081 for the CDC27B gene encoding a CDC27B polypeptide truncated at the NH2 terminal region. The document reports the NH2 terminal region to be conserved in CDC27 homologues of different origin. The CDC27 sequences mentioned in WO01/02430 are described to be useful in modifying endoreduplication.
DNA endoreduplication occurs naturally in flowering plants, for example during seed development. DNA endoreduplication leads to enlarged nuclei with elevated DNA content It has been suggested that the increased DNA content during endoreduplication may provide for increased gene expression during endosperm development and kernel filling, since it coincides with increased enzyme activity and protein accumulation at this time (Kowles et al., (1992) Genet. Eng. 14:65-88). In cereal species, the cellular endosperm stores the reserves of the seed during a phase marked by endoreduplication. The magnitude of DNA endoreduplication is highly correlated with endosperm fresh weight, which implies an important role of DNA endoreduplication in the determination of endosperm mass (Engelen-Eigles et al. (2000) Plant Cell Environ. 23:657-663). In maize for example, the endosperm makes up 70 to 90% of kernel mass; thus, factors that mediate endosperm development to a great extent also determine grain yield of maize, via individual seed weight. Increased endoreduplication is therefore typically indicative of increased seed biomass but is in no way related to increased seed number.
AT-Hook Transcription Factor
An AT-hook domain is found in polypeptides belonging to a family of transcription factors associated with Chromatin remodeling. The AT-hook motif is made up of 13 or so (sometimes about 9) amino acids which participate in DNA binding and which have a preference for A/T rich regions. In Arabidopsis there are at least 34 proteins containing AT-hook domains. These proteins share homology along most of the sequence, with the AT-hook domain being a particularly highly conserved region.
International Patent application WO 2005/030966 describes several plant transcription factors comprising AT-hook domains and the use of these transcription factors to produce plants having increased biomass and increased stress tolerance. The application concerns members of the G1073 lade of transcription factors and states that, “Use of tissue-specific or inducible promoters mitigates undesirable morphological effects that may be associated with constitutive overexpression of G1073 clade members (e.g., when increased size is undesirable).” The data provided in this application relate to dicotyledonous plants.
In contrast to these teachings, it has now been found that expression in a monocotyledonous (monocot) plant of a polynucleic acid encoding an AT-hook transcription factor comprising a DUF296 domain (which includes members of lade G1073), gives plants having little or no increase in biomass compared with suitable control plants, regardless of whether that expression is driven by a constitutive promoter or in a tissue-specific manner. This suggests that teachings concerning expression of such transcription factors in dicots may not be so readily applicable to monocots. It has also now been found that the extent or nature of any increase in seed yield obtained is dependent upon the tissue-specific promoter used.
DOF Transcription Factors
Dof domain proteins are plant-specific transcription factors with a highly conserved DNA-binding domain with a single C2-C2 zinc finger. During the past decade, numerous Dof domain proteins have been identified in both monocots and dicots including maize, barley, wheat, rice, tobacco, Arabidopsis, pumpkin, potato and pea. Dof domain proteins have been shown to function as transcriptional activators or repressors in diverse plant-specific biological processes.
Cyclin Dependent Kinase Inhibitors (CKI)
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. One approach to increasing seed yield in plants may be through modification of the inherent growth mechanisms of a plant.
The inherent growth mechanisms of a plant reside in a highly ordered sequence of events collectively known as the ‘cell cycle’. Progression through the cell cycle is fundamental to the growth and development of all multi-cellular organisms and is crucial to cell proliferation. The major components of the cell cycle are highly conserved in yeast, mammals, and plants. The cell cycle is typically divided into the following sequential phases: G0-G1-S-G2-M. DNA replication or synthesis generally takes place during the S phase (“S” is for DNA synthesis) and mitotic segregation of the chromosomes occurs during the M phase (the “M” is for mitosis), with intervening gap phases, G1 (during which cells grow before DNA replication) and G2 (a period after DNA replication during which the cell prepares for division). Cell division is completed after cytokinesis, the last step of the M phase. Cells that have exited the cell cycle and that have become quiescent are said to be in the G0 phase. Cells in this phase can be stimulated to renter the cell cycle at the G1 phase. The “G” in G1, G2 and G0 stands for “gap”. Completion of the cell cycle process allows each daughter cell during cell division to receive a full copy of the parental genome.
Cell division is controlled by two principal cell cycle events, namely initiation of DNA synthesis and initiation of mitosis. Each transition to each of these key events is controlled by a checkpoint represented by specific protein complexes (involved in DNA replication and division). The expression of genes necessary for DNA synthesis at the G1/S boundary is regulated by the E2F family of transcription factors in mammals and plant cells (La Thangue, 1994; Muller et al., 2001; De Veylder et al., 2002). Entry into the cell cycle is regulated/triggered by an E2F/Rb complex that integrates signals and allows activation of transcription of cell cycle genes. The transition between the different phases of the cell cycle, and therefore progression through the cell cycle, is driven by the formation and activation of different heterodimeric serine/threonine protein kinases, generally referred to as cyclin-dependent kinases (CDKs). A prerequisite for activity of these kinases is the physical association with a specific cyclin, the timing of activation being largely dependent upon cyclin expression. Cyclin binding induces conformational changes in the N-terminal lobe of the associating CDK and contributes to the localisation and substrate specificity of the complex. Monomeric CDKs are activated when they are associated with cyclins and thus have kinase activity. Cyclin protein levels usually fluctuate in the cell cycle and therefore represent a major factor in determining timing of CDK activation. The periodic activation of these complexes containing cyclins and CDK during cell cycle mediates the temporal regulation of cell-cycle transitions (checkpoints). Other factors regulating CDK activity include cyclin dependent kinase inhibitors (CKIs or ICKs, KIPs, CIPs, INKs), CDK activating kinases (CAKs), a CDK phosphatase (Cdc25) and a CDK subunit (CKS) (Mironov et al. 1999; Reed 1996).
The existence of an inhibitor of mitotic CDKs was inferred from experiments with endosperm of maize seed (Grafi and Larkins (1995) Science 269, 1262-1264). Since then, several CKIs have been identified in various plant species, such as Arabidopsis (Wang et al. (1997) Nature 386(6624): 451-2; De Veylder et al. (2001) Plant Cell 13: 1653-1668; Lui et al. (2000) Plant J 21: 379-385), tobacco (Jasinski et al. (2002) Plant Physiol 2002 130(4): 871-82), Chenopodium rubrum (Fountain et al. (1999) Plant Phys 120: 339) or corn (Coelho et al. (2005) Plant Physiol 138: 2323-2336). The encoded proteins are characterized by a stretch of approximately 45 carboxy-terminal amino acids showing homology to the amino-terminal cyclin/Cdk binding domain of animal CKIs of the p21Clp1/p27Klp2/p57Klp2-types. Outside this carboxy-terminal region, plant CKIs show little homology.
Published International patent application WO 2005/007829 in the name of Monsanto Technology LLC describes various isolated nucleic acid molecules encoding polypeptides having cyclin dependent kinase inhibitor activity.
Published International patent applications, WO 02/28893 and WO 99/14331, both in the name of CropDesign N.V., describe various plant cyclin dependent kinase inhibitors. The use of these inhibitors to increase yield is mentioned in these applications.
SUMMARY OF THE INVENTION
It has now surprisingly been found that increasing activity of a SYR protein and/or expression of a nucleic acid encoding a SYR protein in plants results in plants having increased seed yield and or increased growth rate, relative to corresponding wild type plants. It has also now surprisingly been found that overexpression of SYR in rice primarily increases seed yield, whereas the leafy biomass and flowering time are not obviously affected (in contrast to the major phenotypes of ARGOS overexpression in Arabidopsis, which were shown to be increased leafy biomass and delayed flowering (Hu et al., Plant Cell 15, 1951-1961, 2003; US 2005/0108793)).
According to one embodiment of the present invention there is provided a method for increasing seed yield and/or growth rate of a plant comprising increasing activity of a SYR polypeptide or a homologue thereof in a plant and/or expression of a nucleic acid encoding such a protein; and optionally selecting for plants having improved growth characteristics.
Advantageously, performance of the methods of the invention insofar as they concern SYR, result in plants having a variety of improved growth characteristics, such as improved seed yield without effect on the biomass of vegetative plant parts, when compared to corresponding control plants, and a life cycle comparable to corresponding control plants, without delay in flowering time. Further advantageously, performance of the methods according to the present invention result in plants having improved tolerance to abiotic stress relative to corresponding wild type (or other control) plants.
It has now surprisingly been found that modulating activity of an FG-GAP protein and/or expression of a nucleic acid encoding an FG-GAP protein in plants results in plants having improved growth characteristics, and in particular increased yield, relative to corresponding wild type plants.
According to another embodiment of the present invention there is provided a method for improving growth characteristics of a plant comprising modulating activity of an FG-GAP polypeptide or a homologue thereof and/or modulating expression of a nucleic acid encoding an FG-GAP polypeptide or a homologue thereof in a plant and optionally selecting for plants having improved growth characteristics.
Advantageously, performance of the methods according to the present invention, insofar as they concern an FG-GAP polypeptide or a homologue thereof, result in plants having a variety of improved growth characteristics, such as improved growth, improved yield, improved biomass, improved architecture or improved cell division, each relative to corresponding wild type plants. Preferably, the improved growth characteristics comprise at least increased yield relative to corresponding wild type plants.
It has now surprisingly been found that increasing non-constitutive expression in a plant of a nucleic acid encoding a CYP90B polypeptide or a homologue thereof gives plants having increased yield relative to suitable control plants.
According to a further embodiment of the present invention, there is provided a method for increasing plant yield comprising increasing non-constitutive expression in a plant of a nucleic acid encoding a CYP90B polypeptide or a homologue thereof.
It has now been found that preferentially increasing expression in the shoot apical meristem tissue of plants of a nucleic acid encoding a CDC27 polypeptide having at least one inactive TPR domain in the NH2 terminal region of the polypeptide gives plants having increased seed number relative to suitable control plants.
The invention therefore provides a method for increasing the seed number of plants relative to that of suitable control plants, comprising preferentially increasing expression in plant shoot apical meristem tissue of a nucleic acid encoding a CDC27 polypeptide having at least one inactive TPR domain in the NH2 terminal region of the polypeptide.
It has now been found that preferentially increasing expression of a nucleic acid encoding a polypeptide comprising an AT-hook domain and a DUF296 domain in endosperm tissue of a monocotyledonous plant gives plants having increased seed yield relative to suitable control plants.
A further embodiment of the present invention therefore provides a method for increasing seed yield in monocotyledonous plants relative to suitable control plants, comprising preferentially increasing expression in endosperm tissue of a monocotyledonous plant of a nucleic acid encoding a polypeptide comprising an AT-hook domain and a DUF296 domain.
It has now been found that increasing expression in a plant of a nucleic acid encoding a DOF transcription factor polypeptide gives plants having increased yield relative to suitable control plants.
According to a further embodiment of the present invention, there is provided a method for increasing plant yield comprising increasing expression in a plant of a nucleic acid encoding a DOF transcription factor polypeptide.
It has now been found that preferential reduction in expression of an endogenous CKI gene in endosperm tissue of a plant gives plants with better seed yield than seed yield in plants where there is no preferential reduction in expression of an endogenous CKI gene in plant endosperm tissue. The present invention therefore provides a method for increasing seed yield in plants relative to suitable control plants, comprising preferentially reducing expression of an endogenous CKI gene in endosperm tissue of a plant.
DETAILED DESCRIPTION OF THE INVENTION
The term “increased yield” as defined herein is taken to mean an increase in biomass (weight) of one or more parts of a plant (particularly harvestable parts) relative to corresponding wild type or other control plants, which increase in biomass may be aboveground or underground. An increase in biomass underground may be due to an increase in the biomass of plant parts, such as tubers, rhizomes, bulbs etc. Particularly preferred is an increase in any one or more of the following: increased root biomass, increased root volume, increased root number, increased root diameter and increased root length. The term increased yield also encompasses an increase in seed yield.
The term “increased seed 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 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; (ii) increased number of flowers (“florets”) per panicle (iii) increased number of filled seeds; (iv) increased seed size; (v) increased seed volume; (vi) increased individual seed area; (vii) increased individual seed length and/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; (ix) increased fill rate, (which is the number of filled seeds divided by the total number of seeds and multiplied by 100); and (x) increased thousand kernel weight (TKW), which is extrapolated from the number of filled seeds counted and their total weight. An increased TKW may result from an increased seed size and/or seed weight. An increased TKW may result from an increase in embryo size and/or endosperm size.
Taking corn as an example, a yield increase may be manifested as one or more of the following: an increase in the number of ears per plant, an increase in the number of rows, number of kernels per row, kernel weight, TKW, ear length/diameter, among others. Taking rice as an example, a yield increase may be manifested by an increase in one or more of the following: number of panicles per plant, number of spikelets per panicle, number of flowers per panicle, increase in the seed filling rate, increase in TKW, among others. An increase in yield may also result in modified architecture, or may occur as a result of modified architecture.
The improved growth characteristics obtained by performing the methods of the invention, insofar as they concern use of CDC27, result in plants having increased seed number. An increased seed number encompasses an increase in the total number of seeds and/or the number of filled seeds and/or an increase in the seed filling rate (which is the number of filled seeds divided by the total number of seeds and multiplied by 100), each relative to suitable control plants, which increase may be per plant and/or per hectare or acre. Taking corn as an example, an increase in the number of seeds is typically manifested by an increase in the number of ears per plant, an increase in the number of rows, number of kernels per row, increase in the seed filling rate, among others. Taking rice as an example, an increase in the number of seeds is typically manifested by an increase in 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.
The invention therefore provides a method for increasing the seed number of plants relative to that of suitable control plants, comprising preferentially increasing expression in plant shoot apical meristem tissue of a nucleic acid encoding a CDC27 polypeptide having at least one inactive TPR domain in the NH2 terminal region of the polypeptide.
Insofar as the methods of the invention concern SYR, preferably performance of the methods result in plants having increased seed yield. Further preferably, the increased seed yield comprises an increase in one or more of number of (filled) seeds, total seed weight, seed size, thousand kernel weight, fill rate and harvest index, each relative to control plants. Therefore, according to the present invention, there is provided a method for increasing plant seed yield, which method comprises increasing activity of a SYR polypeptide and/or expression in a plant of a nucleic acid encoding a SYR polypeptide or a homologue thereof.
Insofar as the methods of the invention concern FG-GAP, preferably performance of the methods result in plants having increased yield and, more particularly, increased biomass and/or increased seed yield. Preferably, the increased seed yield comprises an increase in one or more of number of (filled) seeds, total seed weight, seed size, thousand kernel weight and harvest index, each relative to control plants. Therefore, according to the present invention, there is provided a method for increasing plant yield, particularly, increased biomass and/or increased seed yield, which method comprises modulating activity of an FG-GAP polypeptide and/or expression in a plant of a nucleic acid encoding an FG-GAP polypeptide or a homologue thereof.
Insofar as the methods of the invention concern CYP90B, preferably the increased yield includes one or more of the following: increased HI, increased TKW, increased seed area and increased seed length, each relative to suitable control plants. Therefore, according to the present invention, there is provided a method for increasing plant yield, particularly seed yield, relative to suitable control plants, which method comprises increasing non-constitutive expression in a plant of a nucleic acid encoding a CYP90B polypeptide or a homologue thereof.
Insofar as methods of the invention concern AT-hook transcription factors, seed yield in monocotyledonous plants is increased. There is therefore provided a method for increasing seed yield in monocotyledonous plants relative to suitable control plants, comprising preferentially increasing expression in endosperm tissue of a monocotyledonous plant of a nucleic acid encoding a polypeptide comprising an AT-hook domain and a DUF296 domain.
Insofar as the methods of the invention concern DOF transcription factors, preferably the increased yield is increased seed yield. According to a preferred feature of the present invention, there is provided a method for increasing plant seed yield relative to seed yield of suitable control plants, which method comprises increasing expression in a plant of a nucleic acid encoding a DOF transcription factor polypeptide.
Insofar as the methods of the invention concern CKIs, the improved growth characteristic is increased seed yield. The present invention therefore provides a method for increasing seed yield in plants relative to suitable control plants, comprising preferentially reducing expression of an endogenous CKI gene in endosperm tissue of a plant.
Since the improved plants according to the present invention have increased yield (seed 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 or cell types of a plant (including seeds), or may be throughout substantially the whole plant. Plants having an increased growth rate may have a shorter life cycle. The life cycle of a plant is taken to mean the time needed to grow from a dry mature seed up to the stage where the plant has produced dry mature seeds, similar to the starting material. This life cycle may be influenced by factors such as early vigour, growth rate, flowering time and speed of seed maturation. An increase in growth rate may take place at one or more stages in the life cycle of a plant or during substantially the whole plant life cycle. Increased growth rate during the early stages in the life cycle of a plant may reflect enhanced vigour. The increase in growth rate may alter the harvest cycle of a plant allowing plants to be sown later and/or harvested sooner than would otherwise be possible. If the growth rate is sufficiently increased, it may allow for the sowing of further seeds of the same plant species (for example sowing and harvesting of rice plants followed by sowing and harvesting of further rice plants all within one conventional growing period). Similarly, if the growth rate is sufficiently increased, it may allow for the further sowing of seeds of different plants species (for example the sowing and harvesting of rice plants followed by, for example, the sowing and optional harvesting of soy bean, potatoes or any other suitable plant). Harvesting additional times from the same rootstock in the case of some plants may also be possible. Altering the harvest cycle of a plant may lead to an increase in annual biomass production per acre (due to an increase in the number of times (say in a year) that any particular plant may be grown and harvested). An increase in growth rate may also allow for the cultivation of transgenic plants in a wider geographical area than their wild-type counterparts, since the territorial limitations for growing a crop are often determined by adverse environmental conditions either at the time of planting (early season) or at the time of harvesting (late season). Such adverse conditions may be avoided if the harvest cycle is shortened. The growth rate may be determined by deriving various parameters from growth curves plotting growth experiments, such parameters may be: T-Mid (the time taken for plants to reach 50% of their maximal size) and T-90 (time taken for plants to reach 90% of their maximal size), amongst others. The term “flowering time” as used herein shall mean the time period between the start of seed germination and the start of flowering.
Performance of the methods of the invention gives plants having an increased growth rate.
Therefore, according to the present invention, there is provided a method for increasing the growth rate of plants, which method comprises increasing activity in a plant of a SYR polypeptide or a homologue thereof and/or expression of a nucleic acid encoding such a protein.
According to the present invention, there is provided a method for increasing the growth rate of plants, which method comprises modulating (preferably increasing) activity in a plant of an FG-GAP polypeptide or a homologue thereof and/or modulating (preferably increasing) expression of a nucleic acid encoding such protein.
According to the present invention, there is provided a method for increasing the growth rate of plants which method comprises increasing non-constitutive expression in a plant of a nucleic acid encoding a CYP90B polypeptide or a homologue thereof.
According to the present invention, there is provided a method for increasing the growth rate of plants, which method comprises increasing expression in a plant of a nucleic acid encoding a DOF transcription factor polypeptide.
According to the present invention, there is provided a method for increasing the growth rate of plants relative to suitable control plants, which method comprises preferentially reducing expression of an endogenous Cyclin Dependent Kinase Inhibitor (CKI) gene in endosperm tissue of a plant.
An increase in yield and/or 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 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. Mild stress in the sense of the invention leads to a reduction in the growth of the stressed plants of less than 40%, 35% or 30%, preferably less than 25%, 20% or 15%, more preferably less than 14%, 13%, 12%, 11% or 10% or less in comparison to the control plant under non-stress conditions. 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), anaerobic stress, chemical toxicity and oxidative stress. The abiotic stress may be an osmotic stress caused by a water stress (particularly due to drought), salt stress, oxidative stress or an ionic stress. Chemicals may also cause abiotic stresses (for example too high or, too low concentrations of minerals or nutrients). Biotic stresses are typically those stresses caused by pathogens, such as bacteria, viruses, fungi and insects. The term “non-stress conditions” as used herein are those environmental conditions that do not significantly go beyond the everyday climatic and other abiotic conditions that plants may encounter, and which allow optimal growth of the plant. Persons skilled in the art are aware of normal soil conditions and climatic conditions for a given geographic location.
Insofar as the methods of the invention concern SYR, performance of the methods result in plants having increased tolerance to abiotic stress. As reported in Wang et al. (Planta (2003) 218: 1-14), abiotic stress leads to a series of morphological, physiological, biochemical and molecular changes that adversely affect plant growth and productivity. Drought, salinity, extreme temperatures and oxidative stress are known to be interconnected and may induce growth and cellular damage through similar mechanisms. For example, drought and/or salinisation are manifested primarily as osmotic stress, resulting in the disruption of homeostasis and ion distribution in the cell. Oxidative stress, which frequently accompanies high or low temperature, salinity or drought stress may cause denaturation of functional and structural proteins. As a consequence, these diverse environmental stresses often activate similar cell signaling pathways and cellular responses, such as the production of stress proteins, up-regulation of anti-oxidants, accumulation of compatible solutes and growth arrest.
Since diverse environmental stresses activate similar pathways, the exemplification of the present invention with drought stress (insofar as the invention concerns the use of SYR polypeptides and their encoding nucleic acids) should not be seen as a limitation to drought stress, but more as a screen to indicate the involvement of SYR polypeptides or homologues thereof in abiotic stresses in general. Furthermore, the methods of the present invention may be performed under non-stress conditions or under conditions of mild drought to give plants having improved growth characteristics (particularly increased yield) relative to corresponding wild type or other control plants.
A particularly high degree of “cross talk” is reported between drought stress and high-salinity stress (Rabbani et al. (2003) Plant Physiol 133: 1755-1767). Therefore, it would be apparent that a SYR polypeptide or a homologue thereof would, along with its usefulness in conferring drought-tolerance in plants, also find use in protecting the plant against various other abiotic stresses. Similarly, it would be apparent that a SYR protein (as defined herein) would, along with its usefulness in conferring salt-tolerance in plants, also find use in protecting the plant against various other abiotic stresses. Furthermore, Rabbani et al. (2003, Plant Physiol 133: 1755-1767) report that similar molecular mechanisms of stress tolerance and responses exist between dicots and monocots. The methods of the invention are therefore advantageously applicable to any plant.
The term “abiotic stress” as defined herein is taken to mean any one or more of: water stress (due to drought or excess water), anaerobic stress, salt stress, temperature stress (due to hot, cold or freezing temperatures), chemical toxicity stress and oxidative stress. According to one aspect of the invention, the abiotic stress is an osmotic stress, selected from water stress, salt stress, oxidative stress and ionic stress. Preferably, the water stress is drought stress. The term salt stress is not restricted to common salt (NaCl), but may be any one or more of: NaCl, KCl, LiCl, MgCl2, CaCl2, amongst others.
Increased tolerance to abiotic stress is manifested by increased plant yield in abiotic stress conditions. Insofar as the invention concerns the use of SYR polypeptides and their encoding nucleic acids, such increased yield may include one or more of the following: increased number of filled seeds, increased total seed yield, increased number of flowers per panicle, increased seed fill rate, increased Harvest Index, increased Thousand Kernel Weight, increased root length or increased root diameter, each relative to corresponding wild type plants.
Performance of the methods of the invention gives plants having increased tolerance to abiotic stress. Performance of the methods of the invention gives plants grown under non-stress conditions or under mild drought conditions improved growth characteristics (particularly increased yield and/or increased emergence vigour (or early vigour)) relative to corresponding wild type plants or other control plants grown under comparable conditions.
According to the present invention, there is provided a method for increasing abiotic stress tolerance in plants-which method comprises modulating expression in a plant of a nucleic acid encoding a SYR polypeptide or a homologue thereof. According to one aspect of the invention, the abiotic stress is osmotic stress, selected from one or more of the following: water stress, salt stress, oxidative stress and ionic stress. Preferably, the water stress is drought stress.
The present invention also provides a method for improving abiotic stress tolerance in plants, comprising increasing activity in a plant of a SYR protein or a homologue thereof.
Insofar as the methods of the invention concern DOF transcription factors, the methods may be performed under conditions of mild drought to give plants having increased yield relative to suitable control plants. As reported in Wang et al. (Planta (2003) 218: 1-14), abiotic stress leads to a series of morphological, physiological, biochemical and molecular changes that adversely affect plant growth and productivity. Drought, salinity, extreme temperatures and oxidative stress are known to be interconnected and may induce growth and cellular damage through similar mechanisms. Rabbani et al. (Plant Physiol (2003) 133: 1755-1767) describes a particularly high degree of “cross talk” between drought stress and high-salinity stress. For example, drought and/or salinisation are manifested primarily as osmotic stress, resulting in the disruption of homeostasis and ion distribution in the cell. Oxidative stress, which frequently accompanies high or low temperature, salinity or drought stress, may cause denaturing of functional and structural proteins. As a consequence, these diverse environmental stresses often activate similar cell signaling pathways and cellular responses, such as the production of stress proteins, up-regulation of anti-oxidants, accumulation of compatible solutes and growth arrest.
Performance of the methods of the invention gives plants grown under mild drought conditions increased yield relative to suitable control plants grown under comparable conditions. Therefore, according to the present invention, there is provided a method for increasing yield in plants grown under mild drought conditions, which method comprises increasing expression in a plant of a nucleic acid encoding a DOF transcription factor polypeptide.
The abovementioned improved growth characteristics may advantageously be improved in any plant. Insofar as the methods of the invention concern the use of AT-hook transcription factors, the methods are applicable to monocotyledonous plants.
The term “plant” as used herein encompasses whole plants, ancestors and progeny of the plants and plant parts, including seeds, shoots, stems, leaves, roots (including tubers), flowers, and tissues and organs, wherein each of the aforementioned comprise the gene/nucleic acid of interest or the genetic modification in the gene/nucleic acid of interest. The term “plant” also encompasses plant cells, suspension cultures, callus tissue, embryos, meristematic regions, gametophytes, sporophytes, pollen and microspores, again wherein each of the aforementioned comprise the gene/nucleic acid of interest.
Plants that are particularly useful in the methods of the invention include 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 Acer spp., Actinidia spp., Abelmoschus spp., Agropyron spp., Allium spp., Amaranthus spp., Ananas comosus, Annona spp., Apium graveolens, Anachis spp, Artocarpus spp., Asparagus offlcinalis, Avena spp. (e.g. Avena sativa, Avena fatua, Avena byzantina, Avena fatua var. sativa, Avena hybrida), Averrhoa carambola, Benincasa hispida, Bertholletia excelsea, Beta vulgaris, Brassica spp. (e.g. Brassica napus, Brassica rapa ssp. [canola, oilseed rape, turnip rape]), Cadaba farinosa, Camellia sinensis, Canna indica, Capsicum spp., Carex elata, Carica papaya, Carissa macrocarpa, Carya spp., Catthamus tinctorius, Castanea spp., Cichorium endivia, Cinnamomum spp., Citrullus lanatus, Citrus spp., Cocos spp., Coffea spp., Colocasia esculenta, Cola spp., Coriandrum sativum, Corylus spp., Crataegus spp., Crocus sativus, Cucurbita spp., Cucumis spp., Cynara spp., Daucus carota, Desmodium spp., Dimocarpus longan, Dioscorea spp., Diospyros spp., Echinochloa spp., Elaeis (e.g. Elaeis guineensis, Elaeis oleifera), Eleusine coracana, Eiobotrya japonica, Eugenia unifiora, Fagopyrum spp., Fagus spp., Ficus carica, Fortuneila spp., Fragana spp., Ginkgo biloba, Glycine spp. (e.g. Glycine max, Soja hispida or Soja max), Gossypium hirsutum, Helianthus spp. (e.g. Helianthus annuus), Hemerocallis fulva, Hibiscus spp., Hordeum spp. (e.g. Hordeum vulgare), Ipomoea batatas, Juglans spp., Lactuca sativa, Lathyrus spp., Lens culinaris, Linum usitatissimum, Litchi chinensis, Lotus spp., Luffa acutangula, Lupinus spp., Luzula sylvatica, Lycopersicon spp. (e.g. Lycopersicon esculentum, Lycopersicon lycopersicum, Lycopersicon pyriforme), Macrotyloma spp., Malus spp., Malpighia emarginata, Mammea americana, Mangifera indica, Manihot spp., Manilkara zapota, Medicago sativa, Melilotus spp., Mentha spp., Momordica spp., Morus nigra, Musa spp., Nicotiana spp., Olea spp., Opuntia spp., Ornithopus spp., Oryza spp. (e.g. Oryza sativa, Oryza latifolia), Panicum miliaceum, Passiflora edulis, Pastinaca sativa, Persea spp., Petroselinum crispum, Phaseolus spp., Phoenix spp., Physalis spp., Pinus spp., Pistacia vera, Pisum spp., Poa spp., Populus spp., Prosopis spp., Prunus spp., Psidium spp., Punica granatum, Pyrus communis, Quercus spp., Raphanus sativus, Rheum rhabarbarum, Ribes spp., Ricinus communis, Rubus spp., Saccharum spp., Sambucus spp., Secale cereale, Sesamum spp., Sinapis sp., Solanum spp. (e.g. Solanum tuberosum, Solanum integrifolium or Solanum lycopersicum), Sorghum bicolor, Spinacia spp., Syzygium spp., Tagetes spp., Tamarindus indica, Theobroma cacao, Trnfolium spp., Triticosecale rimpaui, Triticum spp. (e.g. Triticum aestivum, Triticum durum, Triticum turgidum, Triticum hybemum, Triticum macha, Triticum sativum or Triticum vulgare), Tropaeolum minus, Tropaeolum majus, Vaccinium spp., Vicia spp., Vigna spp., Viola odorata, Vitis spp., Zea mays, Zizania palustnis, Vziphus spp., amongst others.
Preferably, the plant is a crop plant such as soybean, sunflower, canola, alfalfa, rapeseed, cotton, tomato, potato or tobacco. 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.
Where the methods of the invention concern use of an AT-hook transcription factor, the monocotyledonous plant is a cereal, such as rice, maize, sugarcane, wheat, barley, millet, rye, sorghum, grasses or oats.
DEFINITIONS
Polypeptide
The terms “polypeptide” and “protein” are used interchangeably herein and refer to amino acids in a polymeric form of any length. The terms “polynucleotide(s)”, “nucleic acid sequence(s)”, “nucleotide sequence(s)” are used interchangeably herein and refer to nucleotides, either ribonucleotides or deoxyribonucleotides or a combination of both, in a polymeric form of any length.
Control Plant
The choice of suitable control plants is a routine part of an experimental setup and may include corresponding wild type plants or corresponding plants without the gene of interest. The control plant is typically of the same plant species or even of the same variety as the plant to be assessed. The control plant may also be a nullizygote of the plant to be assessed. A control plant as used herein refers not only to whole plants, but also to plant parts, including seeds and seed parts.
Increase, Improve
The terms “increase”, “improving” or “improve” are used interchangeably herein and are taken to mean at least a 5%, 6%, 7%, 8%, 9% or 10%, preferably at least 15% or 20%, more preferably 25%, 30%, 35% or 40% more yield and/or growth in comparison to corresponding wild type or other control plants as defined herein.
Hybridisation
The term “hybridisation” as defined herein is a process wherein substantially homologous complementary nucleotide sequences anneal to each other. The hybridisation process may occur entirely in solution, i.e. both complementary nucleic acids are in solution. The hybridisation process may also occur with one of the complementary nucleic acids immobilised to a matrix such as magnetic beads, Sepharose beads or any other resin. The hybridisation process may furthermore occur with one of the complementary nucleic acids immobilised to a solid support such as a nitro-cellulose or nylon membrane or immobilised by e.g. photolithography to, for example, a siliceous glass support (the latter known as nucleic acid arrays or microarrays or as nucleic acid chips). In order to allow hybridisation to occur, the nucleic acid molecules are generally thermally or chemically denatured to melt a double strand into two single strands and/or to remove hairpins or other secondary structures from single stranded nucleic acids. The stringency of hybridisation is influenced by conditions such as temperature, salt concentration, ionic strength and hybridisation buffer composition.
“Stringent hybridisation conditions” and “stringent hybridisation wash conditions” in the context of nucleic acid hybridisation experiments such as Southern and Northern hybridisations are sequence dependent and are different under different environmental parameters. The skilled artisan is aware of various parameters which may be altered during hybridisation and washing and which will either maintain or change the stringency conditions.
The Tm is the temperature under defined ionic strength and pH, at which 50% of the target sequence hybridises to a perfectly matched probe. The Tm is dependent upon the solution conditions and the base composition and length of the probe. For example, longer sequences hybridise specifically at higher temperatures. The maximum rate of hybridisation is obtained from about 16° C. up to 32° C. below Tm. The presence of monovalent cations in the hybridisation solution reduce the electrostatic repulsion between the two nucleic acid strands thereby promoting hybrid formation; this effect is visible for sodium concentrations of up to 0.4M. Formamide reduces the melting temperature of DNA-DNA and DNA-RNA duplexes with 0.6 to 0.7° C. for each percent formamide, and addition of 50% formamide allows hybridisation to be performed at 30 to 45° C., though the rate of hybridisation will be lowered. Base pair mismatches reduce the hybridisation rate and the thermal stability of the duplexes. On average and for large probes, the Tm decreases about 1° C. per % base mismatch. The Tm may be calculated using the following equations, depending on the types of hybrids:

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

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

        DNA-RNA or RNA-RNA hybrids:

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

        oligo-DNA or oligo-RNAd hybrids:
        For <20 nucleotides: Tm=2 (In)
        For 20-35 nucleotides: Tm=22+1.46 (In)
        a or for other monovalent cation, but only accurate in the 0.01-0.4 M range.
        b only accurate for % GC in the 30% to 75% range.
        cL=length of duplex in base pairs.
        d Oligo, oligonucleotide; In, effective length of primer=(no. of G/C)+(no. of A/T).

Note: for each 1% formamide, the Tm is reduced by about 0.6 to 0.7° C., while the presence of 6M urea reduces the Tm by about 30° C.

    
    


Specificity of hybridisation is typically the function of post-hybridisation washes. To remove background resulting from non-specific hybridisation, samples are washed with dilute salt solutions. Critical factors of such washes include the ionic strength and temperature of the final wash solution: the lower the salt concentration and the higher the wash temperature, the higher the stringency of the wash. Wash conditions are typically performed at or below hybridisation stringency. Generally, suitable stringent conditions for nucleic acid hybridisation assays or gene amplification detection procedures are as set forth above. More or less stringent conditions may also be selected. Generally, low stringency conditions are selected to be about 50° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. Medium stringency conditions are when the temperature is 20° C. below Tm, and high stringency conditions are when the temperature is 10° C. below Tm. For example, stringent conditions are those that are at least as stringent as, for example, conditions A-L; and reduced stringency conditions are at least as stringent as, for example, conditions M-R. Non-specific binding may be controlled using any one of a number of known techniques such as, for example, blocking the membrane with protein containing solutions, additions of heterologous RNA, DNA, and SDS to the hybridisation buffer, and treatment with Rnase.
Examples of hybridisation and wash conditions are listed in Table 1:











TABLE 1









Wash


Stringency
Polynucleotide
Hybrid Length
Hybridization Temperature
Temperature


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







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




equal to 50
and 50% formamide


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


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




equal to 50
and 50% formamide


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


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




equal to 50
and 50% formamide


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


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




equal to 50
and 50% formamide


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


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




equal to 50
and 50% formamide


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


K
RNA:RNA
> or
70° C. 4xSSC; or 40° C., 6xSSC
67° C.; 1xSSC




equal to 50
and 50% formamide


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


M
DNA:DNA
> or
50° C. 4xSSC; or 40° C., 6xSSC
50° C.; 2xSSC




equal to 50
and 50% formamide


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


O
DNA:RNA
> or
55° C. 4xSSC; or 42° C., 6xSSC
55° C.; 2xSSC




equal to 50
and 50% formamide


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


Q
RNA:RNA
> or
60° C. 4xSSC; or 45° C., 6xSSC
60° C.; 2xSSC




equal to 50
and 50% formamide


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





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


†SSPE (1xSSPE is 0.15M NaCl, 10 mM NaH2PO4, and 1.25 mM EDTA, pH7.4) may be substituted for SSC (1xSSC is 0.15 M NaCl and 15 mM sodium citrate) in the hybridisation and wash buffers; washes are performed for 15 minutes after hybridisation is complete. The hybridisations and washes may additionally include 5 × Denhardt's reagent, 0.5-1.0% SDS, 100 μg/ml denatured, fragmented salmon sperm DNA, 0.5% sodium pyrophosphate, and up to 50% formamide.


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


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






For the purposes of defining the level of stringency, reference may conveniently be made to Sambrook et al. (2001) Molecular Cloning: a laboratory manual, 3rd Edition Cold Spring Harbor Laboratory Press, CSH, New York or to Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989).
T-DNA Activation Tagging
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 down stream of the coding region of a gene in a configuration such that the promoter directs expression of the targeted gene. Typically, regulation of expression of the targeted gene by its natural promoter is disrupted and the gene 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.
Tilling
TILLING (Targeted Induced Local Lesions In Genomes) is a mutagenesis technology useful to generate and/or identify and/or to eventually isolate mutagenised variant nucleic acids. TILLING also allows selection of plants carrying such mutant variants. These mutant variants may even exhibit higher activity than that exhibited by the gene in its natural form. TILLING combines high-density mutagenesis with high-throughput screening methods. The steps typically followed in TILLING are: (a) EMS mutagenesis (Redei G P and Koncz C (1992) In Methods in Arabidopsis Research, Koncz C, Chua N H, Schell J, eds. Singapore, World Scientific Publishing Co, pp. 1682; Feldmann et al., (1994) In Meyerowitz E M, Somerville C R, eds, Arabidopsis. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 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, N.J., pp 91-104); (b) DNA preparation and pooling of individuals; (c) PCR amplification of a region of interest; (d) denaturation and annealing to allow formation of heteroduplexes; (e) DHPLC, where the presence of a heteroduplex in a pool is detected as an extra peak in the chromatogram; (f) identification of the mutant individual; and (g) sequencing of the mutant PCR product. Methods for TILLING are well known in the art (McCallum et al., (2000) Nat Biotechnol 18: 455-457; reviewed by Stemple (2004) Nat Rev Genet. 5(2): 145-50).
Site-Directed Mutagenesis
Site-directed mutagenesis may be used to generate variants of SYR 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).
Transposon Mutagenesis
Transposon mutagenesis is a mutagenesis technique based on the insertion of transposons in genes, which frequently results in gene-knockout. The technique has been used for several plant species, including rice (Greco et al., Plant Physiol, 125, 1175-1177, 2001), corn (McCarty et al., Plant J. 44, 5261, 2005) and Arabidopsis (Parinov and Sundaresan, Curr. Opin. Biotechnol. 11, 157-161, 2000).
Directed Evolution
Directed evolution or gene shuffling consists of iterations of DNA shuffling followed by appropriate screening and/or selection to generate variant nucleic acids or portions thereof, or polypeptides or homologues thereof having a modified biological activity (Castle et al., (2004) Science 304(5674): 11514; U.S. Pat. Nos. 5,811,238 and 6,395,547).
Homologous Recombination
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 (Offring a 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; Iida and Terada (2004) Curr Opin Biotech 15(2):132-8). The nucleic acid to be targeted (which may be any of the nucleic acids or variant defined herein) needs to be targeted to the particular gene locus. 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.
Homologues
“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 2 below).
Orthologues and Paralogues
Encompassed by the term “homologues” are orthologous sequences and paralogous sequences, two special forms of homology which encompass evolutionary concepts used to describe ancestral relationships of genes.
The term “paralogous” relates to gene-duplications within the genome of a species leading to paralogous genes. Paralogues may easily be identified by performing a BLAST analysis against a set of sequences from the same species as the query sequence.
The term “orthologous” relates to homologous genes in different organisms due to speciation. Orthologues in, for example, dicot 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: 1 or SEQ ID NO: 2) against any sequence database, such as the publicly available NCBI database which may be found at: http://www.ncbi.nim.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: 1 or SEQ ID NO: 2 the second blast would therefore be against Oryza sativa 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 probability 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. Less conserved substitutions may be made in case the above-mentioned amino acid properties are not so critical. Conservative substitution tables are readily available in the art. The table below gives examples of conserved amino acid substitutions.







TABLE 2







Examples of conserved amino acid substitutions:










Residue
Conservative Substitutions







Ala
Ser



Arg
Lys



Asn
Gln; His



Asp
Glu



Gln
Asn



Cys
Ser



Glu
Asp



Gly
Pro



His
Asn; Gln



Ile
Leu, Val



Leu
Ile; Val



Lys
Arg; Gln



Met
Leu; Ile



Phe
Met; Leu; Tyr



Ser
Thr; Gly



Thr
Ser; Val



Trp
Tyr



Tyr
Trp; Phe



Val
Ile; Leu










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)-tag, glutathione S-transferase-tag, protein A, maltose-binding protein, dihydrofolate reductase, Tag-100 epitope, c-myc epitope, FLAG®-epitope, lacZ, CMP (calmodulin-binding peptide), HA epitope, protein C epitope and VSV epitope.
Homologues in the form of “deletion variants” of a protein are characterised by the removal of one or more amino acids from a protein.
Amino acid variants of a protein may readily be made using peptide synthetic techniques well known in the art, such as solid phase peptide synthesis and the like, or by recombinant DNA manipulations. Methods for the manipulation of DNA sequences to produce substitution, insertion or deletion variants of a protein are well known in the art. For example, techniques for making substitution mutations at predetermined sites in DNA are well known to those skilled in the art and include M13 mutagenesis, T7-Gen in vitro mutagenesis (USB, Cleveland, Ohio), QuickChange Site Directed mutagenesis (Stratagene, San Diego, Calif.), PCR-mediated site-directed mutagenesis or other site-directed mutagenesis protocols.
Derivatives
“Derivatives” are polypeptides or proteins which may comprise naturally modified and/or non-naturally modified amino acid residues compared to the amino acid sequence of a naturally-occurring form (that is not having undergone post-translational modifications) of the protein, for example, as presented in SEQ ID NO: 2. “Derivatives” of a protein encompass polypeptides or proteins 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.
Alternative Splice Variants
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 known in the art.
Allelic Variant
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.
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.
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.
A tissue-preferred or tissue-specific promoter is one that is capable of preferentially initiating transcription in certain tissues, such as the leaves, roots, seed tissue etc, or even in specific cells.
The term “constitutive” as defined herein refers to a promoter that is expressed predominantly in at least one tissue or organ and predominantly at any life stage of the plant. Preferably the promoter is expressed predominantly throughout the plant.
Examples of other constitutive promoters are shown in Table 3 below.







TABLE 3







Examples of constitutive promoters








Gene Source
Reference





Actin
McElroy et al, Plant Cell, 2: 163-171, 1990


CAMV 35S
Odell et al, Nature, 313: 810-812, 1985


CaMV 19S
Nilsson et al., Physiol. Plant. 100: 456-462, 1997


GOS2
de Pater et al, Plant J Nov; 2(6): 837-44, 1992, WO 2004/065596


Ubiquitin
Christensen et al, Plant Mol. Biol. 18: 675-689, 1992


Rice cyclophilin
Buchholz et al, Plant Mol Biol. 25(5): 837-43, 1994


Maize H3 histone
Lepetit et al, Mol. Gen. Genet. 231: 276-285, 1992


Alfalfa H3 histone
Wu et al. Plant Mol. Biol. 11: 641-649, 1988


Actin 2
An et al, Plant J. 10(1); 107-121, 1996


34S FMV
Sanger et al., Plant. Mol. Biol., 14, 1990: 433-443


Rubisco small subunit
U.S. Pat. No. 4,962,028


OCS
Leisner (1988) Proc Natl Acad Sci USA 85(5): 2553


SAD1
Jain at al., Crop Science, 39 (6), 1999: 1696


SAD2
Jain et al., Crop Science, 39 (6), 1999: 1696


nos
Shaw et al. (1984) Nucleic Acids Res. 12(20): 7831-7846


V-ATPase
WO 01/14572


Super promoter
WO 95/14098


G-box proteins
WO 94/12015
















TABLE 4







Examples of non-constitutive promoters









Gene source and name
Expression Pattern
Reference





Rice RP6
Endosperm-specific
Wen et al. (1993) Plant Physiol




101(3): 1115-6


Sorghum kafirin
Endosperm-specific
DeRose et al. (1996) Plant Molec




Biol 32: 1029-35


Corn zein
Endosperm-specific
Matzke et al. (1990) Plant Mol Biol




14(3): 323-32


Rice Oleosin 18 kDa
Embryo (and
Chuang et al. (1996) J Biochem



aleurone) specific
120(1): 74-81


Rice Oleosin 16 kDa
Embryo (and
Chuang et al. (1996) J Biochem



aleurone) specific
120(1): 74-81


Soybean beta-conglycinin
Embryo
Chiera et al. (2005) Plant Molec Biol




56(6): 895-904


Rice Wsi18
Whole seed
Joshee et al. (1998) Plant Cell




Physiol 39(1): 64-72.


Rice
Whole seed
Sasaki et al. (2002) NCBI accession




number BAA85411


Rice OSH1
Early shoot apical
Sato et al. (1996) Proc Natl Acad Sci



meristem
93 (15): 8117-8122


Rice Rcc2
Root-specific
Xu et al. (1995) Plant Mol Biol 27(2):




237-48


Rice Rcc3
Root-specific
Xu et al. (1995) Plant Mol Biol 27(2):




237-48


Arabidopsis Pyk10
Root-specific
Nitz et al. (2001) Plant Sci 161(2):




337-346
















TABLE 5







Examples of early shoot apical meristem promoters










Gene source
Gene family
Plant source
Reference





OSH1
KNOX family class
Oryza sativa
Matsuoka et al., (1993) Plant Cell



1 homeobox

5: 1039-1048





Sato et al., (1996) PNAS 93: 8117-8122


Knotted1
KNOX family class
Zea mays
Hake et al., (1989) EMBO Journal



1 homeobox

8: 15-22


KNAT1
KNOX family class
Arabidopsis
Lincoln et al., (1994) Plant Cell 6:



1 homeobox
thaliana
1859-1876


Oskn2
KNOX family class
Oryza sativa
Postma-Haarsma et al., (1999)



1 homeobox

Plant Mol Biol 39(2): 257-71


Oskn3
KNOX family class
Oryza sativa
Postma-Haarsma et al., (1999)



1 homeobox

Plant Mol Biol 39(2): 257-71
















TABLE 6







Examples of endosperm-specific promoters for use in the present invention










Expression



Gene source
pattern
Reference





wheat LMW and
endosperm
Mol Gen Genet 216: 81-90,


HMW

1989;


glutenin-1

NAR 17: 461-2, 1989.


wheat α, β, γ-gliadins
endosperm
EMBO 3: 1409-15, 1984.


barley ltr1 promoter
endosperm


barley B1, C, D,
endosperm
Theor Appl Gen 98: 1253-62,


hordein

1999; Plant J 4: 343-55, 1993; Mol




Gen Genet 250: 750-60, 1996.


barley DOF
endosperm
Mena et al., The Plant Journal,




116(1): 53-62, 1998.


blz2
endosperm
EP99106056.7


synthetic promoter
endosperm
Vicente-Carbajosa et al., Plant J.




13: 629-640, 1998.


rice prolamin NRP33
endosperm
Wu et al., Plant Cell Physiology




39(8) 885-889, 1998.


rice α-globulin Glb-1
endosperm
Wu et al., Plant Cell Physiology




39(8) 885-889, 1998.


rice α-globulin
endosperm
Nakase et al., Plant Mol. Biol.


REB/OHP-1

33: 513-522, 1997.


rice ADP-glucose PP
endosperm
Trans Res 6: 157-68, 1997.


maize ESR gene
endosperm
Plant J 12: 235-46, 1997.


family


sorgum γ-kafirin
endosperm
PMB 32: 1029-35, 1996.
















TABLE 7







Examples of seed-specific promoters for use in the present invention









Gene source
Expression pattern
Reference





seed-specific genes
seed
Simon, et al., Plant Mol. Biol. 5: 191,




1985; Scofield, et al., J. Biol. Chem.




262: 12202, 1987; Baszczynski, et




al., Plant Mol. Biol. 14: 633, 1990.


Brazil Nut albumin
seed
Pearson, et al., Plant Mol. Biol. 18:




235-245, 1992.


legumin
seed
Ellis, et al., Plant Mol. Biol. 10: 203-214,




1988.


glutelin (rice)
seed
Takaiwa, et al., Mol. Gen. Genet.




208: 15-22, 1986; Takaiwa, et al.,




FEBS Letts. 221: 43-47, 1987.


zein
seed
Matzke et al., Plant Mol Biol, 14(3):




323-32, 1990.


napA
seed
Stalberg, et al., Planta 199: 515-519,




1996.


wheat LMW and HMW
endosperm
Mol Gen Genet 216: 81-90, 1989;


glutenin-1

NAR 17: 461-2, 1989.


wheat SPA
seed
Albani et al., Plant Cell, 9: 171-184,




1997.


wheat α, β, γ-gliadins
endosperm
EMBO 3: 1409-15, 1984.


barley Itr1 promoter
endosperm


barley B1, C, D,
endosperm
Theor Appl Gen 98: 1253-62, 1999;


hordein

Plant J 4: 343-55, 1993; Mol Gen




Genet 250: 750-60, 1996.


barley DOF
endosperm
Mena et al., The Plant Journal,




116(1): 53-62, 1998.


blz2
endosperm
EP99106056.7


synthetic promoter
endosperm
Vicente-Carbajosa et al., Plant J.




13: 629-640, 1998.


rice prolamin NRP33
endosperm
Wu et al., Plant Cell Physiology




39(8) 885-889, 1998.


rice α-globulin Glb-1
endosperm
Wu et al., Plant Cell Physiology




39(8) 885-889, 1998.


rice OSH1
embryo
Sato et al., Proc. Natl. Acad. Sci.




USA, 93: 8117-8122, 1996.


rice α-globulin
endosperm
Nakase et al., Plant Mol. Biol. 33:


REB/OHP-1

513-522, 1997.


rice ADP-glucose PP
endosperm
Trans Res 6: 157-68, 1997.


maize ESR gene
endosperm
Plant J 12: 235-46, 1997.


family


sorgum γ-kafirin
endosperm
PMB 32: 1029-35, 1996.


KNOX
embryo
Postma-Haarsma et al., Plant Mol.




Biol. 39: 257-71, 1999.


rice oleosin
embryo and aleurone
Wu et al., J. Biochem., 123: 386,




1998.


sunflower oleosin
seed (embryo and dry seed)
Cummins et al., Plant Mol. Biol. 19:




873-876, 1992.









Terminator Sequence

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.
Selectable Marker
The term “selectable marker gene” as referred to herein 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 nptit that phosphorylates neomycin and kanamycin, or hpt, phosphorylating hygromycin), to herbicides (for example bar which provides resistance to BastaTM; 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).
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 Mol 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) Mol. Gen. Genet. 202: 179-185); DNA or RNA-coated particle bombardment (Klein T M et al., (1987) Nature 327: 70) infection with (non-integrative) viruses and the like. Transgenic rice plants are preferably produced via Agrobacterum-mediated transformation using any of the well known methods for rice transformation, such as described in any of the following: published European patent application EP 1198985 A1, Aldemita and Hodges (Planta 199: 612-617, 1996); Chan et al. (Plant Mol Biol 22 (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 Ishida 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, both techniques being well known to persons having ordinary skill in the art.
The generated transformed plants may be propagated by a variety of means, such as by clonal propagation or classical breeding techniques. For example, a first generation (or T1) transformed plant may be selfed and homozygous second-generation (or T2) transformants selected, and the T2 plants may then further be 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).
Detailed Description Seed Yield Regulator (SYR)
The activity of a SYR protein may be increased by increasing levels of the SYR polypeptide. Alternatively, activity may also be increased when there is no change in levels of a SYR, or even when there is a reduction in levels of a SYR protein. This may occur when the intrinsic properties of the polypeptide are altered, for example, by making a mutant or selecting a variant that is more active that the wild type.
The term “SYR protein or homologue thereof” as defined herein refers to a polypeptide of about 65 to about 200 amino acids, comprising (i) a leucine rich domain that resembles a leucine zipper in the C-terminal half of the protein, which leucine rich domain is (ii) preceded by a tripeptide with the sequence YFS (conserved motif 1a, SEQ ID NO: 6), or YFT (conserved motif 1b, SEQ ID NO: 7), or YFG (conserved motif 1c, SEQ ID NO: 8) or YLG (conserved motif 1d, SEQ ID NO: 9), and (iii) followed by a conserved motif 2 ((V/A/I)LAFMP(T/S), SEQ ID NO: 10). Preferably, the conserved motif 2 is (A/V)LAFMP(T/S) (SEQ ID NO: 10), most preferably, the conserved motif is VLAFMPT (SEQ ID NO: 10). The “SYR protein or homologue thereof” preferably also has a conserved C-terminus peptide ending with the conserved motif 3 (SYL or PYL, SEQ ID NO: 11). The leucine rich domain of the SYR protein or its homologue is about 38 to 48 amino acids long, starting immediately behind the conserved motif 1 and stopping immediately before the conserved motif 2, and comprises at least 30% of leucine. The Leu rich domain preferably has a motif that resembles the Leucine Zipper motif (L-X6-L-X6-L-X6-L, (SEQ ID NO: 287) wherein X6 is a sequence of 6 consecutive amino acids). A preferred example of a SYR protein is represented by SEQ ID NO: 2, an overview of its domains is given in FIG. 1. It should be noted that the term “SYR protein or homologue thereof” does not encompass the ARGOS protein from Arabidopsis thaliana (SEQ ID NO: 26).
Further preferably, SYR proteins have two transmembrane domains, with the N-terminal part and C-terminal part of the protein located inside and the part between the transmembrane domains located outside.
Alternatively, the homologue of a SYR protein has in increasing order of preference at least 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% overall sequence identity to the amino acid represented by SEQ ID NO: 2, provided that the homologous protein comprises the conserved motifs 1 (a, b, c or d), 2 and 3, and the leucine rich domain as outlined above. The overall sequence identity is determined using a global alignment algorithm, such as the Needleman Wunsch algorithm in the program GAP (GCG Wisconsin Package, Accelrys), preferably with default parameters.
The various structural domains in a SYR protein may be identified using specialised databases e.g. SMART (Schultz et al. (1998) Proc. Natl. Acad. Sci. USA 95, 5857-5864; Letunic et al. (2002) Nucleic Acids Res 30, 242-244; http://smart.embl-heidelberg.de/), InterPro (Mulder et al., (2003) Nucl. Acids. Res. 31, 315-318; http://www.ebi.ac.uk/interpro/), Prosite (Bucher and Bairoch (1994), A generalized profile syntax for biomolecular sequences motifs and its function in automatic sequence interpretation. (In) ISMB-94; Proceedings 2nd International Conference on Intelligent Systems for Molecular Biology. Altman R., Brutlag D., Karp P., Lathrop R., Searls D., Eds., pp 53-61, AAAIPress, Menlo Park; Hulo et al., Nucl. Acids. Res. 32:D134-D137, (2004), http://www.expasy.org/prosite/) or Pfam (Bateman et al., Nucleic Acids Research 30(1):276-280 (2002), http://www.sanger.ac.uktSoftware/Pfam/).
Methods for the search and identification of SYR homologues would be well within the realm of persons skilled in the art. Such methods comprise comparison of the sequences represented by SEQ ID NO: 1 or 2, in a computer readable format, with sequences that are available in public databases such as MIPS (http://mips.gsf.deI), GenBank (http://www.ncbi.nim.nih.gov/Genbank/index.html) or EMBL Nucleotide Sequence Database (http://www.ebi.ac.uk/embl/index.html), using algorithms well known in the art for the alignment or comparison of sequences, such as GAP (Needleman and Wunsch, J. Mol. Biol. 48; 443-453 (1970)), BESTFIT (using the local homology algorithm of Smith and Waterman (Advances in Applied Mathematics 2; 482-489 (1981))), BLAST (Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J., J. Mol. Biol. 215:403-410 (1990)), FASTA and TFASTA (W. R. Pearson and D. J. Lipman Proc.Natl. Acad. Sci. USA 85:2444-2448 (1988)). The software for performing BLAST analysis is publicly available through the National Centre for Biotechnology Information (NCBI).
Transmembrane domains are about 15 to 30 amino acids long and are usually composed of hydrophobic residues that form an alpha helix. They are usually predicted on the basis of hydrophobicity (for example Klein et al., Biochim. Biophys. Acta 815, 468, 1985; or Sonnhammer et al., In J. Glasgow, T. Littlejohn, F. Major, R. Lathrop, O, Sankoff, and C. Sensen, editors, Proceedings of the Sixth International Conference on Intelligent Systems for Molecular Biology, pages 175-182, Menlo Park, Calif., 1998. AAAI Press).
Examples of proteins falling under the definition of “SYR polypeptide or a homologue thereof” are listed in Table A of Example 1 and include sequences from various monocotyledonous plants, such as rice (SEQ ID NO: 2, SEQ ID NO: 12 and SEQ ID NO: 13), corn (SEQ ID NO: 14 and SEQ ID NO: 44), wheat (SEQ ID NO: 15), barley (SEQ ID NO: 16), sugarcane (SEQ ID NO: 17 and SEQ ID NO: 18), sorghum (SEQ ID NO: 19); and from dicotyledonous plants such as Arabidopsis (SEQ ID NO: 20 and SEQ ID NO: 21), grape (SEQ ID NO: 22), citrus (SEQ ID NO: 23) or tomato (SEQ ID NO: 24 and SEQ ID NO: 25). It is envisaged that the Leu rich domain is important for the function of the protein, hence proteins with the Leu rich domain but without the conserved motifs 1 or 2 may be useful as well in the methods of the present invention; examples of such proteins are given in SEQ ID NO: 34 and 35.
It is to be understood that the term “SYR polypeptide or a homologue thereof” is not to be limited to the sequence represented by SEQ ID NO: 2 or to the homologues listed as SEQ ID NO: 12 to SEQ ID NO: 25, but that any polypeptide of about 65 to about 200 amino acids meeting the criteria of comprising a leucine rich domain as defined above, preceded by the conserved tripeptide motif 1 (a, b, c or d) and followed by the conserved motif 2 and preferably also by the conserved motif 3; or having at least 38% sequence identity to the sequence of SEQ ID NO: 2, may be suitable for use in the methods of the invention.
In another embodiment, the present invention provides an isolated SYR protein selected from the group consisting of:

    
    
        (a) a polypeptide as given in SEQ ID NO 44,
        (b) a polypeptide with an amino acid sequence which has at least, in increasing order of preference, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as given in SEQ ID NO 44,
        (c) a derivative of a protein as defined in (a) or (b).
    
    


The sequence represented by SEQ ID NO: 43 was hitherto unknown as a SYR encoding gene. There is therefore provided an isolated nucleic acid sequence comprising:

    
    
        (i) a nucleic acid sequence represented by SEQ ID NO: 43, or the complement strand thereof;
        (ii) a nucleic acid sequence encoding the amino acid sequence represented by SEQ ID NO: 44;
        (iii) a nucleic acid sequence capable of hybridising (preferably under stringent conditions) with a nucleic acid sequence of (i) or (ii) above, which hybridising sequence preferably encodes a SYR protein;
        (iv) a nucleic acid which is an allelic variant to the nucleic acid sequences according to (i) or (ii);
        (v) a nucleic acid which is a splice variant to the nucleic acid sequences according to (i) or (ii);
        (vi) a nucleic acid sequence which has 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence defined in (i) or (ii).
    
    


The activity of a SYR protein or homologue thereof may be assayed by expressing the SYR protein or homologue thereof under control of a GOS2 promoter in Oryza sativa, which results in plants with increased increased seed yield without a delay in flowering time when compared to corresponding wild type plants. This increase in seed yield may be measured in several ways, for example as an increase of total seed weight, number of filled seeds or harvest index.
A SYR protein or homologue thereof is encoded by a SYR nucleic acid/gene. Therefore the term “SYR nucleic acid/gene” as defined herein is any nucleic acid/gene encoding a SYR protein or a homologue thereof as defined above.
Examples of SYR nucleic acids include but are not limited to those represented by any one of SEQ ID NO: 1, SEQ ID NO: 27 to SEQ ID NO: 32, SEQ ID NO: 36 to 42 and SEQ ID NO: 44. See also the list of nucleic acids mentioned in Table A of Example 1.
SYR nucleic acids/genes and variants thereof may be suitable in practising the methods of the invention. Variant SYR nucleic acid/genes include portions of a SYR nucleic acid/gene and/or nucleic acids capable of hybridising with a SYR nucleic acid/gene.
The term portion as defined herein refers to a piece of DNA encoding a polypeptide of about 65 to about 200 amino acids, comprising a leucine rich domain as defined above, preceded by the conserved tripeptide motif 1 (a, b, c or d) and followed by the conserved motif 2 and preferably also by the conserved motif 3. Preferably, the portion comprises one or more of the conserved motifs defined above. A portion may be prepared, for example, by making one or more deletions to a SYR 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 SYR fragment. Preferably, the portion is a portion of a nucleic acid as represented by any one of SEQ ID NO: 1, SEQ ID NO: 27 to SEQ ID NO: 32, SEQ ID NO: 36 to SEQ ID NO: 42 and SEQ ID NO: 44. Most preferably the portion of a nucleic acid is as represented by SEQ ID NO: 1.
Another variant of a SYR nucleic acid/gene is a nucleic acid capable of hybridising under reduced stringency conditions, preferably under stringent conditions, with a SYR nucleic acid/gene as hereinbefore defined, which hybridising sequence encodes a polypeptide of about 65 to about 200 amino acids, comprising a leucine rich domain as defined above, preceded by the conserved tripeptide motif 1 (a, b, c or d) and followed by the conserved motif 2 and preferably also by the conserved motif 3; or having at least 38% sequence identity to the sequence of SEQ ID NO: 2.
Preferably, the hybridising sequence is one that is capable of hybridising to a nucleic acid as represented by SEQ ID NO: 1, SEQ ID NO: 27 to SEQ ID NO: 32, SEQ ID NO: 36 to SEQ ID NO: 42 and SEQ ID NO: 44, or to a portion of any of the aforementioned sequences. Most preferably the hybridising sequence is capable of hybridising to SEQ ID NO: 1. The term “hybridisation” is as defined herein.
The SYR nucleic acid or variant thereof may be derived from any natural or artificial source. The nucleic acid/gene or variant thereof may be isolated from a microbial source, such as yeast or fungi, or from a plant, algal or animal (including human) source. This nucleic acid may be modified from its native form in composition and/or genomic environment through deliberate human manipulation. The nucleic acid is preferably of plant origin, whether from the same plant species (for example to the one in which it is to be introduced) or whether from a different plant species. The nucleic acid may be isolated from a monocotyledonous species, preferably from the family Poaceae, further preferably from Oryza sativa. More preferably, the SYR nucleic acid is isolated from Oryza sativa and is represented by SEQ ID NO: 1, and the SYR amino acid sequence is as represented by SEQ ID NO: 2.
The expression of a nucleic acid encoding a SYR polypeptide or a homologue thereof may be modulated by introducing a genetic modification (preferably in the locus of a SYR 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 down stream 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, transposon mutagenesis, directed evolution and homologous recombination or by introducing and expressing in a plant a nucleic acid encoding a SYR polypeptide or a homologue thereof. The aforementioned methods are defined herein in the section headed “Definitions”. Following introduction of the genetic modification, there follows a step of selecting for modified expression of a nucleic acid encoding a SYR polypeptide or a homologue thereof, which modification in expression gives plants having increased seed yield.
T-DNA activation, TILLING, site-directed mutagenesis, transposon mutagenesis and directed evolution are examples of technologies that enable the generation of novel alleles and SYR variants.
A preferred method for introducing a genetic modification (which in this case need not be in the locus of a SYR gene) is to introduce and express in a plant a nucleic acid encoding a SYR polypeptide or a homologue thereof, as defined herein in. The nucleic acid to be introduced into a plant may be a full-length nucleic acid or may be a portion or a hybridising sequence as hereinbefore defined.
“Homologues” of a protein are defined herein in the section headed “Definitions”. The SYR polypeptide or homologue thereof may be a derivative. For a definition of the term “derivative” see the section herein headed “Definitions”.
The SYR polypeptide or homologue thereof may be encoded by an alternative splice variant of a SYR nucleic acid/gene. The term “alternative splice variant” is defined in the “Definitions” section. Preferred splice variants are splice variants of the nucleic acid encoding a polypeptide of about 65 to about 200 amino acids, comprising a leucine rich domain as defined above, preceded by the conserved tripeptide motif 1 (a, b, c or d) and followed by the conserved motif 2 and preferably also by the conserved motif 3; or having at least 38% sequence identity to the sequence of SEQ ID NO: 2. Further preferred are splice variants represented by SEQ ID NO: 1, SEQ ID NO: 27 to SEQ ID NO: 32, SEQ ID NO: 36 to SEQ ID NO: 42 and SEQ ID NO: 44. Most preferred is the splice variant represented by SEQ ID NO: 1.
The homologue may also be encoded by an allelic variant of a nucleic add encoding a SYR polypeptide or a homologue thereof, preferably an allelic variant of a nucleic acid encoding a polypeptide of about 65 to about 200 amino acids, comprising a leucine rich domain as defined above, preceded by the conserved tripeptide motif 1 (a, b, c or d) and followed by the conserved motif 2 and preferably also by the conserved motif 3; or having at least 38% sequence identity to the sequence of SEQ ID NO: 2. Further preferably, the allelic variant encoding the SYR polypeptide is represented by any one of SEQ ID NO: 1, or SEQ ID NO: 12 to SEQ ID NO: 25. Most preferably, the allelic variant encoding the SYR polypeptide is as represented by SEQ ID NO: 1. The term “allelic variant” is defined in the “Definitions” section.
According to a preferred aspect of the present invention, increased expression of the SYR nucleic acid or variant thereof is envisaged. 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 SYR nucleic acid or variant thereof. For example, endogenous promoters may be altered in vivo by mutation, deletion, and/or substitution (see, Klmiec, U.S. Pat. No. 5,565,350; Zarling et al., PCT/US93/03868), or isolated promoters may be introduced into a plant cell in the proper orientation and distance from a gene of the present invention so as to control the expression of the gene. 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 may be derived from the natural gene, from a variety of other plant genes, or from T-DNA. The 3′ end sequence to be added may be derived from, for example, the nopaline synthase or octopine synthase genes, or alternatively from another plant gene, or less preferably from any other eukaryotic gene.
An intron sequence may also be added to the 5′ untranslated region or the coding sequence of the partial coding sequence to increase the amount of the mature message that accumulates in the cytosol. Inclusion of a spliceable intron in the transcription unit in both plant and animal expression constructs has been shown to increase gene expression at both the mRNA and protein levels up to 1000-fold, Buchman and Berg, Mol. Cell. biol. 8:4395-4405 (1988); Callis et al., Genes Dev. 1:1183-1200 (1987). Such intron enhancement of gene expression is typically greatest when placed near the 5′ end of the transcription unit. Use of the maize introns Adh1-S intron 1, 2, and 6, the Bronze-1 intron are known in the art. See generally, The Maize Handbook, Chapter 116, Freeling and Walbot, Eds., Springer, N.Y. (1994).
The invention also provides genetic constructs and vectors to facilitate introduction and/or expression of the nucleotide sequences useful in the methods according to the invention.
Therefore, there is provided a gene construct comprising:

    
    
        (i) a SYR 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;

with the proviso that the gene construct does not comprise a nucleic acid sequence encoding the protein of SEQ ID NO: 26.

    
    


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 SYR 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 defined herein in the section headed “Definitions”.
Advantageously, any type of promoter may be used to drive expression of the nucleic acid sequence. Preferably, the SYR nucleic acid or functional variant thereof is operably linked to a constitutive promoter. Preferably, the constitutive promoter capable of preferentially expressing the nucleic acid throughout the plant has a comparable expression profile to a GOS2 promoter. More preferably, the constitutive promoter has the same expression profile as the rice GOS2 promoter, most preferably, the promoter capable of preferentially expressing the nucleic acid throughout the plant is the GOS2 promoter from rice (SEQ ID NO: 5).
It should be clear that the applicability of the present invention is not restricted to the SYR nucleic acid represented by SEQ ID NO: 1, nor is the applicability of the invention restricted to expression of a SYR nucleic acid when driven by a GOS2 promoter. An alternative constitutive promoter that is useful in the methods of the present invention is the High Mobility Group Protein (HMGP) promoter (SEQ ID NO: 33). Examples of other constitutive promoters that may also be used to drive expression of a SYR nucleic acid are shown in Table 3 in the section headed “Definitions”.
Optionally, one or more terminator sequences may also be used in the construct introduced into a plant. The term “terminator” is defined in the “Definitions” section.
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 defined in the “Definitions” section.
The present invention also encompasses plants obtainable by the methods according to the present invention. The present invention therefore provides plants obtainable by the method according to the present invention, which plants have introduced therein a SYR nucleic acid or variant thereof, as defined above.
The invention also provides a method for the production of transgenic plants having increased seed yield, comprising introduction and expression in a plant of a SYR nucleic acid or a variant thereof as defined above.
More specifically, the present invention provides a method for the production of transgenic plants having increased seed yield, which method comprises:

    
    
        (i) introducing and expressing in a plant or plant cell a SYR nucleic acid or variant thereof, and
        (ii) cultivating the plant cell under conditions promoting plant growth and development;

with the proviso that the SYR nucleic acid or variant thereof is not a nucleic acid sequence encoding the protein of SEQ ID NO: 26.

    
    


The nucleic acid may be introduced directly into a plant cell or into the plant itself (including introduction into a tissue, organ or any other part of a plant). According to a preferred feature of the present invention, the nucleic acid is preferably introduced into a plant by transformation. The term “transformation” is defined in the “Definitions” section.
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 SYR 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 directly derived from a harvestable part of such a plant, such as dry pellets or powders, oil, fat and fatty acids, starch or proteins.
The present invention also encompasses use of SYR nucleic acids or variants thereof and use of SYR polypeptides or homologues thereof.
One such use relates to improving the growth characteristics of plants, in particular in improving seed yield. The seed yield may include one or more of the following: increased total weight of seeds, increased number of filled seeds, fill rate and increased harvest index.
SYR nucleic acids or variants thereof, or SYR polypeptides or homologues thereof may find use in breeding programmes in which a DNA marker is identified which may be genetically linked to a SYR gene or variant thereof. The SYR nucleic acids/genes or variants thereof, or SYR 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 seed yield. The SYR gene or variant thereof may, for example, be a nucleic acid as represented by any one of SEQ ID NO: 1, SEQ ID NO: 27 to SEQ ID NO: 32, SEQ ID NO: 36 to SEQ ID NO: 42 and SEQ ID NO: 44.
Allelic variants of a SYR 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 seed 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: 1, SEQ. ID NO: 27 to SEQ ID NO: 32, SEQ ID NO: 36 to SEQ ID NO: 42 and SEQ ID NO: 44. 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 SYR 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 SYR nucleic acids or variants thereof requires only a nucleic acid sequence of at least 15 nucleotides in length. The SYR nucleic acids or variants thereof may be used as restriction fragment length polymorphism (RFLP) markers. Southern blots (Sambrook J, Fritsch E F and Maniatis T (1989) Molecular Cloning, A Laboratory Manual) of restriction-digested plant genomic DNA may be probed with the SYR 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 SYR nucleic acid or variant thereof in the genetic map previously obtained using this population (Botstein et al. (1980) Am. J. Hum. Genet. 32: 314-331).
The production and use of plant gene-derived probes for use in genetic mapping is described in Bernatzky and Tanksley (GENETICS 112 (4): 887-898, 1986). Numerous publications describe genetic mapping of specific cDNA clones using the methodology outlined above or variations thereof. For example, F2 intercross populations, backcross populations, randomly mated populations, near isogenic lines, and other sets of individuals may be used for mapping. Such methodologies are well known to those skilled in the art.
The nucleic acid probes may also be used for physical mapping (i.e., placement of sequences on physical maps; see Hoheisel et at 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 favour 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 seed yield, as described hereinbefore. These advantageous growth characteristics may also be combined with other economically advantageous traits, such as further yield-enhancing traits, tolerance to various stresses in addition to the abiotic stress resistance, traits modifying various architectural features and/or biochemical and/or physiological features.
Detailed Description FG-GAP
The activity of an FG-GAP protein may be modulated by modulating levels of the FG-GAP polypeptide. Alternatively, activity may also be modulated when there is no change in levels of an FG-GAP. This may occur when the intrinsic properties of the polypeptide are altered, for example, by making a mutant or selecting a variant that is more active or less active than the wild type.
The term “FG-GAP protein or homologue thereof” as defined herein refers to a polypeptide comprising (i) an N-terminal secretion signal peptide, (ii) one or more FG-GAP domains followed by (iii) a transmembrane domain in the C-terminal half of the protein. An example is given in FIG. 6.
Signal peptides are typical for proteins that are directed to the secretory pathway. The presence of a secretion signal may be easily predicted using computer algorithms (for example SignalP 3.0, Bendtsen et al., J. Mol. Biol., 340:783-795, 2004). A typical secretion signal consists of a positively charged n-region, followed by a hydrophobic n-region and a neutral, polar c-region. Furthermore, the amino acid residues at position −3 and −1 relative to the cleavage site are usually small and neutral.
Transmembrane domains are about 15 to 30 amino acids long and are usually composed of hydrophobic residues that form an alpha helix. They are usually predicted on the basis of hydrophobicity (for example Klein et al., Biochim. Biophys. Acta 815, 468, 1985; or Sonnhammer et al., In J. Glasgow, T. Littlejohn, F. Major, R. Lathrop, D. Sankoff, and C. Sensen, editors, Proceedings, of the Sixth International Conference on Intelligent Systems for Molecular Biology, pages 175-182, Menlo Park, Calif., 1998. AMI Press).
The FG-GAP domain (Pfam accession number PF01839, INTERPRO entry IPR000413) is typically found in integrins where it is present as a repeat (up to 7 copies) in the extracellular part of the protein. So far, only integrins from animal origin have been well characterised. The consensus sequence for the FG-GAP domain is given in SEQ ID NO: 53:
fgssvaagDlnGDGrpDlvvgaPgadggtdgsvyll,
wherein the capital letters represent the single letter amino acid code for highly conserved amino acids and the other letters represent the single letter amino acid code for less conserved amino acids. The domain often comprises a Phe-Gly-Xn-Gly-Ala-Pro (SEQ ID NO: 288) motif wherein Xn represents a variable number of amino acids. Because this consensus sequence is derived form animal proteins, it does not entirely match with the plant FG-GAP domain sequences. For example, the hexapeptide “Pgadgg” (SEQ ID NO: 289) may not be present in plant FG-GAP domains. Therefore, the term “FG-GAP domain” as used herein encompasses SEQ ID NO: 53 and sequences that have at least 40% sequence similarity to SEQ ID NO: 53, upon alignment of SEQ ID NO: 53 and the corresponding matching sequence, using the Needleman & Wunsch algorithm with a gap opening penalty of 10 and a gap elongation penalty of 0.5.
The FG-GAP domain may also comprise a Ca2+ binding site.
Preferably, the FG-GAP protein also comprises a FDGYLYLI(D/E)G motif 1 (SEQ ID NO: 50). More preferably, the conserved motif 1 is FDGYLYLIDG (SEQ ID NO: 50).
Additionally and/or alternatively, the FG-GAP protein may comprise one or more DGXX(D/E) motifs (conserved motif 2, SEQ ID NO: 51), wherein X may be any amino acid. This conserved motif may be part of a larger motif DXDXDGXX(D/E) (conserved motif 3, SEQ ID NO: 52), wherein X may be any amino acid. Thus, the FG-GAP protein preferably comprises one or more copies of the conserved motif 3.
Alternatively, the homologue of an FG-GAP protein has in increasing order of preference 50%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% overall sequence identity to the amino acid represented by SEQ ID NO: 46, provided that the homologous protein comprises a signal peptide sequence, one or more FG-GAP domains, and a transmembrane domain in the C-terminal half of the protein, and preferably also one or more of the conserved motifs 1, 2 or 3. The overall sequence identity is determined using a global alignment algorithm, such as the Needleman Wunsch algorithm in the program GAP (GCG Wisconsin Package, Accelrys), preferably with default parameters and full-length protein sequences.
The various structural domains in an FG-GAP protein may be identified using specialised databases e.g. SMART (Schultz et al. (1998) Proc. Natl. Acad. Sci. USA 95, 5857-5864; Letunic et al. (2002) Nucleic Acids Res 30, 242-244), InterPro (Mulder et al., (2003) Nucl. Acids. Res. 31, 31 5318), Prosite (Bucher and Balroch (1994), A generalized profile syntax for biomolecular sequences motifs and its function in automatic sequence interpretation. (In) ISMB-94; Proceedings 2nd International Conference on Intelligent Systems for Molecular Biology. Altman R., Brutlag D., Karp P., Lathrop R., Searis D., Eds., pp 53-61, AAAIPress, Menlo Park; Hulo et al., Nucl. Acids. Res. 32:D134-D137, (2004),) or Pfam (Bateman et al., Nucleic Acids Research 30(1):276-280 (2002)).
Methods for the search and identification of FG-GAP homologues would be well within the realm of persons skilled in the art. Such methods comprise comparison of the sequences represented by SEQ ID NO: 45 or 46, in a computer readable format, with sequences that are available in public databases such as MIPS, GenBank or EMBL Nucleotide Sequence Database, using algorithms well known in the art for the alignment or comparison of sequences, such as GAP (Needleman and Wunsch, J. Mol. Biol. 48; 443-453 (1970)), BESTFIT (using the local homology algorithm of Smith and Waterman (Advances in Applied Mathematics 2; 482-489 (1981))), BLAST (Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J., J. Mol. Biol. 215:403-410 (1990)), FASTA and TFASTA (W. R. Pearson and D. J. Lipman Proc. Natl. Acad. Sci. USA 85:2444-2448 (1988)). The software for performing BLAST analysis is publicly available through the National Centre for Biotechnology Information (NCBI).
Examples of proteins falling under the definition of “FG-GAP polypeptide or a homologue thereof” include an Arabidopsis protein (SEQ ID NO: 55) and two rice proteins (SEQ ID NO: 57 and 59). The presence of FG-GAP proteins has also been demonstrated in other plant species of the Magnoliophyta, including Titicum aestivum, Zea mays, Solanum tuberosum, Aquilegia sp., Brassica napus, Citrus sinensis, Asparagus officinalis, Populus sp., Euphorbia esula and also in other plant taxa such as ferns (Ceratoptedis richardii) or in Welwitschia mirabilis. A non-limiting list of examples of ESTs encoding FG-GAP proteins is given in Table 8:











TABLE 8







Species
GenBank accession
SEQ ID NO:









Triticum aestivum
CK207217
16



Zea mays
AY111316
17



Solanum tuberosum
BG598275
18



Aquilegia sp.
DT735817
19



Brassica napus
CX192752
20



Citrus sinensis
CX674859
21



Asparagus officinalis
CV288972
22



Populus sp.
CN520999
23



Populus sp.
CX176799
24



Euphorbia esula
DV130386
25



Ceratopteris richardii
CV736049
26



Welwitschia mirabilis
DT601669
27










The proteins encoded by the genes from which these EST's are derived are also useful for practising the methods of the present invention and fall within the scope of this invention. A person skilled in the art would be able to isolate the full length coding sequence of these genes using standard methods.
The invention furthermore provides an isolated FG-GAP protein selected from the group consisting of:

    
    
        (a) a protein encoded by the nucleic acid of SEQ ID NO: 72;
        (b) a protein comprising a signal sequence, one or more FG-GAP domains and a transmembrane domain located in the C-terminal half of the protein, wherein said protein comprises at least one of SEQ ID NO: 73 to SEQ ID NO: 72;
        (c) an active fragment of an amino acid sequence as defined in (a) or (b), which active fragment comprises a signal sequence, one or more FG-GAP domains and a transmembrane domain located in the C-terminal half of the protein.
    
    


It is to be understood that the term “FG-GAP polypeptide or a homologue thereof” is not to be limited to the sequence represented by SEQ ID NO: 46 or to the homologues listed as SEQ ID NO: 55, 57 and 59, but that any polypeptide meeting the criteria of comprising a signal peptide, one or more FG-GAP domains and a transmembrane domain located in the C-terminal half of the protein, and preferably also one or more of the conserved motifs of SEQ ID NO: 50 to 52; or having at least 50% sequence identity to the sequence of SEQ ID NO: 46, may be suitable for use in the methods of the Invention.
Plant FG-GAP proteins play a role during pollen development (Paxson-Sowders et al. 2001). In dex1 mutant plants, primexine deposition is delayed and significantly reduced. The normal rippling of the plasma membrane and production of spacers observed in wild-type plants is also absent in the mutant. FG-GAP proteins are able to complement this mutation and to restore the normal phenotype.
Alternatively, the activity of an FG-GAP protein or homologue thereof may be assayed by expressing the FG-GAP protein or homologue thereof under control of a constitutive promoter in Oryza sativa, which results in plants with increased aboveground biomass and/or increased seed yield compared to corresponding wild type plants. This increase in seed yield may be measured in several ways, for example as an increase of total seed weight, number of filled seeds or total number of seeds.
An FG-GAP protein or homologue thereof is encoded by an FG-GAP nucleic acid/gene. Therefore the term “FG-GAP nucleic acid/gene” as defined herein is any nucleic acid/gene encoding an FG-GAP protein or a homologue thereof as defined above.
Examples of FG-GAp nucleic acids include but are not limited to those represented by any one of SEQ ID NO: 45, SEQ ID NO: 54, SEQ ID NO: 56 or SEQ ID NO: 58. Examples of partial FG-GAP nucleic acids are listed in Table 8.
The invention also provides an isolated nucleic acid encoding an FG-GAP protein, selected from the group consisting of:

    
    
        (i) the nucleic acid as represented in SEQ ID NO: 72;
        (ii) a nucleic acid encoding a protein as defined in (a) to (c) above;
        (iii) a nucleic acid sequence capable of hybridising (preferably under stringent conditions) with a nucleic acid sequence of (i) or (ii) above, which hybridising sequence preferably encodes a protein comprising a signal peptide, one or more FG-GAP domains and a transmembrane domain located in the C-terminal half of the protein;
        (iv) a nucleic acid which is an allelic variant to the nucleic acid sequences according to (i) to (iii);
        (v) a nucleic acid which is an alternative splice variant to the nucleic acid sequences according to (i) to (iii);
        (vi) a portion of a nucleic acid sequence according to any of (i) to (v) above, which portion preferably encodes a protein comprising a signal peptide, one or more FG-GAP domains and a transmembrane domain located in the C-terminal half of the protein.
    
    


FG-GAP nucleic acids/genes and variants thereof may be suitable in practising the methods of the invention. Variant FG-GAP nucleic acid/genes include portions of an FG-GAP nucleic acid/gene, allelic variants, splice variants and/or nucleic acids capable of hybridising with an FG-GAP nucleic acid/gene.
The term portion as defined herein refers to a piece of DNA encoding a polypeptide comprising a signal peptide, one or more FG-GAP domains and a transmembrane domain located in the C-terminal half of the protein, and preferably also one or more of the conserved motifs of SEQ ID NO: 50 to 52. Preferably, the portion comprises one or more of the conserved motifs defined above. A portion may be prepared, for example, by making one or more deletions to an FG-GAP 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 FG-GAP fragment. Preferably, the portion is a portion of a nucleic acid as represented by any one of SEQ ID NO: 45, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58 or SEQ ID NO: 72. The portion may also be a portion of the coding sequences from which the sequences of Table 8 are derived. Most preferably the portion of a nucleic acid is as represented by SEQ ID NO: 45.
Another variant of an FG-GAP nucleic acid/gene is a nucleic acid capable of hybridising under reduced stringency conditions, preferably under stringent conditions, with an FG-GAP nucleic acid/gene as hereinbefore defined, which hybridising sequence encodes a polypeptide comprising a signal peptide, one or more FG-GAP domains and a transmembrane domain located in the C-terminal half of the protein, and preferably also one or more of the conserved motifs of SEQ ID NO: 50 to 52.
Preferably, the hybridising sequence is one that is capable of hybridising to a nucleic acid as represented by SEQ ID NO: 45, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58 or SEQ ID NO: 72, or to a portion of any of the aforementioned sequences, including the EST's listed in Table 8. Most preferably the hybridising sequence is capable of hybridising to SEQ ID NO: 45. The term “hybridisation” is as defined in the section headed “Definitions”.
The FG-GAP nucleic add or variant thereof may be derived from any natural or artificial source. The nucleic acid/gene or variant thereof may be isolated from a microbial source, such as yeast or fungi, or from a plant, algal or animal (including human) source. This nucleic acid may be modified from its native form in composition and/or genomic environment through deliberate human manipulation. The nucleic acid is preferably of plant origin, whether from the same plant species (for example to the one in which it is to be introduced) or whether from a different plant species. The nucleic acid may be isolated from a dicotyledonous species, preferably from the family Brassicaceae, further preferably from Arabidopsis thaliana. More preferably, the FG-GAP nucleic acid is isolated from Arabidopsis thaliana and is represented by SEQ ID NO: 45, and the FG-GAP amino acid sequence is as represented by SEQ ID NO: 46.
The expression of a nucleic acid encoding an FG-GAP polypeptide or a homologue thereof may be modulated by introducing a genetic modification (preferably in the locus of an FG-GAP 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 down stream 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, transposon mutagenesis, directed evolution and homologous recombination or by introducing and expressing in a plant a nucleic acid encoding an FG-GAP polypeptide or a homologue thereof. These methods are defined in the section headed “Definitions”. Following introduction of the genetic modification, there follows a step of selecting for modified expression of a nucleic acid encoding an FG-GAP polypeptide or a homologue thereof, which modification in expression gives plants having increased yield.
T-DNA activation, TILLING, site-directed mutagenesis, transposon mutagenesis and directed evolution are examples of technologies that enable the generation of novel alleles and FG-GAP variants.
A preferred method for introducing a genetic modification (which in this case need not be in the locus of an FG-GAP gene) is to introduce and express in a plant a nucleic acid encoding an FG-GAP polypeptide or a homologue thereof, as defined above. The nucleic acid to be introduced into a plant may be a full-length nucleic acid or may be a portion or a hybridising sequence as hereinbefore defined. Preferably, the plant in which the genetic modification is introduced is not a dex1 mutant plant, in which the DEX1 gene is not functional (Paxson-Sowders et al. 2001).
“Homologues” of a protein are defined in the section headed “Definitions”. The FG-GAP polypeptide or homologue thereof may be a derivative, as defined in the “Definitions” section.
The FG-GAP polypeptide or homologue thereof may be encoded by an alternative splice variant of an FG-GAP nucleic acid/gene. The term “alternative splice variant” is as defined herein. Preferred are splice variants of the nucleic acid encoding a polypeptide comprising a signal peptide, one or more FG-GAP domains and a transmembrane domain located in the C-terminal half of the protein, and preferably also one or more of the conserved motifs of SEQ ID NO: 50 to 52. Further preferred are splice variants represented by SEQ ID NO: 45, SEQ ID NO: 54, SEQ ID NO: 56 or SEQ ID NO: 58, or a splice variant of the nucleic acid represented by SEQ ID NO: 72, or a splice variant of one of the genes from which the sequences in Table 8 are derived. Most preferred is the splice variant represented by SEQ ID NO: 45.
The homologue may also be encoded by an allelic variant of a nucleic acid encoding an FG-GAP polypeptide or a homologue thereof, preferably an allelic variant of a nucleic acid encoding a polypeptide comprising a signal peptide, one or more FG-GAP domains and a transmembrane domain located in the C-terminal half of the protein, and preferably also one or more of the conserved motifs of SEQ ID NO: 50 to 52. Further preferably, the allelic variant encoding the FG-GAP polypeptide is represented by any one of SEQ ID NO: 45, SEQ ID NO: 54, SEQ ID NO: 56 or SEQ ID NO: 58. Most preferably, the allelic variant encoding the FG-GAP polypeptide is as represented by SEQ ID NO: 45. Allelic variants are defined in the “Defintions” section.
According to a preferred aspect of the present invention, modulated expression of the FG-GAP nucleic acid or variant thereof is envisaged. Preferably, the modulated expression is overexpression. Methods for overexpression of genes or gene products are well documented in the art and include, for example, overexpression driven by appropriate promoters, the use of transcription enhancers or translation enhancers. Isolated nucleic acids which serve as promoter or enhancer elements may be introduced in an appropriate position (typically upstream) of a non-heterologous form of a polynucleotide so as to upregulate expression of an FG-GAP nucleic acid or variant thereof. For example, endogenous promoters may be altered in vivo by mutation, deletion, and/or substitution (see, Kmiec, U.S. Pat. No. 5,565,350; Zarling et al., PCT/US93/03868), or isolated promoters may be introduced into a plant cell in the proper orientation and distance from a gene of the present invention so as to control the expression of the gene. Methods for reducing the expression of genes or gene products are also 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 may be derived from the natural gene, from a variety of other plant genes, or from T-DNA. The 3′ end sequence to be added may be derived from, for example, the nopaline synthase or octopine synthase genes, or alternatively from another plant gene, or less preferably from any other eukaryotic gene.
An intron sequence may also be added to the 5′ untranslated region or the coding sequence of the partial coding sequence to increase the amount of the mature message that accumulates in the cytosol. Inclusion of a spliceable intron in the transcription unit in both plant and animal expression constructs has been shown to increase gene expression at both the mRNA and protein levels up to 1000-fold, Buchman and Berg, Mol. Cell. biol. 8:4395-4405 (1988); Callis et al., Genes Dev. 1:1183-1200 (1987). Such intron enhancement of gene expression is typically greatest when placed near the 5′ end of the transcription unit. Use of the maize introns Adh1-S intron 1, 2, and 6, the Bronze-1 intron are known in the art. See generally, The Maize Handbook, Chapter 116, Freeling and Walbot, Eds., Springer, N.Y. (1994).
The invention also provides genetic constructs and vectors to facilitate introduction and/or expression of the nucleotide sequences useful in the methods according to the invention.
Therefore, there is provided a gene construct comprising:


    
    
        (i) an FG-GAP 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;

with the proviso that the gene construct is not a pPZP-type gene construct as described by Hajdukiewicz et al. (Plant Mol. Biol. 25, 989-994) and Paxson-Sowders (2001).

    
    


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 an FG-GAP 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 defined in the section headed “Definitions”.
Advantageously, any type of promoter may be used to drive expression of the nucleic acid sequence. Preferably, the FG-GAP nucleic acid or functional variant thereof is operably linked to a constitutive promoter. The term “constitutive” is as defined herein. Preferably, the constitutive promoter capable of preferentially expressing the nucleic acid throughout the plant has a comparable expression profile to a GOS2 promoter. More preferably, the constitutive promoter has the same expression profile as the rice GOS2 promoter, most preferably, the promoter capable of preferentially expressing the nucleic acid throughout the plant is the GOS2 promoter from rice (nucleotides 1 to 2193 of the sequence represented in SEQ ID NO: 49). It should be clear that the applicability of the present invention is not restricted to the FG-GAP nucleic acid represented by SEQ ID NO: 45, nor is the applicability of the invention restricted to expression of an FG-GAP nucleic acid when driven by a GOS2 promoter. Examples of other constitutive promoters that may also be used to drive expression of an FG-GAP nucleic acid are shown in Table 3 in the “Definition” section.
Optionally, one or more terminator sequences may also be used in the construct introduced into a plant. The term “terminator” being defined in the “Definitions” section.
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 defined in the “Definitions” section herein.
The present invention also encompasses plants obtainable by the methods according to the present invention. The present invention therefore provides plants obtainable by the method according to the present invention, which plants have introduced therein an FG-GAP nucleic acid or variant thereof, as defined above.
The invention also provides a method for the production of transgenic plants having increased yield, comprising introduction and expression in a plant of an FG-GAP nucleic acid or a variant thereof as defined above.
More specifically, the present invention provides a method for the production of transgenic plants having increased yield, which method comprises:

    
    
        (i) introducing and expressing in a plant or plant cell an FG-GAP nucleic acid or variant thereof; and
        (ii) cultivating the plant cell under conditions promoting plant growth and development.
    
    


The nucleic acid may be introduced directly into a plant cell or into the plant itself (including introduction into a tissue, organ or any other part of a plant). According to a preferred feature of the present invention, the nucleic acid is preferably introduced into a plant by transformation.
The term “transformation” is as defined in the “Definitions” section.
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 FG-GAP 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, oil, fat and fatty acids, starch and proteins.
The present invention also encompasses use of FG-GAP nucleic acids or variants thereof and use of FG-GAP polypeptides or homologues thereof.
One such use relates to improving the growth characteristics of plants, in particular in improving yield, especially seed yield. The seed yield may include one or more of the following: increased total weight of seeds, increased number of filled seeds and increased total number of seeds.
FG-GAP nucleic acids or variants thereof, or FG-GAP polypeptides or homologues thereof may find use in breeding programmes in which a DNA marker is identified which may be genetically linked to an FG-GAP gene or variant thereof. The FG-GAP nucleic acids/genes or variants thereof, or FG-GAP 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 FG-GAP gene or variant thereof may, for example, be a nucleic acid as represented by any one of SEQ ID NO: 45, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, and SEQ ID NO: 72, or genes from which the sequences listed in Table 8 were derived.
Allelic variants of an FG-GAP 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: 45, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, and SEQ ID NO: 72, or of one of the coding sequences from which the sequences listed in Table 8 were derived. 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.
An FG-GAP 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 FG-GAP nucleic acids or variants thereof requires only a nucleic acid sequence of at least 15 nucleotides in length. The FG-GAP nucleic acids or variants thereof may be used as restriction fragment length polymorphism (RFLP) markers. Southern blots (Sambrook J, Fritsch E F and Maniatis T (1989) Molecular Cloning, A Laboratory Manual) of restriction-digested plant genomic DNA may be probed with the FG-GAP 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 FG-GAP nucleic acid or variant thereof in the genetic map previously obtained using this population (Botstein et al. (1980) Am. J. Hum. Genet. 32: 314-331).
The production and use of plant gene-derived probes for use in genetic mapping is described in Bernatzky and Tanksley (Plant Mol. Biol. Reporter 4: 37-41, 1986). Numerous publications describe genetic mapping of specific cDNA clones using the methodology outlined above or variations thereof. For example, F2 intercross populations, backcross populations, randomly mated populations, near isogenic lines, and other sets of individuals may be used for mapping. Such methodologies are well known to those skilled in the art.
The nucleic acid probes may also be used for physical mapping (i.e., placement of sequences on physical maps; see Hoheisel et al. In: 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 favour 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. 17:6795-4807). 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 advantageous growth characteristics may also be combined with other economically advantageous traits, such as further yield-enhancing traits, tolerance to various stresses, traits modifying various architectural features and/or biochemical and/or physiological features.
Detailed Description CYP90B
The term “CYP90B polypeptide or homologue thereof” as defined herein refers to a polypeptide comprising the following: (a) CYP domains A to D; (b) an N-terminal hydrophobic anchor domain; (c) a transition domain; and (d) within the A domain, the consensus sequence Phe-Ala-Gly-His-Glu-Thr-Ser-Ser (SEQ ID NO: 290), allowing for one amino acid change at any position.
Furthermore, the CYP90B polypeptide or homologue thereof may additionally comprise (i) a sequence with more than 50% identity to SEQ ID NO: 78 and (ii) steroid 22-alpha hydroxylase enzymatic activity.
Examples of a CYP90B polypeptide as defined hereinabove are given in Table 9a herein.
A CYP90B polypeptide or homologue thereof is encoded by a CYP90B nucleic acid/gene. Therefore the term “CYP90B nucleic acid/gene” as defined herein is any nucleic acid/gene encoding a CYP90B polypeptide or a homologue thereof as defined hereinabove.
The various structural domains found in the CYP superfamily of proteins, including in CYP90B polypeptides of the present invention, are well known in the art and may be identified using general databases e.g. SMART (Schultz et al. (1998) Proc. Natl. Acad. Sci. USA 95, 5857-5864; Letunic et al. (2002) Nucleic Acids Res 30, 242-244; smart.embl-heidelberg.de/), InterPro (Mulder et al., (2003) Nucl. Acids. Res. 31, 315-318; at webpage ebi.ac.uk/interpro/), Prosite (Bucher and Bairoch (1994), A generalized profile syntax for biomolecular sequences motifs and its function in automatic sequence interpretation, in ISMB-94; Proceedings 2nd International Conference on Intelligent Systems for Molecular Biology. Altman R., Brutlag D., Karp P., Lathrop R., Searls D., Eds., pp 53-61, AAAI Press, Menlo Park; Hulo et al., Nucl. Acids. Res. 32:D134-D137, (2004), at webpage expasy.org/prosite/) or Pfam (Bateman et al., Nucleic Acids Research 30(1):276-280 (2002), at webpage sanger.ac.uk/Software/Pfam/).
Specialized databases may also be searched at arabidopsis-P450.biotec.uiuc.edu/cgi-bin/p450.pl for Arabidopsis, or more generally on the CYP Homepage drnelson.utmem.edu/CytochromeP450.html. Typical structural domains found in CYP may be the four A to D domains as originally described by Kalb & Loper ((1988) Proc Natl Acad Sci 85: 7221-7225). The A domain (also called helix I) comprises the consensus sequence Ala/Gly-Gly-X-Asp/Glu-Thr-Thr/Ser (SEQ ID NO: 291), and is proposed to bind dioxygen. The B domain is the steroid binding domain. The D domain corresponds to the heme binding domain and comprises the most characteristic CYP amino acid consensus sequence (Phe-X-X-Gly-X-Arg-X-Cys-X-Gly) (SEQ ID NO: 292) (FIGS. 10 and 13).
The presence of consensus sequences may be identified using methods for the alignment of sequences for comparison as described hereinabove. In some instances, the default parameters may be adjusted to modify the stringency of the search. For example using BLAST, the statistical significance threshold (called “expect” value) for reporting matches against database sequences may be increased to show less stringent matches. This way, short nearly exact matches may be identified. The consensus sequence Phe-Ala-Gly-His-Glu-Thr-Ser-Ser (SEQ ID NO: 290) within the A domain of the CYP90B polypeptide (comprising the consensus sequence Ala/Gly-Gly-X-Asp/Glu-Thr-Thr/Ser (SEQ ID NO: 293) as defined hereinabove) as defined herein may be identified in this manner, as a person skilled in the art would be well aware of.
Another domain identified in CYP P450 proteins, and in particular in the CYP90B polypeptide of the invention, may be the anchor domain at the N terminus of the protein for membrane-targeting, rich in hydrophobic residues such as Leu, Ile, Val, Phe and Ala. The N-terminal anchor domain is typically between 20 to 40 amino acids long, but may be shorter (down to 10 amino acids) or longer (up to 100 amino acids). The N-terminal anchor domain is separated from the rest of the protein (globular domain) by a transition domain comprising a cluster of basic residues (at least two, either Lys or Arg, called the halt-transfer signal) preceding a proline cluster that forms a hinge between the abovementioned anchor domain and the globular domain of the protein. A typical consensus sequence for the transition domain is Lys/Arg-Lys/Arg-(X)3-9-Pro-Pro-Gly (SEQ ID NO: 295) (FIGS. 10 and 13). Such a consensus sequence may be identified as mentioned hereinabove.
The presence of an N-terminal hydrophobic anchor domain may readily be identified. 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, in particular the ProtParam tool (Gasteiger E et at (2003) ExPASy: the proteomics server for in-depth protein knowledge and analysis. Nucleic Acids Res 31:3784-3788). 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 addition of the averages of Leu (L), Ile (1), Val (V), Phe (F) and Ala (A) is of 34.04%. As an example, the N-terminal hydrophobic anchor domain of SEQ ID NO: 78 contains 62.5% of the same hydrophobic residues. As defined herein, a N-terminal hydrophobic anchor domain has a hydrophobic amino acid content (in % terms) above that found in the average amino acid composition (in % terms) of the proteins in the Swiss-Prot Protein Sequence database.
Special softwares such as ProtScale (Gasteiger et al. (2005) Protein Identification and Analysis Tools on the ExPASy Server. In John M. Walker, ed: The Proteomics Protocols Handbook, Humana Press pp. 571-607) compute and represent the profile produced by any amino acid scale on a selected protein. An amino acid scale is defined by a numerical value assigned to each type of amino acid. The most frequently used scales are the hydrophobicity or hydrophilicity scales and the secondary structure conformational parameters scales. One of the most frequently used hydrophobicity amino acid scale has been produced by Kyte & Doolittle ((1982) J. Mol. Biol. 157:105-132), in which hydrophobic amino acids have been attributed a positive number, and hydrophilic amino acids a negative number. For example, the ProtScale output profile for hydrophobicity of the CYP90B polypeptide of the invention clearly shows that approximately the first N-terminal 34 amino acids represent a hydrophobic domain, as these are located above the zero delimiting line (FIG. 12). This region corresponds to the N-terminal anchor domain. A person skilled in the art would be well aware of such analyses.
CYP90B 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 Mol 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 Mol 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 CYP90B comprising comprising a sequence with more than 50% identity to SEQ ID NO: 78 may readily be identified using, for example, the ClustalW multiple sequence alignment algorithm (version 1.83) available at http://clustalw.genomejp/sit-bin/nph-ClustalW, with the default pairwise alignment parameters, and a scoring method in percentage. Minor manual editing may be performed to optimise alignment between conserved motifs, as would be apparent to a person skilled in the art.
Examples of CYP90B polypeptides or homologues thereof (encoded by polynucleotide sequence accession number in parenthesis) are given in Table 9a. Table 9b provides for partial CYP90B sequences encoding partial CYP90B open reading frames (ORF).







TABLE 9a







Examples of CYP90B homologues













NCBI or TIGR







nucleotide

Translated



accession
Nucleotide
polypeptide
Insert


Name
number
SEQ ID NO
SEQ ID NO
status
Source















Orysa_CYP90B
AB206579.1
77
78
Full length
Oryza sativa






ORF


Arath_CYP90B1
NM_114926.2
79
80
Full length
Arabidopsis






ORF
thaliana


Sacof_CYP90B**
CA092707.1
81
82
Full length
Saccharum



CF574030.1


ORF
officinarum



CA217329.1


Allce_CYP90B
TC2113
83
84
Full length
Allium cepa






ORF


Zinel_CYP90B
AB231155
85
86
Full length
Zinnia elegans






ORF


Medtr_CYP90B*
AC147964.10
87
88
Full length
Medicago






ORF
trunculata


Poptr_CYP90B′′**
CK090847.1
89
90
Full length
Populus



CV280598.1


ORF
trichocarpa



DT503533.1
















TABLE 9b







Examples of CYP90B with a partial open reading frame (ORF)













NCBI or TIGR







nucleotide
Nucleotide
Translated



accession
SEQ
polypeptide
Insert


Name
number
ID NO
SEQ ID NO
status
Source















Aqufo_CYP90B**
DR940523.1
91
92
Partial
Aquilegia



DR940522.1


ORF
formosa x







Aquilegia







pubescens


Triae_CYP90B
BQ620306.1
93
94
Partial
Triticum


5′ end



ORF
aestivum


Triae_CYP90B
BQ619714.1
95
96
Partial
Triticum


3′ end**
CA715360.1


ORF
aestivum


Eupes_CYP90B
DV141872.1
97
98
Partial
Euphorbia esula






ORF


Goshi_CYP90B
CO125422
99
100
Partial
Gossypium


5′ end**
DT568185.1


ORF
hirsutum


Lyces_CYP90B
BF050501
101
102
Partial
Lycopersicon


5′ end**
AW221826.1


ORF
esculentum



BM409833


Soltu_CYP90B
BQ045917
103
104
Partial
Solanum


5′ end**
BQ114367


ORF
tuberosum


Soltu_CYP90B
BQ114368
105
106
Partial
Solanum


3′ end**



ORF
tuberosum





*Manual splicing from genomic clone


**Contig compiled from several EST accessions (main ones shown); EST sequencing quality being usually lower, a few nucleic acid substitutions may be expected.






It is to be understood that sequences falling under the definition of “CYP90B polypeptide or homologue thereof” are not to be limited to the sequences represented by SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88 or SEQ ID NO: 90, but that any polypeptide comprising the following: (a) CYP domains A to D; (b) an N-terminal hydrophobic anchor domain; (c) a transition domain; and (d) within the A domain, the consensus sequence Phe-Ala-Gly-His-Glu-Thr-Ser-Ser (SEQ ID NO: 290), allowing for one amino acid change at any position may be suitable for use in performance of the invention.
The sequences falling under the definition of “CYP90B polypeptide or homologue thereof” may additionally comprise (i) a sequence with more than 50% identity to SEQ ID NO: 78 and (ii) steroid 22-alpha hydroxylase enzymatic activity.
CYP90B polypeptides or homologues thereof have 22-alpha hydroxylase enzymatic activity, which may be determined by complementation testing using plants having a mutation in DWF4. Such mutant plants are described in Arabidopsis (dwf4 mutant) by Choe et al. ((1998) Plant Cell 10:231-243) and in rice (Tos2091 mutant) by Tanaka et al (US2004/0060079). The size of these mutant plants is several fold smaller than that of their corresponding wild types, i.e., the mutant plants are super-dwarfed. The isolated polypeptide is placed under the control of a promoter capable of expressing this polypeptide in plants, in a recombinant DNA vector suitable for plant transformation. The mutant plants are then transformed with this vector, using techniques that are well known in the art. If the transformed plants no longer display the super-dwarfed phenotype that is indicative that the isolated polypeptide is capable displaying 22-alpha hydroxylase enzymatic activity. Such a polypeptide may be suitable for use in performance of the methods of the invention.
Examples of CYP90B nucleic acids include but are not limited to those represented by any one of 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 SEQ ID NO: 0.89. CYP90B nucleic acids/genes and variants thereof may be suitable in practising the methods of the invention. Variants of CYP90B nucleic acid/genes include portions of a CYP90B nucleic acid/gene and/or nucleic acids capable of hybridising with a CYP90B nucleic acid/gene.
The term portion as defined herein refers to a piece of DNA encoding a polypeptide comprising the following: (a) CYP P450 domains A to D; (b) an N-terminal hydrophobic anchor domain; (c) a transition domain; and (d) within the A domain, the consensus sequence Phe-Ala-Gly-His-Glu-Thr-Ser-Ser, allowing for one amino acid change at any position. A portion may be prepared, for example, by making one or more deletions to a CYP90B 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 CYP90B portion. Preferably, the portion is a portion of a nucleic acid as represented by any one of SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81,
SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87 and SEQ ID NO: 89. Most preferably the portion is a portion of a nucleic acid as represented by SEQ ID NO: 77. Another variant of a CYP90B nucleic acid/gene is a nucleic acid capable of hybridising under reduced stringency conditions, preferably under stringent conditions, with a CYP90B nucleic acid/gene as hereinbefore defined, which hybridising sequence encodes a polypeptide comprising the following: (a) CYP domains A to D; (b) an N-terminal hydrophobic anchor domain; (c) a transition domain; and (d) within the A domain, the consensus sequence Phe-AIa-Gly-His-Glu-Thr-Ser-Ser, allowing for one amino acid change at any position. Preferably, the hybridising sequence is one that is capable of hybridising to a nucleic acid as represented by any one of SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87 and SEQ ID NO: 89, or to a portion of any of the aforementioned sequences as defined hereinabove. Most preferably the hybridising sequence is one that is capable of hybridising to a nucleic acid as represented by SEQ ID NO: 77. The term “hybridisation” is as defined herein in the “Definitions” section.
The CYP90B nucleic acid or variant thereof may be derived from any natural or artificial source. The nucleic acid/gene or variant thereof may be isolated from a microbial source, such as yeast or fungi, or from a plant, algae or animal (including human) source. This nucleic acid may be modified from its native form in composition and/or genomic environment through deliberate human manipulation. The nucleic acid is preferably of plant origin, whether from the same plant species (for example to the one in which it is to be introduced) or whether from a different plant species. The nucleic acid may be isolated from a monocotyledonous species, preferably from the family Poaceae, further preferably from Oryza genus, most preferably from Oryza saliva. More preferably, the CYP90B nucleic acid isolated from Oryza sativa is represented by SEQ ID NO: 77 and the CYP90B amino acid sequence is as represented by SEQ ID NO: 78.
The invention furthermore provides an isolated CYP90B protein selected from the group consisting of:
a protein encoded by the nucleic acid of SEQ ID NO: 117;
a protein comprising comprising the following: (i) CYP domains A to D; (ii) an N-terminal hydrophobic anchor domain; (iii) a transition domain; and (iv) within the A domain, the consensus sequence Phe-Ala-Gly-His-Glu-Thr-Ser-Ser (SEQ ID NO: 290), allowing for one amino acid change at any position, and having in increasing order of preference at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence of SEQ ID NO: 118.
The invention also provides an isolated nucleic acid encoding a CYP90B protein, selected from the group consisting of:
a nucleic acid as represented by SEQ ID NO: 117;
a nucleic acid encoding a protein as defined in (a) and (b) above;
a nucleic acid having in increasing order of preference at least 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to the nucleic acid represented by SEQ ID NO: 117;
a nucleic acid sequence capable of hybridising under stringent conditions with a nucleic acid sequence of (i) to (iii) above, which hybridising sequence encodes a protein comprising (a) CYP domains A to D; (b) an N-terminal hydrophobic anchor domain; (c) a transition domain; and (d) within the A domain, the consensus sequence Phe-Ala-Gly-His-Glu-Thr-Ser-Ser (SEQ ID NO: 290), allowing for one amino acid change at any position, and having in increasing order of preference at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more to the amino acid sequence of SEQ ID NO: 118;

    
    
        a nucleic acid which is an allelic variant or a splice variant of the nucleic acid sequences according to (i) to (iv);
        a portion of a nucleic acid sequence according to any of (i) to (v) above, which portion encodes a protein comprising: (i) CYP domains A to D; (ii) an N-terminal hydrophobic anchor domain; (iii) a transition domain; and (iv) within the A domain, the consensus sequence Phe-Ala-Gly-His-Glu-Thr-Ser-Ser (SEQ ID NO: 290), allowing for one amino acid change at any position, and having in increasing order of preference at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% to the amino acid sequence of SEQ ID NO: 118.
    
    


Furthermore, the CYP90B polypeptide or homologue thereof may additionally comprise (i) a sequence with more than 50% identity to SEQ ID NO: 78 and (ii) steroid 22-alpha hydroxylase enzymatic activity.
The expression of a nucleic acid encoding a CYP90B polypeptide or a homologue thereof may be increased non-constitutive by introducing a genetic modification (preferably in the locus of a CYP90B 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 CYP90B polypeptide or a homologue thereof. The aforementioned methods are defined in the “Definitions” section. Following introduction of the genetic modification, there follows a step of selecting for increased non-constitutive expression of a nucleic acid encoding a CYP90B polypeptide or a homologue thereof, which increase in non-constitutive expression gives plants having increased yield.
T-DNA activation, TILLING, site-directed mutagenesis and directed evolution are examples of technologies that enable the generation of novel alleles and CYP90B variants.
A preferred method for introducing a genetic modification (which in this case need not be in the locus of a CYP90B gene) is to introduce and express in a plant a nucleic acid encoding a CYP90B polypeptide or a homologue thereof. A CYP90B polypeptide or a homologue thereof is defined as polypeptide comprising comprising the following: (a) CYP domains A to D; (b) an N-terminal hydrophobic anchor domain; (c) a transition domain; and (d) within the A domain, the consensus sequence Phe-Ala-Gly-His-Glu-Thr-Ser-Ser (SEQ ID NO: 290), allowing for one amino acid change at any position. The nucleic acid to be introduced into a plant may be a full-length nucleic acid or may be a portion or a hybridising sequence as hereinbefore defined. Furthermore, the nucleic acid encoding a CYP90B polypeptide or a homologue thereof may additionally comprise (i) a sequence with more than 50% identity to SEQ ID NO: 78 and (ii) steroid 22-alpha hydroxylase enzymatic activity.
“Homologues” of a protein are defined herein in the “Definitions” section. The CYP90B polypeptide or homologue thereof may be a derivative, as defined in the “Definitions” section.
The CYP90B polypeptide or homologue thereof may be encoded by an alternative splice variant of a CYP90B nucleic acid/gene. The term “alternative splice variant” is defined in the “Definitions” section. Preferred splice variants are splice variants of the nucleic acid encoding a polypeptide comprising the following: (a) CYP domains A to D; (b) an N-terminal hydrophobic anchor domain; (c) a transition domain; and (d) within the A domain, the consensus sequence Phe-Ala-Gly-His-Glu-Thr-Ser-Ser (SEQ ID NO: 290), allowing for one amino acid change at any position. Additionally, the CYP90B polypeptide or a homologue thereof may additionally comprise (i) a sequence with more than 50% identity to SEQ ID NO: 78 and (ii) steroid 22-alpha hydroxylase enzymatic activity. Further preferred are splice variants of nucleic acid sequences represented by SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87 and SEQ ID NO: 89. Most preferred is a splice variant of a nucleic acid sequence as represented by SEQ ID NO: 77.
The homologue may also be encoded by an allelic variant of a nucleic acid encoding a CYP90B polypeptide or a homologue thereof, preferably an allelic variant of the nucleic acid encoding a polypeptide comprising the following: (a) CYP domains A to D; (b) an N-terminal hydrophobic anchor domain; (c) a transition domain; and (d) within the A domain, the consensus sequence Phe-Ala-Gly-His-Glu-Thr-Ser-Ser (SEQ ID NO: 290), allowing for one amino acid change at any position. Additionally, the CYP90B polypeptide or a homologue thereof may additionally comprise (i) a sequence with more than 50% identity to SEQ ID NO: 78 and (ii) steroid 22-alpha hydroxylase enzymatic activity. Further preferred are allelic variants of nucleic acid sequences represented by SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87 and SEQ ID NO: 89. Most preferred is an allelic variant of a nucleic acid sequence as represented by SEQ ID NO: 77. Allelic variants are also defined in the “Definitions” section.
According to a preferred aspect of the present invention, increased non-constitutive expression of the CYP90B nucleic acid or variant thereof is envisaged. 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 CYP90B nucleic acid or variant thereof. For example, endogenous promoters may be altered in vivo by mutation, deletion, and/or substitution (see, Kmiec, U.S. Pat. No. 5,565,350; Zarling et al., PCT/US93/03868), or isolated promoters may be introduced into a plant cell in the proper orientation and distance from a gene of the present invention so as to control the expression of the gene. 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 3′ end sequence to be added may be derived from, for example, the nopaline synthase or octopine synthase genes, or alternatively from another plant gene, or less preferably from any other eukaryotic gene.
An intron sequence may also be added to the 5′ untranslated region or the coding sequence of the partial coding sequence to increase the amount of the mature message that accumulates in the cytosol. Inclusion of a spliceable intron in the transcription unit in both plant and animal expression constructs has been shown to increase gene expression at both the mRNA and protein levels up to 1000-fold (Buchman and Berg (1988) Mol. Cell. biol. 8: 4395-4405; Callis et al. (1987) Genes Dev 1:1183-1200). Such intron enhancement of gene expression is typically greatest when placed near the 5′ end of the transcription unit. Use of the maize introns Adh1-S intron 1, 2, and 6, the Bronze-1 intron are known in the art. See generally, The Maize Handbook, Chapter 116, Freeling and Walbot, Eds., Springer, N.Y. (1994).
The invention also provides genetic constructs and vectors to facilitate introduction and/or expression of the nucleotide sequences useful in the methods according to the invention.
Therefore, there is provided a gene construct comprising:

    
    
        (i) A CYP90B nucleic acid or variant thereof, as defined hereinabove;
        (ii) One or more control sequences capable of driving non-constitutive expression of the nucleic acid sequence of (i); and optionally
        (iii) A transcription termination sequence.
    
    


Constructs useful in the methods according to the present invention may be constructed using recombinant DNA technology well known to persons skilled in the art. The gene constructs may be inserted into vectors, which may be commercially available, suitable for transforming into plants and suitable for expression of the gene of interest in the transformed cells. The invention therefore provides use of a gene construct as defined hereinabove in the methods of the invention.
Plants are transformed with a vector comprising the sequence of interest (i.e., a nucleic acid encoding a CYP90B 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 defined in the “Definitions” section.
Advantageously, any non-constitutive type of promoter may be used to drive expression of the nucleic acid sequence. The non-constitutive 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 non-constitutive 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”.
According to the methods of the invention, the CYP90B nucleic acid or variant thereof is operably linked to a non-constitutive promoter. A non-constitutive promoter is transcriptionally active only during some phases of plant growth and development and is not ubiquitously expressed. The non-constitutive promoter may be for example a seed-specific promoter, or a root-specific promoter. The seed specific promoter may be an endosperm-specific and/or embryo/aleurone-specific promoter, i.e., transcriptionally active in the seed endosperm and/or seed embryo and aleurone, respectively. The endosperm-specific promoter is preferably a seed-storage protein promoter, further preferably the endosperm-specific promoter is a prolamin promoter, more preferably the endosperm-specific promoter is a rice RP6 prolamin promoter, yet more preferably the endosperm-specific promoter is represented by a nucleic acid sequence substantially similar to SEQ ID NO: 109, most preferably the endosperm-specific promoter is as represented by SEQ ID NO: 109. The embryo/aleurone-specific promoter is preferably a seed-storage protein promoter, further preferably the embryo/aleurone-specific promoter is an oleosin promoter, more preferably the embryo/aleurone-specific promoter is a rice oleosin 18 kDa promoter, yet more preferably the embryo/aleurone-specific promoter is represented by a nucleic acid sequence substantially similar to SEQ ID NO: 110, most preferably the embryo/aleurone-specific promoter is as represented by SEQ ID NO: 110. The root-specific promoter is preferably an Rcc3 promoter, the root-specific promoter is preferably a rice Rcc3 promoter (Xu et al. (1995) Plant Mol Biol 27(2):23748).
It should be clear that the applicability of the present invention is not restricted to the CYP90B nucleic acid represented by SEQ ID NO: 77, nor is the applicability of the invention restricted to expression of a CYP90B nucleic acid when driven by a RP6 prolamin or 18 kDa oleosin promoter. Examples of other non-constitutive promoters that may also be used to perform the methods of the invention are shown in Table 4 in the “Definitions” section.
In contrast to the above-described promoters, a constitutive promoter is transcriptionally active during most phases of plant growth and development and is substantially ubiquitously expressed in the plant. Such constitutive promoters are to be excluded for performance of the methods of the invention. Examples of such promoters may also be found in the “Definitions” section (see Table 3).
Optionally, one or more terminator sequences may also be used in the construct introduced into a plant. The term “terminator” is defined in the “Definitions” section.
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 cosrmid 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 defined in the “Definitions” section.
In a preferred embodiment, there is provided a gene construct comprising:

    
    
        (i) A CYP90B nucleic acid or variant thereof, as defined hereinabove;
        (ii) A promoter capable of driving non-constitutive expression of the nucleic add sequence of (i); and optionally
        (iii) A transcription termination sequence.
    
    


The non-constitutive promoter is preferably a seed-specific promoter. The seed specific promoter may be an endosperm-specific and/or embryo/aleurone-specific promoter, i.e., transcriptionally active in the seed endosperm and/or seed embryo and aleurone, respectively. The endosperm-specific promoter is preferably a seed-storage protein promoter, further preferably the endosperm-specific promoter is a prolamin promoter, more preferably the endosperm-specific promoter is a rice RP6 prolamin promoter, more preferably the endosperm-specific promoter is represented by a nucleic acid sequence substantially similar to SEQ ID NO: 109, most preferably the endosperm-specific promoter is as represented by SEQ ID NO: 109. The embryo/aleurone-specific promoter is preferably a seed-storage protein promoter, further preferably the embryo/aleurone-specific promoter is an oleosin promoter, more preferably the embryo/aleurone-specific promoter is a rice oleosin 18 kDa promoter, more preferably the embryo/aleurone-specific promoter is represented by a nucleic acid sequence substantially similar to SEQ ID NO: 110, most preferably the embryo/aleurone-specific promoter is as represented by SEQ ID NO: 110. The invention further provides use of a construct as defined hereinabove in the methods of the invention.
The present invention also encompasses plants obtainable by the methods according to the present invention. The present invention therefore provides plants, plant parts or plant cells thereof obtainable by the method according to the present invention, which plants or parts or cells thereof comprise a transgene CYP90B nucleic acid or variant thereof.
The invention also provides a method for the production of transgenic plants having increased yield relative to suitable control plants comprising introduction and non-constitutive expression in a plant of a CYP90B nucleic acid or a variant thereof.
More specifically, the present invention provides a method for the production of transgenic plants having increased yield which method comprises:

    
    
        (i) introducing and expressing non-constitutively in a plant, plant part or plant cell a CYP90B nucleic acid or variant thereof; and
        (ii) cultivating the plant cell under conditions promoting plant growth and development.
    
    


The nucleic acid may be introduced directly into a plant cell or into the plant itself (including introduction into a tissue, organ or any other part of a plant). According to a preferred feature of the present invention, the nucleic acid is preferably introduced into a plant by transformation.
The term “transformation” is as defined in the “Definitions” section.
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 CYP90B nucleic acid or variant thereof, non-constitutively expressed. 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, stems, rhizomes, tubers and bulbs. The invention furthermore relates to products derived from a harvestable part of such a plant, such as dry pellets or powders, oil, fat and fatty acids, starch or proteins.
The present invention also encompasses use of CYP90B nucleic acids or variants thereof and use of CYP90B polypeptides or homologues thereof. Such uses relate to increasing plant yield as defined hereinabove in the methods of the invention.
CYP90B nucleic acids or variants thereof, or CYP90B polypeptides or homologues thereof may find use in breeding programmes in which a DNA marker is identified which may be genetically linked to a CYP90B gene or variant thereof. The CYP90B nucleic acids/genes or variants thereof, or CYP90B 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 as defined hereinabove in the methods of the invention. The CYP90B gene or variant thereof may, for example, be a nucleic acid as represented by any one of SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87 and SEQ ID NO: 89.
Allelic variants of a CYP90B 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: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87 and SEQ ID NO: 89. 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 CYP90B 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 CYP90B nucleic acids or variants thereof requires only a nucleic acid sequence of at least 15 nucleotides in length. The CYP90B nucleic acids or variants thereof may be used as restriction fragment length polymorphism (RFLP) markers. Southern blots (Sambrook J, Fritsch E F and Maniatis T (1989) Molecular Cloning, A Laboratory Manual) of restriction-digested plant genomic DNA may be probed with the CYP90B 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 CYP90B nucleic acid or variant thereof in the genetic map previously obtained using this population (Botstein et al. (1980) Am. J. Hum. Genet. 32:314-331).
The production and use of plant gene-derived probes for use in genetic mapping is described in Bernatzky and Tanksley (1986) (GENETICS112 (4): 887-898). 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 hybridisation (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. This increased yield may also be combined with other economically advantageous traits, such as further yield-enhancing traits, tolerance to other abiotic and biotic stresses, traits modifying various architectural features and/or biochemical and/or physiological features.
Detailed Description CDC27
CDC27 polypeptides are well known in the art and are easily identifiable by the presence of a conserved NH2 terminal region (see FIG. 16) and of at least 5 TPR domains with at least one TPR domain in the NH2 terminal region. Furthermore, the CDC27 polypeptide may additionally comprise a sequence with more than 30% identity to SEQ ID NO: 130.
TPR motifs are present in a wide variety of proteins functional in yeast and higher eukaryotes in mitosis (including the APC protein components CDC16, CDC23 and CDC27), transcription, splicing, protein import and neurogenesis (Goebl and Yanagida 1991, Trends Biochem Sci 16, 173-177). A suggested minimal consensus sequence of the TPR motif is: X3-W-X2-L-G-X2-Y-X8-A-X3-F-X2-A-X4-P-X2, (SEQ ID NO: 286) where X=any amino acid (Lamb et al. 1994, EMBO J. 13, 4321-4328). The consensus residues may exhibit significant degeneracy and the non-consensus residues exhibit little or no homology. It is the hydrophobicity and size of the consensus residues, rather than their identity, that seems to be important. In a native CDC27 protein, the TPR forms an α-helical structure, tandem repeats organize into a superhelical structure ideally suited as interfaces for protein recognition (Groves and Barford 1999, Curr Opin Struct Biol 9, 383-389). Within the α-helix, two amphipathic domains are usually present, one at the NH2 terminal region and the other near the COOH-terminal region (Sikorski et al. 1990, Cell 60, 307-317). Also individual TPR motifs may be dispersed throughout the protein sequence.
A full length native CDC27 typically comprises at least 5 TPRs, preferably 6 TPRs, more preferably 7 TPRs, the majority of those TPRs being located in the COOH terminal region. As shown in FIG. 16, there is typically one TPR domain in the NH2 terminal region of a native CDC27 polypeptide, although variant CDC27 sequences may exist or may be created to comprise more than one TPR in the NH2 terminal region.
Any CDC27 polypeptide may be rendered useful in the methods of the invention by inactivation of at least one TPR domain in the NH2 terminal region of the polypeptide. Methods for inactivation are well known in the art and include: removal or substitution of amino acids, in this case, removal or substitution of amino acids of at least one TPR domain in the NH2 terminal region; or mutation techniques, such as substituting conserved amino acids by alanine or substituting phosphorylatable amino acids (such as serine, threonine or tyrosine) by non-phosphorylatable amino acids or vice versa (depending if the phosphorylated protein is active or inactive); or any other method for inactivation.
For the purposes of this application, the NH2 terminal region of a CDC27 protein is taken to be the first half of a full length CDC27 sequence (from NH2 terminal to COOH terminal) (see FIG. 16); preferably the NH2 terminal region of a CDC27 protein is taken to be the first third of a full length CDC27 sequence (from NH2 terminal to COOH terminal); and according to another preferred feature of the present invention, the N-terminal region of a CDC27 protein is taken to be the first 166 amino acids (from NH2 terminal to COOH terminal) of a full-length CDC27 sequence.
An example of a CDC27 polypeptide having at least one inactive TPR domain in the NH2 terminal region is the polypeptide represented by SEQ ID NO: 130, with encoding nucleic acid sequence represented by SEQ ID NO: 129.
Table 10 below gives some examples of CDC27 sequences; these sequences may be rendered useful in the methods of the invention by inactivation of at least one TPR domain in the NH2 terminal region of the polypeptide, for example by using any of the inactivation methods discussed hereinabove.







TABLE 10







Examples of CDC27 polypeptides














Translated




NCBI nucleotide

polypeptide



accession
Nucleotide
SEQ ID


Name
number
SEQ ID NO
NO
Source





CDC27B
AC006081
129
130
Arabidopsis thaliana


CDC27B/Hobbit
AJ487669
131
132
Arabidopsis thaliana


CDC27a
NM_112503.2|
133
134
Arabidopsis thaliana


CDC27
AP003539.3
135
136
Oryza sativa


CDC27
BG887406.1*
137
139
Solanum tuberosum



BG590616.1



DN939130.1



CV470643.1


CDC27/nuc2+
NM_001020032.1
139
140
Schizosaccharomyces






pombe


CDC27/BimA
X59269.1
141
142
Aspergillus niger


CDC27
NM_001256.2
143
144
Homo sapiens


CDC27 5′
CA102186.1*
145
146
Saccharum officinarum



CA279358.1


CDC27 3′
CA197669.1*
147
148
Saccharum officinarum



CA197670.1



CA203636.1



CA232307.1





*Contig compiled from several EST accessions (main ones shown); EST sequencing quality being usually lower, a few nucleic acid substitutions may be expected.






The sequences described in Table 10 are given by way of example only. Further examples are given in FIG. 19, encoding either full length or partial polypeptides (which may be used to obtain the full length sequence using routine methods). It is to be understood that any CDC27 polypeptide sequence having at least one inactive TPR domain in the NH2 terminal region of the polypeptide, or a nucleic acid/gene encoding such a polypeptide, may be suitable for use in performing the methods of the invention.
Other CDC27 polypeptides may readily be identified using routine techniques well known in the art, such as by sequence alignment. Sequences so identified may subsequently be rendered useful in the methods of the invention by inactivation of at least one TPR domain in the NH2 terminal region of the polypeptide, for example by using any of the inactivation methods discussed hereinabove. 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 Mol 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 Mol 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 a CDC27 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. Minor manual editing may be performed to optimise alignment between conserved motifs, as would be apparent to a person skilled in the art.
Various structural domains in a CDC27 protein, such as TPR domains, may be identified using specialised databases e.g. SMART (Schultz et al. (1998) Proc. Natl. Acad. Sci. USA 95, 5857-5864; Letunic et al. (2002) Nucleic Acids Res 30, 242-244; http://smart.embl-heidelberg.deI), InterPro (Mulder et al., (2003) Nucl. Acids. Res. 31, 315-318; http://www.ebi.ac.uk/interproo), Prosite (Bucher and Bairoch (1994), A generalized profile syntax for biomolecular sequences motifs and its function in automatic sequence interpretation. (In) ISMB-94; Proceedings 2nd International Conference on Intelligent Systems for Molecular Biology. Altman R., Brutlag D., Karp P., Lathrop R., Searls D., Eds., pp 53-61, AAAIPress, Menlo Park; Hulo et al., Nucl. Acids. Res. 32:D134D137, (2004), http://www.expasy.org/prosite/), Pfam (Bateman et al., Nucleic Acids Research 30(1): 276-280 (2002), http://www.sanger.ac.uk/Software/Pfamo or ProDom (Servant F, Bru C, Carrère S, Courcelle E, Gouzy J, Peyruc D, Kahn D (2002) ProDom:
Automated clustering of homologous domains. Briefings in Bioinformatics. vol 3, no 3:246-251).
The sequences mentioned in Table 10 and FIG. 19 may be considered homologues of a CDC27 polypeptide. “Homologues” of a protein are defined in the “Definitions” section herein. Preferred homologues are amino acid sequences having in increasing order of preference at least 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more sequence identity to the full-length CDC27 protein represented by SEQ ID NO: 132.
Homologues, orthologues and paralogues may be rendered useful in the methods of the invention by inactivation of at least one TPR domain in the NH2 terminal region of the polypeptide, for example by using any of the inactivation methods discussed hereinabove.
Human and yeast CDC27 polypeptides have been shown to interact with two other proteins of the APC complex, CDC16 and CDC23, in vivo via yeast two-hybrid analysis, and in vitro via by co-immunoprecipitation (Lam et al. (1994) EMBO J. 13(18): 4321-4328; Ollendorf & Donoghue (1997) J Biol Chem 272(51): 32011-32018). Such an interaction may be useful to identify CDC27 polypeptides to be rendered useful in the methods of the invention by inactivation of at least one TPR domain in the NH2 terminal region of the polypeptide, for example by using any of the inactivation methods discussed hereinabove
A CDC27 polypeptide having at least one inactive TRP domain in the NH2 terminal region of the polypeptide is encoded by a so-called modified CDC27 nucleic acid/gene. Therefore, the term “modified CDC27 nucleic acid/gene” as defined herein is any nucleic acid/gene encoding a CDC27 polypeptide having at least one inactive TRP domain in the NH2 terminal region of the polypeptide.
The CDC27 nucleic acid or modified CDC27 nucleic acid/gene may be derived from any natural or artificial source. The nucleic acid/gene may be isolated from a microbial source, such as yeast or fungi, or from a plant, algae or animal source. This nucleic acid may be modified from its native form in composition and/or genomic environment through deliberate human manipulation. The nucleic acid is preferably of plant origin, whether from the same plant species (for example to the one in which it is to be introduced) or whether from a different plant species. The nucleic acid may be isolated from a dicotyledonous species, preferably from the family Brassicaceae, further preferably from Arabidopsis thaliana. More preferably, the modified CDC27 nucleic acid isolated from Arabidopsis thaliana is represented by SEQ ID NO: 129 and the CDC27 having at least one inactive TPR in the NH2 terminal region of the amino acid is as represented by SEQ ID NO: 130.
A CDC27 nucleic acid/gene is a nucleic acid capable of hybridising under reduced stringency conditions, preferably under stringent conditions, with a CDC27 nucleic acid/gene as represented by any one of SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 137 or SEQ ID NO: 141. Most preferably the hybridising sequence is one that is capable of hybridising to a nucleic acid as represented by SEQ ID NO: 129 or SEQ ID NO: 131. Such hybridising sequences may be rendered useful in the methods of the invention by inactivation of at least one TPR domain in the NH2 terminal region of the encoded polypeptide, for example by using any of the inactivation methods discussed hereinabove.
The term “hybridisation” is as defined herein in the “Definitions” section.
The CDC27 nucleic acid or modified CDC27 nucleic acid/gene may be in the form of an alternative splice variant. An alternative splice variant is defined in the “Definitions” section. Preferred are splice variants of any of the aforementioned CDC27 nucleic acids sequences, namely SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 137 or SEQ ID NO: 141. Most preferred is a splice variant of a nucleic acid sequence as represented by SEQ ID NO: 129 or SEQ ID NO: 131. Such splice variants may be rendered useful in the methods of the invention inactivation of at least one TPR domain in the NH2 terminal region of the encoded CDC27 polypeptide, for example by using any of the inactivation methods discussed hereinabove.
The CDC27 nucleic acid or modified CDC27 nucleic acid/gene may be in the form of an allelic variant of a nucleic acid encoding a truncated CDC27 polypeptide comprising at least one inactivated TPR domain in the NH2 terminal region. Preferred are allelic variants of nucleic acid sequences represented by SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 137 or SEQ ID NO: 141. Most preferred is an allelic variant of a nucleic acid sequence as represented by SEQ ID NO: 129 or SEQ ID NO: 131. 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. Such allelic variants may be rendered useful in the methods of the invention inactivation of at least one TPR domain in the NH2 terminal region of the encoded CDC27 polypeptide, for example by using any of the inactivation methods discussed hereinabove.
The CDC27 nucleic acid or modified CDC27 nucleic acid/gene may be generated by site-directed mutagenesis. 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).
The CDC27 nucleic acid or modified CDC27 nucleic acid/gene may also be generated by directed evolution (see “Definitions” section for further details).
Such variants produced by site-directed mutagenesis or by directed evolution may be rendered useful in the methods of the invention inactivation of at least one TPR domain in the NH2 terminal region of the encoded CDC27 polypeptide, for example by using any of the inactivation methods discussed hereinabove.
The expression of a modified CDC27 nucleic acid/gene encoding a CDC27 polypeptide having at least one inactive TPR domain in the NH2 terminal region of the polypeptide may be increased by introducing a genetic modification (preferably in the locus of a CDC27 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 down stream of the coding region.
The genetic modification is preferably introduced by introducing and expressing in a plant a nucleic acid encoding a CDC27 polypeptide having at least one inactive TPR domain in the NH2 terminal region of the polypeptide. Following introduction of the genetic modification, there follows an optional step of selecting for increased expression (in shoot apical meristem tissue) of a modified nucleic acid encoding a CDC27 polypeptide having at least one inactive TPR domain in the NH2 terminal region of the polypeptide, which increase in expression gives plants having increased yield.
According to a preferred aspect of the present invention, increased expression of the CDC27 nucleic acid is envisaged. Methods for increasing expression of genes or gene products are well documented in the art and include, 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 CDC27 nucleic acid. For example, endogenous promoters may be altered in vivo by mutation, deletion, and/or substitution (see, Kmiec, U.S. Pat. No. 5,565,350; Zarling et al., PCT/US93/03868), or isolated promoters may be introduced into a plant cell in the proper orientation and distance from a gene of the present invention to control the expression of the gene.
If polypeptide expression is desired, it is generally desirable to include a polyadenylation region at the 3′-end of a polynucleotide coding region. The polyadenylation region may be derived from the natural gene, from a variety of other plant genes, or from T-DNA. The 3′ end sequence to be added may be derived from, for example, the nopaline synthase or octopine synthase genes, or alternatively from another plant gene, or less preferably from any other eukaryotic gene.
An intron sequence may also be added to the 5′ untranslated region or the coding sequence of the partial coding sequence to increase the amount of the mature message that accumulates in the cytosol. Inclusion of a spliceable intron in the transcription unit in both plant and animal expression constructs has been shown to increase gene expression at both the mRNA and protein levels up to 1000-fold (Buchman and Berg (1988) Mol. Cell. biol. 8: 4395-4405; Callis et al. (1987) Genes Dev 1:1183-1200). Such intron enhancement of gene expression is typically greatest when placed near the 5′ end of the transcription unit. Use of the maize introns Adh1-S intron 1, 2, and 6, the Bronze-1 intron are known in the art. See generally, The Maize Handbook, Chapter 116, Freeling and Walbot, Eds., Springer, N.Y. (1994).
The invention also provides genetic constructs and vectors to facilitate introduction and/or expression of the nucleotide sequences useful in the methods according to the invention.
Therefore, there is provided a gene construct comprising:

    
    
        (i) A CDC27 nucleic acid encoding a CDC27 polypeptide having at least one inactivated TPR domain in the NH2 terminal region of the polypeptide;
        (ii) One or more control sequences capable of preferentially driving expression of the nucleic acid sequence of (i) in shoot apical meristem tissue; and optionally
        (iii) A transcription termination sequence.
    
    


Such genetic constructs 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. The invention therefore provides use of a gene construct as defined hereinabove in the methods of the invention.
Plants are transformed with a vector comprising the sequence of interest (i.e., a nucleic acid encoding a CDC27 polypeptide having at least one inactive TPR domain in the NH2 terminal region of the polypeptide. The sequence of interest is operably linked to one or more control sequences (at least to a promoter) capable of preferentially driving expression in shoot apical meristem tissue of a plant. The terms “regulatory elemen”, “control sequence” and “promoter” are all used interchangeably herein and are defined in the “Definitions” section.
The CDC27 nucleic acid encoding a CDC27 polypeptide having at least one inactive TPR domain in the NH2 terminal region of the polypeptide or variant is operably linked to a shoot apical meristem promoter, preferably to an early shoot apical meristem promoter. An “early shoot apical meristem promoter” as defined herein is a promoter that is transcriptionally active in the shoot apical meristem from the embryo globular stage up to the young seedling stage, these stages being well known to persons skilled in the art. Reference herein to preferentially increasing expression in shoot apical meristem tissue is taken to mean increasing expression in shoot apical meristem tissue substantially to the exclusion of expression elsewhere in the plant, apart from any residual expression due to leaky promoters. Preferably, the early shoot apical meristem promoter is an OSH1 promoter (from rice; SEQ ID NO: 151 (Matsuoka et al., (1993) Plant Cell 5: 1039-1048; Sato et al., (1996) Proc Natl Acad Sci USA 93(15): 8117-22). It should be clear that the applicability of the present invention is not restricted to the modified CDC27 nucleic acid represented by SEQ ID NO: 129, nor is the applicability of the invention restricted to expression of a modified CDC27 nucleic acid when driven by an OSH1 promoter. Examples of other early shoot apical meristem promoters are shown in Table 5 in the “Definitions” section. These are members of the KNOX family class 1 homeobox, from paralogous or orthologous genes. It should be understood that the list below is non-exhaustive.
Optionally, one or more terminator sequences may also be used in the construct introduced into a plant. The term “terminator” is defined herein in the “Definitions” section.
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 defined in the “Definitions” section.
The present invention also encompasses plants obtainable by the methods according to the present invention. The present invention therefore provides plants or parts thereof, including plant cells, obtainable by the method according to the present invention, which plants or plant parts comprise a CDC27 nucleic acid encoding a CDC27 polypeptide having at least one inactive TPR domain in the NH2 terminal region of the polypeptide and which nucleic acid is operably linked to a shoot apical meristem promoter.
The invention also provides a method for the production of transgenic plants having increased seed number relative to suitable control plants, comprising introduction and expression in a plant of a CDC27 nucleic acid encoding a CDC27 polypeptide having at least one inactive TPR domain in the NH2 terminal region of the polypeptide, which CDC27 nucleic acid is under the control of a shoot apical meristem promoter.
More specifically, the present invention provides a method for the production of transgenic plants having increased seed number relative to suitable control plants, which method comprises:

    
    
        (i) introducing and expressing in a plant, plant part or plant cell a CDC27 nucleic acid encoding a CDC27 polypeptide having at least one inactive TPR domain in the NH2 terminal region of the polypeptide, which nucleic acid is under the control of a shoot apical meristem promoter; and
        (ii) cultivating the plant cell under conditions promoting plant growth and development.
    
    


The nucleic acid may be introduced directly into a plant cell or into the plant itself (including introduction into a tissue, organ or any other part of a plant). According to a preferred feature of the present invention, the nucleic acid is preferably introduced into a plant by transformation.
The term “transformation” is defined in the “Definitions” section.
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 CDC27 nucleic acid encoding a CDC27 polypeptide having at least one inactive TPR domain in the NH2 terminal region of the polypeptide and which nucleic acid is under the control of a shoot apical meristem promoter. 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, stems, rhizomes, tubers and bulbs. The invention furthermore relates to products derived, preferably directly derived, from a harvestable part of such a plant, such as dry pellets or powders, oil, fat and fatty adds, starch or proteins.
The present invention also encompasses use of CDC27 nucleic acids encoding CDC27 polypeptides having at least one inactive TPR domain in the NH2 terminal region of the polypeptide, which nucleic acids are under the control of a shoot apical meristem promoter. Such uses relate to increasing plant yield as defined hereinabove in the methods of the invention.
Performance of the methods according to the present invention result in plants having increased seed number relative to suitable control plants. This increase in seed number may also be combined with other economically advantageous traits, such as further yield-enhancing traits, tolerance to other abiotic and biotic stresses, traits modifying various architectural features and/or biochemical and/or physiological features.
Detailed Description AT-hook
AT-hook domains are well known in the art and are typically found in polypeptides belonging to a family of transcription factors associated with Chromatin remodeling. The AT-hook motif is made up of 13 or so (sometimes about 9) amino acids which participate in DNA binding and which have a preference for A/T rich regions. In Arabidopsis there are at least 34 proteins containing AT-hook domains. These proteins share homology along most of the sequence, with the AT-hook domain being a particularly highly conserved region. The AT-hook domain is illustrated in FIG. 23 and Table 11 hereinafter, see also the appropriate annotation of SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO: 159, SEQ ID NO: 161, SEQ ID NO: 163, SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO: 169 and SEQ ID NO: 171 where the position of the AT-hook domain is specified. As shown in the alignment of FIG. 23, some variation within the AT-hook domain is allowed. Typically, one or two AT-hook domains precede the DUF296 domain. Reference herein to an AT-hook domain is taken to mean a polypeptide sequence having in increasing order of preference at least 70%, 75%, 80%, 85%, 90% or 95% identity to the AT-hook domain of SEQ ID NO: 153, which is repeated here for convenience: RRPRGRPAGSKNK (AT-hook domain of SEQ ID NO: 153).
DUF296 domains (referred to in Interpro as IPRO05175) are also well known in the art. The DUF296 domain is illustrated in FIG. 23 and Table 11 hereinafter; see also the appropriate annotation of SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO: 159, SEQ ID NO: 161, SEQ ID NO: 163, SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO: 169 and SEQ ID NO: 171, where the position of the DUF296 domain is specified. As shown in the alignment of FIG. 23, variation within the DUF296 domain is allowed whilst still being easily identified as a DUF296 domain due to the presence of some highly conserved amino acid residues. Typically, the DUF296 domain is preceded by one or two AT-hook domains.
According to a preferred feature of the present invention, polypeptides comprising an AT-hook domain and a DUF296 domain additionally comprise one of the following motifs:
Motif 1 (SEQ ID NO: 190): QGQ V/I GG; or
Motif 2 (SEQ ID NO: 191): ILSLSGSFLPPPAPP; or
Motif 3 (SEQ ID NO: 192): NATYERLP; or
Motif 4 (SEQ ID NO: 193): SFTNVAYERLPL with zero or one amino acid change at any position; or
Motif 5 (SEQ ID NO: 194): GRFEILSLTGSFLPGPAPPGSTGLTIYLAGGQGQWGGSWG with zero, one or two amino acid changes at any position.
According to a preferred feature of the present invention, sequences suitable for use in the methods of the invention are polypeptides comprising an AT-hook domain (as defined hereinabove) and a DUF296 domain (as defined hereinabove) and Motif 2 (as defined hereinabove), or nucleic acids encoding such polypeptides.
It is to be understood that the sequences detailed in Table 1 and those shown in the alignment of FIG. 23 are only examples of sequences useful in the methods of the invention and that any polypeptide having an AT-hook domain and a DUF296 domain, or any nucleic acid encoding the same, may be suitable for use in performing the methods of the invention.








TABLE 11







Examples of amino acid sequences comprising an AT-hook domain and a



DUF296 domain with details of the sequences of these domains and their


respective positions

















AT Hook
sequence
Duf296







domain
AT Hook
domain
sequence DUF296



SEQ ID NO
Species
position
domain
position
domain


















 1
SEQ ID NO: 153
ORYSA
 97-109
rrprgrpagsknk
124-241
lrthvmevaggcdisesitt




CDS3129




farrrqrgvcvlsgagtvtn








vtlrqpasqgavvalhgrfe








ilslsgsflpppappeatgl








tvylaggqgqvvggsvvgal








taagpvvimaasfanavy


 


 2
SEQ ID NO: 155
ORYSA
 97-109
RRPRGRPPGSKNK
109-227
lrahilevgsgcdvfecvst



CDS3128




yarrrqrgvcvlsgsgvvtn








vtlrqpsapagavvslhgrf








eilslsgsflpppappgats








ltiflaggqgqvvggnvvga








lyaagpviviaasfanvay


 


 3
SEQ ID NO: 157
LOTUS
 81-93
rrprgrpagsknk
108-225
lkthvmevadgcdivdsvsn



LOTCO-




farrrqrgvcimsgtgtvtn



AP006863.1




vtlrqpassgavvtlhgrfe



(gi68264919)




ilslagsflpppappaasgl








tiylaggqgqvvggsvvgal








iasgpvvimaasfsnaay


 


 4
SEQ ID NO: 159
ARATH
119-131
RRPRGRPAGSKNK
145-263
lrthvmeigdgcdivdcmat



NP_192942




farrrqrgvcvmsgtgsvtn








vtirqpgsppgsvvslhgrf








eilslsgsflpppappaatg








lsvylaggqgqvvggsvvgp








llcsgpvvvmaasfsnaay


 


 5
SEQ ID NO: 161
ARATH
105-117
rrprgrpagsknk
132-252
farrrqrgvcvmsgtgnvtn



NP_194012




vtirqpgshpspgsvvslhg








rfeilslsgsflpppappta








tglsvylaggqgqvvggsvv








gpllcagpvvvmaasfsna


 


 6
SEQ ID NO: 163
ARATH
 89-101
rrprgrpagsknk
116-237
lkshvmevangcdvmesvtv



NP_182067




farrrqrgicvlsgngavtn








vtirqpasvpgggssvvnlh








grfeilslsgsflpppappa








asgltiylaggqgqvvggsv








vgplmasgpvvimaasfgna








ay


 


 7
SEQ ID NO: 165
ARATH
 59-71
rrprgrpagsknk
 86-201
frchvmeitnacdvmeslav



At3g60870/At




farrrqrgvcvltgngavtn



_NP_191646




vtvrqpgggvvslhgrfeil








slsgsflpppappaasglkv








ylaggqgqviggsvvgplta








sspvvvmaasfgnasy


 


 8
SEQ ID NO: 167
ARATH
 88-100
rrprqrppgsknk
115-233
lqshvleiatgadvaeslna



CDS0185




farrrgrgvsvlSgsglvtn








vtlrqpaasggvvslrgqfe








ilsmcgaflptsgspaaaag








ltiylagaqgqvvgggvagp








liasgpviviaatfcnaty


 


 9
SEQ ID NO: 169
ORYSA
111-123
rrprgrpagsknk
138-256
lrahvlevasgcdlvdsvat



PROT encoded




farrrqvgvcvlsatgavtn



by AK107405




vsvrqpgaqpgavvnltgrf








dilslsgsflpppappsatg








ltvyvsggqgqvvggtvagp








liavgpvvimaasfgnaay


 


10
SEQ ID No: 171
ORYSA
 45-57
rrprgrppgsknk
 72-190
mrshvleiasgadiveaiag



NP_912386.1




fsrrrqrgvsvlsgsgavtn








vtlrqpagtgaaavalrgrf








eilsmsgaflpapappgatg








lavylaggqgqvvggsvmge








liasgpvmviaatfgnaty


 


11
SEQ ID NO: 173
LYCES
 54-66
rrprgrpagsknk
 81-198
lrahilevssghdvfesvat



Le_BT013387




yarkrqrgicilsgsgtvnn








vtirqpqaagsvvtlhgrfe








ilslsgsflpppappgatsl








tiylaggqgqvvggnvvgal








iasgpviviassftnvay


 


12
SEQ ID NO: 175
ARATH
 34-46
rrprgrpagsknk
 61-180
lrshvlevtsgsdiseavst



CDS3125




yatrrgcgvciisgtgavtn








vtirgpaapagggvitlhgr








fdilsltgtalpppappgag








gltvylaggqgqvvggnvag








sliasgpvvlmaasfanavy


 


13
SEQ ID NO: 177
ARATH
 80-92
rrprgrpagsknk
107-232
lkshvmeiasgtdvietlat



CDS3399




farrrqrgicilsgngtvan








vtlrqpstaavaaapggaav








lalqgrfeilsltgsflpgp








appgstgltiylaggqgqvv








ggsvvgplmaagpvmliaat








fsnaty


 


14
SEQ ID NO: 179
ORYSA
 35-47
rrprgrppgsknk
 62-179
lrshvmevaggadvaesiah



PRO AK110263




farrrqrgvcvlsgagtvtd








valrcqpaapsavvlrgrfe








ilsltgtflpgpappgstgl








tvylaggqgqvvggsvvgtl








taagpvmv


 


15
SEQ ID NO: 191
ARATH
 67-79
rrprgrppgsknk
 94-211
lrshvleisdgsdvadtiah



At4g14465/NP




fsrrrqrgvcvlsgtgsvan



_567432




vtlrqaaapggvvslqgrfe








ilsltgaflpgpsppgstgl








tvylagvqgqvvggsvvgpl








laigsvmviaatfsnaty


 


16
SEQ ID NO: 183
ARATH
 82-94
rrprgrppgsknk
109-226
lrahilevtngcdvfdcvat



CDS4145




yarrrqrgicvlsgsgtvtn








vsirqpsaagavvtlqgtfe








ilslsgsflpppappgatsl








tiflaggqgqvvggsvvgel








taagpviviaasftnvay


 


17
SEQ ID NO: 185
ORYSA
 82-94
rrprgrppgsknk
109-227
lrahilevgsgcdvfecvst



XP_473716




yarrrqrgvcvlsgsgvvtn








vtlrqpsapagavvslhgrf








eilslsgsflpppappgats








ltiflaggqgqvvggnvvga








lyaagpviviaasfanvay


 


18
SEQ ID NO: 187
ARATH
 78-90
rrprgrpagsknk
105-222
lrahilevgsgcdvfecist



NP_181070




yarrrqrgicvlsgtgtvtn








vsirqptaagavvtlrgtfe








ilslsgsflpppappgatsl








tiflagaqgqvvggnvvgel








maagpvmvmaasftnvay


 


19
SEQ ID NO: 189
TC102931
 86-98
rrprgrpagsknk
113-230
lrshvmevangcdimesvtv



TC102931




farrrgrgvcilsgsgtvtn








vtlrqpaspgavvtlhgrfe








ilslsgsflpppappaasgl








aiylaggqgqvvggsvvgpl








lasgpvvimaasfgnaay









A person skilled in the art will readily be able to identify polypeptides comprising an AT-hook domain and a DUF296 domain using techniques and tools well known in the art. Such identification may be by sequence alignment for comparison of sequences using GAP, BESTFIT, BLAST, FASTA and TFASTA. GAP uses the algorithm of Needleman and Wunsch ((1970) J Mol 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 Mol 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. Polypeptides comprising an AT-hook domain and a DUF296 domain 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. Minor manual editing may be performed to optimise alignment between conserved motifs, as would be apparent to a person skilled in the art.
The AT-hook domain and the DUF296 domain may be identified using specialised databases e.g. SMART (Schultz et al. (1998) Proc. Natl. Acad. Sd. USA 95, 5857-5864; Letunic et al. (2002) Nucleic Acids Res 30, 242-244; http://smart.embl-heidelberg.de/), InterPro (Mulder et al., (2003) Nucl. Acids. Res. 31, 315318; http://www.ebi.ac.uk/interpro/), Prosite (Bucher and Bairoch (1994), A generalized profile syntax for biomolecular sequences motifs and its function in automatic sequence interpretation. (In) ISMB-94; Proceedings 2nd International Conference on Intelligent Systems for Molecular Biology. Altman R., Brutlag D., Karp P., Lathrop R., Searls D., Eds., pp 53-61, AAAIPress, Menlo Park; Hulo et al., Nucl. Acids. Res. 32:D134-D137, (2004), http://www.expasy.org/prosite/) or Pfam (Bateman et al., Nucleic Acids Research 30(1):276-280 (2002), http://www.sanger.ac.uk/Software/Pfam/).
The sequences mentioned in Table 11, or as identified using the techniques mentioned above (such as sequence alignment), may be considered homologues of a polypeptide comprising an AT-hook domain and a DUF296 domain, which homologues also comprise an AT-hook domain and a DUF296 domain but which may vary elsewhere in the sequence. “Homologues” of a protein are defined in the “Definitions” section herein. Preferred homologues are amino acid sequences having in increasing order of preference at least 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more sequence identity to the amino acid sequence represented by SEQ ID NO: 153, which homologues comprise an AT-hook domain and a DUF296 domain and further preferably comprise Motif 2.
The polypeptide comprising an AT-hook domain and a DUF296 domain, or a homologue of such polypeptide, may be a derivative, as defined in the “Definitions” section herein.
Any nucleic acid encoding a polypeptide comprising an AT-hook domain and a DUF296 domain may be suitable for use in the methods of the invention. Examples of such sequences include those nucleotide sequences represented by SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO: 162, SEQ ID NO: 164, SEQ ID NO: 166, SEQ ID NO: 168 and SEQ ID NO: 170.
Variants of a nucleic acid encoding a polypeptide comprising an AT-hook domain and a DUF296 domain may also be suitable for use in practising the methods of the invention so long as the variants encode polypeptides comprising an AT-hook domain and a DUF296 domain. Such nucleic acid variants may be portions of a nucleic acid encoding a polypeptide comprising an AT-hook domain and a DUF296 domain and/or nucleic acids capable of hybridising with a nucleic acid encoding a polypeptide comprising an AT-hook domain and a DUF296 domain.
A portion may be prepared, for example, by making one or more deletions to a nucleic acid encoding a polypeptide comprising an AT-hook domain and a DUF296 domain. 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 portion. Preferably, the portion is a portion of a nucleic acid as represented by any one of SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO: 162, SEQ ID NO: 164, SEQ ID NO: 166, SEQ ID NO: 168 and SEQ ID NO: 170. Most preferably the portion is a portion of a nucleic acid as represented by SEQ ID NO: 152, which portion encodes a polypeptide comprising an AT-hook domain and a DUF296 domain and further preferably comprises Motif 2.
Another nucleic acid variant is a nucleic acid capable of hybridising under reduced stringency conditions, preferably under stringent conditions, with a nucleic acid encoding a polypeptide comprising an AT-hook domain and a DUF296 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: 152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO: 162, SEQ ID NO: 164, SEQ ID NO: 166, SEQ ID NO: 168 and SEQ ID NO: 170, or to a portion of any of the aforementioned sequences as defined hereinabove. Most preferably, the hybridising sequence is one that is capable of hybridising to a nucleic acid as represented by SEQ ID NO: 152, which hybridizing sequence encodes a polypeptide comprising an AT-hook domain and a DUF296 domain and further preferably comprises Motif 2.
The term “hybridisation” is as defined herein in the “Definitions” section.
Another nucleic acid variant is an alternative splice variant, as defined in the “Definitions” section. Preferred are splice variants of nucleic acid sequences represented by SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO: 162, SEQ ID NO: 164, SEQ ID NO: 166, SEQ ID NO: 168 and SEQ ID NO: 170. Most preferred is a splice variant of a nucleic acid sequence as represented by SEQ ID NO: 152, which splice variant encodes a polypeptide comprising an AT-hook domain and a DUF296 domain and further preferably comprising Motif 2.
Another nucleic acid variant is an allelic variant as defined in the “Definitions” section. Preferred are allelic variants of nucleic acid sequences represented by SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO: 162, SEQ ID NO: 164, SEQ ID NO: 166, SEQ ID NO: 168 and SEQ ID NO: 170. Most preferred is an allelic variant of a nucleic acid sequence as represented by SEQ ID NO: 152, which allelic variant encodes a polypeptide comprising an AT-hook domain and a DUF296 domain and further preferably comprises Motif 2.
Nucleic acid variants may also be obtained through directed evolution (see “Definitions” section).
Site-directed mutagenesis may also be used to generate variants of a nucleic acid encoding a polypeptide comprising an AT-hook domain and a DUF296 domain. See “Definitions” section.
The nucleic acid encoding a polypeptide comprising an AT-hook domain and a DUF296 domain may be derived from any natural or artificial source. The nucleic acidigene or variant thereof may be isolated from a microbial source, such as yeast or fungi, or from a plant, algae or animal source. This nucleic acid may be modified from its native form in composition and/or genomic environment through deliberate human manipulation. The nucleic acid is preferably of plant origin, whether from the same plant species (for example to the one in which it is to be introduced) or whether from a different plant species. The nucleic acid may be isolated from a dicotyledonous species, preferably from a monocotyledonous species such as rice. More preferably, the rice nucleic acid encoding a polypeptide comprising an AT-hook domain and a DUF296 domain is represented by SEQ ID NO: 152 and the encoded polypeptide is as represented by SEQ ID NO: 153.
The expression of an AT-hook-encoding nucleic acid may be modulated by introducing a genetic modification (preferably in the locus of a gene encoding a polypeptide comprising an AT-hook domain and a DUF296 domain). 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, homologous recombination and by introducing and expressing in a monocotyledonous plant a nucleic acid encoding a polypeptide comprising an AT-hook domain and a DUF296 domain. See the “Definitions” section for details of T-DNA activation, TILLING and homologous recombination. Following introduction of the genetic modification, there may follow a step of selecting for increased expression in endosperm tissue of a nucleic acid encoding a polypeptide comprising an AT-hook domain and a DUF296 domain, which targeted expression gives plants having increased seed yield.
The choice of promoter for T-DNA activation tagging in the case of the present invention would be any promoter capable of preferentially directing expression in endosperm tissue of a monocotyledonous plant.
T-DNA activation and TILLING are examples of technologies that enable the generation of novel alleles and variants of a nucleic acid encoding a polypeptide comprising an AT-hook domain and a DUF296 domain.
A preferred method for introducing a genetic modification (which in this case need not be in the locus of a nucleic acid/gene encoding a polypeptide comprising an AT-hook domain and a DUF296 domain) is to introduce and express in a plant a nucleic acid encoding a polypeptide comprising an AT-hook domain and a DUF296 domain. The nucleic acid to be introduced into a plant may be a full-length nucleic acid or may be a portion or any other variant nucleic acid so long as the variant nucleic-acid encodes a polypeptide comprising an AT-hook domain and a DUF296 domain.
The methods of the present invention rely on preferentially increasing expression in endosperm tissue of a monocotyledonous plant of a nucleic acid encoding a polypeptide comprising an AT-hook domain and a DUF296 domain. This may be achieved by 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 gene/nucleic acid or variant thereof encoding a polypeptide comprising an AT-hook domain and a DUF296 domain. For example, endogenous promoters may be altered in vivo by mutation, deletion, and/or substitution (see, Kmiec, U.S. Pat. No. 5,565,350; Zarling et al., PCT/US93/03868), or isolated promoters may be introduced into a plant cell in the proper orientation and distance from a gene of the present invention to control expression of the gene.
If polypeptide expression is desired, it is generally desirable to include a polyadenylation region at the 3′-end of a polynucleotide coding region. The polyadenylation region can be derived from the natural gene, from a variety of other plant genes, or from T-DNA. The 3′ end sequence to be added may be derived from, for example, the nopaline synthase or octopine synthase genes, or alternatively from another plant gene, or less preferably from any other eukaryotic gene.
An intron sequence may also be added to the 5′ untranslated region or the coding sequence of the partial coding sequence to increase the amount of the mature message that accumulates in the cytosol. Inclusion of a spliceable intron in the transcription unit in both plant and animal expression constructs has been shown to increase gene expression at both the mRNA and protein levels up to 1000-fold (Buchman and Berg (1988) Mol. Cell. biol. 8: 4395-4405; Callis et al. (1987) Genes Dev 1:1183-1200). Such intron enhancement of gene expression is typically greatest when placed near the 5′ end of the transcription unit. Use of the maize introns Adh1-S intron 1, 2, and 6, the Bronze-1 intron are known in the art. See generally, The Maize Handbook, Chapter 116, Freeling and Walbot, Eds., Springer, N.Y. (1994).
The invention also provides genetic constructs and vectors to facilitate introduction and/or expression of the nucleotide sequences useful in the methods according to the invention.
Therefore, there is provided a gene construct comprising:

    
    
        (i) A nucleic acid encoding a polypeptide comprising an AT-hook domain and a DUF296 domain;
        (ii) One or more control sequences capable of driving expression of the nucleic acid sequence of (i) in endosperm tissue of a monocotyledonous plant; and optionally
        (iii) A transcription termination sequence.
    
    


The invention also provides use of a construct as defined hereinabove in methods for increasing seed yield of 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. The invention also provides use of a construct as defined hereinabove in methods for increasing seed yield in a monocotyledonous plant.
Monocotyledonous plants are transformed with a vector comprising the sequence of interest (i.e., a nucleic acid encoding a polypeptide comprising an AT-hook domain and a DUF296 domain). The sequence of interest is operably linked to one or more control sequences (at least to a promoter) capable of preferentially increasing expression in endosperm tissue of a monocotyledonous plant. The terms “regulatory element”, “control sequence” and “promoter” are all used interchangeably herein and are defined in the “Definitions” section.
An endosperm-specific promoter refers to any promoter able to preferentially drive expression of the gene of interest in endosperm tissue. Reference herein to preferentially increasing expression in endosperm tissue is taken to mean increasing expression in endosperm tissue substantially to the exclusion of expression elsewhere in the plant, apart from any residual expression due to leaky promoters. For example, the prolamin promoter shows strong expression in the endosperm, with leakiness in meristem, more specifically the shoot meristem and/or discrimination centre in the meristem.
Preferably, the endosperm-specific promoter is a promoter isolated from a prolamin gene, such as a rice prolamin RP6 (Wen et al., (1993) Plant Physiol 101(3):1115-6) promoter as represented by SEQ ID NO: 195 or a promoter of similar strength and/or a promoter with a similar expression pattern as the rice prolamin promoter. Similar strength and/or similar expression pattern may be analysed, for example, by coupling the promoters to a reporter gene and checking the function of the reporter gene in tissues of the plant. One well-known reporter gene is beta-glucuronidase and the calorimetric GUS stain used to visualize beta-glucuronidase activity in plant tissue. It should be clear that the applicability of the present invention is not restricted to the nucleic acid represented by SEQ ID NO: 152, nor is the applicability of the invention restricted to expression of a nucleic acid encoding an AT-hook domain and a DUF296 domain when driven by a prolamin promoter. Examples of other endosperm-specific promoters which may also be used perform the methods of the invention are shown in Table 6 in the “Definitions” section.
Optionally, one or more terminator sequences may also be used in the construct introduced into a plant. The term “terminator” is defined in the “Definitions” section.
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 defined herein.
In a preferred embodiment, there is provided a gene construct comprising:

    
    
        (i) A nucleic acid encoding a polypeptide comprising an AT-hook domain and a DUF296 domain;
        (ii) A prolamin promoter capable of preferentially driving expression of the nucleic acid sequence of (i) in endosperm tissue of a monocotyledonous plant; and optionally
        (iii) A transcription termination sequence.
    
    


The present invention also encompasses monocotyledonous plants obtainable by the methods according to the present invention. The present invention therefore provides monocotyledonous plants, parts thereof (including plant cells) obtainable by the methods according to the present invention, which plants or parts thereof comprise a transgene encoding a polypeptide comprising an AT-hook domain and a DUF296 domain operably linked to an endosperm-specific promoter, preferably to a prolamin promoter.
The invention also provides a method for the production of transgenic monocotyledonous plants having increased seed yield relative to suitable control plants, comprising introduction and expression in a monocotyledonous plant of a nucleic acid encoding a polypeptide comprising an AT-hook domain and a DUF296 domain, wherein said expression is preferentially increased in endosperm tissue of a monocotyledonous plant.
More specifically, the present invention provides a method for the production of transgenic monocotyledonous plants having increased seed yield which method comprises:

    
    
        (i) introducing and preferentially increasing expression in endosperm tissue of a monocotyledonous plant of a nucleic acid encoding a polypeptide comprising an AT-hook domain and a DUF296 domain; and
        (ii) cultivating the plant cell under conditions promoting plant growth and development.
    
    


The nucleic acid may be introduced directly into a plant cell of a monocotyledonous plant or into the plant itself (including introduction into a tissue, organ or any other part of a plant). According to a preferred feature of the present invention, the nucleic acid is preferably introduced into a plant by transformation.
The term “transformation” is defined in the “Definitions” section herein.
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 a nucleic acid encoding a polypeptide comprising an AT-hook domain and a DUF296 domain operably linked to an endosperm-specific promoter. Preferred host cells according to the invention are monocotyledonous plant cells.
The invention also extends to harvestable parts of a monocotyledonous plant such. as, but not limited to seeds, leaves, fruits, flowers, stems, rhizomes, tubers and bulbs. The invention furthermore relates to products derived from, preferably directly derived from, a harvestable part of such a plant, such as dry pellets or powders, oil, fat and fatty acids, starch or proteins.
The present invention also encompasses use of a nucleic acid encoding a polypeptide comprising an AT-hook domain and a DUF296 domain in Increasing seed yield of a monocotyledonous plant using the methods of the invention.
Detailed Description DOF Transcription Factors
The term “DOF transcription factor polypeptide” as defined herein refers to any polypeptide comprising feature (i) as follows, and additionally either feature (ii) or (iii) as follow:

    
    
        (i) in increasing order of preference at least 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% sequence identity to either the DOF domain represented by SEQ ID NO: 200 or SEQ ID NO: 228; and
        (ii) in increasing order of preference at least 70%, 75%, 80%, 85%, 90% or 95% sequence identity to the DOF domain represented by SEQ ID NO: 200; or
        (iii) Motif I: KALKKPDKILP (SEQ ID NO: 229) with no changes; or with one or more conservative change at any position; or with one, two or three non-conservative change(s) at any position; and/or
        
            Motif II: DDPGIKLFGKTIPF (SEQ ID NO: 230) with no changes; or with one or more conservative change at any position; or with one, two or three non-conservative change(s) at any position.
        
        
    
    


Additionally, polypeptides comprising feature (i) and feature (iii) above may comprise any one, any two or all three of the following motifs:

    
    
        Motif III: SPTLGKHSRDE (SEQ ID NO: 231) with no changes; or with one or more conservative change at any position; or with one, two or three non-conservative change(s) at any position; and/or
    
    


Motif IV: LQANPAALSRSQNFQE (SEQ ID NO: 232) with no changes; or with one or more conservative change at any position; or with one, two or three non-conservative change(s) at any position; and/or
Motif V: KGEGCLWVPKTLRIDDPDEAAKSSIWTTLGIK (SEQ ID NO: 233) with no changes; or with one or more conservative change at any position; or with one, two, three, four or five non-conservative change(s) at any position.
A preferred polypeptide comprising feature (i) and feature (iii) above comprises both Motif I and II.
Furthermore, DOF transcription factor polypeptides (at least in their native form) typically have DNA-binding activity and have an activation domain. The presence of an activation domain and DNA-binding activity may easily be determined by a person skilled in the art using routine techniques and procedures.
SEQ ID NO: 199 (encoded by SEQ ID NO: 198) is an example of a DOF transcription factor polypeptide comprising features (i) and (ii) as defined hereinabove, i.e. at least 60% sequence identity to either the DOF domain represented by SEQ ID NO: 200 or SEQ ID NO: 228; and at least 70% sequence identity to the DOF domain represented by SEQ ID NO: 200. Further examples of DOF transcription factor polypeptides comprising features (i) and (ii) as defined hereinabove are given in SEQ ID NO: 202 (encoded by SEQ ID NO: 201), SEQ ID NO: 204 (encoded by SEQ ID NO: 203). SEQ ID NO: 206 (encoded by SEQ ID NO: 205), SEQ ID NO: 208 (encoded by SEQ ID NO: 207), SEQ ID NO: 210 (encoded by SEQ ID NO: 209), SEQ ID NO: 212 (encoded by SEQ ID NO: 211), SEQ ID NO: 214 (encoded by SEQ ID NO: 213), SEQ ID NO: 216 (encoded by SEQ ID NO: 215), SEQ ID NO: 218 (encoded by SEQ ID NO: 217), SEQ ID NO: 220 (encoded by SEQ ID NO: 219), SEQ ID NO: 222 (encoded by SEQ ID NO: 221).
SEQ ID NO: 227 (encoded by SEQ ID NO: 226) is an example of a DOF transcription factor polypeptide comprising features (i) and (iii) as defined hereinabove, i.e. at least 60% sequence identity to either the DOF domain represented by SEQ ID NO: 200 or SEQ ID NO: 228; and Motif I and/or Motif II as defined hereinabove. Further examples of DOF transcription factor polypeptides comprising features (i) and (iii) as defined hereinabove are given in SEQ ID NO: 235 (encoded by SEQ ID NO: 234), SEQ ID NO: 237 (encoded by SEQ ID NO: 236), SEQ ID NO: 239 (encoded by SEQ ID NO: 238), SEQ ID NO: 241 (encoded by SEQ ID NO: 240), SEQ ID NO: 243 (encoded by SEQ ID NO: 242), SEQ ID NO: 245 (encoded by SEQ ID NO: 244), SEQ ID NO: 247 (encoded by SEQ ID NO: 246), SEQ ID NO: 249 (encoded by SEQ ID NO: 248), SEQ ID NO: 251 (encoded by SEQ ID NO: 250), SEQ ID NO: 253 (encoded by SEQ ID NO: 252), SEQ ID NO: 255 (encoded by SEQ ID NO: 254).
The further examples represented by SEQ ID NO: 202, SEQ ID NO: 204, SEQ ID NO: 206, SEQ ID NO: 208, SEQ ID NO: 210, SEQ ID NO: 212, SEQ ID NO: 214, SEQ ID NO: 216, SEQ ID NO: 218, SEQ ID NO: 220, SEQ ID NO: 222 are examples of “homologues” of a DOF transcription factor polypeptide represented by SEQ ID NO: 199.
The further examples represented by SEQ ID NO: 235, SEQ ID NO: 237, SEQ ID NO: 239, SEQ ID NO: 241, SEQ ID NO: 243, SEQ ID NO: 245, SEQ ID NO: 247, SEQ ID NO: 249, SEQ ID NO: 251, SEQ ID NO: 253, SEQ ID NO: 255 are examples of “homologueso of a DOF transcription factor polypeptide represented by SEQ ID NO: 227.
“Homologues” of a protein are as defined herein in the uDefinitions” section.
The DOF transcription factor polypeptide or homologue thereof may be a derivative. “Derivabves” are defined in the “Definitions” section herein.
The various structural domains in a DOF transcription factor protein, such as the DOF domain, may be identified using specialised databases e.g. SMART (Schultz et al. (1998) Proc. Natl. Acad. Sci. USA 95, 5857-5864; Letunic et al. (2002) Nucleic Acids Res 30, 242-244; http://smart.embl-heidelberg.de/), InterPro (Mulder et al., (2003) Nucl. Acids. Res. 31, 315-318; http://www.ebi.ac.uk/interpro/), Prosite (Bucher and Bairoch (1994), A generalized profile syntax for biomolecular sequences motifs and its function in automatic sequence interpretation. (In) ISMB-94; Proceedings 2nd International Conference on Intelligent Systems for Molecular Biology. Altman R., Brutlag D., Karp P., Lathrop R., Searis D., Eds., pp 5361, AAAIPress, Menlo Park; Hulo et al., Nucl. Acids. Res. 32:D134-D137, (2004), http://www.expasy.org/prositeq or Pfam (Bateman et al., Nucleic Acids Research 30(1): 276-280 (2002), http://www.sanger.ac.uk/Software/Pfam/).
Examples of nucleic acids encoding DOF transcription factor polypeptides (and homologues thereof) include those represented by any one of: SEQ ID NO: 198, SEQ ID NO: 201, SEQ ID NO: 203, SEQ ID NO: 205, SEQ ID NO: 207, SEQ ID NO: 209, SEQ ID NO: 211, SEQ ID NO: 213, SEQ ID NO: 215, SEQ ID NO: 217, SEQ ID NO: 219, SEQ ID NO: 221, SEQ ID NO: 226, SEQ ID NO: 234, SEQ ID NO: 236, SEQ ID NO: 238, SEQ ID NO: 240, SEQ ID NO: 242, SEQ ID NO: 244, SEQ ID NO: 246, SEQ ID NO: 248, SEQ ID NO: 250, SEQ ID NO: 252 and SEQ ID NO: 254. Variants of nucleic acids encoding DOF transcription factor polypeptides may be suitable for use in the methods of the invention. Suitable variants include portions of nucleic acids encoding DOF transcription factor polypeptides and/or nucleic acids capable of hybridising with nucleic acids/genes encoding DOF transcription factor polypeptides. Further variants include splice variants and allelic variants of nucleic acids encoding DOF transcription factor polypeptides (and homologues thereof).
The term “portion” as defined herein refers to a piece of DNA encoding a polypeptide comprising feature (i) as follows, and additionally either feature (ii) or (iii) as follow:

    
    
        (i) in increasing order of preference at least 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% sequence identity to either the DOF domain represented by SEQ ID NO: 200 or SEQ ID NO: 228; and
        (ii) in increasing order of preference at least 70%, 75%, 80%, 85%, 90% or 95% sequence identity to the DOF domain represented by SEQ ID NO: 200; or
        (iii) Motif I: KALKKPDKILP (SEQ ID NO: 229) with no changes; or with one or more conservative change at any position; or with one, two or three non-conservative change(s) at any position; and/or
        
            Motif II: DDPGIKLFGKTIPF (SEQ ID NO: 230) with no changes; or with one or more conservative change at any position; or with one, two or three non-conservative change(s) at any position.
        
        
    
    


Additionally feature (iii) above may comprise any one, any two or all three of the following motifs:

    
    
        Motif III: SPTLGKHSRDE (SEQ ID NO: 231) with no changes; or one or more conservative change at any position; or with one, two or three non-conservative change(s) at any position; and/or
        Motif IV: LQANPAALSRSQNFQE (SEQ ID NO: 232) with no changes; or with one or more conservative change at any position; or with one, two or three non-conservative change(s) at any position; and/or
        Motif V: KGEGCLWVPKTLRIDDPDEAAKSSIWTrLGIK (SEQ ID NO: 233) with no changes; or with one or more conservative change at any position; or with one, two, three, four or five non-conservative change(s) at any position.
    
    


A portion may be prepared, for example, by making one or more deletions to a nucleic acid encoding a DOF transcription factor polypeptide. 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 DOF transcription factor portion.
Nucleic acid portions encoding DOF transcription factor polypeptides comprising features (i) and (ii) as defined hereinabove are preferably portions of a nucleic acid as represented by any one of: SEQ ID NO: 198, SEQ ID NO: 201, SEQ ID NO: 203, SEQ ID NO: 205, SEQ ID NO: 207, SEQ ID NO: 209, SEQ ID NO: 211, SEQ ID NO: 213, SEQ ID NO: 215, SEQ ID NO: 217, SEQ ID NO: 219 and SEQ ID NO: 221.
Nucleic acid portions encoding DOF transcription factor polypeptides comprising features (i) and (iii) as defined hereinabove are preferably portions of a nucleic acid as represented by any one of: SEQ ID NO: 226, SEQ ID NO: 234, SEQ ID NO: 236, SEQ ID NO: 238, SEQ ID NO: 240, SEQ ID NO: 242, SEQ ID NO: 244, SEQ ID NO: 246, SEQ ID NO: 248, SEQ ID NO: 250, SEQ ID NO: 252 and SEQ ID NO: 254.
Another variant of a DOF transcription factor nucleic acid/gene is a nucleic acid capable of hybridising under reduced stringency conditions, preferably under stringent conditions, with a DOF transcription factor nucleic acid/gene as hereinbefore defined, which hybridising sequence encodes a polypeptide comprising feature (i) as follows and additionally either feature (ii) or (iii) as follow:

    
    
        (i) in increasing order of preference at least 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% sequence identity to either the DOF domain represented by SEQ ID NO: 200 or SEQ ID NO: 228; and
        (ii) in increasing order of preference at least 70%, 75%, 80%, 85%, 90% or 95% sequence identity to the DOF domain represented by SEQ ID NO: 200; or
        (iii) Motif I: KALKKPDKILP (SEQ ID NO: 229) with no changes; or with one or more conservative change at any position; or with one, two or three non-conservative change(s) at any position; and/or
        
            Motif II: DDPGIKLFGKTIPF (SEQ ID NO: 230) with no changes; or with one or more conservative change at any position; or with one, two or three non-conservative change(s) at any position.
        
        
    
    


Additionally feature (iii) above may comprise any one, any two or all three of the following motifs:

    
    
        Motif III: SPTLGKHSRDE (SEQ ID NO: 231) with no changes; or with one or more conservative change at any position; or with one, two or three non-conservative change(s) at any position; and/or
        Motif IV: LQANPAALSRSQNFQE (SEQ ID NO: 232) with no changes; or with one or more conservative change at any position; or with one, two or three non-conservative change(s) at any position; and/or.
        Motif V: KGEGCLWVPKTLRIDDPDEMKSSIWrrLGIK (SEQ ID NO: 233) with no changes; or with one or more conservative change at any position; or with one, two, three, four or five non-conservative change(s) at any position.
    
    


Preferably, the hybridising sequence encoding DOF transcription factor polypeptides comprising features (i) and (ii) as defined hereinabove is a sequence capable of hybridising to a nucleic acid as represented by any one of: SEQ ID NO: 201, SEQ ID NO: 203, SEQ ID NO: 205, SEQ ID NO: 207, SEQ ID NO: 209, SEQ ID NO: 211, SEQ ID NO: 213, SEQ ID NO: 215, SEQ ID NO: 217, SEQ ID NO: 219 and SEQ ID NO: 221.
Preferably, the hybridising sequence encoding DOF transcription factor polypeptides comprising features (i) and (iii) as defined hereinabove is a sequence capable of hybridising to a nucleic acid as represented by any one of: SEQ ID NO: 234, SEQ ID NO: 236, SEQ ID NO: 238, SEQ ID NO: 240, SEQ ID NO: 242, SEQ ID NO: 244, SEQ ID NO: 246, SEQ ID NO: 248, SEQ ID NO: 250, SEQ ID NO: 252 and SEQ ID NO: 254.
The term “hybridisation” is as defined herein in the “Definitions” section.
The DOF transcription factor polypeptide may be encoded by an alternative splice variant. The term “alternative splice variant” is as defined in the “Definitions” section herein.
Preferred splice variants are splice variants of the nucleic acid encoding a polypeptide comprising feature (i) as follows and additionally either feature (ii) or (iii) as follow:

    
    
        (i) in increasing order of preference at least 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% sequence identity to either the DOF domain represented by SEQ ID NO: 200 or SEQ ID NO: 228; and
        (ii) in increasing order of preference at least 70%, 75%, 80%, 85%, 90% or 95% sequence identity to the DOF domain represented by SEQ ID NO: 200; or
        (iii) Motif I: KALKKPDKILP (SEQ ID NO: 229) with no changes; or with one or more conservative change at any position; or with one, two or three non-conservative change(s) at any position; and/or
        
            Motif II: DDPGIKLFGKTIPF (SEQ ID NO: 230) with no changes; or with one or more conservative change at any position; or with one, two or three non-conservative change(s) at any position.
        
        
    
    


Preferred splice variants of nucleic acids encoding DOF transcription factor polypeptides comprising features (i) and (ii) as defined hereinabove are splice variants of a nucleic acid as represented by any one of: SEQ ID NO: 201, SEQ ID NO: 203, SEQ ID NO: 205, SEQ ID NO: 207, SEQ ID NO: 209, SEQ ID NO: 211, SEQ ID NO: 213, SEQ ID NO: 215, SEQ ID NO: 217, SEQ ID NO: 219 and SEQ ID NO: 221.
Preferred splice variants of nucleic acids encoding DOF transcription factor polypeptides comprising features (i) and (iii) as defined hereinabove are preferably splice variants of a nucleic acid as represented by any one of: SEQ ID NO: 234, SEQ ID NO: 236, SEQ ID NO: 238, SEQ ID NO: 240, SEQ ID NO: 242, SEQ ID NO: 244, SEQ ID NO: 246, SEQ ID NO: 248, SEQ ID NO: 250, SEQ ID NO: 252 and SEQ ID NO: 254.
The DOF transcription factor polypeptide may also be encoded by an allelic variant, which are also defined in the “Definitions” section herein.
Preferred allelic variants are allelic variants of the nucleic acid encoding a polypeptide comprising feature (i) as follows and additionally either feature (ii) or (iii) as follow:

    
    
        (i) in increasing order of preference at least 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% sequence identity to either the DOF domain represented by SEQ ID NO: 200 or SEQ ID NO: 228; and
        (ii) in increasing order of preference at least 70%, 75%, 80%, 85%, 90% or 95% sequence identity to the DOF domain represented by SEQ ID NO: 200; or
        (iii) Motif I: KALKKPDKILP (SEQ ID NO: 229) with no changes; or with one or more conservative change at any position; or with one, two or three non-conservative change(s) at any position; and/or
        
            Motif II: DDPGIKLFGKTIPF (SEQ ID NO: 230) with no changes; or with one or more conservative change at any position; or with one, two or three non-conservative change(s) at any position.
        
        
    
    


Preferred allelic variants of nucleic acids encoding DOF transcription factor polypeptides comprising features (i) and (ii) as defined hereinabove are splice variants of a nucleic acid as represented by any one of: SEQ ID NO: 201, SEQ ID NO: 203, SEQ ID NO: 205, SEQ ID NO: 207, SEQ ID NO: 209, SEQ ID NO: 211, SEQ ID NO: 213, SEQ ID NO: 215, SEQ ID NO: 217, SEQ ID NO: 219 and SEQ ID NO: 221.
Preferred allelic variants of nucleic acids encoding DOF transcription factor polypeptides comprising features (i) and (iii) as defined hereinabove are preferably portions of a nucleic acid as represented by any one of. SEQ ID NO: 234, SEQ ID NO: 236, SEQ ID NO: 238, SEQ ID NO: 240, SEQ ID NO: 242, SEQ ID NO: 244, SEQ ID NO: 246, SEQ ID NO: 248, SEQ ID NO: 250, SEQ ID NO: 252 and SEQ ID NO: 254.
Further variants of Nucleic acids encoding DOF transcription factor polypeptides as defined hereinabove may be generated using, for example, site-directed mutagenesis as defined in the “Definitions” section herein.
Directed evolution (or gene shuffling) may also be used to generate variants of nucleic acids encoding DOF transcription factor polypeptides. See “definitions” section.
DOF transcription factor polypeptides are plant-specific. Nucleic acids encoding the same may be derived from any natural or artificial source. The nucleic acid or variant thereof may be modified from its native form in composition and/or genomic environment through deliberate human manipulation. Preferably the DOF transcription factor nucleic acid or variant thereof is from a dicotyledonous plant, further preferably from the family Brassicaceae, more preferably the nucleic acid is from Arabidopsis thaliana. 
The expression of a nucleic acid encoding a DOF transcription factor polypeptide may be increased by introducing a genetic modification (preferably in the locus of a DOF transcription factor 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 and homologous recombination or by introducing and expressing in a plant a nucleic acid encoding a DOF transcription factor polypeptide. The methods of T-DNA activation, TILLING and homologous recombination are as defined in the “Definitions” section herein. Following introduction of the genetic modification, there follows an optional step of selecting for increased expression of a nucleic acid encoding a DOF transcription factor polypeptide, which increased expression gives plants having increased yield.
T-DNA activation and TILLING are examples of technologies that enable the generation of novel alleles and DOF transcription factor variants.
A preferred method for introducing a genetic modification (which in this case need not be in the locus of a DOF transcription factor gene) is to introduce and express in a plant a nucleic acid encoding a DOF transcription factor polypeptide as defined hereinabove. The nucleic acid to be introduced into a plant may be a full-length nucleic acid or may be a portion or a hybridising sequence or another nucleic acid variant as hereinbefore defined.
The methods of the invention rely on increased expression of a nucleic acid encoding a DOF transcription factor 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 nucleic acid encoding a DOF transcription factor polypeptide. For example, endogenous promoters may be altered in vivo by mutation, deletion, and/or substitution (see, Kmiec, U.S. Pat. No. 5,565,350; Zarling et al., PCT/US93/03868), or isolated promoters may be introduced into a plant cell in the proper orientation and distance from a gene of the present invention so as to control the expression of the gene.
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 3′ end sequence to be added may be derived from, for example, the nopaline synthase or octopine synthase genes, or alternatively from another plant gene, or less preferably from any other eukaryotic gene.
An intron sequence may also be added to the 5′ untranslated region or the coding sequence of the partial coding sequence to increase the amount of the mature message that accumulates in the cytosol. Inclusion of a spliceable intron in the transcription unit in both plant and animal expression constructs has been shown to increase gene expression at both the mRNA and protein levels up to 1000-fold (Buchman and Berg (1988) Mol. Cell. biol. 8: 4395-4405; Callis et al. (1987) Genes Dev 1:1183-1200). Such intron enhancement of gene expression is typically greatest when placed near the 5′ end of the transcription unit. Use of the maize introns Adh1-S intron 1, 2, and 6, the Bronze-1 intron are known in the art. For general information see: The Maize Handbook, Chapter 116, Freeling and Walbot, Eds., Springer, N.Y. (1994).
The invention also provides genetic constructs and vectors to facilitate introduction and/or expression of the nucleotide sequences useful in the methods according to the invention.
Therefore, there is provided a gene construct comprising:

    
    
        (i) A nucleic acid or variant thereof encoding a DOF transcription factor polypeptide 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.
    
    


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. The Invention therefore provides use of a gene construct as defined hereinabove in the methods of the invention.
Plants are transformed with a vector comprising the sequence of interest (i.e., a nucleic acid encoding a DOF transcription factor polypeptide). 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 defined in the “Definitions” section herein.
Advantageously, any type of promoter, whether natural or synthetic, may be used to drive expression of the nucleic acid sequence.
According to one preferred feature of the invention, the DOF transcription factor nucleic acid or variant thereof is operably linked to a constitutive promoter as defined in the “Definitions” section herein. The constitutive promoter is preferably a GOS2 promoter, more preferably the constitutive promoter is a rice GOS2 promoter, further preferably the constitutive promoter is represented by a nucleic acid sequence substantially similar to SEQ ID NO: 225, most preferably the constitutive promoter is as represented by SEQ ID NO: 225. Preferred is the use of a constitutive promoter to drive expression of a nucleic acid encoding a DOF transcription factor polypeptide comprising features (i) and (ii) as defined hereinabove, i.e. at least 60% sequence identity to either the DOF domain represented by SEQ ID NO: 200 or SEQ ID NO: 228; and at least 70% sequence identity to the DOF domain represented by SEQ ID NO: 200.
It should be clear that the applicability of the present invention is not restricted to the DOF transcription factor nucleic acid represented by SEQ ID NO: 198, nor is the applicability of the invention restricted to expression of a DOF transcription factor nucleic acid when driven by a GOS2 promoter. Examples of other constitutive promoters which may also be used perform the methods of the invention are shown in Table 3 in the “Definitions” section herein.
According to another preferred feature of the invention, the nucleic acid encoding a DOF transcription factor polypeptide is operably linked to a seed-specific promoter, i.e. a promoter that is expressed predominantly in seed tissue, but which may have residual expression elsewhere in the plant due to leaky promoter expression. Further preferably, the seed-specific promoter is isolated from a gene encoding a seed-storage protein, especially an endosperm-specific promoter. Most preferably the endosperm-specific promoter is isolated from a prolamin gene, such as a rice prolamin RP6 (Wen et al., (1993) Plant Physiol 101(3): 1115-6) promoter as represented by SEQ ID NO: 258, or a promoter of similar strength and/or a promoter with a similar expression pattern as the rice prolamin promoter. Similar strength and/or similar expression pattern may be analysed, for example, by coupling the promoters to a reporter gene and checking the function of the reporter gene in tissues of the plant. One well-known reporter gene is beta-glucuronidase and the calorimetric GUS stain used to visualize betaglucuronidase activity in plant tissue. The prolamin promoter shows strong expression in the endosperm, with leakiness in meristem, more specifically the shoot meristem and/or discrimination centre in the meristem.
Preferred according to the invention is the use of a seed-specific promoter, especially an endosperm-specific promoter, to drive expression of a nucleic acid encoding a DOF transcription factor polypeptide comprising features (i) and (iii) as defined hereinabove, i.e. at least 60% sequence identity to either the DOF domain represented by SEQ ID NO: 200 or SEQ ID NO: 228; and Motif I and/or Motif II.
It should be clear that the applicability of the present invention is not restricted to the DOF transcription factor nucleic acid represented by SEQ ID NO: 226, nor is the applicability of the invention restricted to expression of a DOF transcription factor nucleic acid when driven by a prolamin promoter.
Examples of seed-specific promoters are presented in Table 7 in the “Definitions” section herein, which promoters or derivatives thereof are useful in performing the methods of the present invention.
Optionally, one or more terminator sequences may also be used in the construct introduced into a plant. The term “terminator” is as defined in the “Definitions” section herein.
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 defined herein in the “Definitions” section.
The present invention also encompasses plants obtainable by the methods according to the present invention. The present invention therefore provides plants, plant parts or plant cells thereof obtainable by the method according to the present invention, which plants or parts or cells thereof comprise a nucleic acid transgene (or variant thereof as define above) encoding a DOF transcription factor polypeptide.
The invention also provides a method for the production of transgenic plants having increased yield relative to suitable control plants, comprising introduction and expression in a plant of a nucleic acid or a variant thereof encoding a DOF transcription factor polypeptide.
More specifically, the present invention provides a method for the production of transgenic plants having increased yield which method comprises:

    
    
        (i) introducing and expressing in a plant, plant part or plant cell a nucleic acid or variant thereof encoding a DOF transcription factor polypeptide; and
        (ii) cultivating the plant cell under conditions promoting plant growth and development.
    
    


The nucleic acid may be introduced directly into a plant cell or into the plant itself (including introduction into a tissue, organ or any other part of a plant). According to a preferred feature of the present invention, the nucleic acid is preferably introduced into a plant by transformation.
The term “transformation” is as defined herein in the “Definitions” section.
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 nucleic acid or variant thereof encoding a DOF transcription factor polypeptide. 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, stems, rhizomes, tubers and bulbs. The invention furthermore relates to products derived, preferably directly derived, from a harvestable part of such a plant, such as dry pellets or powders, oil, fat and fatty acids, starch or proteins.
The present invention also encompasses use of nucleic acids or variants thereof encoding DOF transcription factor polypeptides and use of DOF transcription factor polypeptides in increasing plant yield as defined hereinabove in the methods of the invention.
Nucleic acids or variants thereof encoding DOF transcription factor polypeptides, or DOF transcription factor polypeptides, may find use in breeding programmes in which a DNA marker is identified which may be genetically linked to a DOF transcription factor gene or variant thereof. The nucleic acids/genes or variants thereof, or the DOF transcription factor polypeptides 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 as defined hereinabove in the methods of the invention.
Allelic variants of a DOF transcription factor 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. 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 nucleic acid or variant thereof encoding a DOF transcription factor polypeptide 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 DOF transcription factor nucleic acids or variants thereof requires only a nucleic acid sequence of at least 15 nucleotides in length. The DOF transcription factor nucleic acids or variants thereof may be used as restriction fragment length polymorphism (RFLP) markers. Southern blots (Sambrook J, Fritsch E F and Maniatis T (1989) Molecular Cloning, A Laboratory Manual) of restriction-digested plant genomic DNA may be probed with the DOF transcription factor 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 DOF transcription factor nucleic acid or variant thereof in the genetic map previously obtained using this population (Botstein et al. (1980) Am. J. Hum. Genet. 32:314-331).
The production and use of plant gene-derived probes for use in genetic mapping is described in Bernatzky and Tanksley (1986) Plant Mol. Biol. Reporter 4: 37-41. Numerous publications describe genetic mapping of specific cDNA clones using the methodology outlined above or variations thereof. For example, F2 intercross populations, backcross populations, randomly mated populations, near isogenic lines, and other sets of individuals may be used for mapping. Such methodologies are well known to those skilled in the art.
The nucleic acid probes may also be used for physical mapping (i.e., placement of sequences on physical maps; see Hoheisel et 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 hybridisation (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. This increased yield may also be combined with other economically advantageous traits, such as further yield-enhancing traits, tolerance to other abiotic and biotic stresses, traits modifying various architectural features and/or biochemical and/or physiological features.
Detailed Description CKI
Reference herein to a preferential “reduction” in expression of an endogenous CKI gene in endosperm tissue of a plant is taken to mean a reduction or substantial elimination of expression of an endogenous CKI gene (in endosperm tissue) relative to endogenous CKI gene expression levels found in endosperm tissue of wild type plants. This reduction or substantial elimination of endogenous CKI gene expression may result in reduced or substantially eliminated CKI protein levels and/or activity in endosperm tissue of a plant.
Reference herein to an “endogenous” CKI gene not only refers to CKI genes as found in a plant in its natural form (i.e., without there being any human intervention), but also refers to isolated CKI genes subsequently introduced into a plant. For example, a transgenic plant containing a CKI transgene may encounter a reduction or substantial elimination of the CKI transgene and/or a reduction or substantial elimination of an endogenous CKI gene (in endosperm tissue).
This reduction (or substantial elimination) of endogenous CKI gene expression may be achieved using any one or more of several well-known gene silencing methods. “Gene silencing” or “downregulation” of expression, as used herein, refers to a reduction or the substantial elimination of CKI gene expression and/or CKI polypeptide levels and/or CKI polypeptide activity.
One such method for reduction or substantial elimination of endogenous CKI gene expression is RNA-mediated downregulation of gene expression (RNA silencing). Silencing in this case is triggered in a plant by a double stranded RNA molecule (dsRNA) that is substantially homologous to a target CKI gene. This dsRNA is further processed by the plant into about 21 to about 26 nucleotides called short interfering RNAs (siRNAs). The siRNAs are incorporated into an RNA-induced silencing complex (RISC) that cleaves the mRNA of a CKI target gene, thereby reducing or substantially eliminating the number of CKI mRNAs to be translated into a CKI protein.
One example of an RNA silencing method involves the introduction of coding sequences or parts thereof in a sense orientation into a plant. “Sense orientation” refers to DNA that is homologous to an mRNA transcript thereof. Introduced into a plant would therefore be at least an additional copy (in full or in part) of a CKI gene already present in the host plant. The additional gene, or part thereof, will silence an endogenous CKI gene, giving rise to a phenomenon known as co-suppression. The reduction of CKI gene expression will be more pronounced if several additional copies are introduced into the plant, as there is a positive correlation between high transcript levels and the triggering of co-suppression.
Another example of an RNA silencing method involves the use of antisense CKI nucleic acid sequences. An “antisense” nucleic acid comprises a nucleotide sequence that is complementary to a “sense” nucleic acid encoding a protein, e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence. Accordingly, an antisense nucleic acid can hydrogen bond to a sense nucleic acid. The antisense nucleic acid can be complementary to an entire CKI coding strand or only to a portion thereof. The antisense nucleic acid molecule may be antisense to a “coding region” or antisense to a “non-coding region” of the coding strand of a nucleotide sequence encoding CKI. The term “coding region” refers to the region of the nucleotide sequence comprising codons that are translated into amino acid residues. The term “non-coding region” refers to 5′ and 3′ sequences that flank the coding region that are not translated into amino acids (I.e., also referred to as 5′ and 3′ untranslated regions).
Antisense nucleic acids can be designed according to the rules of Watson and Crick base pairing. The antisense nucleic acid molecule may be complementary to the entire coding region of CKI mRNA, but is preferably an oligonucleotide which is antisense to only a portion of the coding or non-coding region of CKI mRNA. For example, the antisense oligonucleotide may be complementary to the region surrounding the translation start site of CKI mRNA. The length of a suitable antisense oligonucleotide would be known in the art and may be start from about 20 nucleotides in length or less. An antisense nucleic acid of the invention can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art. For example, an antisense nucleic acid (e.g., an antisense oligonucleotide) can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used. Examples of modified nucleotides which can be used to generate the antisense nucleic acid are well known in the art.
Other known nucleotide modifications include methylation, cyclization and ‘caps’ and substitution of one or more of the naturally occurring nucleotides with an analog such as inosine. Other modifications of nucleotides are well known to a person skilled in the art.
Alternatively, the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic add of interest, described further in the following subsection). Preferably, production of antisense nucleic acids in plants occurs by means of a stably integrated transgene comprising a promoter operative for preferential expression in endosperm tissue plants, an antisense oligonucleotide, and a terminator.
A preferred method for reduction or substantial elimination of endogenous CKI gene expression via RNA silencing is by using an expression vector into which a CKI gene or fragment thereof has been cloned as an inverted repeat (in part or completely), separated by a spacer (non-coding DNA). After transcription of the inverted repeat, a chimeric CKI RNA with a self-complementary structure is formed (partial or complete). This double-stranded RNA structure is referred to as the hairpin RNA (hpRNA). The hpRNA is processed by the plant into siRNAs that are incorporated into a RISC. The RISC further cleaves the mRNA of a CKI target gene, thereby reducing or substantially eliminating the number of CKI mRNAs to be translated into a CKI protein. See for example, Grierson et al. (1998) WO 98/53083; Waterhouse et al. (1999) WO 99/53050).
The nucleic acid molecules used for silencing in the methods of the invention (whether introduced into a plant or generated in situ) hybridize with or bind to cellular mRNA and/or genomic DNA encoding an CKI protein to thereby inhibit expression of the protein, e.g., by inhibiting transcription and/or translation. The hybridization can be by conventional nucleotide complementarity to form a stable duplex, or, for example, in the case of an antisense nucleic acid molecule which binds to DNA duplexes, through specific interactions in the major groove of the double helix. Antisense nucleic acid molecules may be introduced into a plant by transformation or direct injection at a specific tissue site. Alternatively, antisense nucleic acid molecules can be modified to target selected cells and then administered systemically. For example, for systemic administration, antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, e.g., by linking the antisense nucleic acid molecules to peptides or antibodies which bind to cell surface receptors or antigens. The antisense nucleic acid molecules can also be delivered to cells using the vectors described herein.
According to a further aspect, the antisense nucleic acid is an α-anomeric nucleic acid molecule. An α-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual β-units, the strands run parallel to each other (Gaultier et al., (1987) Nucleic Acids. Res. 15:6625-6641). The antisense nucleic acid molecule may also comprise a 2′-o-methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res. 15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987) FEBS Lett. 215:327-330).
In still another embodiment, an antisense nucleic acid of the invention is a ribozyme. Ribozymes are catalytic RNA molecules with ribonuclease activity which are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region. Thus, ribozymes (e.g., hammerhead ribozymes (described in Haselhoff and Gerlach (1988) Nature 334:585-591)) can be used to catalytically cleave CKI mRNA transcripts to thereby inhibit translation of CKI mRNA. A ribozyme having specificity for a CKI-encoding nucleic acid can be designed based upon the nucleotide sequence of a CKI cDNA. For example, a derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in an CKI-encoding mRNA. See, e.g., Cech et al. U.S. Pat. No. 4,987,071; and Cech et at. U.S. Pat. No. 5,116,742. Alternatively, CKI mRNA can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, e.g., Bartel, D. and Szostak, J. W. (1993) Science 261:1411-1418. The use of ribozymes for gene silencing in plants is known in the art (e.g., Atkins et al. (1994) WO 94/00012; Lenne et at. (1995) WO 95/03404; Lutziger et al. (2000) WO 00/00619; Prinsen et al. (1997) WO 97113865 and Scott et al. (1997) WO 97/38116).
Gene silencing may also be achieved by insertion mutagenesis (for example, T-DNA insertion or transposon insertion) or by gene silencing strategies as described by, among others, Angell and Baulcombe 1998 (Amplicon VIGS WO 98/36083); Baulcombe (WO 99/15682).
Gene silencing may also occur if there is a mutation on the endogenous CKI gene and/or a mutation on an isolated CKI gene subsequently introduced into a plant. The reduction or substantial elimination of CKI expression may be caused by a non-functional CKI. CKI binds to both CDK and cyclins (Verkest et at., (2005) Plant Cell 17: 1723-1736). For example, mutation of the cyclin binding site within a CKI, provides for a CKI that can still bind to a CDK but that cannot inhibit the active CDK-cyclin complex.
A further approach to gene silencing is by targeting nucleotide sequences complementary to the regulatory region of the CKI (e.g., the CKI promoter and/or enhancers) to form triple helical structures that prevent transcription of the CKI gene in target cells. See Helene, C. (1991) Anticancer Drug Des. 6(6): 56984; Helene, C. et al. (1992) Ann. N.Y. Acad. Sci. 660:27-36; and Maher, L. J. (1992) Bioassays 14(12): 807-15.
Described above are examples of various methods for gene silencing (for the reduction or substantial elimination of endogenous CKI gene expression. The methods of the invention rely on the preferential reduction of expression of an endogenous CKI gene in endosperm tissue of a plant. A person skilled in the art would readily be able to adapt the aforementioned methods for silencing so as to achieve preferential gene silencing in endosperm tissue, through the use of an appropriate promoter, for example.
It should be noted that the essence of the present invention resides in the advantageous and surprising results found upon reduction or substantial elimination of endogenous CKI gene expression in endosperm tissue of a plant, and is not limited to any particular method for such reduction or substantial elimination of endogenous CKI gene expression. Other such methods will be well known to the skilled man.
For optimal performance, the gene silencing techniques used for the reduction or substantial elimination of endogenous CKI gene expression requires the use of CKI nucleic acid sequences from monocotyledonous plants for transformation into monocotyledonous plants. Preferably, a CKI nucleic acid from any given plant species is introduced into that same species. For example, a CKI nucleic acid from rice (be it a full length CKI sequence or a fragment) is transformed into a rice plant. The CKI nucleic acid need not be introduced into the same plant variety.
Reference herein to a “CKI gene” or a CKI nucleic acid” is taken to mean a polymeric form of a deoxyribonucleotide or a ribonucleotide polymer of any length, either double- or single-stranded, or analogues thereof, that have the essential characteristic of a natural ribonucleotide in that they can hybridise to nucleic acids in a manner similar to naturally occurring polynucleotides. A “CKI gene” or a CKI nucleic acid” refers to a sufficient length of substantially contiguous nucleotides of a CKI-encoding gene to perform gene silencing; this may be as little as 20 or fewer nucleotides. A gene encoding a (functional) protein is not a requirement for the various methods discussed above for the reduction or substantial elimination of expression of an endogenous CKI gene.
The methods of the invention may be performed using a sufficient length of substantially contiguous nucleotides of a CKI gene/nucleic acid, which may consist of 20 or fewer nucleotides, which may be from any part of the CKI gene/nucleic acid, such as the 3′ end of the coding region that is well conserved amongst the CKI gene family.
CKI genes are well known in the art and useful in the methods of the invention are substantially contiguous nucleotides of any of the plant CKI genes/nucleic acid described in published International patent application WO 2005/007829 in the name of Monsanto Technology LLC and Published International patent applications, WO 02/28893 and WO 99114331 in the name of CropDesign N.V, which CKI gene/nucleotide sequences are incorporated herein as if fully set forth.
Other CKI gene/nucleic acid sequences may also be used in the methods of the invention, and may readily be identified by a person skilled in the art. CKI polypeptides may be identified by the presence of one or more of several well-known features (see below). Upon identification of a CKI polypeptide, a person skilled in the art could easily derive, using routine techniques, the corresponding encoding nucleic acid sequence and use a sufficient length of contiguous nucleotides of the same to perform any one or more of the gene silencing methods described above (for the reduction or substantial elimination of an endogenous CKI gene expression, in the endosperm).
One distinguishing feature of a CKI polypeptide is a C-terminal region comprising between about 40 and about 55 highly conserved amino acids. As a guide, polypeptides comprising in increasing order of preference at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to the C-terminal region of a CKI as represented by SEQ ID NO: 262 may be taken to be CKI homologues. A person skilled in the art may easily derive the corresponding nucleic acid encoding such homologues, and use a sufficient length of contiguous nucleotides of the same to perform any one or more of the gene silencing methods described above (for the reduction or substantial elimination of an endogenous CKI gene expression).
A person skilled in the art will be well aware of what is meant by a “C-terminal” of a protein; for the purposes of this application, the C-terminal region of a CKI may be taken to be the second half (from N-terminal to C-terminal) of a full length CKI polypeptide.
Homologues, as defined above, i.e. polypeptides comprising at least 50% identity to the C-terminal region of a CKI as represented by SEQ ID NO: 262, 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 Mol 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 Mol 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. Homologous sequences 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. Minor manual editing may be performed to optimise alignment between conserved motifs (see below), as would be apparent to a person skilled in the art.
Plant CKI polypeptides may also be identified by the presence of certain conserved motifs (see Table 12 below). The presence of these conserved motifs may be identified using methods for the alignment of sequences for comparison as described hereinabove. In some instances, the default parameters may be adjusted to modify the stringency of the search. For example using BLAST, the statistical significance threshold (called “expect” value) for reporting matches against database sequences may be increased to show less stringent matches. This way, short nearly exact matches may be identified. Upon identification of a CKI polypeptide by the presence of these motifs, a person skilled in the art may easily derive the corresponding nucleic acid encoding the polypeptide comprising the relevant motifs, and use a sufficient length of contiguous nucleotides of the same to perform any one or more of the gene silencing methods described above (for the reduction or substantial elimination of an endogenous CKI gene expression).
Typically, the presence of at least one of the motifs 1 to 5 (for example motif 2 is particularly well conserved) should be sufficient to identify any query sequence as a CKI, however for increased certainty, the presence of at least Motifs 1, 2 and 3 is preferred. The consensus sequence provided is based on the sequences displayed in Table 12 below. A person skilled in the art would be well aware that the consensus sequence may vary somewhat if further or different sequences were used for comparison.
Motif 1: FXXKYNFD (SEQ ID NO: 261), wherein X is any amino acid
Motif 2: [P/L]LXGRYEW (SEQ ID NO: 262), wherein X is any amino acid and [P/L] means that either a proline or a leucine appear at the indicated position
Motif 3: EXE[D/E]FFXXXE (SEQ ID NO: 263), wherein X is any amino acid and [D/E] means that either an aspartate or a glutamate appear at the indicated position
Motif 4: YXQLRSRR (SEQ ID NO: 264), wherein X is any amino acid
Motif 5: MGKY[M/I][K/R]KX[(KR] (SEQ ID NO: 265), wherein X is any amino acid, [M/I] means that either a methionine or an isoleucine appear at the indicated position, and [K/R] means that either a lysine or an arginine appear at the indicated position
Motif 6: SXGVRTRA (SEQ ID NO: 266), wherein X is any amino acid
Motifs 1, 2, and 3 are typically found in the carboxyl-terminal region of plant CKI proteins. This region is believed to be involved in the interaction of CKIs with both CDKs and cyclins (Chen et al. (1996) Mol. Cell Biol 16, 46734682, Matsuoka et al. (1995) Genes Dev. 9, 650-662, and Nakayama and Nakayama (1998) Bioessays 20, 1020-1029). Motifs 4, 5, and 6 are typically found in the amino-terminal region of plant CKI proteins.
CKI proteins from monocot plants, particularly rice, are characterized by extensive α-helical stretches especially between motifs 5 and 6 and between motifs 6 and 4.







TABLE 12







Conserved motifs in plant CKI proteins. CKI1 to CKI7 denote Arabidopsis thaliana


CKIs. Os: Oryza sativa, Zm: Zea mays, Sb: Sorghum bicolor














Motif 1
Motif 2
Motif 3
Motif 4
Motif 5
Motif 6

















Alfalfa CKI
198-
211-
182-
74-
1-
45-



FMEKYNFD
PLPGRYET
EFEEFCAK
YLQLRNRR
MGKYMKKLK
SDGVRTRA





HE


CKI1
167-
180-
151-
20-


AC003040
FKKKYNFD
PLEGRYEW
EIEDFFVE
YMQLRSRR





AE


CKI2
183-
197-
164-


AL132979
CSMKYNFD
LGGGRYEW
ELEDFFQV





AE


CKI3
197-
210-
181-
58-
1-
26-


AB012242
FMEKYNFD
PLSGRYEW
EMEEFFAY
YLQLRSRR
MGKYMKKSK
SPGVRTRA





AE


CKI4
264-
277-
248-
102-
1-
44-


AC003974
FIEKYNFD
PLPGRFEW
EMDEFFSG
YLQLRSRR
MGKYIRKSK
SLGVLTRA





AE


CKI5
164-
177-
148-
54-
1-
24-


AB028609
FIQKYNFD
PLPGRYEW
EIEDFFAS
YLQLRSRR
MGKYIKKSK
ALGFRTRA





AE


CKI6
173-
186-
155-


AP000419
FIEKYNFD
PLEGRYKW
EIEDLFSE





LE


CKI7
170-
183-
154-


AC011807
FTEKYNYD
PLEGRYQW
ELDDFFSA





AE


Chenopodium
171-
184-
155-
25-


CKI
FSEKYNFD
PLKGRYDW
EIEEFFAV
IPQLRSRR


AJ002173


AE


OsCKI2
233-
247-
217-
75-
1-
24-



FAAKYNFD
LDAGRFEW
EIEAFFAA
YLQLRSRM
MGKYMRKFR
VVGVRTRS





AE


OsCKI1
----YNYD
PLQGRYEW


OsCKI3
FAEKY---

EIEAFFAA





AE


OsCKI4
170-
183-
154-
48-
1-
28-



FIDKYNFD
PLPGRFEW
ELEAFFAA
YLELRSRR
MGKYMRKAK
PLGVRTRA





EE


OsCKI5
196-
209-
180-
63-
1-
20-



FAAKYNFD
PLDAGGAG
EIEEFLAA
YLRLRSRR
MGKKKKRDG
VGGVRTRA




RFEW
AE


ZmCKI1
FASKYNFD
LDAGRFEW
EIQEFFAA





AE


ZmCKI2
FIDKYNFD
PLPGRFEW
EMNEYFAA





EQ


SbCKI
FAEAYNYD
PLEGRFEW
EIEAFFAA





AE


CONSENSUS
FX2KYNFD
[P/L]LXG
EXE[D/E]
YXQLRSRR
MGKY[M/I]
SXGVRTRA




R[Y/F]EW
FFX3E

[K/R]KX







[K/R]









In addition to the abovementioned features, a CKI protein may also comprise any one or more of the following: a Cy-box, a nuclear localization sequence and a PEST sequence.
The term “Cy-Box” refers to an amino acid sequence of about 5 amino acid residues in length having the consensus sequence RXHuF, wherein X is any amino acid and Hu is a hydrophobic uncharged amino acid, such as M, I, L or V. Cy-boxes are typically involved in the interaction of CKIs with cyclins.
A “nuclear localization sequence” refers to an amino acid sequence of about 4-20 amino acid residues in length, which serves to direct a protein to the nucleus. Typically, the nuclear localization sequence is rich in basic amino acids, such as arginine (R) and lysine (K). Nuclear localization signals are described in, for example, Gorlich D. (1998) EMBO 5.17:2721-7. The Os CKI4 protein comprises multiple nuclear localization sequences.
A “PEST sequence” refers to an amino acid sequence which is enriched in the amino acid residues proline (P), glutamate (E), serine (S) and threonine M and which is present in proteins with a high proteolytic turnover rate. PEST sequences are described in, for example, Rogers et al., (1986) Science 234, 364-368.
The various structural domains in a CKI protein may be identified using specialised databases e.g. SMART (Schultz et al. (1998) Proc. Natl. Acad. Sci. USA 95, 5857-5864; Letunic et al.
(2002) Nucleic Acids Res 30, 242-244; http://smart.embl-heidelberg.deI), InterPro (Mulder et al., (2003) Nucl. Acids. Res. 31, 315-318; http://www.ebi.ac.ukfinterpro/), Prosite (Bucher and Bairoch (1994), A generalized profile syntax for biomolecular sequences motifs and its function in automatic sequence interpretation. (In) ISMB-94; Proceedings 2nd International Conference on Intelligent Systems for Molecular Biology. Altman R., Brutlag D., Karp P., Lathrop R., Searls D., Eds., pp 53-61, AAAIPress, Menlo Park; Hulo et al., Nucl. Acids. Res. 32:D134-D137, (2004), http://www.expasy.org/prosite/) or Pfam (Bateman et al., Nucleic Acids Research 30(1): 276-280 (2002), http://www.sanger.ac.uk/Software/Pfam/).
Furthermore, a CKI protein may also be identifiable by its ability to inhibit the activity of a Cyclin Dependent Kinase (CDK), e.g., a plant CDK. CDKs are a group of serinelthreonine kinases which regulate the progression of the cell cycle in eukaryotes, e.g., plants. CDKs are typically complexed with cyclins forming an enzyme complex, CDK being the catalytic subunit and cyclin being the regulatory subunit of the enzyme complex (VWang, H. (1997) The Plant Journal 15(4): 501-510).
Therefore upon identification of a CKI polypeptide using one or several of the features described above, a person skilled in the art may easily derive the corresponding nucleic acid encoding the polypeptide, and use a sufficient length of substantially contiguous nucleotides of the same to perform any one or more of the gene silencing methods described above (for the reduction or substantial elimination of an endogenous CKI gene expression).
Preferred for use in the methods of the invention is a sufficient length of substantially contiguous nucleotides of SEQ ID NO: 267 (OsCKI4), or the use of a sufficient length of substantially contiguous nucleotides of a nucleic acid sequence encoding an orthologue or paralogue of OsCKI4 (SEQ ID NO: 267). Examples of such orthologues and paralogues of OsCKI4 are provided in Table 13 below.
Orthologues and paralogoues are homologues that 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: 267 or SEQ ID NO: 268) 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: 267 or SEQ ID NO: 268 the second blast would therefore be against rice 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.







TABLE 13







Orthologues and Paralogues of OsCKI4 (SEQ ID NO: 267 and 268)














SEQ ID





SEQ ID nucleic
polypeptide


Name
NCBI
acid sequence
sequence
Source





Zeama_CKI4
AY986792
269
270
Zea mays


like


Triae_CKI4 like
Contig of
271
272
Triticum aestivum



BG908519.1



and



CA640135.1


Orysa_CKI3
AK064723.1
273
274
Oryza sativa


Zeama_CKI3
DV174570.1
275
276
Zea mays


like


Sorbi_CKI3 like
contig of
277
278
Sorghum bicolor



CN152732.1



and



CD224882.1


Sacof_CKI4 like
CO373621.1
279
280
Saccharum officinarum









The source of the substantially contiguous nucleotides of a CKI gene/nucleic acid may be any plant source or artificial source. For optimal performance, the gene silencing techniques used for the reduction or substantial elimination of endogenous CKI gene expression requires the use of CKI sequences from monocotyledonous plants for transformation into monocotyledonous plants. Preferably, CKI sequences from the family Poaceae are transformed into plants of the family Poaceae. Further preferably, a CKI nucleic acid from rice (be it a full length CKI sequence or a fragment) is transformed into a rice plant. The CKI nucleic acid need not be introduced into the same plant variety. Most preferably, the CKI nucleic acid from rice is a sufficient length of substantially contiguous nucleotides of SEQ ID NO: 267 (OsCKI4) or a sufficient length of substantially contiguous nucleotides of a nucleic acid sequence encoding an orthologue or paralogue of OsCKI4 (SEQ ID NO: 267). As mentioned above, a person skilled in the art would be well aware of what would constitute a sufficient length of substantially contiguous nucleotides to perform any of the gene silencing methods defined hereinabove, this may be as little as 20 or fewer substantially contiguous nucleotides in some cases.
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 one or more control sequences capable of preferentially driving expression of a sense and/or antisense CKI nucleic acid sequence in plant endosperm tissue so as to silence an endogenous CKI gene in endosperm tissue of a plant; and optionally a transcription termination sequence.
A preferred construct for gene silencing is one comprising an inverted repeat of a CKI gene or fragment thereof, preferably capable of forming a hairpin structure, which inverted repeat is under the control of an endosperm-specific promoter.
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. The invention therefore provides use of a gene construct as defined hereinabove in the methods of the invention.
The sequence of interest is operably linked to one or more control sequences (at least to a promoter) capable of preferentially increasing expression in endosperm tissue of a plant. The terms “regulatory element”, “control sequence” and “promoter” are all used interchangeably herein and are defined in the “Definitions” section herein.
An endosperm-specific promoter refers to any promoter able to preferentially drive expression of the gene of interest in endosperm tissue. Reference herein to “preferentially” driving expression in endosperm tissue is taken to mean driving expression of any sequence operably linked thereto in endosperm tissue substantially to the exclusion of driving expression elsewhere in the plant, apart from any residual expression due to leaky promoter expression. For example, the prolamin promoter shows strong expression in the endosperm, with leakiness in meristem, more specifically the shoot meristem and/or discrimination centre in the meristem.
Preferably, the endosperm-specific promoter is a promoter isolated from a prolamin gene, such as a rice prolamin RP6 (Wen et al., (1993) Plant Physiol 101(3): 1115-6) promoter as represented by SEQ ID NO: 281 or a promoter of similar strength and/or a promoter with a similar expression pattern as the rice prolamin promoter. Similar strength and/or similar expression pattern may be analysed, for example, by coupling the promoters to a reporter gene and checking the function of the reporter gene in tissues of the plant. One well-known reporter gene is beta-glucuronidase and the colorimetric GUS stain used to visualize beta-glucuronidase activity in plant tissue. Examples of other endosperm-specific promoters which may also be used perform the methods of the invention are shown in Table 6 in the “Definitions” section herein.
Optionally, one or more terminator sequences may also be used in the construct introduced into a plant. The term “terminator” is as defined herein in the “Definitions” section.
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 defined herein in the “Definitions” section.
The present invention also encompasses plants including plant parts obtainable by the methods according to the present invention having increased seed yield relative to suitable control plants and which have reduced or substantially eliminated expression of an endogenous CKI gene in plant endosperm tissue.
The invention also provides a method for the production of transgenic plants having increased seed yield relative to suitable control plants, which transgenic plants have reduced or substantially eliminated expression of an endogenous CKI gene in plant endosperm tissue.
More specifically, the present invention provides a method for the production of transgenic plants having increased seed yield which method comprises:

    
    
        (i) introducing and expressing in a plant, plant part or plant cell a gene construct comprising one or more control sequences capable of preferentially driving expression of a sense and/or antisense CKI nucleic acid sequence in plant endosperm tissue so as to silence an endogenous CKI gene in endosperm tissue of a plant; and
        (ii) cultivating the plant, plant part or plant cell under conditions promoting plant growth and development.
    
    


Preferably, the construct introduced into a plant is one comprising an inverted repeat (in part or complete) of a CKI gene or fragment thereof, preferably capable of forming a hairpin structure.
According to a preferred feature of the present invention, the construct is introduced into a plant by transformation.
The term “transformation” is as defined in the “Definitions” section herein.
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 extends to harvestable parts of a plant such as seeds and products derived, preferably directly derived, from a harvestable part of such a plant, such as dry pellets or powders, oil, fat and fatty acids, starch or proteins.
The present invention also encompasses use of CKI nucleic acids for the reduction or substantial elimination of endogenous CKI gene expression in plant endosperm tissue for increasing plant seed yield as defined hereinabove.



DESCRIPTION OF FIGURES
The present invention will now be described with reference to the following figures in which:
FIG. 1 gives an overview of the conserved motifs present in SEQ ID NO: 2. The leucine rich domain is underlined, the conserved motifs 1, 2 and 3 are indicated in bold and the sequence in italics represents the putative N-glycosylation site with the putative protein kinase C phosphorylation site.
FIG. 2 shows a multiple alignment of various SYR proteins. The asterisks indicate identical amino acid residues, the colons represent highly conserved substitutions and the dots represent less conserved substitutions. With the information from FIG. 1, the various domains and conserved motifs in SEQ ID NO: 2 can be easily identified in the other SYR proteins.
FIG. 3 shows binary vectors for transformation and expression in Oryza sativa of an Oryza sativa SYR nucleic acid. In pGOS2::SYR, the SYR coding sequence is under the control of a rice GOS2 promoter.
FIG. 4 shows binary vectors for transformation and expression in Oryza sativa of an Oryza sativa SYR nucleic acid. In pHMGP::SYR, the SYR coding sequence is under the control of a rice HMGP promoter (SEQ ID NO: 18 in WO 2004/070039, which SEQ ID NO: 18 of WO 2004/070039 is incorporated herein as if fully set forth).
FIG. 5 details examples of sequences useful in performing the methods according to the present invention. SEQ ID NO: 1 and SEQ ID NO: 2 represent the nucleotide and protein sequence of SYR used in the examples. The start and stop codons in SEQ ID NO: 1 are given in bold. SEQ ID NO: 3 and SEQ ID NO: 4 are primer sequences used for isolating the SYR nucleic acid. SEQ ID NO: 5 is the sequence of the GOS2 promoter and SEQ ID NO: 33 of the PRO0170 promoter as used in the examples, SEQ ID NO: 6 to SEQ ID NO: 11 represent consensus sequences of conserved parts in the SYR proteins. SEQ ID NO: 12 to 25, 27 to 32 and 36 to 42 are nucleotide (full length or partial) and protein sequences of homologues of the SYR gene and protein as given in SEQ ID NO: 1 and SEQ ID NO: 2. SEQ ID NO: 26 represents the ARGOS protein sequence (GenBank accession AY305869).
FIG. 6 gives an overview of FG-GAP protein domains. The protein of SEQ ID NO: 46 comprises secretion signal (boxed N-terminal part), an FG-GAP domain starting at P73 and ending with L98, indicated in bold and underlined, and a transmembrane domain (bold and boxed). The conserved motif DXDXDGXX(D/E) (SEQ ID NO: 52) is boxed and underlined, wherein the motif DGXX(D/E) (SEQ ID NO: 51) is in italics. The conserved FDGYLYLID (SEQ ID NO: 294) domain is underlined.
FIG. 7 shows a multiple alignment of full length FG-GAP proteins (SEQ ID NO: 46, SEQ ID NO: 55, SEQ ID NO: 57 and SEQ ID NO: 59), the asterisks indicate identical amino acids, the colons indicate highly conserved substitutions and the dots indicate less conserved substitutions. The partial sequences listed in Table G of Example 12 may be useful in such a multiple alignment for the identification of additional motifs.
FIG. 8 shows a binary vector for transformation and expression in Oryza sativa of an Arabidopsis thaliana FG-GAP encoding nucleic acid under the control of a rice GOS2 promoter.
FIG. 9 details examples of sequences useful in performing the methods according to the present invention. SEQ ID NO: 45 and SEQ ID NO: 46 represent the nucleotide and protein sequence of FG-GAP used in the examples; the start and stop codons in SEQ ID NO: 45 are given in bold. SEQ ID NO: 47 and SEQ ID NO: 48 are primer sequences used for isolating the FG-GAP nucleic acid. SEQ ID NO: 49 is the sequence of the promoter-gene combination as used in the examples, SEQ ID NO: 50 to SEQ ID NO: 53 represent consensus sequences of conserved parts in the FG-GAP proteins. SEQ ID NO: 54 to 71 are nucleotide (full length or partial) and protein sequences of homologues of the FG-GAP gene and protein as given in SEQ ID NO: 45 and SEQ ID NO: 46. SEQ ID NO: 72 is the genomic sequence encoding a Medicago sativa FG-GAP protein which protein comprises the peptide sequences represented by SEQ ID NO: 72 to 76.
FIG. 10 shows the important features found in CYP90B polypeptides or homologues thereof: the N-terminal hydrophobic domain, the transition domain (with the K/R-K/R-X3-9-P-P-G (SEQ ID NO: 295) the A to D domains. Within the A domain the consensus sequence Ala/Gly-Gly-X-Asp/Glu-Thr-Thr/Ser (SEQ ID NO: 293) is identified. The consensus sequence Phe-Ala-Gly-His-Glu-Thr-Ser-Ser (SEQ ID NO: 290) of the CYP90B polypeptides comprises this consensus sequence Ala/Gly-Gly-X-Asp/Glu-Thr-Thr/Ser (SEQ ID NO: 293). Phe-X-X-Gly-X-Arg-X-Cys-X-Gly is SEQ ID NO: 292.
FIG. 11 shows the branched brassinosteroid biosynthetic pathway. In Arabidopsis, the CYP90B1/DWF4 polypeptide comprises the steroid 22-alpha hydroxylase enzymatic activity.
FIG. 12 shows the ProtScale output profile for hydrophobicity of the CYP90B polypeptide of the invention. The first N-terminal 34 amino acids (boxed) represent a hydrophobic domain, as these are located above the zero delimiting line. This region corresponds to the N-terminal anchor domain.
FIG. 13 shows a multiple alignment of several plant CYP90B polypeptides, using VNTI AlignX multiple alignment program, based on a modified ClustalW algorithm (InforMax, Bethesda, Md., informaxinc.com), with default settings for gap opening penalty of 10 and a gap extension of 0.05). The N-terminal hydrophobic domain, the transition domain (with the K/R-KJR-X3-9-P-P-G (SEQ ID NO: 295) and the A to D domains are indicated. The consensus sequence Phe-Ala-Gly-His-Glu-Thr-Ser-Ser (SEQ ID NO: 290) is boxed within the A domain. The accession numbers of the CYP90B polypeptides may be found in Table 9a and 9b. The Arabidopsis Arath_CYP90A1_CPD (At5g05690), Arath_CYP90C1_ROT3 (At4g36380) and Arath_CYP90D1 (At3g13730) are shown as non-CYP90B polypeptides. Sequences shown are: Orysa_CYP90B (SEQ ID NO: 78); Arath_CYP90B1_DWF4 (SEQ ID NO: 80); Sacof_CYP90B (SEQ ID NO: 82); Allce_CYP90B (SEQ ID NO: 84); Zinel_CYP90B (SEQ ID NO: 86); Medtr_CYP90B (SEQ ID NO: 88); Poptr_CYP90B (SEQ ID NO: 90); Aqufo_CYP90B partial (SEQ ID NO: 92); Triae_CYP90B partial (SEQ ID NO: 94); Eupes_CYP90B partial (SEQ ID NO: 98 Goshi_CYP90B partial (SEQ ID NO: 100); Lyces_CYP90B partial (SEQ ID NO: 102); Arath_CYP90A1 CPD (SEQ ID NO: 296); Arath_CYP90C1_ROT3 (SEQ ID NO: 297); Arath_CYP90D1 (SEQ ID NO: 298); and Consensus (SEQ ID NO: 314).
FIG. 14 shows a plant transformation vector for expression in Oryza sativa of an Oryza sativa CYP90B nucleic acid under the control of a plant promoter, which may be a non-constitutive promoter (such as endosperm or embryo/aleurone specific) or a constitutive promoter (such as GOS2 and HMGB1).
FIG. 15 details examples of sequences useful in performing the methods according to the present invention. Several sequences result from public EST assemblies (see Table 9a), with lesser quality sequencing. As a consequence, a few nucleic acid substitutions may be expected. The start (ATG) and stop codons delimit the nucleic acid sequences when these are full length.
FIG. 16 represents a schematic figure of a full length CDC27 polypeptide (more specifically the Arabidopsis thaliana CDC27B hobbit polypeptide). The tetratrico peptide repeats (TPR) are represented as black boxes. The NH2 terminal region of the polypeptide is represented as a black bar.
FIG. 17 shows the multiple alignment of CDC27 polypeptides from different sources, using VNTI AlignX multiple alignment program, based on a modified ClustalW algorithm (InforMax, Bethesda, Md., webpage at informaxinc.com), with default settings for gap opening penalty of 10 and a gap extension of 0.05). The tetratrico peptide repeats (TPR) are boxed across the alignment. The conserved NH2 domain PD011373 (as defined in ProDom, ribosome.toulouse.inra.fr/prodom/current/cgi-bin/ProDomBlast3.p1) is double-underlined. Sequences shown are: Arath_CDC27A (SEQ ID NO: 134); Arath_CDC27B_Hobbit (SEQ ID NO: 132); Soltu_CDC27 (SEQ ID NO: 138); Orysa_CDC27 (SEQ ID NO: 136); Sacof_CDC27 partial (SEQ ID NO: 146); Aspni_BIMA (SEQ ID NO: 142); Schpo_nuc2+ (SEQ ID NO: 140); Homsa_CDC27 (SEQ ID NO: 144); and Consensus (SEQ ID NO: 315).
FIG. 18 shows a binary vector pOSH1::CDC27 for expression in Orysa sativa of a modified Arabidopsis thaliana CDC27 nucleic acid under the control of a plant promoter which is a shoot apical meristem promoter.
FIG. 19 shows a table listing partial and full length CDC27 orthologs and paralogs from different sources, produced by TIGR (Institute for Genomic Research at webpage tigr.org). TC895803 may be found webpage tigr.org/tigr-scripts/tgi/ego/ego_report.pl?ego=895803.
FIG. 20 details examples of sequences useful in performing the methods according to the present invention, or useful in isolating such sequences. Several sequences result from public EST assemblies (see Table 10), with lesser quality sequencing. As a consequence, a few nucleic acid substitutions may be expected. The start (ATG) and stop codons delimit the nucleic acid sequences when these encode full length CDC27 polypeptides.
FIG. 21 shows a phylogenetic tree of various polypeptide sequences comprising an AT-hook domain and a DUF296 domain. The phylogenetic tree was made using VNTI AlignX multiple alignment program, based on a modified ClustalW algorithm (InforMax, Bethesda, Md., webpage at informaxinc.com), with default settings for gap opening penalty of 10 and a gap extension of 0.05).
FIG. 22 shows a binary vector pPROLAMIN::AT-hook, for expression in Oryza sativa of an Oryza sativa nucleic acid encoding a polypeptide comprising an AT-hook domain and a DUF296 domain and Motif 2 under the control of a prolamin promoter.
FIG. 23 shows a multiple alignment of a polypeptide comprising an AT-hook domain and a DUF296 domain, prepared using VNTI AlignX multiple alignment program, based on a modified ClustalW algorithm (InforMax, Bethesda, Md., webpage at informaxinc.com), with default settings for gap opening penalty of 10 and a gap extension of 0.05. Shown in the alignment is the AT-hook domain and the DUF296 domain and Motif 2 in bold, italics and underlined. Sequences shown are: Os_NP—912386 (SEQ ID NO: 171); PROT_Os_AK110263 (SEQ ID NO: 179); PROT_Os_AK107405 (SEQ ID NO: 169); PROT_Le_BT013387(SEQ ID NO: 173); PROT_Lc_AP006863.1 (SEQ ID NO: 157); PRO_Mt_TC102931 (SEQ ID NO: 189); Os_XP—473716 (SEQ ID NO: 155); Os_CDS3129 (SEQ ID NO: 153); Os_CDS3128 (SEQ ID NO: 185); At_NP—567432 (SEQ ID NO: 181); At_NP—194012 (SEQ ID NO: 161); At_NP—192942 (SEQ ID NO: 159); At_NP—191646 (SEQ ID NO: 165); At_NP—182067 (SEQ ID NO: 163); AT_NP—181070 (SEQ ID NO: 187); At_CDS4145 (SEQ ID NO: 183); At_CDS3399 (SEQ ID NO: 177); At_CDS3125 (SEQ ID NO: 175); At_CDS0185 (SEQ ID NO: 167); and Consensus (SEQ ID NO: 316).
FIG. 24 details examples of sequences useful in performing the methods according to the present invention.
FIG. 25 shows a phylogenetic tree of DOF transcription factors. The box nearest the top shows the major clustering of sequences sharing homology to SEQ ID NO: 227 (and comprising features (i) and (iii) as defined hereinabove, i.e. at least 60% sequence identity to either the DOF domain represented by SEQ ID NO: 200 or SEQ ID NO: 228; and Motif I and/or Motif II as defined hereinabove). The box nearest the bottom shows the major clustering of sequences sharing homology to SEQ ID NO: 199 (and comprising features (i) and (ii) as defined hereinabove, i.e. at least 60% sequence identity to either the DOF domain represented by SEQ ID NO: 200 or SEQ ID NO: 228; and at least 70% sequence identity to the DOF domain represented by SEQ ID NO: 200).
FIG. 26 shows a binary vector pGOS2::DOF, for expression in Oryza sativa of an Arabidopsis thaliana DOF transcription factor under the control of a GOS2 promoter.
FIG. 27 shows a binary vector pPROLAMIN:DOF, for expression in Oryza sativa of an Arabidopsis thaliana DOF transcription factor under the control of a prolamin promoter.
FIG. 28 details examples of sequences useful in performing the methods according to the present invention.
FIG. 29 is a schematic representation of a full-length plant CKI polypeptide. The typical motifs 1 to 5 (SEQ ID NO: 261 to SEQ ID NO: 265) useful in identifying CKIs are boxed and numbered accordingly (motif 6 not shown).
FIG. 30 shows a neighbour-joining tree from a multiple alignment of CKI polypeptides from different sources, and made using the ClustalW public software available at clustalw.genome.jp, with the default settings. A subgroup of monocot and dicot CKI4s is indicated by the large bracket. Within this subgroup, monocot CKIs cluster together, as indicated by the medium bracket. The monocot CKI4 branch is indicated by the small bracket.
FIG. 31 is a multiple alignment of CKI polypeptides from different plant sources, made using VNTI AlignX multiple alignment program, based on a modified ClustalW algorithm (InforMax, Bethesda, Md., webpage at informaxinc.com), with default settings for gap opening penalty of 10 and a gap extension of 0.05). The conserved C-terminal end of CKIs is boxed, as well as motifs 1 to 5 (SEQ ID NO: 261 to SEQ ID NO: 265) useful in identifying plant CKIs (motif 6 not shown). Sequences shown are: AtKRP1 (SEQ ID NO: 299); AtKRP2 (SEQ ID NO: 300); AtKRP3 (SEQ ID NO: 301); AtKRP4 (SEQ ID NO: 302); AtKRP5 (SEQ ID NO: 303); AtKRP6 (SEQ ID NO: 304); AtKRP7 (SEQ ID NO: 305); Cheru CKI (SEQ ID NO: 306); Orysa_CKI1 (SEQ ID NO: 307); Glyma_CKI4 like (SEQ ID NO: 308); Medsa CKI (SEQ ID NO: 309); Orysa_CKI2 (SEQ ID NO: 310); Orysa_CKI3 (SEQ ID NO: 274); Orysa_CKI4 (SEQ ID NO: 268); Orysa_CKI5 (SEQ ID NO: 311);Sorbi_ICK3 like (SEQ ID NO: 278); Triae_ICK4 like (SEQ ID NO: 272); Zeama_CKI1 like (SEQ ID NO: 312); Zeama_CKI2 like (SEQ ID NO: 313); Zeama_CKI3 like (SEQ ID NO: 276); Zeama_CKI4 like (SEQ ID NO: 270); and Consensus (SEQ ID NO: 317).
FIG. 32 shows a binary vector for CKI RNA silencing in Oryza sativa, using a hairpin construct, under the control of an endosperm-specific promoter and under the control of a shoot-specific promoter.
FIG. 33 details examples of sequences useful in performing the methods according to the present invention, or useful in isolating such sequences. Several sequences result from public EST assemblies, with lesser quality sequencing. As a consequence, a few nucleic acid substitutions may be expected. The start (ATG) and stop codons delimit the nucleic acid sequences when these encode full-length CKI polypeptides. However both 5′ and 3′ UTR may also be used for the performing the methods of the invention.



EXAMPLES
The present invention will now be described with reference to the following examples, which are by way of illustration alone. The following examples are not intended to completely define or to otherwise limit the scope of the invention.
DNA Manipulation
Unless otherwise stated, recombinant DNA techniques are performed according to standard protocols described in (Sambrook (2001) Molecular Cloning: a laboratory manual, 3rd Edition, Cold Spring Harbor Laboratory Press, CSH, New York) or in Volumes 1 and 2 of Ausubel et al. (1994), Current Protocols in Molecular Biology, Current Protocols (http://www.4ulr.com/products/currentprotocols/index.html). Standard materials and methods for plant molecular work are described in Plant Molecular Biology Labfax (1993) by R.D.D. Croy, published by BIOS Scientific Publications Ltd (UK) and Blackwell Scientific Publications (UK).
Statistical Analysis
A two factor ANOVA (analyses of variance) corrected for the unbalanced design was used as statistical model for the overall evaluation of plant phenotypic characteristics. An F-test was carried out on all the parameters measured of all the plants of all the events transformed with that gene. The F-test was carried out to check for an effect of the gene over all the transformation events and to verify for an overall effect of the gene, also named herein “global gene effect”. If the value of the F test shows that the data are significant, than it is concluded that there is a “gene” effect, meaning that not only presence or the position of the gene is causing the effect. The threshold for significance for a true global gene effect is set at 5% probability level for the F test.
To check for an effect of the genes within an event, i.e., for a line-specific effect, a t-test was performed within each event using data sets from the transgenic plants and the corresponding null plants. “Null plants” or “null segregants” or “nullizygotes” are the plants treated in the same way as the transgenic plant, but from which the transgene has segregated. Null plants can also be described as the homozygous negative transformed plants. The threshold for significance for the t-test is set at 10% probability level. The results for some events can be above or below this threshold. This is based on the hypothesis that a gene might only have an effect in certain positions in the genome, and that the occurrence of this position-dependent effect is not uncommon. This kind of gene effect is also named herein a “line effect of the gene”. The p-value Is obtained by comparing the t-value to the t-distribution or alternatively, by comparing the F-value to the F-distribution. The p-value then gives the probability that the null hypothesis (i.e., that there is no effect of the transgene) is correct.
Example A
SYR
Example 1
Identification of Sequences Related to SEQ ID NO: 1 and SEQ ID NO: 2
Sequences (full length cDNA, ESTs or genomic) related to SEQ ID NO: 1 and/or protein sequences related to SEQ ID NO: 2 were identified amongst those maintained in the Entrez Nucleotides database at the National Center for Biotechnology Information (NCBI) using database sequence search tools, such as the Basic Local Alignment Tool (BLAST) (Altschul et al., (1990) J. Mol. Biol. 215:403-410; and Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402). The program was used to find regions of local similarity between sequences by comparing nucleic acid or polypeptide sequences to sequence databases and by calculating the statistical significance of matches. The polypeptide encoded by SEQ ID NO: 1 was used for the TBLASTN algorithm, with default settings and the filter to ignore low complexity sequences set off. The output of the analysis was viewed by pairwise comparison, and ranked according to the probability score (E-value), where the score reflects the probability that a particular alignment occurs by chance (the lower the E-value, the more significant the hit). In addition to E-values, comparisons were also scored by percentage identity. Percentage identity refers to the number of identical nucleotides (or amino acids) between the two compared nucleic acid (or polypeptide) sequences over a particular length. In some instances, the default parameters were adjusted to modify the stringency of the search.
In addition to the publicly available nucleic acid sequences available at NCBI, other sequence databases may also searched following the same procedure as described above.
Table A provides a list of nucleic acid and protein sequences related to the nucleic acid sequence as represented by SEQ ID NO: 1 and the protein sequence represented by SEQ ID NO: 2.







TABLE A







Nucleic acid sequences related to the nucleic acid sequence (SEQ ID NO: 1) useful


in the methods of the present invention, and the corresponding deduced polypeptides.















Poly-
Database




Source
Nucleic acid
peptide SEQ
accession


Name
organism
SEQ ID NO:
ID NO:
number
Status















OsSYR
Oryza sativa
1
2
/
Full length







or partial


rice SYR
Oryza sativa
12
27
XP_472637
Full length


homologue 1


rice SYR
Oryza sativa
13

AP008218
Full length


homologue 2


corn SYR
Zea mays
14
28
AY110705
partial


homologue


wheat SYR
Triticum
15

/
Full length


homologue
aestivum


barley SYR
Hordeum
16
36
CB871444
Full length


homologue
vulgare


sugar cane SYR
Saccharum
17
37
CA165713
partial


homologue 1
officinarum


sugar cane SYR
Saccharum
18
38
CA242805
Full length


homologue 2
officinarum


sorghum SYR
Sorghum bicolor
19
39
CX611532
Full length


homologue


AtSYR
Arabidopsis
20
40
NM_115853
Full length


homologue 1
thaliana


AtSYR
Arabidopsis
21
41
NM_180078
Full length


homologue 2
thaliana


grape SYR
Vitis vinifera
22
29
CF404276
Full length


homologue


Citrus SYR
Citrus reticulata
23
30
CF830612
partial


homologue


tomato SYR
Lycopersicon
24
32
AI774560
Full length


homologue 1
esculentum


tomato SYR
Lycopersicon
25
31
BG125370
Full length


homologue 2
esculentum









Example 2
Alignment of Relevant Polypeptide Sequences
AlignX from the Vector NTI (Invitrogen) is based on the popular Clustal algorithm of progressive alignment (Thompson et al (1997) Nucleic Acids Res 25:4876-4882; Chema et al. (2003). Nucleic Acids Res 31:3497-3500). A phylogenetic tree can be constructed using a neighbour joining clustering algorithm. Default values are for the gap open penalty of 10, for the gap extension penalty of 0.1 and the selected weight matrix is Blosum 62 (if polypeptides are aligned).
The result of the multiple sequence alignment using polypeptides relevant in identifying the ones useful in performing the methods of the invention is shown in FIG. 2. The leucine rich repeat and the conserved motifs can be easily discriminated in the various sequences.
Example 3
Calculation of Global Percentage Identity between Polypeptide Sequences Useful in Performing the Methods of the Invention
Global percentages of similarity and identity between full length polypeptide sequences useful in performing the methods of the invention were determined using one of the methods available in the art, the MatGAT (Matrix Global Alignment Tool) software (BMC Bioinformatics. 2003 4:29. MatGAT: an application that generates similarity/identity matrices using protein or DNA sequences. Campanella J J, Bitincka L, Smalley J; software hosted by Ledion Bitincka). MatGAT software generates similarity/identity matrices for DNA or protein sequences without needing pre-alignment of the data. The program performs a series of pair-wise alignments using the Myers and Miller global alignment algorithm (with a gap opening penalty of 12, and a gap extension penalty of 2), calculates similarity and identity using for example Blosum 62 (for polypeptides), and then places the results in a distance matrix. Sequence similarity is shown in the bottom half of the dividing line and sequence identity is shown in the top half of the diagonal dividing line.
Parameters used in the comparison were:


    
    
        Scoring matrix: Blosum62
        First Gap: 12
        Extending gap: 2
    
    


Results of the software analysis are shown in Table B for the global similarity and identity over the full length of the polypeptide sequences (excluding the partial polypeptide sequences). Percentage identity is given above the diagonal and percentage similarity is given below the diagonal.
The percentage identity between the polypeptide sequences useful in performing the methods of the invention can be as low as 27% amino acid identity compared to SEQ ID NO: 2.







TABLE B







MatGAT results for global similarity and identity over the full length of the polypeptide sequences.

























1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17




























 1. SEQID2

29.8
46.8
55.2
67.0
66.1
66.7
71.4
63.6
36.8
34.6
35.5
39.7
39.0
41.0
27.6
32.1


 2. SEQID12
40.4

29.8
23.0
26.8
28.1
23.6
25.3
28.7
30.3
28.1
30.9
32.0
28.1
24.7
16.3
17.4


 3. SEQID13
57.9
39.3

42.9
46.0
47.6
44.4
47.6
45.2
31.9
33.3
33.1
34.1
37.3
34.1
24.8
28.3


 4. SEQID14
59.0
32.0
50.8

57.1
55.4
77.4
77.4
83.2
25.4
26.7
26.6
30.2
32.2
33.3
21.6
23.9


 5. SEQID15
80.9
41.0
57.9
69.1

89.1
63.4
67.9
66.1
36.9
31.9
33.1
40.5
37.3
40.9
24.8
27.9


 6. SEQID16
79.1
38.2
59.5
65.5
95.5

61.6
66.1
62.5
36.4
32.6
36.0
40.5
38.8
38.2
24.0
28.8


 7. SEQID17
69.5
34.8
57.1
78.1
72.7
69.1

94.9
81.3
30.8
29.6
31.7
34.1
34.7
39.4
25.5
29.0


 8. SEQID18
74.3
37.1
60.3
80.0
77.3
73.6
94.9

85.0
33.1
31.9
33.8
36.5
37.3
42.4
28.2
32.0


 9. SEQID19
69.2
39.3
56.3
86.0
78.2
74.5
84.1
88.8

36.9
32.6
36.7
38.1
39.8
40.2
28.8
29.6


10. SEQID20
54.6
41.6
56.9
46.2
57.7
60.8
50.0
53.1
54.6

66.2
46.9
51.9
44.3
42.7
26.3
26.9


11. SEQID21
51.9
44.4
56.3
47.4
54.8
54.8
50.4
53.3
52.6
77.8

49.0
46.8
41.1
39.3
28.7
27.2


12. SEQID22
54.0
43.8
54.7
45.3
53.2
54.0
49.6
51.8
54.7
65.5
65.5

61.9
45.1
40.3
24.0
22.9


13. SEQID23
58.7
45.5
55.6
50.0
60.3
59.5
54.8
57.1
63.5
66.9
66.7
77.7

53.8
44.4
27.0
27.6


14. SEQID24
61.9
42.7
57.9
55.1
58.5
63.6
61.0
63.6
62.7
66.9
64.4
68.3
77.0

73.7
27.9
29.4


15. SEQID25
62.9
35.4
50.0
53.3
60.0
58.2
66.7
69.7
61.7
56.2
54.8
54.7
60.3
73.7

36.7
38.6


16. SEQID34
45.7
25.3
38.1
38.1
39.1
40.0
45.5
48.5
44.9
40.0
40.7
36.0
41.3
41.5
56.3

42.0


17. SEQID35
50.5
30.3
45.2
40.0
46.4
44.5
47.5
50.5
45.8
34.6
42.2
36.7
40.5
42.4
55.2
57.7









Example 4
Topology Prediction of the Polypeptide Sequences Useful in Performing the Methods of the Invention
TargetP 1.1 was used to predict the subcellular location of eukaryotic proteins. According to the program, the location assignment is based on the predicted presence of any of the N-terminal pre-sequences: chloroplast transit peptide (cTP), mitochondrial targeting peptide (mTP) or secretory pathway signal peptide (SP). Scores on which the final prediction is based are not really probabilities, and they do not necessarily add to one. However, the location with the highest score is the most likely according to TargetP, and the relationship between the scores (the reliability class) may be an indication of how certain the prediction is. The reliability class (RC) ranges from 1 to 5, where 1 indicates the strongest prediction. TargetP is maintained at the server of the Technical University of Denmark.
For the sequences predicted to contain an N-terminal presequence a potential cleavage site may also be present.
A number of parameters were selected, such as organism group (non-plant or plant), cutoff sets (none, predefined set of cutoffs, or user-specified set of cutoffs), and the calculation of prediction of cleavage sites (yes or no).
The results of TargetP 1.1 analysis of the polypeptide sequence as represented by SEQ ID NO: 2 are presented Table C below. The “plant” organism group was been selected, no cutoffs defined, and the predicted length of the transit peptide requested. According to the results, the subcellular localization of the polypeptide sequence as represented by SEQ ID NO: 2 may be the mitochondrion; however the reliability class of 5 (i.e. the lowest reliability class) should be considered.







TABLE C





TargetP 1.1 analysis of the polypeptide sequence as represented by


SEQ ID NO: 2


















Length (AA)
105



Chloroplastic transit peptide
0.025



Mitochondrial transit peptide
0.552



Secretory pathway signal peptide
0.009



Other subcellular targeting
0.416



Predicted Location
mitochondrion



Reliability class
5










Two transmembrane domains were identified by the TMHMM program, hosted on the server of the Center for Biological Sequence Analysis, Technical University of Denmark. The results below show that the probability that the N-terminus is located inside is 0.997. Further details on the orientation are given in Table D below.







TABLE D







results of TMHMM 2.0











Orientation
begin-end residue















inside
1
42



TMhelix
43
65



outside
66
74



TMhelix
75
92



inside
93
105










Many other algorithms can be used to perform such analyses, including:

    
    
        ChloroP 1.1 hosted on the server of the Technical University of Denmark;
        Protein Prowler Subcellular Localisation Predictor version 1.2 hosted on the server of the Institute for Molecular Bioscience, University of Queensland, Brisbane, Australia;
        PENCE Proteome Analyst PA-GOSUB 2.5 hosted on the server of the University of Alberta, Edmonton, Alberta, Canada;
    
    


Example 5
Gene Cloning
The Oryza sativa SYR gene was amplified by PCR using as template an Oryza sativa 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 1.59×107 cfu. Original titer was determined to be 9.6×105 cfu/ml after first amplification of 6×1011 cfu/ml. After plasmid extraction, 200 ng of template was used in a 50 μl PCR mix. Primers prm08170 (SEQ ID NO: 3; sense, start codon in bold, AftB1 site in italic: 5′-ggggacaagtttgtacaaaaaagcag gcttaaacaatggaaggtgtaggtgctagg-3′) and prm08171 (SEQ ID NO: 4; reverse, complementary, AttB2 site in italic: 5′-ggggaccacmtgtacaagaaagctgggtcaaaaacaaaaataaattcco-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 the correct size 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”, PSYR. Plasmid pDONR201 was purchased from Invitrogen, as part of the Gateway® technology.
Example 6
Vector Construction
The entry clone pSYR was subsequently used in an LR reaction with 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: 5) for constitutive expression was located upstream of this Gateway cassette. A similar vector construct was prepared, but with the high mobility group protein promoter (HMGP, SEQ ID NO: 33) instead of the GOS promoter
After the LR recombination step, the resulting expression vectors, pGOS2::SYR (with the GOS2 promoter) and pHMGP::SYR (with the HMGP promoter), both for constitutive SYR expression (FIG. 2) were transformed into Agrobacterium strain LBA4044 and subsequently to Oryza sativa plants.
Example 7
Rice Transformation
The Agrobacterium containing the expression vector was used to transform Oryza sativa plants. Mature dry seeds of the rice japonica cultivar Nipponbare were dehusked. Sterilization was carried out by incubating for one minute in 70% ethanol, followed by 30 minutes in 0.2% HgCl2, followed by a 6 times 15 minutes wash with sterile distilled water. The sterile seeds were then germinated on a medium containing 2,4-D (callus induction medium). After incubation in the dark for four weeks, embryogenic, scutellum-derived calli were excised and propagated on the same medium. After two weeks, the calli were multiplied or propagated by subculture on the same medium for another 2 weeks. Embryogenic callus pieces were sub-cultured on fresh medium 3 days before co-cultivabon (to boost cell division activity).
Agrobacterium strain LBA4404 containing the expression vector was used for cocultivation. Agrobacterium was inoculated on AB medium with the appropriate antibiotics and cultured for 3 days at 28° C. The bacteria were then collected and suspended in liquid co-cultivation medium to a density (OD600) of about 1. The suspension was then transferred to a Petri dish and the calli immersed in the suspension for 15 minutes. The callus tissues were then blotted dry on a filter paper and transferred to solidified, co-cultivation medium and incubated for 3 days in the dark at 25° C. Co-cultivated calli were grown on 2,4-D-containing medium for 4 weeks in the dark at 28° C. in the presence of a selection agent. During this period, rapidly growing resistant callus islands developed. After transfer of this material to a regeneration medium and incubation in the light, the embryogenic potential was released and shoots developed in the next four to five weeks. Shoots were excised from the calli and incubated for 2 to 3 weeks on an auxin-containing medium from which they were transferred to soil. Hardened shoots were grown under high humidity and short days in a greenhouse.
Approximately 35 independent TO rice transformants were generated for one construct. The primary transformants were transferred from a tissue culture chamber to a greenhouse. After a quantitative PCR analysis to verify copy number of the T-DNA insert, only single copy transgenic plants that exhibit tolerance to the selection agent were kept for harvest of T1 seed. Seeds were then harvested three to five months after transplanting. The method yielded single locus transformants at a rate of over 50% (Aldemita and Hodges 1996, Chan et al. 1993, Hiei et al. 1994).
For transformation of other crops see Example 40.
Example 8
Evaluation Methods of Plants Transformed with SYR under the Control of the Rice GOS2 Promoter or the HMGP Promoter
Evaluation Set-Up
Approximately 15 to 20 independent T0 rice transformants were generated. The primary transformants were transferred from a tissue culture chamber to a greenhouse for growing and harvest of T1 seed. Eight 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. The selected T1 plants were transferred to a greenhouse. Each plant received a unique barcode label to link unambiguously the phenotyping data to the corresponding plant. The selected T1 plants were grown on soil in 10 cm diameter pots under the following environmental settings: photoperiod=11.5 h, daylight intensity=30,000 lux or more, daytime temperature=28° C. or higher, night time temperature=22° C., relative humidity=60-70%. Transgenic plants and the corresponding nullizygotes were grown side-by-side at random positions. From the stage of sowing until the stage of maturity the plants were passed several times through a digital imaging cabinet. At each time point digital images (2048×1536 pixels, 16 million colours) were taken of each plant from at least 6 different angles.
Salt Stress Screen
Plants from 4 events (T2 seeds) were grown on a substrate made of coco fibers and argex (3 to 1 ratio). A normal nutrient solution was used during the first two weeks after transplanting the plantlets in the greenhouse. After the first two weeks, 25 mM of salt (NaCl) was added to the nutrient solution, until the plants were harvested.
Drought Screen
Plants from five events (T2 seeds) were grown in potting soil under normal conditions until they approached the heading stage. They were then transferred to a “dry” section where irrigation was withheld. Humidity probes were inserted in randomly chosen pots to monitor the soil water content (SWC). When SWC went below certain thresholds, the plants were automatically re-watered continuously until a normal level was reached again. The plants were then retransferred again to normal conditions. The rest of the cultivation (plant maturation, seed harvest) was the same as for plants not grown under abiotic stress conditions. A confirmation round was performed consisting of repeating the screen with T2 seeds not harvested from plants of the first drought screen, but from plants grown under normal conditions.
Parameters Measured
The plant aboveground area (or leafy biomass) was determined by counting the total number of pixels on the digital images from aboveground plant parts discriminated from the background. This value was averaged for the pictures taken on the same time point from the different angles and was converted to a physical surface value expressed in square mm by calibration. Experiments show that the aboveground plant area measured this way correlates with the biomass of plant parts above ground. The Areamax is the above ground area at the time point at which the plant had reached its maximal leafy biomass.
The mature primary panicles were harvested, bagged, barcode-labelled and then dried for three days in the oven at 37° C. The panicles were then threshed and all the seeds collected. The filled husks were separated from the empty ones using an air-blowing device. After separation, both seed lots were then counted using a commercially available counting machine. The empty husks were discarded. The filled husks were weighed on an analytical balance and the cross-sectional area of the seeds was measured using digital imaging. This procedure resulted in the set of the following seed-related parameters:
The flowers-per-panicle estimates the average number of florets per panicle on a plant, derived from the number of total seeds divided by the number of first panicles. The tallest panicle and all the panicles that overlapped with the tallest panicle when aligned vertically, were considered as first panicles and were counted manually. The number of filled seeds was determined by counting the number of filled husks that remained after the separation step.
The total seed yield (total seed weight) 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 and corresponds to the number of florets per plant. Thousand Kernel Weight (TKW) is extrapolated from the number of filled seeds counted and their total weight. Harvest index is defined as the ratio between the total seed weight and the above-ground area (mm2, multiplied by a factor 108. The parameter EmerVigor is an indication of the seedling vigour. It is calculated from the area (in mm2) covered by leafy biomass in the first imaging. The seed fill rate (fillrate) is an indication of the filling of the seeds. It is expressed as a proportion (in %) of the number of filled seeds over the number of florets (nrtotalseed).
These parameters were derived in an automated way from the digital images using image analysis software and were analysed statistically. 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.
Example 9
Measurement of Yield-Related Parameters for pGOS2::SYR Transformants Grown Under Normal Growth Conditions
Upon analysis of the seeds as described above, the inventors found that plants transformed with the pGOS2::SYR gene construct had a higher seed yield, expressed as number of filled seeds, total weight of seeds and harvest index, compared to plants lacking the SYR transgene. The p-values show that the increases were significant. Methods for statistical analysis are as given in the introductory section to the Examples.
The results obtained for plants in the T1 generation are summarised in Table E, which represent the mean values for all the tested lines:










TABLE E







% difference
p-value




















Nr filled seeds
+47
0.0000



Total weight seeds
+52
0.0000



Harvest Index
+54
0.0000










The data obtained for SYR in the first experiment were confirmed in a second experiment with T2 plants. Four lines that had the correct expression pattern were selected for further analysis. Seed batches from the positive plants (both hetero- and homozygotes) in T1 were screened by monitoring marker expression. For each chosen event, the heterozygote seed batches were then retained for T2 evaluation. Within each seed batch an equal number of positive and negative plants were grown in the greenhouse for evaluation. Measurement of the seed yield parameters again showed increase in number of filled seeds, total weight of seeds and harvest index, compared to plants lacking the SYR transgene.
Example 10
Measurement of Yield-Related Parameters for pGOS2::SYR Transformants Grown Under Stress Conditions
Upon analysis of the seeds as described above, the inventors found that plants transformed with the pGOS2::SYR gene construct and grown under salt stress, had a higher seed yield, expressed as number of filled seeds, total weight of seeds, fill rate and harvest index, compared to plants lacking the SYR transgene. Furthermore, these salt-stressed plants had a higher seedling vigour compared to the control plants. When the plants were grown under drought stress, the transgenic plants had a higher total weight of seeds and an increased harvest index compared to plants lacking the SYR transgene. These differences were significant, with a P value from the F test below 0.05.
Example 11
Measurement of Yield-Related Parameters for pHMGP::SYR Transformants
Similarly as for the plants transformed with the pGOS2::SYR gene construct, the inventors found that plants transformed with the pHMGP::SYR gene construct had a higher seed yield, expressed as number of filled seeds, total weight of seeds and harvest index, compared to plants lacking the SYR transgene. The p-values show that the increases were significant. The results obtained for plants in the T1 generation are summarised in Table F, which represent the mean values for all the tested lines:










TABLE F







% difference
p-value




















Nr filled seeds
+34
0.0000



Total weight seeds
+33
0.0000



Harvest Index
+37
0.0000










Example B
FG-GAP
Example 12
Identification of Sequences Related to SEQ ID NO: 45 and SEQ ID NO: 46
Sequences (full length cDNA, ESTs or genomic) related to SEQ ID NO: 45 and/or protein sequences related to SEQ ID NO: 46 were identified amongst those maintained in the Entrez Nucleotides database at the National Center for Biotechnology Information (NCBI) using database sequence search tools, such as the Basic Local Alignment Tool (BLAST) (Altschul et al. (1990) J. Mol. Biol. 215:403-410; and Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402). The program was used to find regions of local similarity between sequences by comparing nucleic acid or polypeptide sequences to sequence databases and by calculating the statistical significance of matches. The polypeptide encoded by SEQ ID NO: 45 was used for the TBLASTN algorithm, with default settings and the filter to ignore low complexity sequences set off. The output of the analysis was viewed by pairwise comparison, and ranked according to the probability score (E-value), where the score reflects the probability that a particular alignment occurs by chance (the lower the E-value, the more significant the hit). In addition to E-values, comparisons were also scored by percentage identity. Percentage identity refers to the number of identical nucleotides (or amino acids) between the two compared nucleic acid (or polypeptide) sequences over a particular length. In some instances, the default parameters may be adjusted to modify the stringency of the search.
In addition to the publicly available nucleic acid sequences available at NCBI, other sequence databases may also searched following the same procedure as described above.
Table G provides a list of nucleic acid and protein sequences related to the nucleic acid sequence as represented by SEQ ID NO: 45 and the protein sequence represented by SEQ ID NO: 46.







TABLE G







Nucleic acid sequences related to the nucleic acid sequence (SEQ ID NO: 45) useful


in the methods of the present invention, and the corresponding deduced polypeptides.
















Database




Source
Nucleic acid
Polypeptide
accession


Name
organism
SEQ ID NO:
SEQ ID NO:
number
Status





AtFG-GAP
Arabidopsis
45
46

Full length



thaliana


AtFG-GAP
Arabidopsis
54
55
NM_114965
Full length


homologue
thaliana


OsFG-GAP
Oryza sativa
56
57
NM_185137
Full length


homologue 1


OsFG-GAP
Oryza sativa
58
59
AK068943
Full length


homologue 2


TaFG-GAP
Triticum
60
/
CK207217
partial


homologue
aestivum


ZmFG-GAP
Zea mays
61
/
AY111316
Partial


homologue


StFG-GAP
Solanum
62
/
BG598275
Partial


homologue
tuberosum


AFG-GAP
Aquilegia sp.
63
/
DT735817
Partial


homologue


BnFG-GAP
Brassica napus
64
/
CX192752
Partial


homologue


CsFG-GAP
Citrus sinensis
65
/
CX674859
Partial


homologue


AoFG-GAP
Asparagus
66
/
CV288972
Partial


homologue
officinalis


PFG-GAP
Populus sp.
67
/
CN520999
Partial


homologue 1


PFG-GAP
Populus sp.
68
/
CX176799
Partial


homologue 2


EeFG-GAP
Euphorbia esula
69
/
DV130386
Partial


homologue


CrFG-GAP
Ceratopteris
70
/
CV736049
Partial


homologue
richardii


WmFG-GAP
Welwitschia
71
/
DT601669
Partial


homologue
mirabilis


Ms FG-GAP
Medicago sativa
72
SEQ ID NO: 73

partial


homologue


to SEQ ID NO:





76









Example 13
Alignment of Relevant Polypeptide Sequences
AlignX from the Vector NTI (Invitrogen) is based on the popular Clustal algorithm of progressive alignment (Thompson et al. (1997) Nucleic Acids Res 25:4876-4882; Chema et al. (2003). Nucleic Acids Res 31:3497-3500). A phylogenetic tree may be constructed using a neighbour-joining clustering algorithm. Default values are for the gap open penalty of 10, for the gap extension penalty of 0,1 and the selected weight matrix is Blosum 62 (if polypeptides are aligned).
The result of the multiple sequence alignment using polypeptides relevant in identifying the ones useful in performing the methods of the invention is shown in FIG. 7. one can clearly see that despite some gaps in the alignment, sequence conservation is found throughout most of the protein sequence.
Example 14
Calculation of Global Percentage Identity between Polypeptide Sequences Useful in Performing the Methods of the Invention
Global percentages of similarity and identity between full length polypeptide sequences useful in performing the methods of the invention were determined using one of the methods available in the art, the MatGAT (Matrix Global Alignment Tool) software (BMC Bioinformatics. 2003 4:29. MatGAT: an application that generates similarity/identity matrices using protein or DNA sequences. Campanella J J, Bitincka L, Smalley J; software hosted by Ledion Bitincka). MatGAT software generates similarity/identity matrices for DNA or protein sequences without needing pre-alignment of the data. The program performs a series of pair-wise alignments using the Myers and Miller global alignment algorithm (with a gap opening penalty of 12, and a gap extension penalty of 2), calculates similarity and identity using for example Blosum 62 (for polypeptides), and then places the results in a distance matrix. Sequence similarity is shown in the bottom half of the dividing line and sequence identity is shown in the top half of the diagonal dividing line.
Parameters used in the comparison were:

    
    
        Scoring matrix: Blosum62
        First Gap: 12
        Extending gap: 2
    
    


Results of the software analysis are shown in Table H for the global similarity and identity over the full length of the polypeptide sequences (excluding the partial polypeptide sequences). Percentage identity is given above the diagonal and percentage similarity is given below the diagonal.
The percentage identity between the polypeptide sequences useful in performing the methods of the invention can be as low as 17% amino acid identity compared to SEQ ID NO: 46.







TABLE H







MatGAT results for global similarity and identity


over the full length of the polypeptide sequences.












1
2
3
4

















1. AtFGAP1

18.1
65.5
17.4



2. AtFGGAP2
31.4

17.9
67.7



3. OsFGGAP1
76.7
33.5

16.9



4. OsFGGAP2
32.8
83.6
33










Example 15
Identification of Domains Comprised in Polypeptide Sequences Useful in Performing the Methods of the Invention
The Integrated Resource of Protein Families, Domains and Sites (InterPro) database is an integrated interface for the commonly used signature databases for text- and sequence-based searches. The InterPro database combines these databases, which use different methodologies and varying degrees of biological information about well-characterized proteins to derive protein signatures. Collaborating databases include SWISS-PROT, PROSITE, TrEMBL, PRINTS, Propom and Pfam, Smart and TIGRFAMs. Interpro is hosted at the European Bioinformatics Institute in the United Kingdom.
The results of the InterPro scan of the polypeptide sequence as represented by SEQ ID NO: 46 are presented in Table I.







TABLE I







InterPro scan results of the polypeptide sequence as


represented by SEQ ID NO: 46











Database
Accession number
Accession name







Pfam
PF01839
FG-GAP



INTERPRO
IPR013517
FG-GAP



INTERPRO
IPR000413
Integrins alpha chain










Example 16
Topology Prediction of the Polypeptide Sequences Useful in Performing the Methods of the Invention
TargetP 1.1 predicts the subcellular location of eukaryotic proteins. The location assignment is based on the predicted presence of any of the N-terminal pre-sequences: chloroplast transit peptide (cTP), mitochondrial targeting peptide (mTP) or secretory pathway signal peptide (SP). Scores on which the final prediction is based are not really probabilities, and they do not necessarily add to one. However, the location with the highest score is the most likely according to TargetP, and the relationship between the scores (the reliability class) may be an indication of how certain the prediction is. The reliability class (RC) ranges from 1 to 5, where 1 indicates the strongest prediction. TargetP is maintained at the server of the Technical University of Denmark.
For the sequences predicted to contain an N-terminal presequence a potential cleavage site can also be predicted.
A number of parameters were selected, such as organism group (non-plant or plant), cutoff sets (none, predefined set of cutoffs, or user-specified set of cutoffs), and the calculation of prediction of cleavage sites (yes or no).
The results of TargetP 1.1 analysis of the polypeptide sequence as represented by SEQ ID NO: 46 are presented in Table J. The “plant” organism group has been selected, no cutoffs defined, and the predicted length of the transit peptide requested. The subcellular localization of the polypeptide sequence as represented by SEQ ID NO: 46 is probably not intracellular, there is a slight preference for the secretory pathway (though with a reliability score of 5) and the predicted length of the putative transit peptide is 24 amino acids starting from the N-terminus (not as reliable as the prediction of the subcellular localization itself, may vary in length of a few amino acids).







TABLE J





TargetP 1.1 analysis of the polypeptide sequence as


represented by SEQ ID NO: 46


















Length (AA)
896



Chloroplastic transit peptide
0.010



Mitochondrial transit peptide
0.546



Secretory pathway signal peptide
0.643



Other subcellular targeting
0.038



Predicted Location
secreted



Reliability class
5



Predicted transit peptide length
24










When analysed with SignalP (Bendtsen et al., J. Mol. Biol., 340:783-795, 2004), there is a reliable (probability of 0.998) positive identification for the presence of an N-terminal secretion signal peptide with a length of 24 amino acids. Furthermore, when using the THMM algorithm (Center for Biological Sequence Analysis, Technical University of Denmark), the protein is predicted to be located at the outer side of the cell with only a C-terminal tail in the cytoplasm: residues 1-859: outside; residues 860-879: transmembrane domain, residues 880-896: inside. Many other algorithms can be used to perform such analyses, including:

    
    
        ChloroP 1.1 hosted on the server of the Technical University of Denmark;
        Protein Prowler Subcellular Localisation Predictor version 1.2 hosted on the server of the Institute for Molecular Bioscience, University of Queensland, Brisbane, Australia;
        PENCE Proteome Analyst PA-GOSUB 2.5 hosted on the server of the University of Alberta, Edmonton, Alberta, Canada;
    
    


Example 17
Gene Cloning
The Arabidopsis thaliana FG-GAP gene was amplified by PCR using as template an Arabidopsis thafiana 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 1.59×107 cfu. Original titer was determined to be 9.6×10 cfu/ml after first amplification of 6×1011 cfu/ml. After plasmid extraction, 200 ng of template was used in a 50 ul PCR mix. Primers prm06643 (SEQ ID NO: 47; sense, start codon in bold, AttB1 site in italic: 5′-ggggacaagtttgtacaaaaaagcaggcttaaacaatgaaatctcgagcgagg-3′) and prm06644 (SEQ ID NO: 48; reverse, complementary, AttB2 site in italic: 5′-ggggaccactttgtacaagaaagctgggtcctg tttacagatggtacctagt-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 3.2 kb (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”, pFG-GAP. Plasmid pDONR201 was purchased from Invitrogen, as part of the Gateway® technology.
Example 18
Vector Construction
The entry clone pFG-GAP was subsequently used in an LR reaction with pGOS2, 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 (nucleotides 1 to 2193 of SEQ ID NO: 49, the promoter-gene combination) for constitutive expression was located upstream of this Gateway cassette.
After the LR recombination step, the resulting expression vector, pGOS2::FG-GAP for FG-GAP (FIG. 7) was transformed into Agrobacterium strain LBA4044 and subsequently to
Oryza sativa plants. Transformed rice plants were allowed to grow and were then examined for the parameters described in Example 19.
For transformation of other crops see Example 40.
Example 19
Evaluation Methods for Plants Transformed with FG-GAP under the Control of the rice GOS2 Promoter
Approximately 15 to 20 independent T0 rice transformants were generated. The primary transformants were transferred from a tissue culture chamber to a greenhouse for growing and harvest of T1 seed. Seven 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. The selected T1 plants were transferred to a greenhouse. Each plant received a unique barcode label to unambiguously link the phenotyping data to the corresponding plant. The selected T1 plants were grown on soil in 10 cm diameter pots under the following environmental settings: photoperiod=11.5 h, daylight intensity=30,000 lux or more, daytime temperature=28° C. or higher, night time temperature=22° C., relative humidity=60-70%. Transgenic plants and the corresponding nullizygotes were grown side-by-side at random positions. From the stage of sowing until the stage of maturity the plants were passed several times through a digital imaging cabinet. At each time point digital images (2048×1536 pixels, 16 million colours) were taken of each plant from at least 6 different angles.
The plant aboveground area (or leafy biomass) was determined by counting the total number of pixels on the digital images from aboveground plant parts discriminated from the background. This value was averaged for the pictures taken on the same time point from the different angles and was converted to a physical surface value expressed in square mm by calibration. Experiments show that the aboveground plant area measured this way correlates with the biomass of plant parts above ground. The Areamax is the above ground area at the time point at which the plant had reached its maximal leafy biomass.
The mature primary panicles were harvested, bagged, barcode-labelled and then dried for three days in the oven at 37° C. The panicles were then threshed and all the seeds collected. The filled husks were separated from the empty ones using an air-blowing device. After separation, both seed lots were then counted using a commercially available counting machine. The empty husks were discarded. The filled husks were weighed on an analytical balance and the cross-sectional area of the seeds was measured using digital imaging. This procedure resulted in the set of the following seed-related parameters:
The flowers-per-panicle is a parameter estimating the average number of florets per panicle on a plant, derived from the number of total seeds divided by the number of first panicles. The tallest panicle and all the panicles that overlapped with the tallest panicle when aligned vertically, were considered as first panicles and were counted manually. The number of filled seeds was determined by counting the number of filled husks that remained after the separation step. The total seed yield (total seed weight) 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 and corresponds to the number of florets per plant. Thousand Kernel Weight (TKW) is extrapolated from the number of filled seeds counted and their total weight. Harvest index is defined as the ratio between the total seed weight and the above-ground area (mm2), multiplied by a factor 106. These parameters were derived in an automated way from the digital images using image analysis software and were analysed statistically. 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.
A two factor ANOVA (analyses of variance) corrected for the unbalanced design was used as statistical model for the overall evaluation of plant phenotypic characteristics. An F-test was carried out on all the parameters measured of all the plants of all the events transformed with that gene. The F-test was carried out to check for an effect of the gene over all the transformation events and to verify for an overall effect of the gene, also referred to herein as a “global gene effect”. If the value of the F test showed that the data were significant, than it was concluded that there was a “gene” effect, meaning that it was not only presence or the position of the gene that was causing the effect. The threshold for significance for a true global gene effect was set at a 5% probability level for the F test.
To check for an effect of the genes within an event, i.e., for a line-specific effect, a t-test was performed within each event using data sets from the transgenic plants and the corresponding null plants. “Null plants” or “null segregants” or “nullizygotes” refer to plants treated in the same way as the transgenic plant, but from which the transgene has segregated. Null plants can also be described as the homozygous negative transformed plants. The threshold for significance for the t-test was set at a 10% probability level. The results for some events can be above or below this threshold. This is based on the hypothesis that a gene might only have an effect in certain positions in the genome, and that the occurrence of this position-dependent effect is not uncommon. This kind of gene effect is also referred to herein as a “line effect of the gene”. The p-value was obtained by comparing the t-value to the t-distribution or alternatively, by comparing the F-value to the F-distribution. The p-value then gives the probability that the null hypothesis (i.e., that there is no effect of the transgene) is correct.
The data obtained for FG-GAP in the first experiment were confirmed in a second experiment with T2 plants. Four lines were selected for further analysis. Seed batches from the positive plants (both hetero- and homozygotes) in T1, were screened by monitoring marker expression. For each chosen event, the heterozygote seed batches were then retained for T2 evaluation. Within each seed batch an equal number of positive and negative plants were grown in the greenhouse for evaluation.
A total number of 120 FG-GAP transformed plants were evaluated in the T2 generation, that is 30 plants per event of which 15 were positive for the transgene, and 15 negative.
Because two experiments with overlapping events were carried out, a combined analysis was performed. This is useful to check consistency of the effects over the two experiments, and if this is the case, to accumulate evidence from both experiments in order to increase confidence in the conclusion. The method used was a mixed-model approach that takes into account the multilevel structure of the data (i.e. experiment-event-segregants). P-values were obtained by comparing likelihood ratio test to chi square distributions.
Example 20
Evaluation of FG-GAP Transformants
Measurement of Yield-Related Parameters
Upon analysis of the seeds as described above, the inventors found that plants transformed with the FG-GAP gene construct had a higher seed yield, expressed as number of filled seeds and total weight of seeds, compared to plants lacking the FG-GAP transgene. The p-values show that the increases were significant. Also the harvest index was increased (+9%). The results obtained for plants in the T1 generation are summarised in Table K:










TABLE K







% difference
p-value of F-test




















Nr filled seeds
+19
0.0051



Total weight seeds
+17
0.0199










These positive results were again obtained in the T2 generation. In Table L, data show the overall % increases for the number of filled seeds, total weight of seeds and harvest index, calculated from the data of the individual lines of the T2 generation, and the respective p-values. These T2 data were re-evaluated in a combined analysis with the results for the T1 generation, and the obtained p-values show that the observed effects were highly significant.










TABLE L









T2 generation












%

Combined analysis



difference
p-value of F-test
p-value














Nr filled seeds
+17
0.0247
0.0004


Total weight seeds
+17
0.0283
0.0014


Harvest Index
+20
0.0030
0.0007









Example C
CYP90B
Example 21
Gene Cloning of Oryza sativa CYP90B cDNA
The Oryza sativa CYP90B cDNA was amplified by PCR using as template an Oryza sativa 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 1.6 kb and the original number of clones was of the order of 1.67×107 cfu. Original titer was determined to be 3.34×106 cfu/ml after first amplification of 6×1010 cfu/ml. After plasmid extraction, 200 ng of template was used in a 50 μl PCR mix. Primers (SEQ ID NO: 107; sense, start codon in bold, AftB1 site in italic: 5′ GGGGACAAGTTTGTACAAAAAAGCAGGCTTAAACAATGGCCGCCATGATGGC 3′) and (SEQ ID NO: 108; reverse, complementary, AttB2 site in italic: 5′ GGGGACCACTTTGTACMGAAAGCTGGGT TTACTCCTGCTCATCATCC 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 1585 bp (including attB sites; from start to stop 1521 bp) was amplified and purified also using standard methods. The first step of the Gateway procedure, the BP reaction, was then performed, during which the PCR fragment recombines in vivo with the pDONR201 plasmid to produce, according to the Gateway terminology, an “entry clone”. Plasmid pDONR201 was purchased from Invitrogen, as part of the Gateway® technology.
Example 22
Vector Construction
The entry clone was subsequently used in an LR reaction with destination vectors used for Oryza sativa transformation. These vectors contain 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. Four different rice promoters located upstream of this Gateway cassette were used to express the Otyza saliva CYP90B: prolamin RP6, oleosin 18 kDa, GOS2 and HMGB1.
After the LR recombination step, the resulting expression vectors (prolamin RP6 promoter, oleosin 18 kDa, GOS2 and HMGB1—see FIG. 14) 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 the Examples below. For transformation of other crops see Example 40.
Example 23
Description of the Phenotypic Evaluation Procedure
Approximately 15 to 20 independent T0 rice transformants were generated per construct. The primary transformants were transferred from a tissue culture chamber to a greenhouse for growing and harvest of T1 seed. Four or five 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. The transgenic plants and the suitable control plants were grown side-by-side at random positions. From the stage of sowing until the stage of maturity the plants were passed several times through a digital imaging cabinet. At each time point digital images (2048×1536 pixels, 16 million colours) were taken of each plant from at least 6 different angles.
Three T1 events were further evaluated in the T2 generation following the same evaluation procedure as for the T1 generation but with more individuals per event.
Seed-Related Parameter Measurements
The mature primary panicles were harvested, counted, 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 (TKVV) is extrapolated from the number of filled seeds counted and their total weight. The harvest index (HI) in the present invention is defined as the ratio between the total seed yield and the above ground area (mm2), multiplied by a factor 106. The total number of flowers per panicle as defined in the present invention is the ratio between the total number of seeds and the number of mature primary panicles. The seed fill rate as defined in the present invention is the proportion (expressed as a %) of the number of filled seeds over the total number of seeds (or florets). Individual seed parameters (width, length and area) were measured using a custom-made device consisting of two main components, a weighing and imaging device, coupled to software for image analysis. Both husked and dehusked seeds were used for these measurements.
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.
Example 24
Results of Oryza sativa CYP90B under the Control of Non-Constitutive Promoters
24.1 Transgenic plants expressing CYP90B under the control of the endosperm-specific promoter
The seed yield and HI measurement results for transgenic plants expressing CYP90B under the control of the endosperm-specific (prolamin RP6) promoter are shown in Table M and N, respectively. The number of events with an increase is indicated, as well as the p values from the F test for the T1 and T2 generations.







TABLE M







Seed yield measurement results of transgenic plants expressing


CYP90B under the control of the endosperm-specific promoter.











Number of events





showing an increase
% Difference
P value of F test














T1 generation
3 out of 4
11
0.1572


T2 generation
3 out of 3
13
0.0103
















TABLE N







HI measurement results of transgenic plants expressing CYP90B


under the control of the endosperm-specific promoter.











Number of events





showing an increase
% Difference
P value of F test














T1 generation
4 out of 4
11
0.047


T2 generation
3 out of 3
10
0.0392









The transgenic rice plants expressing CYP90B under the control of the endosperm-specific (prolamin RP6) promoter present an increased harvest, due to an increase in seed yield while aboveground plant biomass remains unchanged (data not shown), when compared to control plants.
24.2 Transgenic Plants Expressing CYP90B under the Control of the Embryo/Aleurone-Specific Promoter
The TKW measurement results for transgenic plants expressing CYP90B under the control of an embryo/aleurone (oleosin 18 kDa) promoter are shown in Table 0. The number of events with an increase is indicated as well as the p values from the F test for the T1 and T2 generations.







TABLE O







TKW measurement results of transgenic plants expressing


CYP90B under the control of the embryo/aleurone promoter.











Number of events





showing an increase
% Difference
P value of F test














T1 generation
5 out of 5
4
0.0002


T2 generation
2 out of 3
1
0.2428









The average seed area measurement results for transgenic plants expressing CYP90B under the control of the oleosin 18 kDa promoter are shown in Table P. The number of events with an increase is indicated as well as the p values from the F test for the T1 and T2 generations.







TABLE P







Average seed area measurement results of transgenic plants expressing


CYP90B under the control of the embryo/aleurone promoter.











Number of events





showing an increase
% Difference
P value of F test














T1 generation
5 out of 5
3
<0.0001


T2 generation
3 out of 3
2
0.0272









The average seed length measurement results for transgenic plants expressing CYP90B under the control of the oleosin 18 kDa promoter are shown in Table Q. The number of events with an increase is indicated as well as the p values from the F test for the T1 and T2 generations.







TABLE Q







Average seed length measurement results of transgenic plants


expressing CYP90B under the control of the embryo/aleurone promoter.











Number of events





showing an increase
% Difference
P value of F test














T1 generation
5 out of 5
3
<0.0001


T2 generation
3 out of 3
1
0.0086









Transgenic rice plants expressing CYP90B under the control of an embryo/aleurone (oleosin 18 kDa) promoter have seeds with increased TKW, seed area and seed length. No significant increase in seed yield was observed.
Example 25
Evaluation and Results of Oryza sativa CYP90B under the Control of Constitutive Promoters
25.1 Transgenic Plants Expressing CYP90B under the Control of the GOS2 Constitutive Promoter
The evaluation measurement results for transgenic plants expressing CYP90B under the control of the GOS2 constitutive promoter are shown in Table R. The number of events with an increase is indicated, as well as the p values from the F test for the T1 generation. No T2 generation evaluation is performed when negative results are obtained in the T1 generation.







TABLE R







Evaluation measurement results of transgenic plants expressing


CYP90B under the control of the GOS2 constitutive promoter.











Number of events

P value of



showing an increase
% Difference
F test














Aboveground biomass
5 out of 5
−13
<0.0001


Height
5 out of 5
−7
<0.0001


Number of filled seeds
5 out of 5
−53
<0.0001


Number of seeds
5 out of 5
−32
<0.0001


Seed yield
5 out of 5
−53
<0.0001


HI
5 out of 5
−46
<0.0001









25.2 Transgenic Plants Expressing CYP90B under the Control of the HMBG1 Constitutive Promoter

The evaluation measurement results for transgenic plants expressing CYP90B under the control of the HMGB1 constitutive promoter are shown in Table S. The number of events with an increase is indicated, as well as the p values from the F test for the T1 generation. No T2 generation evaluation is performed when negative results are obtained in the T1 generation.







TABLE S







Evaluation measurement results of transgenic plants expressing


CYP90B under the control of the HMGB1 constitutive promoter.











Number of events

P value of



showing an increase
% Difference
F test














Aboveground biomass
5 out of 5
−18
<0.0001


Height
5 out of 5
−6
<0.0001


Number of filled seeds
5 out of 5
−56
<0.0001


Number of seeds
5 out of 5
−33
<0.0001


Seed yield
5 out of 5
−56
<0.0001


HI
5 out of 5
−46
<0.0001









Transgenic rice plants expressing CYP90B under the control of two different constitutive promoters show strongly reduced aboveground plant biomass, plant height, number of filled seeds, seed yield and HI compared to control plants.
Example D
CDC27
Example 26
Cloning of an Arabidopsis thaliana Gene Encoding a CDC27 Polypeptide having at Least One Inactive TPR Domain in the NH2 Terminal Region of the Polypeptide
The Arabidopsis thaliana gene encoding a CDC27 polypeptide having at least one inactive TPR domain in the NH2 terminal region of the polypeptide (COS0171—2) 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 sie of the bank was 1.5 kb and the original number of clones was of the order of 1.59×107 cfu. Original titer was determined to be 9.6×106 cfu/ml, and after the first amplification of 101° cfu/ml. After plasmid extraction, 200 ng of template was used in a 50 μl PCR mix. Primers (SEQ ID NO: 149; sense, start codon in bold, AttB1 site in italic: 5′-GGGGACMGTTTGTACAAAAAAGCAGGCTTCACAATGCMCMCTGTCMCTTC 3′) and (SEQ ID NO: 150; reverse, complementary, AttB2 site in italic: 5′ GGGGACCACTTTGTACAAGAAAGCTGGGTTGGAGTAGCTATGGTTITCAC-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 1816 bp (including attB sites; from start to stop 1737 bp) was amplified and purified also using standard methods. The first step of the Gateway procedure, the BP reaction, was then performed, during which the PCR fragment recombines in vivo with the pDONR201 plasmid to produce, according to the Gateway terminology, an “entry clone”. Plasmid pDONR201 was purchased from Invitrogen, as part of the Gateway® technology.
Example 27
Vector Construction
The entry clone was subsequently used in an LR reaction with 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 OSH1 promoter (SEQ ID NO: 151) for shoot apical meristem expression was located upstream of this Gateway cassette.
After the LR recombination step, the resulting expression vector shown in FIG. 18 was transformed into Agrobacterium strain LBA4044 and subsequently to Oryza saliva plants. Transformed rice plants were allowed to grow and were then examined for the parameters described in Examples 28 and 29. For transformation of other crops see Example 40.
Example 28
Description of the Phenotypic Evaluation Procedure
Approximately 15 to 20 independent T0 rice transformants were generated. The primary transformants were transferred from a tissue culture chamber to a greenhouse for growing and harvest of T1 seed. Five 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. The transgenic plants and the suitable control plants were grown side-by-side at random positions. From the stage of sowing until the stage of maturity the plants were passed several times through a digital imaging cabinet. At each time point digital images (2048×1536 pixels, 16 million colours) were taken of each plant from at least 6 different angles. Three of the events evaluated in T1 were further evaluated in the T2 generation following the same evaluation procedure as for the T1 generation but with more individuals per event.
Seed-Related Parameter Measurements
The mature primary panicles were harvested, counted, 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 harvest index (HI) in the present invention is defined as the ratio between the total seed yield and the above ground area (mm2), multiplied by a factor 106. The total number of flowers per panicle as defined in the present invention is the ratio between the total number of seeds and the number of mature primary panicles. The seed fill rate as defined in the present invention is the proportion (expressed as a %) of the number of filled seeds over the total number of seeds (or florets).
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.
Example 29
Results of the Evaluation of Transgenic Rice Plants Expressing a Modified Arabidopsis thaliana CDC27 Nucleic Acid under the Control of a Shoot Apical Meristem Promoter
The evaluation measurement results (seed yield, number of filled seeds, and HI) for transgenic plants expressing a modified CDC27 nucleic acid under the control of a shoot apical meristem promoter (OSH1) are shown in Tables T to V. The number of events with an increase, the % difference with suitable control plants, as well as the p values from the F test for the T1 and T2 generations are indicated.







TABLE T







Seed yield measurement results of transgenic plants expressing a


modified CDC27 nucleic acid under the control of a shoot apical


meristem promoter.











Number of events





showing an increase
% Difference
P value of F test














T1 generation
3 out of 5
35
0.0113


T2 generation
2 out of 3
11
0.0083
















TABLE U







Number of filled seeds measurement results of transgenic


plants expressing a modified CDC27 nucleic acid under the


control of a shoot apical meristem promoter.











Number of events





showing an increase
% Difference
P value of F test














T1 generation
3 out of 5
36
0.0083


T2 generation
2 out of 3
10
0.0099
















TABLE V







Harvest index measurement results of transgenic plants


expressing a modified CDC27 nucleic acid under the


control of a shoot apical meristem promoter.











Number of events





showing an increase
% Difference
P value of F test














T1 generation
3 out of 5
34
0.0053


T2 generation
2 out of 3
6
0.0188









Transgenic rice plants expressing a modified CDC27 nucleic acid under the control of shoot apical meristem promoter have significantly increased seed yield, increased number of filled seeds and increased harvest index.
Example E
AT-Hook
Example 30
Gene Cloning of Oryza sativa A T-Hook-Encoding Nucleic Acid
The Oryza sativa gene encoding a polypeptide comprising an AT-hook domain and a DUF296 domain (see SEQ ID NO: 152) was amplified by PCR using as template an Ofyza saliva 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 1.6 kb and the original number of clones was of the order of 1.67×107 cfu.-Original titer was determined to be 3.34×108 cfu/ml after first amplification of 6×1010 cfu/ml. After plasmid extraction, 200 ng of template was used in a 50 μl PCR mix. Primers (SEQ ID NO: 196; sense, AttB1 primer: 5′-ggggacaagtttgtacaaaaaagcaggcttaaacaatggatccggtcacgg-3′) and (SEQ ID NO: 197; reverse, complementary, AttB2 primer 5′-ggggaccactttgtacaagaaagctgggtggaatcgatccatctcagaa-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 (including aftB sites; from start to stop) was amplified and purified using standard methods. The first step of the Gateway procedure, the BP reaction, was then performed, during which the PCR fragment recombined in vivo with the pDONR201 plasmid to produce, according to the Gateway terminology, an “entry clone”. Plasmid pDONR201 was purchased from Invitrogen, as part of the Gateway® technology.
Example 31
Vector Construction
The entry clone was subsequently used in an LR reaction with a destination vector containing the prolamin promoter 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 prolamin promoter (SEQ ID NO: 195) for endosperm-specific expression was located upstream of this Gateway cassette.
After the LR recombination step, the resulting expression vector shown in FIG. 22 was transformed into Agrobacterium strain LBA4044 and subsequently to Oryza saliva plants. Transformed rice plants were allowed to grow and were then examined for the parameters described below. For transformation of other crops see Example 40.
Example 32
Evaluation and Results
Approximately 15 to 20 independent T0 rice transformants were generated. The primary transformants were transferred from a tissue culture chamber to a greenhouse for growing and harvest of T1 seed. Seven 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.
32.1 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 referred to 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.
32.2 Seed-Related Parameter Measurements
The mature primary panicles were harvested, counted, bagged, barcode-labelled 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) was extrapolated from the number of filled seeds counted and their total weight. The harvest Index (HI) was expressed as a ratio between the total seed yield and the aboveground area (mm2), multiplied by a factor 106. The total number of flowers per panicle was expressed as a ratio between the total number of seeds and the number of mature primary panicles. The seed fill rate was expressed as a % of the number of filled seeds over the total number of seeds (or florets).







TABLE W







Comparative data to showing the difference in seed yield obtained using an


endosperm-specific promoter (prolamin) compared with a root-specific promoter (RCc3


promoter)













Total
Number filled
Flowers per
Harvest
Number


CDS3129 = SEQIDNO 1/2
weight
seeds
panicle
index
total seeds

















root-specific
4 events
% difference
9.20%
10.00%
0.00%
11.20%
0.00%


promoter

p No of F-
0.0451
0.0266
0.7744
0.0027
0.4410




test


endosperm
5 events
% difference
46.10%
48.50%
16.40%
39.40%
19.50%


specific

p No of F-
<0.0001
<0.0001
<0.0001
<0.0001
<0.0001


promoter

test









The table shows the % difference in various parameters for transgenic plants compared to corresponding control plants (nullizygotes); also shown in the Table is the p value from the F-test which indicates the overall effect of the gene. As shown in the table, various seed yield parameters were increased in plants expressing an AT-hook-encoding nucleic acid (SEQ ID NO: 152) under the control of an endosperm-specific promoter, whereas no increase (in fact a significant decrease) was obtained for plants expressing the same transgene under the control of a root-specific promoter in transgenic plants.
Example F
DOF Transcription Factors
Example 33
Gene Cloning of Arabidopsis Thaliana DOF Transcription Factor (SEQ ID NO: 198)
The Arabidopsis thaliana DOF transcription factor gene was amplified by PCR using as template an Arabidopsis thaliana seedlirig 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 original number of clones was of 1.59×107 cfu. Original titer was determined to be 9.6×105 cfu/ml after first amplification of 6×1011 cfu/ml. After plasmid extraction, 200 ng of template was used in a 50 μl PCR mix. Primer (SEQ ID NO: 223) (sense AttB1 primer: 5′ ggggacaagtttgtacaaaaaa gcaggcttaaacaatgggtggatcgatggc 3′) and (SEQ ID NO: 224) (reverse complementary AttB2 primer 5′ ggggaccactttgtacaagaaagctgggtcgttaatgatccgacaaaaca 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 (including attB sites; from start to stop) 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 recombined in vivo with the pDONR201 plasmid to produce, according to the Gateway terminology, an “entry clone”. Plasmid pDONR201 was purchased from Invitrogen, as part of the Gateway® technology.
Example 33a
Vector Construction
The entry clone was subsequently used in an LR reaction with a destination vector containing GOS2 used for Oryza sativa transformation. This vector contained 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: 225) for constitutive expression was located upstream of this Gateway cassette.
After the LR recombination step, the resulting expression vector shown in FIG. 26 was transformed into Agrobacterium strain LBA4044 and subsequently to Oryza sativa plants. Transformed rice plants were allowed to grow and were then examined for the parameters described below. For transformation of other crops see Example 40.
Example 34
Gene Cloning of Arabidopsis Thaliana DOF Transcription Factor (SEQ ID NO: 226)
The Arabidopsis thaliana DOF transcription factor 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 original number of clones was of 1.59×107 cfu. Original titer was determined to be 9.6×105 cfu/ml after first amplification of 6×1011 cfu/ml. After plasmid extraction, 200 ng of template was used in a 50 μl PCR mix. Primer (SEQ ID NO: 256) (sense AttB1 primer 5′ ggggacaagtttgtacaaaaaa gcaggcttaaacaatgatgatggagactagagatc3′) and (SEQ ID NO: 257) (reverse complementary AttB2 primer 5′ ggggaccactttgtacaagaaagctgggtcatatgtaactctaaatctgttca3′), 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 (including attB sites; from start to stop) 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 recombined in vivo with the pDONR201 plasmid to produce, according to the Gateway terminology, an “entry clone”. Plasmid pDONR201 was purchased from Invitrogen, as part of the Gateway) technology.
Example 34a
Vector Construction
The entry clone was subsequently used in an LR reaction with a destination vector containing prolamin 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 prolamin promoter (SEQ ID NO: 258) for seed-specific expression was located upstream of this Gateway cassette.
After the LR recombination step, the resulting expression vector shown in FIG. 27 was transformed into Agrobacterium strain LBM044 and subsequently to Oryza sativa plants. Transformed rice plants were allowed to grow and were then examined for the parameters described below. For transformation of other crops see Example 40.
Example 35
Evaluation and Results
Approximately 15 to 20 independent T0 rice transformants were generated. The primary transformants were transferred from a tissue culture chamber to a greenhouse for growing and harvest of T1 seed. Seven 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. Approximately 4 T1 events were further evaluated in the T2 generation following the same evaluation procedure as for the T1 generation but with more individuals per event.
Plants from five events were grown under normal conditions until the heading stage. Soil moisture was monitored continuously using moisture sensors inserted in the pots of several randomly chosen non-transgenic control plants. In a first phase, the pots were saturated to a maximum value of 60% to reduce the pot-to-pot variability. Once the pots were saturated, irrigation was withheld until a soil-moisture content of below 20% was obtained. The plants were then re-watered until the soil moisture reached the maximum level of 60% again. The plants were then imaged to evaluate the following root-related and seed-related parameters.
Root-Related Parameters
Plants were grown in specially designed pots with transparent bottoms to allow visualization of the roots. A digital camera recorded images through the bottom of the pot during plant growth. Root features such as total projected area (which can be correlated to total root volume), average diameter and length of roots above a certain thickness threshold (length of thick roots, or length of thin roots) were deduced from the generated image using appropriate software.
Seed-Related Parameter Measurements
The mature primary panicles were harvested, counted, bagged, barcode-labelled 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) was extrapolated from the number of filled seeds counted and their total weight. The harvest index (HI) In the present invention is defined as the ratio between the total seed yield and the above ground area (mm2), multiplied by a factor 106. The total number of flowers per panicle as defined in the present invention is the ratio between the total number of seeds and the number of mature primary panicles. The seed fill rate as defined in the present invention is the proportion (expressed as a %) of the number of filled seeds over the total number of seeds (or florets).
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.
Table X below shows the results of the T2 evaluation for transgenic plants expressing a nucleic acid encoding a DOF transcription factor under the control of a GOS2 promoter and the results of the T2 evaluation for transgenic plants expressing a nucleic acid encoding a DOF transcription factor under the control of a prolamin promoter. Although not shown, comparable results were obtained for T1 plants). The p-value from the F test is shown for the parameters listed in the table, as well as the percentage difference between transgenic plants verses nullizygotes.







TABLE X







Results of T2 Evaluation












Total seed
Number of Filled





Weight
Seeds
Harvest Index
Fill Rate
















%

%

%

%




Difference
P-Value
Difference
P-Value
Difference
P-Value
Difference
P-Value



















Example 1
34.0%
0.0070
36.0%
0.0061
32%
0.0032
21%
0.0011


(GOS2)


[Internal


reference


CD12148;


CDS3325]


Example 2
  18%
0.0348
  17%
0.0385
19%
0.035 
12%
0.1373


(prolamin)


[Internal


reference


CD12225;


CDS3319]









In addition to the abovementioned seed-related parameters, the following root parameters also were increased in transgenic plants compared to nullizygotes: 14% increase in total root biomass, 7% increase in number of thin roots (internal threshold), 36% increase in number of thick roots (internal threshold) and an 8% increase in average diameter of roots.
The aforementioned results were obtained under conditions of mild drought stress; similar results would be expected under normal or non-stress conditions.
Example G
CKI
Example 36
Cloning of an Oryza Sativa Gene Encoding an CKI4 Polypeptide
The Oryza sativa gene encoding a CKI4 polypeptide was amplified by PCR using as template an Oryza sativa cell suspension culture cDNA library cloned in the pAD-Gal4-2.1 vector of HybriZAP-2.i kit (Stratagene, La Jolla, Calif. USA), according to the manufacturer's instructions. Average insert size of the bank was 1.5 kb and the original number of clones was of the order of 2×108 pfu. Original titer was determined to be 4×106 pfu/ml and after the first amplification of 1010 pfu/ml. After plasmid extraction, 200 ng of template was used in a 50 μl PCR mix. Primers (SEQ ID NO: 284; sense, start codon in bold, AttB1 site in italic: 5′-GGGGACMGTTTGTACAAAAAGCAGGCTTCACAATGGGCAAGTACATGCGCAAGGCC-3′) and (SEQ ID NO: 285; reverse, complementary, AttB2 site in italic: (5′-GGGGACCACTTTGTACAAGAAAGCTGGGTGGAGCAGAGAGGTCCATGGTGCCC-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 662 bp (including attB sites; from start to stop 585 bp) was amplified and purified also using standard methods. The first step of the Gateway procedure, the BP reaction, was then performed, during which the PCR fragment recombines in vivo with the pDONR201 plasmid to produce, according to the Gateway terminology, an “entry clone”. Plasmid pDONR201 was purchased from Invitrogen, as part of the Gateway® technology.
Example 37
Vector Construction
The entry clone was subsequently used in an LR reaction with 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 two Gateway cassettes in opposite orientation intended for LR in vivo recombination with the sequence of interest already cloned in the entry clone. The two Gateway cassettes were separated by non-coding DNA (in this case a 315 bp fragment of a tobacco matrix attachment region (MAR), NCBI reference U67919, fragment from 774 to 1088 bp), to promote formation of a hairpin structure of the mRNA after transcription. A rice RP6 prolamin promoter (SEQ ID NO: 281) for endosperm-specific expression was located upstream of the first Gateway cassette, in opposite orientation with respect to the promoter.
The entry clone was also used in an LR reaction with another destination vector used for Oryza sativa transformation. This vector was identical to the one described above, except that the RP6 prolamin promoter had been replaced with rice beta-expansin promoter of SEQ ID NO: 282.
After the LR recombination step, the two resulting expression vectors (FIG. 32 for both 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 Examples 38 and 39. For transformation of other crops see Example 40.
Example 38
Description of the Phenotypic Evaluation Procedure
Approximately 15 to 20 independent T0 rice transformants were generated. The primary transformants were transferred from a tissue culture chamber to a greenhouse for growing and harvest of T1 seed. Four to five 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. The transgenic plants and the suitable control plants were grown side-by-side at random positions. From the stage of sowing until the stage of maturity the plants were passed several times through a digital imaging cabinet. At each time point digital images (2048×1536 pixels, 16 million colours) were taken of each plant from at least 6 different angles.
The same events evaluated in T1 were further evaluated in the T2 generation following the same evaluation procedure as for the T1 generation.
Seed-Related Parameter Measurements
The mature primary panicles were harvested, counted, bagged, barcode-labelled and then dried for three days in an oven at 37° C. The panicles were then threshed and all the seeds were collected and counted. The filled husks were separated from the empty ones using an air-blowing device. The empty husks were discarded and the remaining fraction was counted again. The filled husks were weighed on an analytical balance. The number of filled seeds was determined by counting the number of filled husks that remained after the separation step.
The total seed yield was measured by weighing all filled husks harvested from a plant. Total seed number per plant was measured by counting the number of husks harvested from a plant. The harvest index (HI) in the present invention is defined as the ratio between the total seed yield and the above ground area (mm2), multiplied by a factor 106. The total number of flowers per panicle as defined in the present invention is the ratio between the total number of seeds and the number of mature primary panicles. The seed fill rate as defined in the present invention is the proportion (expressed as a %) of the number of filled seeds over the total number of seeds (or florets).
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.
Example 39
Results of the Evaluation of Transgenic Rice Plant with Reduced CKI4 Expression in the Endosperm
The evaluation measurement results (seed yield, number of filled seeds, total number of seeds and flowers per panicle) for transgenic plants with reduced CKI4 expression in the endosperm are presented in Table Y below. The number of plants with an increase in a parameter, the average percentage increase as well as the P value of the T2 generation are shown, and compared to results obtained with transgenic plants with reduced CKI4 expression using a beta expansin promoter for preferential expression in shoot tissue.
The results show that reduced expression of CKI4 in the endosperm gives plants with significantly increased seed weight, number of filled seeds, total number of seeds and flowers per panicle, compared to nullizygotes and compared to transgenic plants with preferentially reduced expression of CKI4 in shoot tissue (using a beta expansin promoter).







TABLE Y







Evaluation measurement results for transgenic plants with


reduced CKI4 expression in the endosperm










Endosperm-specific promoter













Number of






plants with


Shoot-specific



increased


promoter



parameter
% increase
P value
% increase















Seed Yield
4 out of 4
17%
0.006
<17%


Number of
4 out of 4
19%
0.0018
<19%


filled seeds


Total number
4 out of 4
16%
0.0014
<16%


of seeds


Flowers per
3 out of 4
9%
0.006
<9%


panicle









Example 40
Transformation of Corn, Wheat, Soybean, Rapseed and Alfalfa
Corn Transformation
Transformation of maize (Zea mays) is performed with a modification of the method described by Ishida et al. (1996) Nature Biotech 14(6): 745-50. Transformation is genotype-dependent in corn and only specific genotypes are amenable to transformation and regeneration. The inbred line A188 (University of Minnesota) or hybrids with A188 as a parent are good sources of donor material for transformation, but other genotypes can be used successfully as well. Ears are harvested from corn plant approximately 11 days after pollination (DAP) when the length of the immature embryo is about 1 to 1.2 mm. Immature embryos are cocultivated with Agrobacterium tumefaciens containing the expression vector, and transgenic plants are recovered through organogenesis. Excised embryos are grown on callus induction medium, then maize regeneration medium, containing the selection agent (for example imidazolinone but various selection markers can be used). The Petri plates are incubated in the light at 25° C. for 2-3 weeks, or until shoots develop. The green shoots are transferred from each embryo to maize rooting medium and incubated at 25° C. for 2-3 weeks, until roots develop. The rooted shoots are transplanted to soil in the greenhouse. T1 seeds are produced from plants that exhibit tolerance to the selection agent and that contain a single copy of the T-DNA insert.
Wheat Transformation
Transformation of wheat is performed with the method described by Ishida et al. (1996) Nature Biotech 14(6): 745-50. The cultivar Bobwhite (available from CIMMYT, Mexico) is commonly used in transformation. Immature embryos are co-cultivated with Agrobacterium tumefaciens containing the expression vector, and transgenic plants are recovered through organogenesis. After incubation with Agrobacterium, the embryos are grown in vitro on callus induction medium, then regeneration medium, containing the selection agent (for example imidazolinone but various selection markers can be used). The Petri plates are incubated in the light at 25° C. for 2-3 weeks, or until shoots develop. The green shoots are transferred from each embryo to rooting medium and incubated at 25° C. for 2-3 weeks, until roots develop. The rooted shoots are transplanted to soil in the greenhouse. T1 seeds are produced from plants that exhibit tolerance to the selection agent and that contain a single copy of the T-DNA insert.
Soybean Transformation
Soybean is transformed according to a modification of the method described in the Texas A&M patent U.S. Pat. No. 5,164,310. Several commercial soybean varieties are amenable to transformation by this method. The cultivar Jack (available from the Illinois Seed foundation) is commonly used for transformation. Soybean seeds are sterilised for in vitro sowing. The hypocotyl, the radicle and one cotyledon are excised from seven-day old young seedlings. The epicotyl and the remaining cotyledon are further grown to develop axillary nodes. These axillary nodes are excised and incubated with Agrobacterium tumefaciens containing the expression vector. After the cocultivation treatment, the explants are washed and transferred to selection media. Regenerated shoots are excised and placed on a shoot elongation medium. Shoots no longer than 1 cm are placed on rooting medium until roots develop. The rooted shoots are transplanted to soil in the greenhouse. T1 seeds are produced from plants that exhibit tolerance to the selection agent and that contain a single copy of the T-DNA insert.
Rapeseed/Canola Transformation
Cotyledonary petioles and hypocotyls of 5-6 day old young seedling are used as explants for tissue culture and transformed according to Babic et al. (1998, Plant Cell Rep 17: 183-188). The commercial cultivar Westar (Agriculture Canada) is the standard variety used for transformation, but other varieties can also be used. Canola seeds are surface-sterilized for in vitro sowing. The cotyledon petiole explants with the cotyledon attached are excised from the in vitro seedlings, and inoculated with Agrobacterium (containing the expression vector) by dipping the cut end of the petiole explant into the bacterial suspension. The explants are then cultured for 2 days on MSBAP-3 medium containing 3 mg/l BAP, 3% sucrose, 0.7% Phytagar at 23° C., 16 hr light. After two days of co-cultivation with Agrobacterium, the petiole explants are transferred to MSBAP-3 medium containing 3 mg/l BAP, cefotaxime, carbenicillin, or timentin (300 mg/l) for 7 days, and then cultured on MSBAP-3 medium with cefotaxime, carbenicillin, or timentin and selection agent until shoot regeneration. When the shoots are 5-10 mm in length, they are cut and transferred to shoot elongation medium (MSBAP-0.5, containing 0.5 mg/l BAP). Shoots of about 2 cm in length are transferred to the rooting medium (MSO) for root induction. The rooted shoots are transplanted to soil in the greenhouse. T1 seeds are produced from plants that exhibit tolerance to the selection agent and that contain a single copy of the T-DNA insert.
Alfalfa Transformation
A regenerating clone of alfalfa (Medicago sativa) is transformed using the method of (McKersie et al., 1999 Plant Physiol 119: 839-847). Regeneration and transformation of alfalfa is genotype dependent and therefore a regenerating plant is required. Methods to obtain regenerating plants have been described. For example, these can be selected from the cultivar Rangelander (Agriculture Canada) or any other commercial alfalfa variety as described by Brown DCW and A Atanassov (1985. Plant Cell Tissue Organ Culture 4: 111-112). Alternatively, the RA3 variety (University of Wisconsin) has been selected for use in tissue culture (Walker et al., 1978 μm J Bot 65:654-659). Petiole explants are cocultivated with an overnight culture of Agrobacterium tumefaciens C58C1 pMP90 (McKersie et al., 1999 Plant Physiol 119: 839-847) or LBA4404 containing the expression vector. The explants are cocultivated for 3 d in the dark on SH induction medium containing 288 mg/L Pro, 53 mg/L thioproline, 4.35 g/L K2SO4, and 100 μm acetosyringinone. The explants are washed in half-strength Murashige-Skoog medium (Murashige and Skoog, 1962) and plated on the same SH induction medium without acetosyringinone but with a suitable selection agent and suitable antibiotic to inhibit Agrobacterium growth. After several weeks, somatic embryos are transferred to BOi2Y development medium containing no growth regulators, no antibiotics, and 50 g/L sucrose. Somatic embryos are subsequently germinated on half-strength Murashige-Skoog medium. Rooted seedlings were transplanted into pots and grown in a greenhouse. T1 seeds are produced from plants that exhibit tolerance to the selection agent and that contain a single copy of the T-DNA insert.

## Claims

1. A method for increasing yield of a plant relative to a corresponding control plant, comprising:
a) increasing expression in a plant of a nucleic acid encoding an FG-GAP polypeptide by introducing and expressing in a plant a nucleic acid comprising:
i) the nucleic acid sequence of SEQ ID NO: 45,
ii) a nucleic acid sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 46, or
iii) a nucleic acid sequence encoding an FG-GAP polypeptide which comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 46; and

b) selecting a plant having increased yield relative to a corresponding control plant on the basis of said plant showing increased yield relative to said control plant.

2. The method of claim 1, wherein the nucleic acid comprises a sequence capable of hybridising to the nucleic acid sequence of SEQ ID NO: 45 under stringent hybridization conditions comprising hybridization at 65° C. or 42° C. in 1×SSC and 50% formamide followed by one or more washes in 0.3×SSC at 65° C. and encodes an FG-GAP polypeptide comprising a signal peptide, one or more FG-GAP domains, and a transmembrane domain located in the C-terminal half of the polypeptide.

3. The method of claim 1, wherein said nucleic acid encodes an FG-GAP polypeptide comprising one or more of the conserved motifs of SEQ ID NO: 50, SEQ ID NO: 51 and SEQ ID NO: 52.

4. The method of claim 1, wherein said nucleic acid is overexpressed in said plant.

5. The method of claim 1, wherein said nucleic acid is of plant origin, from a dicotyledonous plant, from the family Brassicaceae, or from Arabidopsis thaliana.

6. The method of claim 1, wherein said nucleic acid is operably linked to a constitutive promoter.

7. The method of claim 6, wherein the constitutive promoter is a GOS2 promoter.

8. The method of claim 1, wherein the increased yield is increased seed yield.

9. The method of claim 8, wherein the increased seed yield is selected from the group consisting of increased total weight of seeds, increased number of filled seeds, and increased harvest index.

10. A plant, plant part, or plant cell obtained by the method of claim 1, or a progeny of said plant, wherein the plant, plant part, or plant cell, or said progeny, comprises said nucleic acid operably linked to a constitutive promoter, and wherein said progeny has increased yield relative to a corresponding control plant on the basis of said progeny showing increased yield relative to said control plant.

11. A method for the production of a transgenic plant having increased yield compared to a corresponding wild type plant, comprising:
(a) introducing and expressing in a plant or plant cell a nucleic acid encoding an FG-GAP polypeptide, wherein said nucleic acid comprises:
i) the nucleic acid sequence of SEQ ID NO: 45,
ii) a nucleic acid sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 46, or
iii) a nucleic acid sequence encoding an FG-GAP polypeptide which comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 46;

(b) selecting a plant having increased yield relative to a corresponding control plant on the basis of said plant showing increased yield relative to said control plant;
(c) cultivating the plant under conditions promoting plant growth and development; and
(d) optionally obtaining a progeny of said plant having increased yield, wherein said progeny has increased yield relative to a corresponding control plant.

12. The plant, plant part, or plant cell of claim 10, wherein the plant is a monocotyledonous plant, sugarcane, a cereal, rice, maize, wheat, barley, millet, rye, oats or sorghum, or wherein said plant part or plant cell is derived from a monocotyledonous plant, sugarcane, a cereal, rice, maize, wheat, barley, millet, rye, oats or sorghum.

13. Harvestable parts of the plant of claim 10, wherein the harvestable parts comprise said nucleic acid operably linked to a constitutive promoter.

14. The harvestable parts of claim 13, wherein said harvestable parts are seeds.

15. A method for increasing yield of a plant relative to a corresponding control plant comprising transforming a plant or plant cell with a construct comprising:
(a) a nucleic acid comprising:
(i) the nucleic acid sequence of SEQ ID NO: 45,
(ii) a nucleic acid sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 46; or
(iii) a nucleic acid sequence encoding an FG-GAP polypeptide which comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 46;

(b) one or more control se s uences ca s able of drivin ex ression of the nucleic acid of (a); and optionally
(c) a transcription termination sequence,
and selecting a plant having increased yield relative to a corresponding control plant on the basis of said plant showing increased yield relative to said control plant.

16. The method of claim 15, wherein the increased yield is increased total weight of seeds, increased number of filled seeds, or increased harvest index.

17. The method of claim 1, comprising utilizing said nucleic acid or, the FG-GAP polypeptide encoded by said nucleic acid as a molecular marker.

18. A plant, plant part, or plant cell obtained by the method of claim 11, or a progeny of said plant, wherein the plant, plant part, or plant cell, or said progeny, comprises said nucleic acid, and wherein said progeny has increased yield relative to a corresponding control plant.

19. The plant, plant part, or plant cell of claim 18, wherein the plant is a monocotyledonous plant, sugarcane, a cereal, rice, maize, wheat, barley, millet, rye, oats or sorghum, or wherein said plant part or plant cell is derived from a monocotyledonous plant, sugarcane, a cereal, rice, maize, wheat, barley, millet, rye, oats or sorghum.

20. Harvestable parts of the plant of claim 18, wherein the harvestable parts comprise said nucleic acid.

21. The harvestable parts of claim 20, wherein said harvestable parts are seeds.

22. A plant, plant part, or plant cell obtained by the method of claim 15, or a progeny of said plant, wherein the plant, plant part, or plant cell, or said progeny, comprises said nucleic acid, and wherein said progeny has increased yield relative to a corresponding control plant.

23. The plant, plant part, or plant cell of claim 22, wherein the plant is a monocotyledonous plant, sugarcane, a cereal, rice, maize, wheat, barley, millet, rye, oats or sorghum, or wherein said plant part or plant cell is derived from a monocotyledonous plant, sugarcane, a cereal, rice, maize, wheat, barley, millet, rye, oats or sorghum.

24. Harvestable parts of the plant of claim 22, wherein the harvestable parts comprise said nucleic acid.

25. The harvestable parts of claim 24, wherein said harvestable parts are seeds.