# Patent

## Title
Plants having increased yield-related traits by expressing a growth-regulating factor (GRF) polypeptide and method for making the same

## Abstract

The present invention relates generally to the field of molecular biology and concerns a method for increasing various plant yield-related traits by increasing expression in a plant of a nucleic acid encoding a GRF polypeptide. The present invention also concerns plants having increased expression of a nucleic acid encoding a GRF polypeptide, which plants have increased yield-related traits relative to 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/EP2008/062232, filed Sep. 15, 2008, which claims benefit of European application 07116515.3, filed Sep. 14, 2007, European Application 07116520.3, filed Sep. 14, 2007, European Application 07116516.1, filed Sep. 14, 2007, European Application 07116961.9, filed Sep. 21, 2007, European Application 07117490.8, filed Sep. 28, 2007, U.S. Provisional Application 60/975,877, filed Sep. 28, 2007, U.S. Provisional Application 60/975,900, filed Sep. 28, 2007, U.S. Provisional Application 60/975,887, filed Sep. 28, 2007, U.S. Provisional Application 60/976,835, filed Oct. 2, 2007 and U.S. Provisional Application 60/977,121, filed Oct. 3, 2007.


SUBMISSION OF SEQUENCE LISTING
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 Second_Revised_Sequence_List_13311_00065_US. The size of the text file is 1,426 KB, and the text file was created on Nov. 18, 2010.
The present invention relates generally to the field of molecular biology and concerns a method for increasing various plant yield-related traits by increasing expression in a plant of a nucleic acid sequence encoding a polypeptide selected from the group consisting of: GRF polypeptide, RAA1-like polypeptide, SYR polypeptide, ARKL polypeptide, and YTP polypeptide. The present invention also concerns plants having increased expression of a nucleic acid sequence encoding a polypeptide selected from the group consisting of: GRF polypeptide, RAA1-like polypeptide, SYR polypeptide, ARKL polypeptide, and YTP polypeptide, which plants have increased yield-related traits relative to control plants. The invention also provides constructs useful in the methods of the invention.
The ever-increasing world population and the dwindling supply of arable land available for agriculture fuels research towards increasing the efficiency of agriculture. Conventional means for crop and horticultural improvements utilise selective breeding techniques to identify plants having desirable characteristics. However, such selective breeding techniques have several drawbacks, namely that these techniques are typically labour intensive and result in plants that often contain heterogeneous genetic components that may not always result in the desirable trait being passed on from parent plants. Advances in molecular biology have allowed mankind to modify the germplasm of animals and plants. Genetic engineering of plants entails the isolation and manipulation of genetic material (typically in the form of DNA or RNA) and the subsequent introduction of that genetic material into a plant. Such technology has the capacity to deliver crops or plants having various improved economic, agronomic or horticultural traits.
A trait of particular economic interest is increased 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, leaf senescence and more. Root development, nutrient uptake, stress tolerance and early vigour may also be important factors in determining yield. Optimizing the above-mentioned factors may therefore contribute to increasing crop yield.
Seed yield is a particularly important trait, since the seeds of many plants are important for human and animal nutrition. Crops such as corn, 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.
Plant biomass is yield for forage crops like alfalfa, silage corn and hay. Many proxies for yield have been used in grain crops. Chief amongst these are estimates of plant size. Plant size can be measured in many ways depending on species and developmental stage, but include total plant dry weight, above-ground dry weight, above-ground fresh weight, leaf area, stem volume, plant height, rosette diameter, leaf length, root length, root mass, tiller number and leaf number. Many species maintain a conservative ratio between the size of different parts of the plant at a given developmental stage. These allometric relationships are used to extrapolate from one of these measures of size to another (e.g. Tittonell et al 2005 Agric Ecosys & Environ 105: 213). Plant size at an early developmental stage will typically correlate with plant size later in development. A larger plant with a greater leaf area can typically absorb more light and carbon dioxide than a smaller plant and therefore will likely gain a greater weight during the same period (Fasoula & Tollenaar 2005 Maydica 50:39). This is in addition to the potential continuation of the micro-environmental or genetic advantage that the plant had to achieve the larger size initially. There is a strong genetic component to plant size and growth rate (e.g. ter Steege et al 2005 Plant Physiology 139:1078), and so for a range of diverse genotypes plant size under one environmental condition is likely to correlate with size under another (Hittalmani et al 2003 Theoretical Applied Genetics 107:679). In this way a standard environment is used as a proxy for the diverse and dynamic environments encountered at different locations and times by crops in the field.
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. The ability to engineer early vigour into plants would be of great importance in agriculture. For example, poor early vigour has been a limitation to the introduction of maize (Zea mays L.) hybrids based on Corn Belt germplasm in the European Atlantic.
Harvest index, the ratio of seed yield to aboveground dry weight, is relatively stable under many environmental conditions and so a robust correlation between plant size and grain yield can often be obtained (e.g. Rebetzke et al 2002 Crop Science 42:739). These processes are intrinsically linked because the majority of grain biomass is dependent on current or stored photosynthetic productivity by the leaves and stem of the plant (Gardener et al 1985 Physiology of Crop Plants. Iowa State University Press, pp 68-73). Therefore, selecting for plant size, even at early stages of development, has been used as an indicator for future potential yield (e.g. Tittonell et al 2005 Agric Ecosys & Environ 105: 213). When testing for the impact of genetic differences on stress tolerance, the ability to standardize soil properties, temperature, water and nutrient availability and light intensity is an intrinsic advantage of greenhouse or plant growth chamber environments compared to the field. However, artificial limitations on yield due to poor pollination due to the absence of wind or insects, or insufficient space for mature root or canopy growth, can restrict the use of these controlled environments for testing yield differences. Therefore, measurements of plant size in early development, under standardized conditions in a growth chamber or greenhouse, are standard practices to provide indication of potential genetic yield advantages.
Another trait of importance 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. (2003) Planta 218: 1-14). Abiotic stresses may be caused by drought, salinity, extremes of temperature, chemical toxicity, excess or deficiency of nutrients (macroelements and/or microelements), radiation and oxidative stress. The ability to increase 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 vegetative 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 increase yield-related traits (seed yield and/or biomass) in plants may be through modification of the inherent growth mechanisms of a plant, such as the cell cycle or various signalling pathways involved in plant growth or in defense mechanisms.
It has now been found that various yield-related traits may be increased in plants relative to control plants, by increasing expression in a plant of a nucleic acid sequence encoding a Growth-Regulating Factor (GRF) polypeptide. The increased yield-related traits comprise one or more of: increased early vigour, increased aboveground biomass, increased total seed yield per plant, increased seed filling rate, increased harvest index and increased thousand kernel weight.
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 RAA1-like (Root Architecture Associated 1) in a plant.
It has now been found that various growth characteristics, in particular increased abiotic stress resistance, may be improved in plants by modulating expression in a plant of a nucleic acid encoding a Seed Yield Regulator (SYR) protein.
It has now been found that various yield-related traits may be improved in plants by modulating expression in a plant of a nucleic acid encoding an ARKL (ARADIA Like) polypeptide in a plant.
It has now been found that various yield-related traits may be improved in plants by modulating expression in a plant of a nucleic acid encoding a YTP (Yield Transmembrane Protein) in a plant.
BACKGROUND
DNA-binding proteins are proteins that comprise any of many DNA-binding domains and thus have a specific or general affinity to DNA. DNA-binding proteins include for example transcription factors that modulate the process of transcription, nucleases that cleave DNA molecules, and histones that are involved in DNA packaging in the cell nucleus.
Transcription factors are usually defined as proteins that show sequence-specific DNA binding affinity and that are capable of activating and/or repressing transcription. The Arabidopsis thaliana genome codes for at least 1533 transcriptional regulators, accounting for  ̃5.9% of its estimated total number of genes (Riechmann et al. (2000) Science 290: 2105-2109). The Database of Rice Transcription Factors (DRTF) is a collection of known and predicted transcription factors of Oryza sativa L. ssp. indica and Oryza sativa L. ssp. japonica, and currently contains 2,025 putative transcription factors (TF) gene models in indica and 2,384 in japonica, distributed in 63 families (Gao et al. (2006) Bioinformatics 2006, 22(10):1286-7).
One of these families is the Growth-Regulating Factor (GRF) family of transcription factors, which is specific to plants. At least nine GRF polypeptides have been identified in Arabidopsis thaliana (Kim et al. (2003) Plant J 36: 94-104), and at least twelve in Oryza sativa (Choi et al. (2004) Plant Cell Physiol 45(7): 897-904). The GRF polypeptides are characterized by the presence in their N-terminal half of at least two highly conserved domains, named after the most conserved amino acids within each domain: (i) a QLQ domain (InterPro accession IPR014978, PFAM accession PF08880), where the most conserved amino acids of the domain are Gln-Leu-Gln; and (ii) a WRC domain (InterPro accession IPR014977, PFAM accession PF08879), where the most conserved amino acids of the domain are Trp-Arg-Cys. The WRC domain further contains two distinctive structural features, namely, the WRC domain is enriched in basic amino acids Lys and Arg, and further comprises three Cys and one His residues in a conserved spacing (CX9CX10CX2H), designated as the Effector of Transcription (ET) domain (Ellerstrom et al. (2005) Plant Molec Biol 59: 663-681). The conserved spacing of cysteine and histidine residues in the ET domain is reminiscent of zinc finger (zinc-binding) proteins. In addition, a nuclear localisation signal (NLS) is usually comprised in the GRF polypeptide sequences.
Interaction of some GRF polypeptides with a small family of transcriptional coactivators, GRF-interacting factors (GIF1 to GIF3; also called synovial sarcoma translocation (SYT) polypeptide, SYT1 to SYT3), has been demonstrated using a yeast two-hybrid interaction assay (Kim & Kende (2004) Proc Natl Acad Sci 101: 13374-13379).
The name GRF has also been given to another type of polypeptides, belonging to the 14-3-3 family of polypeptides (de Vetten & Ferl (1994) Plant Physiol 106: 1593-1604), that are totally unrelated the GRF polypeptides useful in performing the methods of the invention.
Transgenic Arabidopsis thaliana plants transformed with a rice GRF (OsGRF1) polypeptide under the control of a viral constitutive 35S CaMV promoter displayed curly leaves, severely reduced elongation of the primary inflorescence, and delayed bolting (van der Knapp et al. (2000) Plant Physiol 122: 695-704). Transgenic Arabidopsis thaliana plants transformed with either one of two Arabidopsis GRF polypeptides (AtGRF1 and AtGRF2) developed larger leaves and cotyledons, were delayed in bolting, and were partially sterile (due to lack of viable pollen), compared to wild type plants (Kim et al. (2003) Plant J 36: 94-104).
In US patent application US2006/0048240, an Arabidopsis thaliana GRF polypeptide is identified as SEQ ID NO: 33421. In US patent application US 2007/0022495, an Arabidopsis thaliana GRF polypeptide is identified as SEQ ID NO: 1803 (also therein referred to as G1438). Transgenic Arabidopsis plants overexpressing G1438 using the 35S CaMV promoter present dark green leaves.
Surprisingly, it has now been found that increasing expression of a nucleic acid sequence encoding a GRF polypeptide gives plants having increased yield-related traits relative to control plants.
According to one embodiment, there is provided a method for increasing yield-related traits in plants relative to control plants, comprising increasing expression of a nucleic acid sequence encoding a GRF polypeptide in a plant. The increased yield-related traits comprise one or more of: increased early vigour, increased aboveground biomass, increased total seed yield per plant, increased seed filling rate, increased harvest index and increased thousand kernel weight.
Little is known about the molecular biology of root formation in monocotyledonous plants. So far only a few genes have been identified that affect root development: examples are the rt1 mutant which forms few or no crowns and brace roots (Jenkins, J. Hered. 21: 79-80, 1930), the asr1 mutant, which displays defective seminal roots (De Miranda et al., Maize Genet. Coop. News Lett. 54: 18-19, 1980), the rtcs mutant lacking nodal (adventitious) roots (Hetz et al., Plant J. 10: 845-857, 1996), the slr1 mutant and slr2 mutant with shortened lateral roots (Hochholdinger et al., Plant Physiol 125:1529-1539, 2001), or rum1, which is affected in lateral initiation in the primary root but also in the initiation of seminal root formation (Woll et al., Plant Physiol., 139, 1255-1267, 2005). Liu et al. (Proteomics 6, 4300-4308, 2006) made a proteomic comparison between primary roots of wild-type and rum1 seedlings and identified another 12 genes that were differently regulated and which were involved in lignin biosynthesis, defence, and the citrate cycle.
Another gene involved in root formation in monocotyledonous plants is raa1, first isolated from rice (Ge et al., Plant Physiol. 135, 1502-1513, 2004): the gene encodes a 12.0-kD protein having 58% homology to the Arabidopsis FPF1 (Flowering Promoting Factor 1). In rice, RAA1 was expressed specifically in the apical meristem, the elongation zone of root tip, steles of the branch zone, and the young lateral root. Constitutive overexpression increased the number of adventitious roots, but primary root growth was decreased. In addition, the endogenous auxin content was increased. OsRAA1 was also induced by auxin; suggesting that a positive feedback regulation exists between RAA1 and auxin in rice root development (Ge et al., 2004). Furthermore, plants overexpressing OsRAA1 had longer leaves and sterile florets (Ge et al., 2004). WO 2006/067219 discloses the use of FPF1 and related proteins for increasing the production of carbohydrates in plants, but transgenics overexpressing FPF1 did not show increased seed yield and no effects on root growth were reported.
Surprisingly, it has now been found that modulating expression of a nucleic acid encoding a RAA1-like polypeptide gives plants having enhanced yield-related traits, in particular increased yield relative to control plants.
According one embodiment, there is provided a method for improving yield related traits of a plant relative to control plants, comprising modulating expression of a nucleic acid encoding a RAA1-like polypeptide in a plant. The improved yield related traits comprised increased height, shoot/root index, root thickness, greenness index, number of flowers per panicle and increased thousand kernel weight. Improved yield related traits were observed under normal growth conditions as well as under stress conditions.
Seed Yield Regulator (SYR) is a new protein that hitherto has not been characterised. SYR shows some homology (around 48% sequence identity on DNA level, around 45% at 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. In contrast, overexpression of SYR in rice primarily increases seed yield, whereas the leafy biomass and flowering time are not obviously affected.
Surprisingly, it has now been found that modulating expression in a plant of a nucleic acid encoding a Seed Yield Regulator protein (hereafter named SYR) gives plants, when grown under abiotic stress conditions, having enhanced abiotic stress tolerance relative to control plants.
Therefore, the present invention provides a method for enhancing yield-related traits in plants grown under abiotic stress conditions, relative to control plants, comprising modulating expression in a plant of a nucleic acid encoding a SYR polypeptide.
ARKL polypeptides comprise a RING finger domain which resembles that found in the mouse protein ARKADIA, an E3 ubiquitin ligase involved in Nodal signaling during embryogenesis (Mavrakis et al. 2007; PLoS Biol. 2007 March; 5(3):e67).
Ubiquitilytion, a process by which a protein is modified the by covalent attachment of ubiquitin is a central and essential part of various cellular processes in eukaryotes. In plants, defects in this pathway cause numerous development aberrations, altered response to external stimuli and modify cell cycle and growth patterns. Ubiquitinated proteins are targeted for degradation via a 26S proteasome dependent or independent pathway. Ubiquitin modification plays a role in activation of signalling proteins, endocytosis, sorting, and histone modification.
The fate of the ubiquitinated protein is determined by the nature of the ubiquitin linkage. Single or multiple ubiquitins may be attached to the target (mono and poly ubiquitination; the specific Lys residue used to form the ubiquitin chain can influence the final fate of the modified protein, for example whether that is degradation or activation
The attachment of ubiquitin to proteins occurs in a multistep process involving three enzymes called, E1, E2, E3 (Glikcman and Ciechanover (2000) Physiol Rev 82: 377-482). Initially the ubiquin is linked to protein in an ATP dependent manner which is then transferred to a cystein acceptor in the E2 protein to form a E2-ubiquitin intermediate which acts as a ubiquitin donor to the target protein in a reaction mediated by the ubiquitin ligase, also called E3 ligase or E3 enzyme. There are multiple types of E3 ligases. The RING-type E3 ligases are characterized by the presence of a conserved protein domain called RING finger or RING-ZnF (Really Interesting New Gene-Zinc Finger).
Zinc-binding motifs are stable structures, and they rarely undergo conformational changes upon binding their target. Most ZnF proteins contain multiple finger-like protrusions that make tandem contacts with their target molecule, often recognising extended substrates. The RING finger is a specialized Zinc biding domain which peresumably functions in protein—protein interactions. The RING finger is 40 to 60 residues long and coordinates two zinc atoms. It is distinct from other zinc fingers in that the eight metal ligand amino acid residues that coordinate the zinc ion fall into a specific structure called the cross-brace structure (Borden (2000). J Mol Biol 295: 1103-1112). The spacing of the cysteines/histidines coordinating the Zinc ions in such a domain is C-x(2)-C-x(9 to 39)-C-x(1 to 3)-H-x(2 to 3)-C-x(2)-C-x(4 to 48)-C-x(2)-C. Metal ligand pairs one and three co-ordinate to bind one zinc ion, whilst pairs two and four bind the second. There are two different variants, the C3HC4-type and a C3H2C3-type, which is clearly related despite the different cysteine/histidine pattern. The latter type is sometimes referred to as ‘RING-H2 finger’. In the latter the coordination of the Zinc ion is mediated by 6 cysteins and 2 histidines whilst in the C3HC4 is mediated by 7 cysteins and one histidine.
In Arabidopsis thaliana there are at least 477 putative RING domain comprising proteins. Some contain multiple RING finger domains. The RING domains have been classified into eight types based on of the metal ligand residue present and/or the number of amino acids between them (Stone at al. 2005) Plant Phys. 137, 13-30. The RING-H2 class is the largest class in Arabidopsis. Based on the nature of the domains and their organisation the Arabidopsis RING finger proteins have been further classified in 30 groups, Group 1 to Group 30. Subgroups within some of the groups were also recognized, eg. subgroup 2.1 and 2.2 of group 2 (Stone et al. 2005). Group I was referred to as group of RING finger protein lacking previously described domains. Sequence analysis of those protein revealed regions of similarity between a few proteins outside of the RING domain, which were called DAR1 to DAR3 (Domain Associated with RING). DAR1 and DAR3 are approximately 40 amino acids long and DAR2 120. DAR1 was reported to occur only in proteins of plant origin (Stone et al. 2005). The presence of common conserved domains suggested a related function for the proteins comprising the domains.
Surprisingly, it has now been found that modulating expression of a nucleic acid encoding an ARKL polypeptide gives plants having enhanced yield-related traits in particular increased yield relative to control plants.
According one embodiment, there is provided a method for improving or enhancing yield related traits of a plant relative to control plants, comprising modulating expression of a nucleic acid encoding an ARKL polypeptide in a plant.
All eukaryotic cells contain elaborate systems of internal membranes which set up various membrane-enclosed compartments within the cell. The endomembrane system is collection of membranous structures involved in transport within the cell. The main components of the endomembrane system are the endoplasmic reticulum, Golgi bodies, vesicles, cell membrane and nuclear envelope. Members of the endomembrane system pass materials through each other or though the use of vesicles. A universal feature of all cells is an outer limiting membrane called the plasma membrane.
Cell membranes are built from lipids and proteins. The association of proteins to the membrane may be via a covalent bond, by which the protein is attached to the lipids of the membrane. In the case of the so called transmembrane proteins, polypeptide chains of the protein actually traverse the lipid bilayer. Association to the membrane may also occur via association of the protein, so called peripherial protein, by non-covalent bonds to the protruding portions of integral membrane proteins.
Transmembrane proteins (TM proteins) have an amphiphilic nature with hydrophobic TM segments (TMSs) and hydrophilic loops. In transmembrane proteins, the portion within the lipid bilayer consists primarily of hydrophobic amino acids. These are usually arranged in an alpha helix so that the polar carboxi (—C═O) and amino (—NH) groups at the peptide bonds can interact with each other rather than with their hydrophobic surroundings. Those portions of the polypeptide that project out from the bilayer tend to have a high percentage of hydrophilic amino acids. Furthermore, those that project into the extracellular space are usually glycosilated.
Transmembrane topology of a protein has been determined based on experimental X-ray crystallography, NMR, gene fusion technique, substituted cysteine accessibility method, Asp(N)-linked glycosylation experiment and other biochemical methods. In addition a number transmembrane topology prediction methods have been developed to determine the structure and function of TM proteins from their amino acid sequences (Möller et al., 2001; Ikeda et al., 2002; Chen et al., 2002).
The analysis of protein sequence similarity between proteins has benefited from developments in the genomics field. A number of domains conserved amongst two or more proteins for which no function has yet been assigned can be carried out using specific algorithms. One such conserved domain is the so called DUF221 domain (Domain of Unknown Function 221) as described in Pfam (Finn et al. Nucleic Acids Research (2006) Database Issue 34:D247-D251). This domain is found in a family of hypothetical transmembrane proteins, none of which have any known function, the aligned region is at 538 residues at maximum length. The domain occurs in a number of proteins of eukaryotic origin. Expression of an Arabidopsis gene, EDR4, encoding a protein comprising a DUF221 has been reported to be expressed shortly upon dehydration treatment (Kiyosue et al; Plant Mol Biol. 1994 25(5):791-8). An Arabidopsis knockout mutant, gfs10, in a gene encoding another DUF221 domain-containing protein has been reported to have a phenotype similar to that of vacuolar sorting mutants (Fuji et al; 2007. Plant Cell. 2007. 19(2):597-609).
Surprisingly, it has now been found that modulating expression of a nucleic acid encoding a YTP polypeptide gives plants having enhanced yield-related traits in particular increased yield relative to control plants.
According one embodiment, there is provided a method for enhancing (improving) yield related traits of a plant relative to control plants, comprising modulating expression of a nucleic acid encoding a YTP polypeptide in a plant.
DEFINITIONS
Polypeptide(s)/Protein(s)
The terms “polypeptide” and “protein” are used interchangeably herein and refer to amino acids in a polymeric form of any length, linked together by peptide bonds.
Polynucleotide(s)/Nucleic Acid(s)/Nucleic Acid Sequence(s)/Nucleotide Sequence(s)
The terms “polynucleotide(s)”, “nucleic acid sequence(s)”, “nucleotide sequence(s)”, “nucleic acid(s)”, “nucleic acid molecule” are used interchangeably herein and refer to nucleotides, either ribonucleotides or deoxyribonucleotides or a combination of both, in a polymeric unbranched form of any length.
Control Plant(s)
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. Nullizygotes are individuals missing the transgene by segregation. A “control plant” as used herein refers not only to whole plants, but also to plant parts, including seeds and seed parts.
Homoloque(s)
“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.
A deletion refers to removal of one or more amino acids from a protein.
An insertion refers to one or more amino acid residues being 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)-6-tag, glutathione S-transferase-tag, protein A, maltose-binding protein, dihydrofolate reductase, Tag•100 epitope, c-myc epitope, FLAG®-epitope, lacZ, CMP (calmodulin-binding peptide), HA epitope, protein C epitope and VSV epitope.
A substitution refers to replacement of amino acids of the protein with other amino acids having similar properties (such as similar hydrophobicity, hydrophilicity, antigenicity, propensity to form or break α-helical structures or β-sheet structures). 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. The amino acid substitutions are preferably conservative amino acid substitutions. Conservative substitution tables are well known in the art (see for example Creighton (1984) Proteins. W.H. Freeman and Company (Eds) and Table 1 below).







TABLE 1







Examples of conserved amino acid substitutions













Conservative

Conservative



Residue
Substitutions
Residue
Substitutions







Ala
Ser
Leu
Ile; Val



Arg
Lys
Lys
Arg; Gln



Asn
Gln; His
Met
Leu; Ile



Asp
Glu
Phe
Met; Leu; Tyr



Gln
Asn
Ser
Thr; Gly



Cys
Ser
Thr
Ser; Val



Glu
Asp
Trp
Tyr



Gly
Pro
Tyr
Trp; Phe



His
Asn; Gln
Val
Ile; Leu



Ile
Leu, Val










Amino acid substitutions, deletions and/or insertions 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 manipulation. 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” include peptides, oligopeptides, polypeptides which may, compared to the amino acid sequence of the naturally-occurring form of the protein, such as the protein of interest, comprise substitutions of amino acids with non-naturally occurring amino acid residues, or additions of non-naturally occurring amino acid residues. “Derivatives” of a protein also encompass peptides, oligopeptides, polypeptides which comprise naturally occurring altered (glycosylated, acylated, prenylated, phosphorylated, myristoylated, sulphated etc.) or non-naturally altered 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 or additions 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.
Furthermore, “derivatives” also include fusions of the naturally-occurring form of the protein with tagging peptides such as FLAG, HIS6 or thioredoxin (for a review of tagging peptides, see Terpe, Appl. Microbiol. Biotechnol. 60, 523-533, 2003).
Ortholoque(s)/Paraloque(s)
Orthologues and paralogues encompass evolutionary concepts used to describe the ancestral relationships of genes. Paralogues are genes within the same species that have originated through duplication of an ancestral gene; orthologues are genes from different organisms that have originated through speciation, and are also derived from a common ancestral gene.
Domain
The term “domain” refers to a set of amino acids conserved at specific positions along an alignment of sequences of evolutionarily related proteins. While amino acids at other positions can vary between homologues, amino acids that are highly conserved at specific positions indicate amino acids that are likely essential in the structure, stability or function of a protein. Identified by their high degree of conservation in aligned sequences of a family of protein homologues, they can be used as identifiers to determine if any polypeptide in question belongs to a previously identified polypeptide family.
Motif/Consensus Sequence/Signature
The term “motif” or “consensus sequence” or “signature” refers to a short conserved region in the sequence of evolutionarily related proteins. Motifs are frequently highly conserved parts of domains, but may also include only part of the domain, or be located outside of conserved domain (if all of the amino acids of the motif fall outside of a defined domain).
Hybridisation
The term “hybridisation” as defined herein is a process wherein substantially homologous complementary nucleotide sequences anneal to each other. The hybridisation process can occur entirely in solution, i.e. both complementary nucleic acid molecules are in solution. The hybridisation process can also occur with one of the complementary nucleic acid molecules immobilised to a matrix such as magnetic beads, Sepharose beads or any other resin. The hybridisation process can furthermore occur with one of the complementary nucleic acid molecules 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 sequence arrays or microarrays or as nucleic acid sequence 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 acid molecules.
The term “stringency” refers to the conditions under which a hybridisation takes place. The stringency of hybridisation is influenced by conditions such as temperature, salt concentration, ionic strength and hybridisation buffer composition. Generally, low stringency conditions are selected to be about 30° 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. High stringency hybridisation conditions are typically used for isolating hybridising sequences that have high sequence similarity to the target nucleic acid sequence. However, nucleic acid sequences may deviate in sequence and still encode a substantially identical polypeptide, due to the degeneracy of the genetic code. Therefore medium stringency hybridisation conditions may sometimes be needed to identify such nucleic acid sequence molecules.
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 sequence strands thereby promoting hybrid formation; this effect is visible for sodium concentrations of up to 0.4M (for higher concentrations, this effect may be ignored). 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:
1) DNA-DNA hybrids (Meinkoth and Wahl, Anal. Biochem., 138: 267-284, 1984):

Tm=81.5° C.+16.6×log10[Na+]a+0.41×%[G/Cb]−500×[Lc]−1−0.61×% formamide

2) 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 

3) oligo-DNA or oligo-RNAd hybrids:

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


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. For non-homologous probes, a series of hybridizations may be performed by varying one of (i) progressively lowering the annealing temperature (for example from 68° C. to 42° C.) or (ii) progressively lowering the formamide concentration (for example from 50% to 0%). The skilled artisan is aware of various parameters which may be altered during hybridisation and which will either maintain or change the stringency conditions.
Besides the hybridisation conditions, specificity of hybridisation typically also depends on 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. A positive hybridisation gives a signal that is at least twice of that of the background. Generally, suitable stringent conditions for nucleic acid sequence hybridisation assays or gene amplification detection procedures are as set forth above. More or less stringent conditions may also be selected. The skilled artisan is aware of various parameters which may be altered during washing and which will either maintain or change the stringency conditions.
For example, typical high stringency hybridisation conditions for DNA hybrids longer than 50 nucleotides encompass hybridisation at 65° C. in 1×SSC or at 42° C. in 1×SSC and 50% formamide, followed by washing at 65° C. in 0.3×SSC. Examples of medium stringency hybridisation conditions for DNA hybrids longer than 50 nucleotides encompass hybridisation at 50° C. in 4×SSC or at 40° C. in 6×SSC and 50% formamide, followed by washing at 50° C. in 2×SSC. The length of the hybrid is the anticipated length for the hybridising nucleic acid. When nucleic acid molecules of known sequence are hybridised, the hybrid length may be determined by aligning the sequences and identifying the conserved regions described herein. 1×SSC is 0.15M NaCl and 15 mM sodium citrate; the hybridisation solution and wash solutions may additionally include 5×Denhardt's reagent, 0.5-1.0% SDS, 100 μg/ml denatured, fragmented salmon sperm DNA, 0.5% sodium pyrophosphate.
For the purposes of defining the level of stringency, reference can be made to Sambrook et al. (2001) Molecular Cloning: a laboratory manual, 3rd Edition, Cold Spring Harbor Laboratory Press, CSH, New York or to Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989 and yearly updates).
Splice Variant
The term “splice variant” as used herein encompasses variants of a nucleic acid sequence in which selected introns and/or exons have been excised, replaced, displaced 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 substantially retained; this 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 predicting and isolating such splice variants are well known in the art (see for example Foissac and Schiex (2005) BMC Bioinformatics 6: 25).
Allelic Variant
Alleles or allelic variants are alternative forms of a given gene, located at the same chromosomal position. 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.
Gene Shuffling/Directed Evolution
Gene shuffling or directed evolution consists of iterations of DNA shuffling followed by appropriate screening and/or selection to generate variants of nucleic acid sequences or portions thereof encoding proteins having a modified biological activity (Castle et al., (2004) Science 304(5674): 1151-4; U.S. Pat. Nos. 5,811,238 and 6,395,547).
Regulatory Element/Control Sequence/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. The term “promoter” typically refers to a nucleic acid sequence control sequence located upstream from the transcriptional start of a gene and which is involved in recognising and binding of RNA polymerase and other proteins, thereby directing transcription of an operably linked nucleic acid. 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, increasers 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 increases expression of a nucleic acid sequence molecule in a cell, tissue or organ.
A “plant promoter” comprises regulatory elements, which mediate the expression of a coding sequence segment in plant cells. Accordingly, a plant promoter need not be of plant origin, but may originate from viruses or micro-organisms, for example from viruses which attack plant cells. The “plant promoter” preferably originates from a plant cell, e.g. from the plant which is transformed with the nucleic acid sequence to be expressed in the inventive process and described herein. This also applies to other “plant” regulatory signals, such as “plant” terminators. The promoters upstream of the nucleotide sequences useful in the methods of the present invention can be modified by one or more nucleotide substitution(s), insertion(s) and/or deletion(s) without interfering with the functionality or activity of either the promoters, the open reading frame (ORF) or the 3′-regulatory region such as terminators or other 3′ regulatory regions which are located away from the ORF. It is furthermore possible that the activity of the promoters is increased by modification of their sequence, or that they are replaced completely by more active promoters, even promoters from heterologous organisms. For expression in plants, the nucleic acid sequence molecule must, as described above, be linked operably to or comprise a suitable promoter which expresses the gene at the right point in time and with the required spatial expression pattern.
For the identification of functionally equivalent promoters, the promoter strength and/or expression pattern of a candidate promoter may be analysed for example by operably linking the promoter to a reporter gene and assaying the expression level and pattern of the reporter gene in various tissues of the plant. Suitable well-known reporter genes include for example beta-glucuronidase or beta-galactosidase. The promoter activity is assayed by measuring the enzymatic activity of the beta-glucuronidase or beta-galactosidase. The promoter strength and/or expression pattern may then be compared to that of a reference promoter (such as the one used in the methods of the present invention). Alternatively, promoter strength may be assayed by quantifying mRNA levels or by comparing mRNA levels of the nucleic acid sequence used in the methods of the present invention, with mRNA levels of housekeeping genes such as 18S rRNA, using methods known in the art, such as Northern blotting with densitometric analysis of autoradiograms, quantitative real-time PCR or RT-PCR (Heid et al., 1996 Genome Methods 6: 986-994). Generally by “weak promoter” is intended a promoter that drives expression of a coding sequence at a low level. By “low level” is intended at levels of about 1/10,000 transcripts to about 1/100,000 transcripts, to about 1/500,0000 transcripts per cell. Conversely, a “strong promoter” drives expression of a coding sequence at high level, or at about 1/10 transcripts to about 1/100 transcripts to about 1/1000 transcripts per cell. Generally, by “medium strength promoter” is intended a promoter that drives expression of a coding sequence at a level that is in all instances below that obtained under the control of a 35S CaMV promoter.
Operably Linked
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.
Constitutive Promoter
A “constitutive promoter” refers to a promoter that is transcriptionally active during most, but not necessarily all, phases of growth and development and under most environmental conditions, in at least one cell, tissue or organ. Table 2a below gives examples of constitutive promoters.







TABLE 2a







Examples of plant constitutive promoters








Gene Source
Reference





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


HMGB
WO 2004/070039


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


histone


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


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


subunit


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


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


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


V-ATPase
WO 01/14572


G-box proteins
WO 94/12015


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


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


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


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



7831-7846









Ubiquitous Promoter

A ubiquitous promoter is active in substantially all tissues or cells of an organism.
Developmentally-Regulated Promoter
A developmentally-regulated promoter is active during certain developmental stages or in parts of the plant that undergo developmental changes.
Inducible Promoter
An inducible promoter has induced or increased transcription initiation in response to a chemical (for a review see Gatz 1997, Annu. Rev. Plant Physiol. Plant Mol. Biol., 48:89-108), environmental or physical stimulus, or may be “stress-inducible”, i.e. activated when a plant is exposed to various stress conditions, or a “pathogen-inducible” i.e. activated when a plant is exposed to exposure to various pathogens.
Organ-Specific/Tissue-Specific Promoter
An organ-specific or tissue-specific promoter is one that is capable of preferentially initiating transcription in certain organs or tissues, such as the leaves, roots, seed tissue etc. For example, a “root-specific promoter” is a promoter that is transcriptionally active predominantly in plant roots, substantially to the exclusion of any other parts of a plant, whilst still allowing for any leaky expression in these other plant parts. Promoters able to initiate transcription in certain cells only are referred to herein as “cell-specific”.
Examples of root-specific promoters are listed in Table 2b below:







TABLE 2b







Examples of root-specific promoters








Gene Source
Reference





Rice RCc3
Xu et al (1995) Plant Mol Biol 27(2): 237-48


Arabidopsis phosphate transporter PHT1
Kovama et al., 2005


Medicago phosphate transporter
Xiao et al., 2006


Arabidopsis Pyk10
Nitz et al. (2001) Plant Sci 161(2): 337-346


Tobacco root-specific genes RB7, RD2, RD5,
Conkling et al. (1990) Plant Phys 93(3):


RH12
1203-1211


Barley root-specific lectin
Lerner & Raikhel (1989) Plant Phys 91: 124-129


Root-specific hydroxy-proline rich protein
Keller & Lamb (1989) Genes & Dev 3: 1639-1646


Arabidopsis CDC27B/hobbit
Blilou et al. (2002) Genes & Dev 16: 2566-2575









A seed-specific promoter is transcriptionally active predominantly in seed tissue, but not necessarily exclusively in seed tissue (in cases of leaky expression). The seed-specific promoter may be active during seed development and/or during germination. Examples of seed-specific promoters are shown in Table 2c below. Further examples of seed-specific promoters are given in Qing Qu and Takaiwa (Plant Biotechnol. J. 2, 113-125, 2004), which disclosure is incorporated by reference herein as if fully set forth.







TABLE 2c







Examples of seed-specific promoters








Gene source
Reference





seed-specific genes
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
Pearson et al., Plant Mol. Biol. 18: 235-245, 1992.


Legumin
Ellis et al., Plant Mol. Biol. 10: 203-214, 1988.


glutelin (rice)
Takaiwa et al., Mol. Gen. Genet. 208: 15-22, 1986;



Takaiwa et al., FEBS Letts. 221: 43-47, 1987.


Zein
Matzke et al Plant Mol Biol, 14(3): 323-32 1990


NapA
Stalberg et al, Planta 199: 515-519, 1996.


Wheat LMW and HMW glutenin-1
Mol Gen Genet 216: 81-90, 1989; NAR 17: 461-2, 1989


Wheat SPA
Albani et al, Plant Cell, 9: 171-184, 1997


Wheat α, β, γ-gliadins
EMBO J. 3: 1409-15, 1984


Barley Itr1 promoter
Diaz et al. (1995) Mol Gen Genet 248(5): 592-8


Barley B1, C, D, hordein
Theor Appl Gen 98: 1253-62, 1999; Plant J 4: 343-55,



1993; Mol Gen Genet 250: 750-60, 1996


Barley DOF
Mena et al, The Plant Journal, 116(1): 53-62, 1998


blz2
EP99106056.7


Synthetic promoter
Vicente-Carbajosa et al., Plant J. 13: 629-640, 1998.


rice prolamin NRP33
Wu et al, Plant Cell Physiology 39(8) 885-889, 1998


rice a-globulin Glb-1
Wu et al, Plant Cell Physiology 39(8) 885-889, 1998


rice OSH1
Sato et al, Proc. Natl. Acad. Sci. USA, 93: 8117-8122,



1996


rice α-globulin REB/OHP-1
Nakase et al. Plant Mol. Biol. 33: 513-522, 1997


rice ADP-glucose pyrophosphorylase
Trans Res 6: 157-68, 1997


Maize ESR gene family
Plant J 12: 235-46, 1997


Sorghum α-kafirin
DeRose et al., Plant Mol. Biol 32: 1029-35, 1996


KNOX
Postma-Haarsma et al, Plant Mol. Biol. 39: 257-71, 1999


rice oleosin
Wu et al, J. Biochem. 123: 386, 1998


sunflower oleosin
Cummins et al., Plant Mol. Biol. 19: 873-876, 1992


PRO0117, putative rice 40S
WO 2004/070039


ribosomal protein


PRO0136, rice alanine
Unpublished


aminotransferase


PRO0147, trypsin inhibitor ITR1
Unpublished


(barley)


PRO0151, rice WSI18
WO 2004/070039


PRO0175, rice RAB21
WO 2004/070039


PRO005
WO 2004/070039


PRO0095
WO 2004/070039


α-amylase (Amy32b)
Lanahan et al, Plant Cell 4: 203-211, 1992; Skriver et al,



Proc Natl Acad Sci USA 88: 7266-7270, 1991


Cathepsin β-like gene
Cejudo et al, Plant Mol Biol 20: 849-856, 1992


Barley Ltp2
Kalla et al., Plant J. 6: 849-60, 1994


Chi26
Leah et al., Plant J. 4: 579-89, 1994


Maize B-Peru
Selinger et al., Genetics 149; 1125-38, 1998









A green tissue-specific promoter as defined herein is a promoter that is transcriptionally active predominantly in green tissue, substantially to the exclusion of any other parts of a plant, whilst still allowing for any leaky expression in these other plant parts.
Examples of green tissue-specific promoters which may be used to perform the methods of the invention are shown in Table 2d below.







TABLE 2d







Examples of green tissue-specific promoters









Gene
Expression
Reference





Maize Orthophosphate dikinase
Leaf specific
Fukavama et al., 2001


Maize Phosphoenolpyruvate
Leaf specific
Kausch et al., 2001


carboxylase


Rice Phosphoenolpyruvate
Leaf specific
Liu et al., 2003


carboxylase


Rice small subunit Rubisco
Leaf specific
Nomura et al., 2000


rice beta expansin EXBP9
Shoot
WO 2004/070039



specific


Pigeonpea small subunit Rubisco
Leaf specific
Panguluri et al., 2005


Pea RBCS3A
Leaf specific









Another example of a tissue-specific promoter is a meristem-specific promoter, which is transcriptionally active predominantly in meristematic tissue, substantially to the exclusion of any other parts of a plant, whilst still allowing for any leaky expression in these other plant parts. Examples of meristem-specific promoters which may be used to perform the methods of the invention are shown in Table 2e below.







TABLE 2e







Examples of meristem-specific promoters









Gene source
Expression pattern
Reference





rice OSH1
Shoot apical meristem,
Sato et al. (1996)



from embryo globular
Proc. Natl. Acad.



stage to seedling
Sci. USA, 93: 8117-8122



stage


Rice metallothionein
Meristem specific
BAD87835.1


WAK1 & WAK 2
Shoot and root apical
Wagner & Kohorn



meristems, and in
(2001) Plant Cell



expanding leaves
13(2): 303-318



and sepals









Terminator

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. The terminator can be derived from the natural gene, from a variety of other plant genes, or from T-DNA. The terminator 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.
Modulation
The term “modulation” means in relation to expression or gene expression, a process in which the expression level is changed by said gene expression in comparison to the control plant, the expression level may be increased or decreased. The original, unmodulated expression may be of any kind of expression of a structural RNA (rRNA, tRNA) or mRNA with subsequent translation. The term “modulating the activity” shall mean any change of the expression of the inventive nucleic acid sequences or encoded proteins, which leads to increased yield and/or increased growth of the plants.
Expression
The term “expression” or “gene expression” means the transcription of a specific gene or specific genes or specific genetic construct. The term “expression” or “gene expression” in particular means the transcription of a gene or genes or genetic construct into structural RNA (rRNA, tRNA) or mRNA with or without subsequent translation of the latter into a protein. The process includes transcription of DNA and processing of the resulting mRNA product.
Increased Expression/Overexpression
The term “increased expression” or “overexpression” as used herein means any form of expression that is additional to the original wild-type expression level.
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 the polypeptide of interest. 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., WO9322443), 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 (UTR) 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 enhacement 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).
Endogenous Gene
Reference herein to an “endogenous” gene not only refers to the gene in question as found in a plant in its natural form (i.e., without there being any human intervention), but also refers to that same gene (or a substantially homologous nucleic acid/gene) in an isolated form subsequently (re)introduced into a plant (a transgene). For example, a transgenic plant containing such a transgene may encounter a substantial reduction of the transgene expression and/or substantial reduction of expression of the endogenous gene. The isolated gene may be isolated from an organism or may be manmade, for example by chemical synthesis.
Decreased Expression
Reference herein to “decreased epression” or “reduction or substantial elimination” of expression is taken to mean a decrease in endogenous gene expression and/or polypeptide levels and/or polypeptide activity relative to control plants. The reduction or substantial elimination is in increasing order of preference at least 10%, 20%, 30%, 40% or 50%, 60%, 70%, 80%, 85%, 90%, or 95%, 96%, 97%, 98%, 99% or more reduced compared to that of control plants.
For the reduction or substantial elimination of expression an endogenous gene in a plant, a sufficient length of substantially contiguous nucleotides of a nucleic acid sequence is required. In order to perform gene silencing, this may be as little as 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10 or fewer nucleotides, alternatively this may be as much as the entire gene (including the 5′ and/or 3′ UTR, either in part or in whole). The stretch of substantially contiguous nucleotides may be derived from the nucleic acid sequence encoding the protein of interest (target gene), or from any nucleic acid sequence capable of encoding an orthologue, paralogue or homologue of the protein of interest. Preferably, the stretch of substantially contiguous nucleotides is capable of forming hydrogen bonds with the target gene (either sense or antisense strand), more preferably, the stretch of substantially contiguous nucleotides has, in increasing order of preference, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity to the target gene (either sense or antisense strand). A nucleic acid sequence encoding a (functional) polypeptide is not a requirement for the various methods discussed herein for the reduction or substantial elimination of expression of an endogenous gene.
This reduction or substantial elimination of expression may be achieved using routine tools and techniques. A method for the reduction or substantial elimination of endogenous gene expression is by RNA-mediated silencing using an inverted repeat of a nucleic acid sequence or a part thereof (in this case a stretch of substantially contiguous nucleotides derived from the gene of interest, or from any nucleic acid sequence capable of encoding an orthologue, paralogue or homologue of the protein of interest), preferably capable of forming a hairpin structure. Another example of an RNA silencing method involves the introduction of nucleic acid sequences or parts thereof (in this case a stretch of substantially contiguous nucleotides derived from the gene of interest, or from any nucleic acid sequence capable of encoding an orthologue, paralogue or homologue of the protein of interest) in a sense orientation into a plant. Another example of an RNA silencing method involves the use of antisense nucleic acid sequences. Gene silencing may also be achieved by insertion mutagenesis (for example, T-DNA insertion or transposon insertion) or by strategies as described by, among others, Angell and Baulcombe ((1999) Plant J 20(3): 357-62), (Amplicon VIGS WO 98/36083), or Baulcombe (WO 99/15682). Other methods, such as the use of antibodies directed to an endogenous polypeptide for inhibiting its function in planta, or interference in the signalling pathway in which a polypeptide is involved, will be well known to the skilled man. Artificial and/or natural microRNAs (miRNAs) may be used to knock out gene expression and/or mRNA translation. Endogenous miRNAs are single stranded small RNAs of typically 19-24 nucleotides long.
This reduction or substantial elimination of expression may be achieved using routine tools and techniques. A preferred method for the reduction or substantial elimination of endogenous gene expression is by introducing and expressing in a plant a genetic construct into which the nucleic acid (in this case a stretch of substantially contiguous nucleotides derived from the gene of interest, or from any nucleic acid capable of encoding an orthologue, paralogue or homologue of any one of the protein of interest) is cloned as an inverted repeat (in part or completely), separated by a spacer (non-coding DNA).
In such a preferred method, expression of the endogenous gene is reduced or substantially eliminated through RNA-mediated silencing using an inverted repeat of a nucleic acid or a part thereof (in this case a stretch of substantially contiguous nucleotides derived from the gene of interest, or from any nucleic acid capable of encoding an orthologue, paralogue or homologue of the protein of interest), preferably capable of forming a hairpin structure. The inverted repeat is cloned in an expression vector comprising control sequences. A non-coding DNA nucleic acid sequence (a spacer, for example a matrix attachment region fragment (MAR), an intron, a polylinker, etc.) is located between the two inverted nucleic acids forming the inverted repeat. After transcription of the inverted repeat, a chimeric 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 an RNA-induced silencing complex (RISC). The RISC further cleaves the mRNA transcripts, thereby substantially reducing the number of mRNA transcripts to be translated into polypeptides. For further general details see for example, Grierson et al. (1998) WO 98/53083; Waterhouse et al. (1999) WO 99/53050).
Performance of the methods of the invention does not rely on introducing and expressing in a plant a genetic construct into which the nucleic acid is cloned as an inverted repeat, but any one or more of several well-known “gene silencing” methods may be used to achieve the same effects.
One such method for the reduction of endogenous gene expression is RNA-mediated silencing of gene expression (downregulation). Silencing in this case is triggered in a plant by a double stranded RNA sequence (dsRNA) that is substantially similar to the target endogenous gene. This dsRNA is further processed by the plant into about 20 to about 26 nucleotides called short interfering RNAs (siRNAs). The siRNAs are incorporated into an RNA-induced silencing complex (RISC) that cleaves the mRNA transcript of the endogenous target gene, thereby substantially reducing the number of mRNA transcripts to be translated into a polypeptide. Preferably, the double stranded RNA sequence corresponds to a target gene.
Another example of an RNA silencing method involves the introduction of nucleic acid sequences or parts thereof (in this case a stretch of substantially contiguous nucleotides derived from the gene of interest, or from any nucleic acid capable of encoding an orthologue, paralogue or homologue of the protein of interest) in a sense orientation into a plant. “Sense orientation” refers to a DNA sequence that is homologous to an mRNA transcript thereof. Introduced into a plant would therefore be at least one copy of the nucleic acid sequence. The additional nucleic acid sequence will reduce expression of the endogenous gene, giving rise to a phenomenon known as co-suppression. The reduction of gene expression will be more pronounced if several additional copies of a nucleic acid sequence 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 nucleic acid sequences. An “antisense” nucleic acid sequence comprises a nucleotide sequence that is complementary to a “sense” nucleic acid sequence encoding a protein, i.e. complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA transcript sequence. The antisense nucleic acid sequence is preferably complementary to the endogenous gene to be silenced. The complementarity may be located in the “coding region” and/or in the “non-coding region” of a gene. The term “coding region” refers to a 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 transcribed but not translated into amino acids (also referred to as 5′ and 3′ untranslated regions).
Antisense nucleic acid sequences can be designed according to the rules of Watson and Crick base pairing. The antisense nucleic acid sequence may be complementary to the entire nucleic acid sequence (in this case a stretch of substantially contiguous nucleotides derived from the gene of interest, or from any nucleic acid capable of encoding an orthologue, paralogue or homologue of the protein of interest), but may also be an oligonucleotide that is antisense to only a part of the nucleic acid sequence (including the mRNA 5′ and 3′ UTR). For example, the antisense oligonucleotide sequence may be complementary to the region surrounding the translation start site of an mRNA transcript encoding a polypeptide. The length of a suitable antisense oligonucleotide sequence is known in the art and may start from about 50, 45, 40, 35, 30, 25, 20, 15 or 10 nucleotides in length or less. An antisense nucleic acid sequence according to the invention may be constructed using chemical synthesis and enzymatic ligation reactions using methods known in the art. For example, an antisense nucleic acid sequence (e.g., an antisense oligonucleotide sequence) may 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 acid sequences, e.g., phosphorothioate derivatives and acridine substituted nucleotides may be used. Examples of modified nucleotides that may be used to generate the antisense nucleic acid sequences are well known in the art. Known nucleotide modifications include methylation, cyclization and ‘caps’ and substitution of one or more of the naturally occurring nucleotides with an analogue such as inosine. Other modifications of nucleotides are well known in the art.
The antisense nucleic acid sequence can be produced biologically using an expression vector into which a nucleic acid sequence 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 acid of interest). Preferably, production of antisense nucleic acid sequences in plants occurs by means of a stably integrated nucleic acid construct comprising a promoter, an operably linked antisense oligonucleotide, and a terminator.
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 mRNA transcripts and/or genomic DNA encoding a polypeptide 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 sequence which binds to DNA duplexes, through specific interactions in the major groove of the double helix. Antisense nucleic acid sequences may be introduced into a plant by transformation or direct injection at a specific tissue site. Alternatively, antisense nucleic acid sequences can be modified to target selected cells and then administered systemically. For example, for systemic administration, antisense nucleic acid sequences 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 sequence to peptides or antibodies which bind to cell surface receptors or antigens. The antisense nucleic acid sequences can also be delivered to cells using the vectors described herein.
According to a further aspect, the antisense nucleic acid sequence is an a-anomeric nucleic acid sequence. An a-anomeric nucleic acid sequence forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual b-units, the strands run parallel to each other (Gaultier et al. (1987) Nucl Ac Res 15: 6625-6641). The antisense nucleic acid sequence may also comprise a 2′-o-methylribonucleotide (Inoue et al. (1987) Nucl Ac Res 15, 6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987) FEBS Lett. 215, 327-330).
The reduction or substantial elimination of endogenous gene expression may also be performed using ribozymes. Ribozymes are catalytic RNA molecules with ribonuclease activity that are capable of cleaving a single-stranded nucleic acid sequence, 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 mRNA transcripts encoding a polypeptide, thereby substantially reducing the number of mRNA transcripts to be translated into a polypeptide. A ribozyme having specificity for a nucleic acid sequence can be designed (see for example: Cech et al. U.S. Pat. No. 4,987,071; and Cech et al. U.S. Pat. No. 5,116,742). Alternatively, mRNA transcripts corresponding to a nucleic acid sequence can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules (Bartel and Szostak (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 al. (1995) WO 95/03404; Lutziger et al. (2000) WO 00/00619; Prinsen et al. (1997) WO 97/13865 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 strategies as described by, among others, Angell and Baulcombe ((1999) Plant J 20(3): 357-62), (Amplicon VIGS WO 98/36083), or Baulcombe (WO 99/15682).
Gene silencing may also occur if there is a mutation on an endogenous gene and/or a mutation on an isolated gene/nucleic acid subsequently introduced into a plant. The reduction or substantial elimination may be caused by a non-functional polypeptide. For example, the polypeptide may bind to various interacting proteins; one or more mutation(s) and/or truncation(s) may therefore provide for a polypeptide that is still able to bind interacting proteins (such as receptor proteins) but that cannot exhibit its normal function (such as signalling ligand).
A further approach to gene silencing is by targeting nucleic acid sequences complementary to the regulatory region of the gene (e.g., the promoter and/or enhancers) to form triple helical structures that prevent transcription of the gene in target cells. See Helene, C., Anticancer Drug Res. 6, 569-84, 1991; Helene et al., Ann. N.Y. Acad. Sci. 660, 27-36 1992; and Maher, L. J. Bioassays 14, 807-15, 1992.
Other methods, such as the use of antibodies directed to an endogenous polypeptide for inhibiting its function in planta, or interference in the signalling pathway in which a polypeptide is involved, will be well known to the skilled man. In particular, it can be envisaged that manmade molecules may be useful for inhibiting the biological function of a target polypeptide, or for interfering with the signalling pathway in which the target polypeptide is involved.
Alternatively, a screening program may be set up to identify in a plant population natural variants of a gene, which variants encode polypeptides with reduced activity. Such natural variants may also be used for example, to perform homologous recombination.
Artificial and/or natural microRNAs (miRNAs) may be used to knock out gene expression and/or mRNA translation. Endogenous miRNAs are single stranded small RNAs of typically 19-24 nucleotides long. They function primarily to regulate gene expression and/or mRNA translation. Most plant microRNAs (miRNAs) have perfect or near-perfect complementarity with their target sequences. However, there are natural targets with up to five mismatches. They are processed from longer non-coding RNAs with characteristic fold-back structures by double-strand specific RNases of the Dicer family. Upon processing, they are incorporated in the RNA-induced silencing complex (RISC) by binding to its main component, an Argonaute protein. MiRNAs serve as the specificity components of RISC, since they base-pair to target nucleic acids, mostly mRNAs, in the cytoplasm. Subsequent regulatory events include target mRNA cleavage and destruction and/or translational inhibition. Effects of miRNA overexpression are thus often reflected in decreased mRNA levels of target genes.
Artificial microRNAs (amiRNAs), which are typically 21 nucleotides in length, can be genetically engineered specifically to negatively regulate gene expression of single or multiple genes of interest. Determinants of plant microRNA target selection are well known in the art. Empirical parameters for target recognition have been defined and can be used to aid in the design of specific amiRNAs (Schwab et al., (2005) Dev Cell 8(4):517-27). Convenient tools for design and generation of amiRNAs and their precursors are also available to the public (Schwab et al., (2006) Plant Cell 18(5):1121-33).
For optimal performance, the gene silencing techniques used for reducing expression in a plant of an endogenous gene requires the use of nucleic acid sequences from monocotyledonous plants for transformation of monocotyledonous plants, and from dicotyledonous plants for transformation of dicotyledonous plants. Preferably, a nucleic acid sequence from any given plant species is introduced into that same species. For example, a nucleic acid sequence from rice is transformed into a rice plant. However, it is not an absolute requirement that the nucleic acid sequence to be introduced originates from the same plant species as the plant in which it will be introduced. It is sufficient that there is substantial homology between the endogenous target gene and the nucleic acid sequence to be introduced.
Described above are examples of various methods for the reduction or substantial elimination of expression in a plant of an endogenous gene. A person skilled in the art would readily be able to adapt the aforementioned methods for silencing so as to achieve reduction of expression of an endogenous gene in a whole plant or in parts thereof through the use of an appropriate promoter, for example.
Selectable Marker (Gene)/Reporter Gene
“Selectable marker”, “selectable marker gene” or “reporter gene” includes any gene that confers a phenotype on a cell in which it is expressed to facilitate the identification and/or selection of cells that are transfected or transformed with a nucleic acid sequence construct of the invention. These marker genes enable the identification of a successful transfer of the nucleic acid sequence molecules via a series of different principles. 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 nptII that phosphorylates neomycin and kanamycin, or hpt, phosphorylating hygromycin, or genes conferring resistance to, for example, bleomycin, streptomycin, tetracyclin, chloramphenicol, ampicillin, gentamycin, geneticin (G418), spectinomycin or blasticidin), to herbicides (for example bar which provides resistance to Basta®; aroA or gox providing resistance against glyphosate, or the genes conferring resistance to, for example, imidazolinone, phosphinothricin or sulfonylurea), or genes that provide a metabolic trait (such as manA that allows plants to use mannose as sole carbon source or xylose isomerase for the utilisation of xylose, or antinutritive markers such as the resistance to 2-deoxyglucose). Expression of visual marker genes results in the formation of colour (for example β-glucuronidase, GUS or β-galactosidase with its coloured substrates, for example X-Gal), luminescence (such as the luciferin/luceferase system) or fluorescence (Green Fluorescent Protein, GFP, and derivatives thereof). This list represents only a small number of possible markers. The skilled worker is familiar with such markers. Different markers are preferred, depending on the organism and the selection method.
It is known that upon stable or transient integration of nucleic acid sequences into plant cells, only a minority of the cells takes up the foreign DNA and, if desired, integrates it into its genome, depending on the expression vector used and the transfection technique used. To identify and select these integrants, a gene coding for a selectable marker (such as the ones described above) is usually introduced into the host cells together with the gene of interest. These markers can for example be used in mutants in which these genes are not functional by, for example, deletion by conventional methods. Furthermore, nucleic acid sequence molecules encoding a selectable marker can be introduced into a host cell on the same vector that comprises the sequence encoding the polypeptides of the invention or used in the methods of the invention, or else in a separate vector. Cells which have been stably transfected with the introduced nucleic acid sequence can be identified for example by selection (for example, cells which have integrated the selectable marker survive whereas the other cells die).
Since the marker genes, particularly genes for resistance to antibiotics and herbicides, are no longer required or are undesired in the transgenic host cell once the nucleic acid sequences have been introduced successfully, the process according to the invention for introducing the nucleic acid sequences advantageously employs techniques which enable the removal or excision of these marker genes. One such a method is what is known as co-transformation. The co-transformation method employs two vectors simultaneously for the transformation, one vector bearing the nucleic acid sequence according to the invention and a second bearing the marker gene(s). A large proportion of transformants receives or, in the case of plants, comprises (up to 40% or more of the transformants), both vectors. In case of transformation with Agrobacteria, the transformants usually receive only a part of the vector, i.e. the sequence flanked by the T-DNA, which usually represents the expression cassette. The marker genes can subsequently be removed from the transformed plant by performing crosses. In another method, marker genes integrated into a transposon are used for the transformation together with desired nucleic acid sequence (known as the Ac/Ds technology). The transformants can be crossed with a transposase source or the transformants are transformed with a nucleic acid sequence construct conferring expression of a transposase, transiently or stable. In some cases (approx. 10%), the transposon jumps out of the genome of the host cell once transformation has taken place successfully and is lost. In a further number of cases, the transposon jumps to a different location. In these cases the marker gene must be eliminated by performing crosses. In microbiology, techniques were developed which make possible, or facilitate, the detection of such events. A further advantageous method relies on what is known as recombination systems; whose advantage is that elimination by crossing can be dispensed with. The best-known system of this type is what is known as the Cre/lox system. Cre1 is a recombinase that removes the sequences located between the loxP sequences. If the marker gene is integrated between the loxP sequences, it is removed once transformation has taken place successfully, by expression of the recombinase. Further recombination systems are the HIN/HIX, FLP/FRT and REP/STB system (Tribble et al., J. Biol. Chem., 275, 2000: 22255-22267; Velmurugan et al., J. Cell Biol., 149, 2000: 553-566). A site-specific integration into the plant genome of the nucleic acid sequences according to the invention is possible. Naturally, these methods can also be applied to microorganisms such as yeast, fungi or bacteria.
Transgenic/Transgene/Recombinant
For the purposes of the invention, “transgenic”, “transgene” or “recombinant” means with regard to, for example, a nucleic acid sequence, an expression cassette, gene construct or a vector comprising the nucleic acid sequence or an organism transformed with the nucleic acid sequences, expression cassettes or vectors according to the invention, all those constructions brought about by recombinant methods in which either

    
    
        (a) the nucleic acid sequences encoding proteins useful in the methods of the invention, or
        (b) genetic control sequence(s) which is operably linked with the nucleic acid sequence according to the invention, for example a promoter, or
        (c) a) and b)

are not located in their natural genetic environment or have been modified by recombinant methods, it being possible for the modification to take the form of, for example, a substitution, addition, deletion, inversion or insertion of one or more nucleotide residues. The natural genetic environment is understood as meaning the natural genomic or chromosomal locus in the original plant or the presence in a genomic library. In the case of a genomic library, the natural genetic environment of the nucleic acid sequence is preferably retained, at least in part. The environment flanks the nucleic acid sequence at least on one side and has a sequence length of at least 50 bp, preferably at least 500 bp, especially preferably at least 1000 bp, most preferably at least 5000 bp. A naturally occurring expression cassette—for example the naturally occurring combination of the natural promoter of the nucleic acid sequences with the corresponding nucleic acid sequence encoding a polypeptide useful in the methods of the present invention, as defined above—becomes a transgenic expression cassette when this expression cassette is modified by non-natural, synthetic (“artificial”) methods such as, for example, mutagenic treatment. Suitable methods are described, for example, in U.S. Pat. No. 5,565,350 or WO 00/15815.

    
    


A transgenic plant for the purposes of the invention is thus understood as meaning, as above, that the nucleic acid sequences used in the method of the invention are not at their natural locus in the genome of said plant, it being possible for the nucleic acid sequences to be expressed homologously or heterologously. However, as mentioned, transgenic also means that, while the nucleic acid sequence according to the invention or used in the inventive method are at their natural position in the genome of a plant, the sequence has been modified with regard to the natural sequence, and/or that the regulatory sequences of the natural sequences have been modified. Transgenic is preferably understood as meaning the expression of the nucleic acid sequences according to the invention at an unnatural locus in the genome, i.e. homologous or, preferably, heterologous expression of the nucleic acid sequences takes place. Preferred transgenic plants are mentioned herein.
Transformation
The term “introduction” or “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 there from. 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.
The transfer of foreign genes into the genome of a plant is called transformation. 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. The methods described for the transformation and regeneration of plants from plant tissues or plant cells may be utilized for transient or for stable transformation. 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 plants, including transgenic crop plants, are preferably produced via Agrobacterium-mediated transformation. An advantageous transformation method is the transformation in planta. To this end, it is possible, for example, to allow the agrobacteria to act on plant seeds or to inoculate the plant meristem with agrobacteria. It has proved particularly expedient in accordance with the invention to allow a suspension of transformed agrobacteria to act on the intact plant or at least on the flower primordia. The plant is subsequently grown on until the seeds of the treated plant are obtained (Clough and Bent, Plant J. (1998) 16, 735-743). Methods for Agrobacterium-mediated transformation of rice include well known methods for rice transformation, such as those described in any of the following: 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. Said methods are further described by way of example in B. Jenes et al., Techniques for Gene Transfer, in: Transgenic Plants, Vol. 1, Engineering and Utilization, eds. S. D. Kung and R. Wu, Academic Press (1993) 128-143 and in Potrykus Annu. Rev. Plant Physiol. Plant Molec. Biol. 42 (1991) 205-225). The nucleic acid sequences or the construct to be expressed is preferably cloned into a vector, which is suitable for transforming Agrobacterium tumefaciens, for example pBin19 (Bevan et al., Nucl. Acids Res. 12 (1984) 8711). Agrobacteria transformed by such a vector can then be used in known manner for the transformation of plants, such as plants used as a model, like Arabidopsis (Arabidopsis thaliana is within the scope of the present invention not considered as a crop plant), or crop plants such as, by way of example, tobacco plants, for example by immersing bruised leaves or chopped leaves in an agrobacterial solution and then culturing them in suitable media. The transformation of plants by means of Agrobacterium tumefaciens is described, for example, by Höfgen and Willmitzer in Nucl. Acid Res. (1988) 16, 9877 or is known inter alia from F. F. White, Vectors for Gene Transfer in Higher Plants; in Transgenic Plants, Vol. 1, Engineering and Utilization, eds. S. D. Kung and R. Wu, Academic Press, 1993, pp. 15-38.
In addition to the transformation of somatic cells, which then have to be regenerated into intact plants, it is also possible to transform the cells of plant meristems and in particular those cells which develop into gametes. In this case, the transformed gametes follow the natural plant development, giving rise to transgenic plants. Thus, for example, seeds of Arabidopsis are treated with agrobacteria and seeds are obtained from the developing plants of which a certain proportion is transformed and thus transgenic [Feldman, K A and Marks M D (1987). Mol Gen Genet 208:274-289; Feldmann K (1992). In: C Koncz, N-H Chua and J Shell, eds, Methods in Arabidopsis Research. Word Scientific, Singapore, pp. 274-289]. Alternative methods are based on the repeated removal of the inflorescences and incubation of the excision site in the center of the rosette with transformed agrobacteria, whereby transformed seeds can likewise be obtained at a later point in time (Chang (1994). Plant J. 5: 551-558; Katavic (1994). Mol Gen Genet, 245: 363-370). However, an especially effective method is the vacuum infiltration method with its modifications such as the “floral dip” method. In the case of vacuum infiltration of Arabidopsis, intact plants under reduced pressure are treated with an agrobacterial suspension [Bechthold, N (1993). C R Acad Sci Paris Life Sci, 316: 1194-1199], while in the case of the “floral dip” method the developing floral tissue is incubated briefly with a surfactant-treated agrobacterial suspension [Clough, S J and Bent A F (1998) The Plant J. 16, 735-743]. A certain proportion of transgenic seeds are harvested in both cases, and these seeds can be distinguished from non-transgenic seeds by growing under the above-described selective conditions. In addition the stable transformation of plastids is of advantages because plastids are inherited maternally is most crops reducing or eliminating the risk of transgene flow through pollen. The transformation of the chloroplast genome is generally achieved by a process which has been schematically displayed in Klaus et al., 2004 [Nature Biotechnology 22 (2), 225-229]. Briefly the sequences to be transformed are cloned together with a selectable marker gene between flanking sequences homologous to the chloroplast genome. These homologous flanking sequences direct site specific integration into the plastome. Plastidal transformation has been described for many different plant species and an overview is given in Bock (2001) Transgenic plastids in basic research and plant biotechnology. J Mol Biol. 2001 Sep. 21; 312 (3):425-38 or Maliga, P (2003) Progress towards commercialization of plastid transformation technology. Trends Biotechnol. 21, 20-28. Further biotechnological progress has recently been reported in form of marker free plastid transformants, which can be produced by a transient co-integrated maker gene (Klaus et al., 2004, Nature Biotechnology 22(2), 225-229).
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 increaser or an intron), in the genomic region of the gene of interest or 10 kb up- or downstream of the coding region of a gene in a configuration such that the promoter directs expression of the targeted gene. Typically, regulation of expression of the targeted gene by its natural promoter is disrupted and the gene falls under the control of the newly introduced promoter. The promoter is typically embedded in a T-DNA. This T-DNA is randomly inserted into the plant genome, for example, through Agrobacterium infection and leads to modified expression of genes near the inserted T-DNA. The resulting transgenic plants show dominant phenotypes due to modified expression of genes close to the introduced promoter.
TILLING
The term “TILLING” is an abbreviation of “Targeted Induced Local Lesions In Genomes” and refers to a mutagenesis technology useful to generate and/or identify nucleic acid sequences encoding proteins with modified expression and/or activity. TILLING also allows selection of plants carrying such mutant variants. These mutant variants may exhibit modified expression, either in strength or in location or in timing (if the mutations affect the promoter for example). These mutant variants may 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. 16-82; 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).
Homologous Recombination
Homologous recombination allows introduction in a genome of a selected nucleic acid sequence 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; lida and Terada (2004) Curr Opin Biotech 15(2): 132-8), and approaches exist that are generally applicable regardless of the target organism (Miller et al, Nature Biotechnol. 25, 778-785, 2007).
Yield
The term “yield” in general means a measurable produce of economic value, typically related to a specified crop, to an area, and to a period of time. Individual plant parts directly contribute to yield based on their number, size and/or weight, or the actual yield is the yield per square meter for a crop and year, which is determined by dividing total production (includes both harvested and appraised production) by planted square meters. The term “yield” of a plant may relate to vegetative biomass (root and/or shoot biomass), to reproductive organs, and/or to propagules (such as seeds) of that plant.
Early Vigour
“Early vigour” refers to active healthy well-balanced growth especially during early stages of plant growth, and may result from increased plant fitness due to, for example, the plants being better adapted to their environment (i.e. optimizing the use of energy resources and partitioning between shoot and root). Plants having early vigour also show increased seedling survival and a better establishment of the crop, which often results in highly uniform fields (with the crop growing in uniform manner, i.e. with the majority of plants reaching the various stages of development at substantially the same time), and often better and higher yield. Therefore, early vigour may be determined by measuring various factors, such as thousand kernel weight, percentage germination, percentage emergence, seedling growth, seedling height, root length, root and shoot biomass and many more.
Increase/Improve/Enhance
The terms “increase”, “improve” or “increase” are interchangeable and shall mean in the sense of the application 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 control plants as defined herein.
Seed Yield
Increased seed yield may manifest itself as one or more of the following: a) an increase in seed biomass (total seed weight) which may be on an individual seed basis and/or per plant and/or per hectare or acre; b) increased number of flowers per panicle and/or per plant; c) increased number of (filled) seeds; d) increased seed filling rate (which is expressed as the ratio between the number of filled seeds divided by the total number of seeds); e) increased harvest index, which is expressed as a ratio of the yield of harvestable parts, such as seeds, divided by the total biomass; f) increased number of primary panicles; (g) 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, and may also result from an increase in embryo and/or endosperm size.
An increase in seed yield may also be manifested as an increase in seed size and/or seed volume. Furthermore, an increase in yield may also manifest itself as an increase in seed area and/or seed length and/or seed width and/or seed perimeter. Increased seed yield may also result in modified architecture, or may occur because of modified architecture.
Greenness Index
The “greenness index” as used herein is calculated from digital images of plants. For each pixel belonging to the plant object on the image, the ratio of the green value versus the red value (in the RGB model for encoding color) is calculated. The greenness index is expressed as the percentage of pixels for which the green-to-red ratio exceeds a given threshold. Under normal growth conditions, under salt stress growth conditions, and under reduced nutrient availability growth conditions, the greenness index of plants is measured in the last imaging before flowering. In contrast, under drought stress growth conditions, the greenness index of plants is measured in the first imaging after drought.
Plant
The term “plant” as used herein encompasses whole plants, ancestors and progeny of the plants and plant parts, including seeds, shoots, stems, leaves, roots (including tubers), flowers, and tissues and organs, wherein each of the aforementioned comprise the gene/nucleic acid sequence 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 comprises the gene/nucleic acid sequence 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., Agave sisalana, Agropyron spp., Agrostis stolonifera, Allium spp., Amaranthus spp., Ammophila arenaria, Ananas comosus, Annona spp., Apium graveolens, Arachis spp, Artocarpus spp., Asparagus officinalis, Avena spp. (e.g. Avena sativa, Avena fatua, Avena byzantina, Avena fatua var. sativa, Avena hybrida), Averrhoa carambola, Bambusa sp., 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, Cannabis sativa, Capsicum spp., Carex elate, Carica papaya, Carissa macrocarpa, Carya spp., Carthamus tinctorius, Castanea spp., Ceiba pentandra, Cichorium endivia, Cinnamomum spp., Citrullus lanatus, Citrus spp., Cocos spp., Coffea spp., Colocasia esculenta, Cola spp., Corchorus sp., 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, Erianthus sp., Eriobotrya japonica, Eucalyptus sp., Eugenia uniflora, Fagopyrum spp., Fagus spp., Festuca arundinacea, Ficus carica, Fortunella spp., Fragaria 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., Miscanthus sinensis, Momordica spp., Morus nigra, Musa spp., Nicotiana spp., Olea spp., Opuntia spp., Ornithopus spp., Oryza spp. (e.g. Oryza sativa, Oryza latifolia), Panicum miliaceum, Panicum virgatum, Passiflora edulis, Pastinaca sativa, Pennisetum sp., Persea spp., Petroselinum crispum, Phalaris arundinacea, Phaseolus spp., Phleum pratense, Phoenix spp., Phragmites australis, 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., Salix sp., 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, Trifolium spp., Triticale sp., Triticosecale rimpaui, Triticum spp. (e.g. Triticum aestivum, Triticum durum, Triticum turgidum, Triticum hybernum, Triticum macha, Triticum sativum or Triticum vulgare), Tropaeolum minus, Tropaeolum majus, Vaccinium spp., Vicia spp., Vigna spp., Viola odorata, Vitis spp., Zea mays, Zizania palustris, Ziziphus spp., amongst others.
DETAILED DESCRIPTION OF THE INVENTION
Surprisingly, it has now been found that increasing expression in a plant of a nucleic acid sequence encoding a GRF polypeptide gives plants having increased yield-related traits relative to control plants. According to a first embodiment, the present invention provides a method for increasing yield-related traits in plants relative to control plants, comprising increasing expression in a plant of a nucleic acid sequence encoding a GRF polypeptide.
A preferred method for increasing expression of a nucleic acid sequence encoding a GRF polypeptide is by introducing and expressing in a plant a nucleic acid sequence encoding a GRF polypeptide.
Any reference hereinafter to a “protein useful in the methods of the invention” is taken to mean in one embodiment a GRF polypeptide as defined herein. Any reference hereinafter to a “nucleic acid sequence useful in the methods of the invention” is taken to mean a nucleic acid sequence capable of encoding such a GRF polypeptide. The nucleic acid sequence to be introduced into a plant (and therefore useful in performing the methods of the invention) is any nucleic acid sequence encoding the type of polypeptide, which will now be described, hereafter also named “GRF nucleic acid sequence” or “GRF gene”.
A “GRF polypeptide” as defined herein refers to any polypeptide comprising: (i) a domain having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more amino acid sequence identity to a QLQ domain as represented by SEQ ID NO: 115; and (ii) a domain having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more amino acid sequence identity to a WRC domain as represented by SEQ ID NO: 116.
Alternatively or additionally, a “GRF polypeptide” as defined herein refers to any polypeptide comprising: (i) a QLQ domain with an InterPro accession IPR014978 (PFAM accession PF08880); (ii) a WRC domain with an InterPro accession IPR014977 (PFAM accession PF08879); and (iii) an Effector of Transcription (ET) domain comprising three Cys and one His residues in a conserved spacing (CX9CX10CX2H).
Alternatively or additionally, a “GRF polypeptide” as defined herein refers to any polypeptide having in increasing order of preference at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more amino acid sequence identity to the GRF polypeptide as represented by SEQ ID NO: 2 or to any of the full length polypeptide sequences given in Table A herein.
Alternatively or additionally, a “GRF polypeptide” interacts with GRF-interacting factor (GIF) polypeptides (also called synovial sarcoma translocation (SYT) polypeptides) in a yeast two-hybrid interaction assay.
Surprisingly, it has now been found that modulating expression in a plant of a nucleic acid encoding a RAA1-like polypeptide gives plants having enhanced yield-related traits relative to control plants. According to a first embodiment, the present invention provides a method for enhancing yield-related traits in plants relative to control plants, comprising modulating expression in a plant of a nucleic acid encoding a RAA1-like polypeptide.
A preferred method for modulating (preferably, increasing) expression of a nucleic acid encoding a RAA1-like polypeptide is by introducing and expressing in a plant a nucleic acid encoding a RAA1-like polypeptide.
Any reference hereinafter to a “protein useful in the methods of the invention” is taken to mean in one embodiment a RAA1-like polypeptide as defined herein. Any reference hereinafter to a “nucleic acid useful in the methods of the invention” is taken to mean a nucleic acid capable of encoding such a RAA1-like polypeptide. The nucleic acid to be introduced into a plant (and therefore useful in performing the methods of the invention) is any nucleic acid encoding the type of protein which will now be described, hereafter also named “RAA1-like nucleic acid” or “RAA1-like gene”.
A “RAA1-like polypeptide” as defined herein refers to any polypeptide represented by SEQ ID NO: 121 and to orthologues and paralogues thereof. RAA1-like proteins are small (MW between 10 and 21 kDA) and basic polypeptides (pl above 8.5), and usually have zero or one Cys residue in the sequence that aligns with SEQ ID NO: 121 when using a standard Needleman-Wunsch alignment program with default settings.
Preferably, the RAA1-like polypeptide comprises two or more of the following conserved sequence motifs:









SEQ ID NO: 162 










motif 1:
GVW(V/L)F,



 









SEQ ID NO: 163 










motif 2:
LGW(E/S)RY(Y/F),



 









SEQ ID NO: 164 










motif 3:
(D/H)L(L/I)S(I/V/L)P(R/K/A)(S/D)F,



 









SEQ ID NO: 165 










motif 4:
(H/Y)(F/M)YD(V/I)VVK(N/T)(R/P),






Alternatively, the homologue of a RAA1 protein has in increasing order of preference at least 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: 121, 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.
Preferably, the polypeptide sequence which when used in the construction of a phylogenetic tree, such as the one depicted in FIG. 8 (Ge et al., 2004), clusters with the group of RAA1-like polypeptides comprising the amino acid sequence represented by SEQ ID NO: 121 rather than with any other group.
Surprisingly, it has now been found that modulating expression in a plant of a nucleic acid encoding a SYR polypeptide gives plants, when grown under abiotic stress conditions, having enhanced yield-related traits relative to control plants. According to a first embodiment, the present invention provides a method for enhancing yield-related traits in plants grown under abiotic stress conditions, relative to control plants, comprising modulating expression in a plant of a nucleic acid encoding a SYR polypeptide.
A preferred method for modulating (preferably, increasing) expression of a nucleic acid encoding a SYR polypeptide is by introducing and expressing in a plant a nucleic acid encoding a SYR polypeptide.
Any reference hereinafter to a “protein useful in the methods of the invention” is taken to mean in one embodiment a SYR polypeptide as defined herein. Any reference hereinafter to a “nucleic acid useful in the methods of the invention” is taken to mean a nucleic acid capable of encoding such a SYR polypeptide. The nucleic acid to be introduced into a plant (and therefore useful in performing the methods of the invention) is any nucleic acid encoding the type of protein which will now be described, hereafter also named “SYR nucleic acid” or “SYR gene”.
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 5a, SEQ ID NO: 173), or YFT (conserved motif 5b, SEQ ID NO: 174), or YFG (conserved motif 5c, SEQ ID NO: 175) or YLG (conserved motif 5d, SEQ ID NO: 176), and (iii) followed by a conserved motif 6 ((V/A/I)LAFMP(T/S), SEQ ID NO: 177). Preferably, the conserved motif 6 is (AN)LAFMP(T/S) (SEQ ID NO: 177), most preferably, the conserved motif is VLAFMPT (SEQ ID NO: 177). The “SYR protein or homologue thereof” preferably also has a conserved C-terminal peptide ending with the conserved motif 7 (SYL or PYL, SEQ ID NO: 178). 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 5 and stopping immediately before the conserved motif 6, 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, wherein X6 is a sequence of 6 consecutive amino acids). A preferred example of a SYR protein is represented by SEQ ID NO: 169, an overview of its domains is given in FIG. 11.
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: 169, provided that the homologous protein comprises the conserved motifs 5(a, b, c or d), 6 and 7, 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. Compared to overall sequence identity, the sequence identity will generally be higher when only conserved domains or motifs are considered.
Surprisingly, it has now been found that modulating expression in a plant of a nucleic acid encoding an ARKL polypeptide gives plants having enhanced yield-related traits relative to control plants. According to a first embodiment, the present invention provides a method for enhancing yield-related traits in plants relative to control plants, comprising modulating expression in a plant of a nucleic acid encoding an ARKL polypeptide.
A preferred method for modulating (preferably, increasing) expression of a nucleic acid encoding an ARKL polypeptide is by introducing and expressing in a plant a nucleic acid encoding an ARKL polypeptide.
Any reference hereinafter to a “protein useful in the methods of the invention” is taken to mean an ARKL polypeptide as defined herein. Any reference hereinafter to a “nucleic acid useful in the methods of the invention” is taken to mean a nucleic acid capable of encoding such an ARKL polypeptide. The nucleic acid to be introduced into a plant (and therefore useful in performing the methods of the invention) is any nucleic acid encoding the type of protein which will now be described, hereafter also named “ARKL nucleic acid” or “ARKL gene”.
An “ARKL polypeptide” as defined herein refers to any polypeptide comprising a conserved domain of the zinc finger RING-type and optionally a DAR1 domain. The RING-type zinc finger found in ARKL polypeptide comprises a canonical C3H2C3 zinc finger domain type. It can further be classified into the RING-H2 type within group I as defined by Stone et al. 2005.
A consensus sequence representing the RING-H2 domain has been reported as represented by CX(2)CX(9-39)CX(1-3)HX(2-3)HX(2)CX(4-48)CX(2)C (SEQ ID NO: 400). The length of the variable loops in ARKL polypeptides is typically of 14-15 amino acids between metal ligands 2 and 3 and of 10 amino acids between metal ligands 6 and 7 (FIG. 1). Specific amino acid residues other than those implicated in the direct coordination of Zn2+ ions are highly conserved in the RING-H2 domain of ARKL polypeptides (FIG. 1). SEQ ID NO: 401 represents a consensus sequence conserved amongst the majority of ARKL polypeptides.
A preferred ARKL polypeptide useful in the methods of the invention refers to a polypeptide comprising a ZfC3H2C3 zinc finger RING domain, such domain being represented by SEQ ID NO: 400 or a polypeptide having in increasing order of preference at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more sequence identity to one or more of the ZfC3H2C3 domains as represented by SEQ ID NO: 306 to SEQ ID NO: 351. Further preferably the ARKL polypeptide of the invention comprises a ZfC3H2C3 domain as represented by SEQ ID NO: 401.
ARKL polypeptides typically comprise an additional domain, named DAR1 (Domain Associated with RING), which has been previously described to occur outside of the RING domain in a few RING proteins of plant origin (Stone et al. 2005). DAR1 domain is typically found at the N-terminus of the RING domain. Typically DAR1 domains comprise a conserved amino acid signature as represented by SEQ ID NO: 399 (Motif 8).
A further preferred ARKL polypeptide useful in the methods of the invention refers to a polypeptide comprising a DAR1 domain having in increasing order of preference at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more sequence identity to one or more of the DAR1 domains as represented by SEQ ID NO: 352 to SEQ ID NO. 398. Still more preferably the ARKL polypeptide of the invention comprises Motif 8 as represented by SEQ ID NO: 399.
Zinc finger RING-type and DAR1 domains can be found in protein databases specialized in protein families, domains and functional sites such as Pfam (Finn et al. Nucleic Acids Research (2006) Database Issue 34:D247-D251) or InterPro which integrates the protein signature databases: PROSITE, PRINTS, ProDom, Pfam, SMART, TIGRFAMs, PIRSF, SUPERFAMILY, Gene3D and PANTHER (Mulder et al. 2007 Nucleic Acids Research, 2007, Vol. 35, Database issue D224-D228). Pfam compiles a large collection of multiple sequence alignments and hidden Markov models (HMM) covering many common protein domains and families and is available through the Sanger Institute in the United Kingdom. Trusted matches as considered in the Pfam database are those sequences scoring higher than the gathering cut-off threshold. The gathering cutoff threshold of the RING-H2 domain (Pfam accession number: PF00097) in the Pfam HMM_fs method is 16.0 and in the Pfam HMM_Is method is 15.2. However potential matches, comprising true RING-H2 domain domains, may still fall under the gathering cut-off. Preferably an ARKL polypeptide useful in the methods of the invention is a protein having one or more domains in their sequence that exceed the gathering cutoff of the Pfam protein domain family PF000097, also known as Zinc finger, C3HC4 type (RING finger) family domain.
Alternatively, Zinc finger RING-type and DAR1 domains in a polypeptide may be identified by performing a sequence comparison with known polypeptides comprising a Zinc finger RING-type and/or DAR1 domains and establishing the similarity in the region of said domains. The sequences may be aligned using any of the methods well known in the art such as Blast algorithms. The probability for the alignment to occur with a given sequence is taken as basis for identifying similar polypeptides. A parameter that is typically used to represent such probability is called e-value. The E-value is a measure of the reliability of the S score. The S score is a measure of the similarity of the query to the sequence shown. The e-value describes how often a given S score is expected to occur at random. The e-value cut-off may be as high as 1.0. The typical threshold for a trusted e-value from a BLAST search output using an ARKL polypeptide as query sequence is lower than e−5(=10-5), 1.e-10, 1.e-15, 1.e-20, 1.e-25, 1.e-50, 1.e-75, 1.e-100, 1.e-200, 1.e-300, 1.e-400, 1.e-500, 1.e-600, 1.e-700 and 1.e-800. Preferably ARKL polypeptides useful in the methods of the invention comprise a sequence having in increasing order of preference an e-value lower than e−5(=10-5), 1.e-10, 1.e-15, 1.e-20, 1.e-25, 1.e-50, 1.e-75, 1.e-100, 1.e-200, 1.e-300, 1.e-400, 1.e-500, 1.e-600, 1.e-700 and 1.e-800 in an alignment with a Zinc finger RING-type and/or DAR1 domains as found in a known ARKL polypeptides, such as for example SEQ ID NO: 213.
Examples of ARKL polypeptides useful in the methods of the invention are given in Table A. A sequence comprising the RING-H2 and DAR1 domains as present in the representative ARKL polypeptides of Table A is given in SEQ ID NO: 306 to SEQ ID NO: 351 and SEQ ID NO: 352 to SEQ ID NO: 398, respectively. The amino acid coordinates of the position of RING-H2 and DAR1 domains as present in a selection of ARKL polypeptides of Table A are given in Example 4.
Further preferred ARKL polypeptides useful in the methods of the invention are those having in increasing order of preference at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%; 98% or more sequence identity to any of the polypeptides given in Table A.
Surprisingly, it has now been found that modulating expression in a plant of a nucleic acid encoding a YTP polypeptide gives plants having enhanced yield-related traits relative to control plants. According to a first embodiment, the present invention provides a method for enhancing yield-related traits in plants relative to control plants, comprising modulating expression in a plant of a nucleic acid encoding a YTP polypeptide.
A preferred method for modulating (preferably, increasing) expression of a nucleic acid encoding a YTP polypeptide is by introducing and expressing in a plant a nucleic acid encoding a YTP polypeptide.
Any reference hereinafter to a “protein useful in the methods of the invention” is taken to mean in one embodiment a YTP polypeptide as defined herein. Any reference hereinafter to a “nucleic acid useful in the methods of the invention” is taken to mean a nucleic acid capable of encoding such a YTP polypeptide. The nucleic acid to be introduced into a plant (and therefore useful in performing the methods of the invention) is any nucleic acid encoding the type of protein which will now be described, hereafter also named “YTP nucleic acid” or “YTP gene”.
A “YTP polypeptide” as defined herein refers to a polypeptide comprising at least one transmembrane domain and a portion of at least 50 contiguous amino acids of a DUF221 domain. Additionally the YTP polypeptide may comprise Motif 9 as represented by SEQ ID NO: 546.
Transmembrane proteins have an amphiphilic structure with hydrophobic segments traversing the membranes and hydrophilic loops that may be located at either side of the membrane (see FIG. 20). Loops are the segments (regions) of a protein located between two TM domains. Average sized loops located on the inside side of the membrane are typically more negatively charged than those on the outside of the membrane.
A transmembrane domain forms a secondary structure (usually an alpha or beta helix) of typically 12-35 amino acid residues. The loops between the transmembrane domains are typically shorter than 60 amino acid residues, though long globular regions may also occur. The number of transmembrane domains in a YTP polypeptide is variable, but typically between 2 and 20.
The transmembrane domain found in YTP polypeptides is preferably between 8 and 50 amino acids, most preferably 8, 12, 14, 16, 18, 2, 22, 24, 26, 28, 30, 32, 34, 35, or 36 amino acids. A loop found in YTP polypeptides has preferably above 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100 amino acid residues.
A preferred YTP polypeptide useful in the methods of the invention comprises in increasing order of preference more than 1, 2, 4, 5, 6, 8, 10, 12 transmembrane domains.
Transmembrane domains are highly hydrophobic proteins rich in non-polar amino acids. Table 3 shows a classification of the amino acids according to the side chain properties. Hydrophobic amino acids are indicated. The hydrophobic character of a peptide can be determined by methods well known in the art, as for example reported by Kyte and Doolittle (1982) J. Mol. Biol., 157:105-132.
YTP polypeptides useful in the methods of the invention preferably comprise transmembrane domains having at least 20%, 30%, 40%, 50%, 60%, or more non-polar amino acids. Table 3 gives the polarity of the 20 essential amino acids.







TABLE 3







Classification of amino acids according to the side chain properties.
















Side chain






Side chain
acidity
Hydropathy


Amino Acid
3-Letter
1-Letter
polarity
or basicity
index















Arginine
Arg
R
polar
basic
−4.5


Asparagine
Asn
N
polar
neutral
−3.5


Aspartic acid
Asp
D
polar
acidic
−3.5


Cysteine
Cys
C
polar
neutral
2.5


Glutamic acid
Glu
E
polar
acidic
−3.5


Glutamine
Gln
Q
polar
neutral
−3.5


Histidine
His
H
polar
basic
−3.2


Lysine
Lys
K
polar
basic
−3.9


Serine
Ser
S
polar
neutral
−0.8


Threonine
Thr
T
polar
neutral
−0.7


Tyrosine
Tyr
Y
polar
neutral
−1.3


Alanine
Ala
A
nonpolar
neutral
1.8


Glycine
Gly
G
nonpolar
neutral
−0.4


Isoleucine
Ile
I
nonpolar
neutral
4.5


Leucine
Leu
L
nonpolar
neutral
3.8


Methionine
Met
M
nonpolar
neutral
1.9


Phenylalanine
Phe
F
nonpolar
neutral
2.8


Proline
Pro
P
nonpolar
neutral
−1.6


Tryptophan
Trp
W
nonpolar
neutral
−0.9


Valine
Val
V
nonpolar
neutral
4.2









A transmembrane domain in a protein may be identified using a number of techniques well known in the art such as X-ray crystallography, NMR, gene fusion techniques, substituted cysteine accessibility methods, Asp(N)-linked glycosylation experiments. Additionally or alternatively computer algorithms may be used to predict transmembrane domains. Examples of such domains have been described and are available at institutions providing bioinformatic services (Möller et al. 2001. Bioinformatics 17, 646). Use of one such algorithm to predict the transmembrane domains in a YTP polypeptide is shown in the Examples section herein.
DUF221 domain refers to a conserved amino acid sequence found in some proteins of eukaryotic origin. DUF221 domains are usually 350 to 550 residues in length. Examples of DUF221 domains comprised in YTP polypeptides originating from Arabidopsis thaliana and Oryza sativa are represented by SEQ ID NO: 518 to SEQ ID NO: 543. A consensus sequence representing the sequence SEQ ID NO: 518 to SEQ ID NO: 543 is given in SEQ ID NO: 544.
A preferred YTP polypeptide of the invention comprises a least 50 contiguous amino acids of a DUF221 domain having in increasing order of preference at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with any one of the domains represented by SEQ ID NO: 518 to SEQ ID NO: 544. The sequence similarity is preferably established in a local alignment using algorithms well known in the art such as Blast.
A YTP polypeptide may readily be identified by searching in specialized databases containing conserved protein domains such as Pfam, (Finn et al. Nucleic Acids Research (2006) Database Issue 34:D247-D251). Tools useful in searching such databases are well known in the art, for example INTERPRO (European Bioinformatics institute, UK) which allows searching several protein domain databases simultaneously.
A DUF221 domain may be identified by sequence comparison with known polypeptides comprising a DUF221 domain and establishing the percentage similarity over the region of the DUF221 domain. The sequences may be aligned using any of the methods well known in the art such as Blast (for local alignment) or BestFit (for global alignment) algorithms. The probability of the alignment with a given sequence is taken as basis for identifying similar polypeptides. A parameter that is typically used to represent such probability is called e-value. The e-value is a measure of the reliability of the score “S”. “S” is a measure of the similarity between the two sequences aligned. The e-value describes how often a given “S” score is expected to occur at random. The e-value cut-off may be as high as 1.0. The typical threshold for a trusted (true) hit showing significant sequence homology to the query sequence and resulting from a BLAST search is lower than 1.e−5, in some instance an even lower threshold is taken, for example 1.e-10, or even lower.
Preferably YTP polypeptides useful in the methods of the invention comprise at least 50 contiguous amino acids of a DUF221 domain having in increasing order of preference an e-value lower than 1.e−5, 1.e−10, 1.e−15, 1.e−20, 1.e−25, 1.e−50, 1.e−75, 1.e−100, 1.e−200, 1.e−300, 1.e−400, 1.e−500, 1.e−600, 1.e−700 and 1.e-800 in an local alignment with a DUF221 domain found in a known YTP polypeptide, such as any of the polypeptides of Table A.
It should be understood that the nucleic acids encoding a YTP polypeptide according to the invention it is not restricted to sequences of natural origin. The nucleic acid may encode a “de novo” designed YTP polypeptide.
Alternatively or additionally the YTP protein has in increasing order of preference at least 25%, 26%, 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: 409.
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. Compared to overall sequence identity, the sequence identity will generally be higher when only conserved domains or motifs are considered.
Preferably, a YTP polypeptide sequence when used in the construction of a phylogenetic tree, such as the one depicted in FIG. 21, clusters with the Group 1, comprising the amino acid sequence represented by SEQ ID NO: 409 rather than with the YTP polypeptides in Group 2.
The term “domain” and “motif” is defined in the “definitions” section herein. Specialist databases exist for the identification of domains, for example, 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, 315-318), 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)), or Pfam (Bateman et al., Nucleic Acids Research 30(1): 276-280 (2002). A set of tools for in silico analysis of protein sequences is available on the ExPASy proteomics server (Swiss Institute of Bioinformatics (Gasteiger et al., ExPASy: the proteomics server for in-depth protein knowledge and analysis, Nucleic Acids Res. 31:3784-3788 (2003)).
Analysis of the polypeptide sequence of SEQ ID NO: 2 is presented below in Examples 2 and 4 herein. For example, a GRF polypeptide as represented by SEQ ID NO: 2 comprises a QLQ domain with an InterPro accession IPR014978 (PFAM accession PF08880) and a WRC domain with an InterPro accession IPR014977 (PFAM accession PF08879) in the InterPro domain database. Domains may also be identified using routine techniques, such as by sequence alignment. An alignment of the QLQ domain of the polypeptides of Table A herein, is shown in FIG. 2, and alignment of the WRC domain of the polypeptides of Table A herein, is shown in FIG. 3. Such alignments are useful for identifying the most conserved amino acids between the GRF polypeptides, such as the QLQ and WRC amino acid residues.
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 global (i.e. spanning the complete sequences) alignment of two 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 (NCBI). Homologues may readily be identified using, for example, the ClustalW multiple sequence alignment algorithm (version 1.83), with the default pairwise alignment parameters, and a scoring method in percentage. Global percentages of similarity and identity may also be determined using one of the methods available in the MatGAT software package (Campanella et al., (2003) BMC Bioinformatics, 10: 29. MatGAT: an application that generates similarity/identity matrices using protein or DNA sequences.). Minor manual editing may be performed to optimise alignment between conserved motifs, as would be apparent to a person skilled in the art. Furthermore, instead of using full-length sequences for the identification of homologues, specific domains may also be used. The sequence identity values may be determined over the entire nucleic acid sequence or polypeptide sequence or over selected domains or conserved motif(s), using the programs mentioned above using the default parameters.
For local alignments, the Smith-Waterman algorithm is particularly useful (Smith T F, Waterman M S (1981) J. Mol. Biol 147(1); 195-7).
Outside of the QLQ domain and of the WRC domain, GRF polypeptides reputedly have low amino acid sequence identity. Example 3 herein describes in Table B the percentage identity between the GRF polypeptide as represented by SEQ ID NO: 2 and the GRF polypeptides listed in Table A, which can be as low as 15% amino acid sequence identity. The percentage identity can be substantially increased if the identity calculation is performed between the QLQ domain SEQ ID NO: 2 (as represented by SEQ ID NO: 115 comprised in SEQ ID NO: 2; QLQ domain of the GRF polypeptides of Table A represented in FIG. 2) and the QLQ domains of the polypeptides useful in performing the invention. Similarly, the percentage identity can be substantially increased if the identity calculation is performed between the WRC domain SEQ ID NO: 2 (as represented by SEQ ID NO: 116 comprised in SEQ ID NO: 2; WRC domain of the GRF polypeptides of Table A represented in FIG. 3) and the WRC domains of the polypeptides useful in performing the invention. Percentage identity over the QLQ domain amongst the polypeptide sequences useful in performing the methods of the invention ranges between 25% and 99% amino acid identity, and percentage identity over the WRC domain amongst the polypeptide sequences useful in performing the methods of the invention ranges between 60% and 99% amino acid identity. As can also be observed in FIG. 3, the WRC domain is better conserved amongst the different GRF polypeptides than the QLQ domain, as shown in FIG. 2.
The task of protein subcellular localisation prediction is important and well studied. Knowing a protein's localisation helps elucidate its function. Experimental methods for protein localization range from immunolocalization to tagging of proteins using green fluorescent protein (GFP) or beta-glucuronidase (GUS). Such methods are accurate although labor-intensive compared with computational methods. Recently much progress has been made in computational prediction of protein localisation from sequence data. Among algorithms well known to a person skilled in the art are available at the ExPASy Proteomics tools hosted by the Swiss Institute for Bioinformatics, for example, PSort, TargetP, ChloroP, LocTree, Predotar, LipoP, MITOPROT, PATS, PTS1, SignalP and others.
Furthermore, GRF polypeptides useful in the methods of the present invention (at least in their native form) typically, but not necessarily, have transcriptional regulatory activity and capacity to interact with other proteins. Therefore, GRF polypeptides with reduced transcriptional regulatory activity, without transcriptional regulatory activity, with reduced protein-protein interaction capacity, or with no protein-protein interaction capacity, may equally be useful in the methods of the present invention. DNA-binding activity and protein-protein interactions may readily be determined in vitro or in vivo using techniques well known in the art (for example in Current Protocols in Molecular Biology, Volumes 1 and 2, Ausubel et al. (1994), Current Protocols). GRF polypeptides are capable of transcriptional activation of reporter genes in yeast cells (Kim & Kende (2004) Proc Natl Acad Sci 101(36): 13374-13379). GRF polypeptides are also capable of interacting with GRF-interacting factor polypeptides (GIF1 to GIF3; also called synovial sarcoma translocation (SYT) polypeptides, SYT1 to SYT3) in vivo in yeast cells, using a yeast two-hybrid protein-protein interaction assay (Kim & Kende, supra). In vitro binding assays are also used to show that GRF polypeptides and GIF (also called SYT) polypeptides are interacting partners (Kim & Kende, supra).
The present invention is illustrated in one embodiment by transforming plants with the nucleic acid sequence represented by SEQ ID NO: 1, encoding the GRF polypeptide sequence of SEQ ID NO: 2. However, performance of the invention is not restricted to these sequences; the methods of the invention may advantageously be performed using any nucleic acid sequence encoding a GRF polypeptide as defined herein.
The present invention is illustrated in one embodiment by transforming plants with the nucleic acid sequence represented by SEQ ID NO: 120, encoding the polypeptide sequence of SEQ ID NO: 121. However, performance of the invention is not restricted to these sequences; the methods of the invention may advantageously be performed using any RAA1-like-encoding nucleic acid or RAA1-like polypeptide as defined herein.
In addition, RAA1-like polypeptides, when expressed in rice according to the methods of the present invention as outlined in the examples, give plants having increased yield related traits, in particular increased root/shoot index, increased number of flowers per panicle and increased Thousand Kernel Weight.
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. AAAI Press.).
Examples of proteins falling under the definition of “SYR polypeptide or a homologue thereof” are given in Table A of the examples section and include sequences from various monocotyledonous plants, such as rice (SEQ ID NO: 169, SEQ ID NO: 179 and SEQ ID NO: 180), corn (SEQ ID NO: 181), wheat (SEQ ID NO: 182), barley (SEQ ID NO: 183), sugarcane (SEQ ID NO: 184 and SEQ ID NO: 185), sorghum (SEQ ID NO: 186); and from dicotyledonous plants such as Arabidopsis (SEQ ID NO: 187 and SEQ ID NO: 188), grape (SEQ ID NO: 189), citrus (SEQ ID NO: 190) or tomato (SEQ ID NO: 191 and SEQ ID NO: 192). 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 5 or 6 may be useful as well in the methods of the present invention; examples of such proteins are given in SEQ ID NO: 201 and 202.
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: 169 or to the homologues listed as SEQ ID NO: 179 to SEQ ID NO: 192, 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 5 (a, b, c or d) and followed by the conserved motif 6 and preferably also by the conserved motif 7; or having at least 38% sequence identity to the sequence of SEQ ID NO: 169, may be suitable for use in the methods of the invention.
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 biomass and/or seed yield without a delay in flowering time when grown under conditions of nitrogen deficiency or under drought stress conditions, and 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, fillrate, harvest index or Thousand Kernel Weight.
The present invention is illustrated in one embodiment by transforming plants with the nucleic acid sequence represented by SEQ ID NO: 168, encoding the polypeptide sequence of SEQ ID NO: 169. However, performance of the invention is not restricted to these sequences; the methods of the invention may advantageously be performed using any SYR-encoding nucleic acid or SYR polypeptide as defined herein.
Furthermore, ARKL polypeptides (at least in their native form) typically have E3 ubiquitin-protein ligase activity. Tools and techniques for measuring E3 ubiquitin-protein ligase activity are well known in the art (U.S. Pat. No. 6,737,244; WO/2001/075145; Miura et al. (2005) Proc Natl Acad Sci USA. 102(21): 7760-7765; Kawasaki et al. (2005) The Plant Journal 44, 258-270. Briefly E3 ubiquitin ligase activity of an ARKL polypeptide can be assayed by incubating the ARKL protein with an E1 and E2 proteins and tagged ubiquitin. The ubiquitinated proteins can be detected after SDS-PAGE electrophresis and blotting using an antibody to the tag of the ubiquitin. Examples of E1 and E2 proteins that may be useful in the assay are the Wheat E1 and the Arabidopsis thaliana AtUBC1 E2 protein. Tagged ubiquitin with histidine and antibodies to detect it are commercially available (Calbiochem, San Diego, Calif., USA).
In addition, ARKL polypeptides, when expressed in rice according to the methods of the present invention as outlined in the examples, give plants having increased yield related traits, in particular thousand kernel weight, total seed yield, early vigour and/or harvest index.
The present invention is illustrated in one embodiment by transforming plants with the nucleic acid sequence represented by SEQ ID NO: 212, encoding the polypeptide sequence of SEQ ID NO: 213. However, performance of the invention is not restricted to these sequences; the methods of the invention may advantageously be performed using any ARKL-encoding nucleic acid or ARKL polypeptide as defined herein.
Furthermore, YTP polypeptides typically have seed yield enhancing activity. Tools and techniques for measuring yield enhancing (or improving) activity are well known in the art. Further details are provided in the Examples section herein.
In addition, YTP polypeptides, when expressed and phenotypically evaluated in rice according to the methods of the present invention as outlined in Examples 10 to 15, give plants having increased yield related traits, in particular one or more of total seed weight, thousand kernel weight, number of flowers per panicle, seed filling rate and harvest index.
The present invention is illustrated in one embodiment by transforming plants with the nucleic acid sequence represented by SEQ ID NO: 408, encoding the polypeptide sequence of SEQ ID NO: 409. However, performance of the invention is not restricted to these sequences; the methods of the invention may advantageously be performed using any YTP-encoding nucleic acid or YTP polypeptide as defined herein.
Examples of nucleic acid sequences encoding polypeptides of the invention are given in Table A of Example 1 herein, specially nucleic acid sequences encoding polypeptides selected from the group consisting of:

    
    
        GRF polypeptide are given in Table A1,
        RAA1-like polypeptide are given in Table A2,
        SYR polypeptide are given in Table A3,
        ARKL polypeptide are given in Table A4, and
    
    


YTP polypeptide are given in Table A5 respectively.
Such nucleic acid sequences are useful in performing the methods of the invention. The polypeptide sequences given in Table A of Example 1 are example sequences of orthologues and paralogues of the polypeptide selected from the group consisting of: GRF polypeptide, RAA1-like polypeptide, SYR polypeptide, ARKL polypeptide, and YTP represented by SEQ ID NO: 2, 121, 169, 213 or 409 respectively the terms “orthologues” and “paralogues” being as defined herein. Further orthologues and paralogues may readily be identified by performing a so-called reciprocal blast search. Typically, this involves a first BLAST involving BLASTing a query sequence (for example using any of the sequences listed in Table A of Example 1) against any sequence database, such as the publicly available NCBI database. BLASTN or TBLASTX (using standard default values) are generally used when starting from a nucleotide sequence, and BLASTP or TBLASTN (using standard default values) 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, 120, 168, 212 or 408 respectively or SEQ ID NO: 2, 121, 169, 213 or 409 respectively the second BLAST would therefore be against Arabidopsis thaliana sequences). The results of the first and second BLASTs are then compared. A paralogue is identified if a high-ranking hit from the first blast is from the same species as from which the query sequence is derived, a BLAST back then ideally results in the query sequence amongst the highest hits; an orthologue is identified if a high-ranking hit in the first BLAST is not from the same species as from which the query sequence is derived, and preferably results upon BLAST back in the query sequence being among the highest hits.
The term “table A” used in this specification is to be taken to specify the content of table A1, A2, A3, A4 and/or A5. The term “table A1” used in this specification is to be taken to specify the content of table A1. The term “table A2” used in this specification is to be taken to specify the content of table A2. The term “table A3” used in this specification is to be taken to specify the content of table A3. The term “table A4” used in this specification is to be taken to specify the content of table A4. The term “table A5” used in this specification is to be taken to specify the content of table A5.
In one preferred embodiment, the term “table A” means table A1. In one preferred embodiment, the term “table A” means table A2. In one preferred embodiment, the term “table A” means table A3. In one preferred embodiment, the term “table A” means table A4. In one preferred embodiment, the term “table A” means table A5.
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 addition to E-values, comparisons are 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 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.
Nucleic acid variants may also be useful in practising the methods of the invention. Examples of such variants include nucleic acid sequences encoding homologues and derivatives of any one of the polypeptide sequences given in Table A of Example 1, the terms “homologue” and “derivative” being as defined herein. Also useful in the methods of the invention are nucleic acid sequences encoding homologues and derivatives of orthologues or paralogues of any one of the polypeptide sequences given in Table A of Example 1. Homologues and derivatives useful in the methods of the present invention have substantially the same biological and functional activity as the unmodified protein from which they are derived.
In one embodiment of the invention, a preferred derivative useful in the methods of the invention is an ARKL polypeptide a cystein residue at the position of ligand 5 (see FIG. 15) in the in the RING finger domain coordinating one of the Zinc ions. Another preferred derivative useful in the methods of the invention is an ARKL polypeptide having seven cysteins and one histidine (ZfC3HC4) as residues at the zinc ion ligand positions.
Further nucleic acid variants useful in practising the methods of the invention include portions of nucleic acid sequences encoding polypeptides selected from the group consisting of: GRF polypeptides, RAA1-like polypeptides, SYR polypeptides, ARKL polypeptides, and YTP polypeptides respectively, nucleic acid sequences hybridising to nucleic acid sequences encoding polypeptides selected from the group consisting of: GRF polypeptides, RAA1-like polypeptides, SYR polypeptides, ARKL polypeptides, and YTP polypeptides respectively, splice variants of nucleic acid sequences encoding polypeptides selected from the group consisting of: GRF polypeptides, RAA1-like polypeptides, SYR polypeptides, ARKL polypeptides, and YTP polypeptides respectively, allelic variants of nucleic acid sequences encoding polypeptides selected from the group consisting of: GRF polypeptides, RAA1-like polypeptides, SYR polypeptides, ARKL polypeptides, and YTP polypeptides respectively and variants of nucleic acid sequences encoding polypeptides selected from the group consisting of: GRF polypeptides, RAA1-like polypeptides, SYR polypeptides, ARKL polypeptides, and YTP polypeptides respectively obtained by gene shuffling. The terms hybridising sequence, splice variant, allelic variant and gene shuffling are as described herein.
Nucleic acid sequences encoding polypeptides selected from the group consisting of: GRF polypeptides, RAA1-like polypeptides, SYR polypeptides, ARKL polypeptides, and YTP polypeptides respectively need not be full-length nucleic acid sequences, since performance of the methods of the invention does not rely on the use of full-length nucleic acid sequences. According to the present invention, there is provided a method for increasing yield-related traits, in plants, comprising introducing and expressing in a plant a portion of any one of the nucleic acid sequences given in Table A of Example 1, or a portion of a nucleic acid sequence encoding an orthologue, paralogue or homologue of any of the polypeptide sequences given in Table A of Example 1.
A portion of a nucleic acid sequence may be prepared, for example, by making one or more deletions to the nucleic acid sequence. 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 resultant polypeptide produced upon translation may be bigger than that predicted for the protein portion.
Portions useful in the methods of the invention, encode in one embodiment a GRF polypeptide as defined herein, and have substantially the same biological activity as the polypeptide sequences given in Table A1 of Example 1. Preferably, the portion is a portion of any one of the nucleic acid sequences given in Table A1 of Example 1, or is a portion of a nucleic acid sequence encoding an orthologue or paralogue of any one of the polypeptide sequences given in Table A1 of Example 1. Preferably the portion is, in increasing order of preference at least 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1190 consecutive nucleotides in length, the consecutive nucleotides being of any one of the nucleic acid sequences given in Table A1 of Example 1, or of a nucleic acid sequence encoding an orthologue or paralogue of any one of the polypeptide sequences given in Table A1 of Example 1. Preferably, the portion is a portion of a nucleic sequence encoding a polypeptide sequence polypeptide comprising: (i) a domain having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more amino acid sequence identity to a QLQ domain as represented by SEQ ID NO: 115; and (ii) a domain having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more amino acid sequence identity to a WRC domain as represented by SEQ ID NO: 116. Most preferably the portion is a portion of the nucleic acid sequence of SEQ ID NO: 1.
Portions useful in the methods of the invention, encode in one embodiment a RAA1-like polypeptide as defined herein, and have substantially the same biological activity as the amino acid sequences given in Table A2 of Example 1. Preferably, the portion is a portion of any one of the nucleic acids given in Table A2 of Example 1, or is a portion of a nucleic acid encoding an orthologue or paralogue of any one of the amino acid sequences given in Table A2 of Example 1. Preferably the portion is at least 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200 consecutive nucleotides in length, the consecutive nucleotides being of any one of the nucleic acid sequences given in Table A2 of Example 1, or of a nucleic acid encoding an orthologue or paralogue of any one of the amino acid sequences given in Table A2 of Example 1. Most preferably the portion is a portion of the nucleic acid of SEQ ID NO: 120. Preferably, the portion encodes a fragment of an amino acid sequence which, when used in the construction of a phylogenetic tree, such as the one depicted in FIG. 8, clusters with the group of RAA1-like polypeptides comprising the amino acid sequence represented by SEQ ID NO: 121 rather than with any other group.
Portions useful in the methods of the invention, encode in one embodiment a SYR polypeptide as defined herein, and have substantially the same biological activity as the amino acid sequences given in Table A3 of Example 1. Preferably, the portion is a portion of any one of the nucleic acids given in Table A3 of Example 1, or is a portion of a nucleic acid encoding an orthologue or paralogue of any one of the amino acid sequences given in Table A3 of Example 1. Preferably the portion is at least 150, 200, 250, 300, 350, 400, 450, 500, 550, 600 consecutive nucleotides in length, the consecutive nucleotides being of any one of the nucleic acid sequences given in Table A3 of Example 1, or of a nucleic acid encoding an orthologue or paralogue of any one of the amino acid sequences given in Table A3 of Example 1. Most preferably the portion is a portion of the nucleic acid of SEQ ID NO: 168. Preferably, the portion encodes 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 5 (a, b, c or d) and followed by the conserved motif 6 and preferably also by the conserved motif 7; or having at least 38% sequence identity to the sequence of SEQ ID NO: 169.
Portions useful in the methods of the invention, encode in one embodiment an ARKL polypeptide as defined herein, and have substantially the same biological activity as the amino acid sequences given in Table A4 of Example 1. Preferably, the portion is a portion of any one of the nucleic acids given in Table A4 of Example 1, or is a portion of a nucleic acid encoding an orthologue or paralogue of any one of the amino acid sequences given in Table A4 of Example 1. Preferably the portion is at least 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, consecutive nucleotides in length, the consecutive nucleotides being of any one of the nucleic acid sequences given in Table A4 of Example 1, or of a nucleic acid encoding an orthologue or paralogue of any one of the amino acid sequences given in Table A4 of Example 1. Most preferably the portion is a portion of the nucleic acid of SEQ ID NO: 212. Preferably, the portion encodes a fragment of an amino acid sequence which, when used in the construction of a phylogenetic tree, such as the one depicted in FIG. 17, clusters with the group of ARKL polypeptides comprising the amino acid sequence represented by SEQ ID NO: 213 rather than with any other group such as that represented by the Musmu_Goliath sequence.
Portions useful in the methods of the invention, encode in one embodiment a YTP polypeptide as defined herein, and have substantially the same biological activity as the amino acid sequences given in Table A5 of Example 1. Preferably, the portion is a portion of any one of the nucleic acids given in Table A5 of Example 1, or is a portion of a nucleic acid encoding an orthologue or paralogue of any one of the amino acid sequences given in Table A5 of Example 1. Preferably the portion is at least 300, 400, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1500, 2000, 2500, 3000 consecutive nucleotides in length, the consecutive nucleotides being of any one of the nucleic acid sequences given in Table A5 of Example 1, or of a nucleic acid encoding an orthologue or paralogue of any one of the amino acid sequences given in Table A5 of Example 1. Most preferably the portion is a portion of the nucleic acid of SEQ ID NO: 408. Preferably, the portion encodes a fragment of an amino acid sequence which, when used in the construction of a phylogenetic tree, such as the one depicted in FIG. 21, clusters with the Group 1, comprising the amino acid sequence represented by SEQ ID NO: 409 rather than with the YTP polypeptides in Group 2.
Another nucleic acid sequence variant useful in the methods of the invention is a nucleic acid sequence capable of hybridising, under reduced stringency conditions, preferably under stringent conditions, with a nucleic acid sequence encoding a polypeptide selected from the group consisting of: GRF polypeptide, RAA1-like polypeptide, SYR polypeptide, ARKL polypeptide, and YTP polypeptide respectively as defined herein, or with a portion as defined herein.
According to the present invention, there is provided a method for increasing yield-related traits in plants, comprising introducing and expressing in a plant a nucleic acid sequence capable of hybridizing to any one of the nucleic acid sequences given in Table A of Example 1, or comprising introducing and expressing in a plant a nucleic acid sequence capable of hybridising to a nucleic acid sequence encoding an orthologue, paralogue or homologue of any of the nucleic acid sequences given in Table A of Example 1.
Hybridising sequences useful in the methods of the invention encode a polypeptide selected from the group consisting of: GRF polypeptide, RAA1-like polypeptide, SYR polypeptide, ARKL polypeptide, and YTP polypeptide respectively as defined herein, and have substantially the same biological activity as the polypeptide sequences given in Table A of Example 1. Preferably, the hybridising sequence is capable of hybridising to any one of the nucleic acid sequences given in Table A of Example 1, or to a portion of any of these sequences, a portion being as defined above, or wherein the hybridising sequence is capable of hybridising to a nucleic acid sequence encoding an orthologue or paralogue of any one of the polypeptide sequences given in Table A of Example 1. Preferably, in one embodiment the hybridising sequence is capable of hybridising to a nucleic acid sequence encoding a polypeptide sequence comprising: (i) a domain having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more amino acid sequence identity to a QLQ domain as represented by SEQ ID NO: 115; and (ii) a domain having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more amino acid sequence identity to a WRC domain as represented by SEQ ID NO: 116. Most preferably, the hybridising sequence is capable of hybridising to a nucleic acid sequence as represented by SEQ ID NO: 1 or to a portion thereof.
Preferably, in one embodiment the hybridising sequence is capable of hybridising to a nucleic acid as represented by SEQ ID NO: 120 or to a portion thereof.
Preferably, the hybridising sequence encodes a polypeptide with an amino acid sequence which, when full-length and used in the construction of a phylogenetic tree, such as the one depicted in FIG. 8, clusters with the group of RAA1-like polypeptides comprising the amino acid sequence represented by SEQ ID NO: 121 rather than with any other group.
Preferably, in one embodiment the hybridising sequence is capable of hybridising to a nucleic acid as represented by SEQ ID NO: 168 or to a portion thereof.
Preferably, the 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 5 (a, b, c or d) and followed by the conserved motif 6 and preferably also by the conserved motif 7; or having at least 38% sequence identity to the sequence of SEQ ID NO: 169.
Preferably, in one embodiment the hybridising sequence is capable of hybridising to a nucleic acid as represented by SEQ ID NO: 212 or to a portion thereof.
Preferably, the hybridising sequence encodes a polypeptide with an amino acid sequence which, when full-length and used in the construction of a phylogenetic tree, such as the one depicted in FIG. 17, clusters with the group of ARKL polypeptides comprising the amino acid sequence represented by SEQ ID NO: 213 rather than with any other group such as that represented by the Musmu_Goliath sequence.
Preferably, in one embodiment the hybridising sequence is capable of hybridising to a nucleic acid as represented by SEQ ID NO: 408 or to a portion thereof.
Preferably, the hybridising sequence encodes a polypeptide with an amino acid sequence which, when full-length and used in the construction of a phylogenetic tree, such as the one with in FIG. 21, clusters with the Group 1, comprising the amino acid sequence represented by SEQ ID NO: 409 rather than with the YTP polypeptides in Group 2.
Another nucleic acid sequence variant useful in the methods of the invention is a splice variant encoding a polypeptide selected from the group consisting of: GRF polypeptide, RAA1-like polypeptide, SYR polypeptide, ARKL polypeptide, and YTP polypeptide respectively as defined hereinabove, a splice variant being as defined herein.
According to the present invention, there is provided a method for increasing yield-related traits, comprising introducing and expressing in a plant a splice variant of any one of the nucleic acid sequences given in Table A of Example 1, or a splice variant of a nucleic acid sequence encoding an orthologue, paralogue or homologue of any of the polypeptide sequences given in Table A of Example 1.
Preferred splice variants are in one embodiment splice variants of a nucleic acid sequence represented by SEQ ID NO: 1, or a splice variant of a nucleic acid sequence encoding an orthologue or paralogue of SEQ ID NO: 2. Preferably, the splice variant is a splice variant of a nucleic acid sequence encoding a polypeptide sequence comprising: (i) a domain having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more amino acid sequence identity to a QLQ domain as represented by SEQ ID NO: 115; and (ii) a domain having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more amino acid sequence identity to a WRC domain as represented by SEQ ID NO: 116.
Preferred splice variants are in one embodiment splice variants of a nucleic acid represented by SEQ ID NO: 120, or a splice variant of a nucleic acid encoding an orthologue or paralogue of SEQ ID NO: 121. Preferably, the amino acid sequence encoded by the splice variant, when used in the construction of a phylogenetic tree, such as the one depicted in FIG. 8, clusters with the group of RAA1-like polypeptides comprising the amino acid sequence represented by SEQ ID NO: 121 rather than with any other group.
Preferred splice variants are in one embodiment splice variants of a nucleic acid represented by SEQ ID NO: 168, or a splice variant of a nucleic acid encoding an orthologue or paralogue of SEQ ID NO: 169. Preferably, the amino acid sequence encoded by the splice variant is a polypeptide of about 65 to about 200 amino acids, comprising a leucine rich domain as defined above, preceded by the conserved tripeptide motif 5 (a, b, c or d) and followed by the conserved motif 6 and preferably also by the conserved motif 7; or having at least 38% sequence identity to the sequence of SEQ ID NO: 169.
Preferred splice variants are in one embodiment splice variants of a nucleic acid represented by SEQ ID NO: 212, or a splice variant of a nucleic acid encoding an orthologue or paralogue of SEQ ID NO: 213. Preferably, the amino acid sequence encoded by the splice variant, when used in the construction of a phylogenetic tree, such as the one depicted in FIG. 17, clusters with the group of ARKL polypeptides comprising the amino acid sequence represented by SEQ ID NO: 213 rather than with any other group such as that represented by the Musmu_Goliath sequence.
Preferred splice variants are in one embodiment splice variants of a nucleic acid represented by SEQ ID NO: 408, or a splice variant of a nucleic acid encoding an orthologue or paralogue of SEQ ID NO: 409. Preferably, the amino acid sequence encoded by the splice variant, when used in the construction of a phylogenetic tree, such as the one depicted in FIG. 21, clusters with the Group 1, comprising the amino acid sequence represented by SEQ ID NO: 409 rather than with the YTP polypeptides in Group 2.
Another nucleic acid sequence variant useful in performing the methods of the invention is an allelic variant of a nucleic acid sequence encoding a polypeptide selected from the group consisting of: GRF polypeptide, RAA1-like polypeptide, SYR polypeptide, ARKL polypeptide, and YTP polypeptide respectively as defined hereinabove, an allelic variant being as defined herein.
According to the present invention, there is provided a method for increasing yield-related traits, comprising introducing and expressing in a plant an allelic variant of any one of the nucleic acid sequences given in Table A of Example 1, or comprising introducing and expressing in a plant an allelic variant of a nucleic acid sequence encoding an orthologue, paralogue or homologue of any of the polypeptide sequences given in Table A of Example 1.
The allelic variants useful in one embodiment of the present invention have substantially the same biological activity as the GRF polypeptide of SEQ ID NO: 2 and any of the polypeptide sequences depicted in Table A of Example 1. Allelic variants exist in nature, and encompassed within the methods of the present invention is the use of these natural alleles. Preferably, the allelic variant is an allelic variant of SEQ ID NO: 1 or an allelic variant of a nucleic acid sequence encoding an orthologue or paralogue of SEQ ID NO: 2. Preferably, the allelic variant is an allelic variant of a polypeptide sequence comprising: (i) a domain having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more amino acid sequence identity to a QLQ domain as represented by SEQ ID NO: 115; and (ii) a domain having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more amino acid sequence identity to a WRC domain as represented by SEQ ID NO: 116.
The allelic variants useful in one embodiment of the present invention have substantially the same biological activity as the RAA1-like polypeptide of SEQ ID NO: 121 and any of the amino acids depicted in Table A of Example 1. Allelic variants exist in nature, and encompassed within the methods of the present invention is the use of these natural alleles. Preferably, the allelic variant is an allelic variant of SEQ ID NO: 120 or an allelic variant of a nucleic acid encoding an orthologue or paralogue of SEQ ID NO: 121. Preferably, the amino acid sequence encoded by the allelic variant, when used in the construction of a phylogenetic tree, such as the one depicted in FIG. 8, clusters with the RAA1-like polypeptides comprising the amino acid sequence represented by SEQ ID NO: 121 rather than with any other group.
The allelic variants useful in one embodiment of the present invention have substantially the same biological activity as the SYR polypeptide of SEQ ID NO: 169 and any of the amino acids depicted in Table A of Example 1. Allelic variants exist in nature, and encompassed within the methods of the present invention is the use of these natural alleles. Preferably, the allelic variant is an allelic variant of SEQ ID NO: 168 or an allelic variant of a nucleic acid encoding an orthologue or paralogue of SEQ ID NO: 169. Preferably, the amino acid sequence encoded by the allelic variant is a polypeptide of about 65 to about 200 amino acids, comprising a leucine rich domain as defined above, preceded by the conserved tripeptide motif 5 (a, b, c or d) and followed by the conserved motif 6 and preferably also by the conserved motif 7; or having at least 38% sequence identity to the sequence of SEQ ID NO: 169.
The allelic variants useful in one embodiment of the present invention have substantially the same biological activity as the ARKL polypeptide of SEQ ID NO: 213 and any of the amino acids depicted in Table A of Example 1. Allelic variants exist in nature, and encompassed within the methods of the present invention is the use of these natural alleles. Preferably, the allelic variant is an allelic variant of SEQ ID NO: 212 or an allelic variant of a nucleic acid encoding an orthologue or paralogue of SEQ ID NO: 213. Preferably, the amino acid sequence encoded by the allelic variant, when used in the construction of a phylogenetic tree, such as the one depicted in FIG. 17, clusters with the group of ARKL polypeptides comprising the amino acid sequence represented by SEQ ID NO: 213 rather than with any other group such as that represented by the Musmu_Goliath sequence.
The allelic variants useful in one embodiment of the present invention have substantially the same biological activity as the YTP polypeptide of SEQ ID NO: 409 and any of the amino acids depicted in Table A of Example 1. Allelic variants exist in nature, and encompassed within the methods of the present invention is the use of these natural alleles. Preferably, the allelic variant is an allelic variant of SEQ ID NO: 408 or an allelic variant of a nucleic acid encoding an orthologue or paralogue of SEQ ID NO: 409. Preferably, the amino acid sequence encoded by the allelic variant, when used in the construction of a phylogenetic tree, such as the one depicted in FIG. 21, clusters with the Group 1, comprising the amino acid sequence represented by SEQ ID NO: 409 rather than with the YTP polypeptides in Group 2.
Gene shuffling or directed evolution may also be used to generate variants of nucleic acid sequences encoding a polypeptide selected from the group consisting of: GRF polypeptide, RAA1-like polypeptide, SYR polypeptide, ARKL polypeptide, and YTP polypeptides respectively as defined above, the term “gene shuffling” being as defined herein.
According to the present invention, there is provided a method for increasing yield-related traits, comprising introducing and expressing in a plant a variant of any one of the nucleic acid sequences given in Table A of Example 1, or comprising introducing and expressing in a plant a variant of a nucleic acid sequence encoding an orthologue, paralogue or homologue of any of the polypeptide sequences given in Table A of Example 1, which variant nucleic acid sequence is obtained by gene shuffling.
Preferably in one embodiment, the variant nucleic acid sequence obtained by gene shuffling encodes a polypeptide sequence comprising: (i) a domain having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more amino acid sequence identity to a QLQ domain as represented by SEQ ID NO: 115; and (ii) a domain having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more amino acid sequence identity to a WRC domain as represented by SEQ ID NO: 116.
Preferably in one embodiment, the amino acid sequence encoded by the variant nucleic acid obtained by gene shuffling, when used in the construction of a phylogenetic tree such as the one depicted in FIG. 8, clusters with the group of RAA1-like polypeptides comprising the amino acid sequence represented by SEQ ID NO: 121 rather than with any other group. Preferably in one embodiment, the amino acid sequence encoded by the variant nucleic acid obtained by gene shuffling is a polypeptide of about 65 to about 200 amino acids, comprising a leucine rich domain as defined above, preceded by the conserved tripeptide motif 5 (a, b, c or d) and followed by the conserved motif 6 and preferably also by the conserved motif 7; or having at least 38% sequence identity to the sequence of SEQ ID NO: 169.
Preferably in one embodiment, the amino acid sequence encoded by the variant nucleic acid obtained by gene shuffling, when used in the construction of a phylogenetic tree such as the one depicted in FIG. 17, clusters with the group of ARKL polypeptides comprising the amino acid sequence represented by SEQ ID NO: 213 rather than with any other group such as that represented by the PF00097Musmu_Goliath sequence.
Preferably in one embodiment, the amino acid sequence encoded by the variant nucleic acid obtained by gene shuffling, when used in the construction of a phylogenetic tree such as the one depicted in FIG. 21, clusters with the Group 1, comprising the amino acid sequence represented by SEQ ID NO: 409 rather than with the YTP polypeptides in Group 2.
Furthermore, nucleic acid sequence variants may also be obtained 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.).
Nucleic acid sequences encoding GRF polypeptides may be derived from any natural or artificial source. The nucleic acid sequence may be modified from its native form in composition and/or genomic environment through deliberate human manipulation. Preferably the nucleic acid sequence encoding a GRF polypeptide is from a plant, further preferably from a dicotyledonous plant, more preferably from the family Brassicaceae, most preferably the nucleic acid sequence is from Arabidopsis thaliana. 
Nucleic acids encoding RAA1-like polypeptides may be derived from any natural or artificial source. The nucleic acid may be modified from its native form in composition and/or genomic environment through deliberate human manipulation. Preferably the RAA1-like polypeptide-encoding nucleic acid is from a plant, further preferably from a monocotyledonous plant, more preferably from the family Poaceae, most preferably the nucleic acid is from Oryza sativa. 
Nucleic acids encoding SYR polypeptides may be derived from any natural or artificial source. The nucleic acid may be modified from its native form in composition and/or genomic environment through deliberate human manipulation. Preferably the SYR polypeptide-encoding nucleic acid is from a plant, further preferably from a monocotyledonous plant, more preferably from the family Poaceae, most preferably the nucleic acid is from Oryza sativa. 
Nucleic acids encoding ARKL polypeptides may be derived from any natural or artificial source. The nucleic acid may be modified from its native form in composition and/or genomic environment through deliberate human manipulation. Preferably the ARKL polypeptide-encoding nucleic acid is from a plant, further preferably from a monocotyledonous plant, more preferably from the family Poaceae, most preferably the nucleic acid is from Oryza sativa. 
Nucleic acids encoding YTP polypeptides may also be encoded by a de novo designed YTP polypeptide, i.e. not derived from a natural source. The nucleic acid may be modified from its native form in composition and/or genomic environment through deliberate human manipulation. Preferably the YTP polypeptide-encoding nucleic acid is from a plant, further preferably from a monocotyledonous plant, more preferably from the family Poaceae, most preferably the nucleic acid is from Oryza sativa. 
Performance of the methods of the invention gives in one embodiment plants having enhanced yield-related traits. In particular performance of the methods of the invention gives plants having increased yield, especially increased seed yield relative to control plants. The terms “yield” and “seed yield” are described in more detail in the “definitions” section herein.
Performance of the methods of the invention gives in one embodiment plants having increased abiotic stress resistance (or abiotic stress tolerance, which terms are used interchangeably), effected as enhanced yield-related traits compared to control plants when grown under abiotic stress. In particular, performance of the methods of the invention gives plants having increased yield, especially increased seed yield and increased biomass relative to control plants. The terms “yield” and “seed yield” are described in more detail in the “definitions” section herein.
Reference herein to enhanced yield-related traits is taken to mean an increase in biomass (weight) of one or more parts of a plant, which may include aboveground (harvestable) parts and/or (harvestable) parts below ground.
In one embodiment, such harvestable parts are seeds, and performance of the methods of the invention results in plants having increased seed yield relative to the seed yield of control plants.
In one embodiment such harvestable parts are roots, flowers and/or seeds, and performance of the methods of the invention results in plants having increased biomass and/or seed yield relative to the seed yield of control plants.
In one embodiment, such harvestable parts are seeds, and performance of the methods of the invention results in plants having increased yield, total seed weight, seed filling rate, number of flowers (or florets), harvest index, and thousand kernel weight relative to control plants.
Taking corn as an example, a yield increase may be manifested as one or more of the following: increase in the number of plants established per hectare or acre, an increase in the number of ears per plant, an increase in the number of rows, number of kernels per row, kernel weight, thousand kernel weight, ear length/diameter, increase in the seed filling rate (which is the number of filled seeds divided by the total number of seeds and multiplied by 100), among others. Taking rice as an example, a yield increase may manifest itself as an increase in one or more of the following: number of plants per hectare or acre, number of panicles per plant, number of spikelets per panicle, number of flowers (florets) per panicle (which is expressed as a ratio of the number of filled seeds over the number of primary panicles), increase in the seed filling rate (which is the number of filled seeds divided by the total number of seeds and multiplied by 100), increase in thousand kernel weight, among others.
The present invention provides a method for increasing yield-related traits of plants relative to control plants, which method comprises increasing expression in a plant of a nucleic acid sequence encoding a polypeptide selected from the group consisting of: GRF polypeptide and RAA1-like polypeptide polypeptide respectively as defined herein.
The present invention provides a method for increasing abiotic stress resistance of plants, resulting in increased yield, especially seed yield and/or increased biomass of plants, relative to control plants, when grown under conditions of abiotic stress, which method comprises modulating expression, preferably increasing expression, in a plant of a nucleic acid encoding a SYR polypeptide as defined herein.
The present invention provides a method for increasing yield, especially seed yield of plants, relative to control plants, which method comprises modulating expression in a plant of a nucleic acid encoding an ARKL polypeptide as defined herein.
The present invention provides a method for increasing yield, especially seed yield of plants, relative to control plants, which method comprises modulating expression in a plant of a nucleic acid encoding a YTP polypeptide as defined herein.
Since the transgenic plants according to the present invention have increased yield-related traits, 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 control plants at a corresponding stage in their life cycle.
Besides the increased yield capacity, an increased efficiency of nutrient uptake may also contribute to the increase in yield. It is observed that the plants according to the present invention show a higher efficiency in nutrient uptake. Increased efficiency of nutrient uptake allows better growth of the plant, when the plant is under stress. It is also observed that the transgenic plants according to the present invention have increased drought stress tolerance, allowing the plants to continue growing under conditions that retard or inhibit growth of control plants.
The increased growth rate may be specific to one or more parts of a plant (including seeds), or may be throughout substantially the whole plant. Plants having an increased growth rate may have a shorter life cycle. The life cycle of a plant may be taken to mean the time needed to grow from a dry mature seed up to the stage where the plant has produced dry mature seeds, similar to the starting material. This life cycle may be influenced by factors such as early vigour, growth rate, greenness index, flowering time and speed of seed maturation. The increase in growth rate may take place at one or more stages in the life cycle of a plant or during substantially the whole plant life cycle. Increased growth rate during the early stages in the life cycle of a plant may reflect increased (early) 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 (a similar effect may be obtained with earlier flowering time; delayed flowering is usually not a desirede trait in crops). If the growth rate is sufficiently increased, it may allow for the further sowing of seeds of the same plant species (for example sowing and harvesting of rice plants followed by sowing and harvesting of further rice plants all within one conventional growing period). Similarly, if the growth rate is sufficiently increased, it may allow for the further sowing of seeds of different plants species (for example the sowing and harvesting of corn plants followed by, for example, the sowing and optional harvesting of soybean, potato or any other suitable plant). Harvesting additional times from the same rootstock in the case of some crop plants may also be possible. Altering the harvest cycle of a plant may lead to an increase in annual biomass production per acre (due to an increase in the number of times (say in a year) that any particular plant may be grown and harvested). An increase in growth rate may also allow for the cultivation of transgenic plants in a wider geographical area than their wild-type counterparts, since the territorial limitations for growing a crop are often determined by adverse environmental conditions either at the time of planting (early season) or at the time of harvesting (late season). Such adverse conditions may be avoided if the harvest cycle is shortened. The growth rate may be determined by deriving various parameters from growth curves, such parameters may be: T-Mid (the time taken for plants to reach 50% of their maximal size) and T-90 (time taken for plants to reach 90% of their maximal size), amongst others.
According to a preferred feature of the present invention, performance of the methods of the invention gives plants having an increased growth rate relative to control plants. Therefore, 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 sequence encoding a GRF polypeptide or a RAA1-like polypeptide or a YTP polypeptide or a ARKL polypeptide as defined herein.
According to a preferred feature of the present invention, performance of the methods of the invention gives plants having an increased growth rate relative to control plants when grown under abiotic stress conditions. Therefore, according to the present invention, there is provided a method for increasing the growth rate of plants under abiotic stress conditions, which method comprises modulating expression, preferably increasing expression, in a plant of a nucleic acid encoding a SYR polypeptide as defined herein.
Increased yield-related traits occur whether the plant is under non-stress conditions or whether the plant is exposed to various stresses compared to control plants grown under comparable conditions. 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 everyday biotic and/or abiotic (environmental) stresses to which a plant is exposed.
Abiotic stresses may be due to drought or excess water, anaerobic stress, salt stress, chemical toxicity, oxidative stress and hot, cold or freezing temperatures. 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. Biotic stresses are typically those stresses caused by pathogens, such as bacteria, viruses, fungi, nematodes, and insects. The term “non-stress” conditions as used herein are those environmental conditions that allow optimal growth of plants. Persons skilled in the art are aware of normal soil conditions and climatic conditions for a given location.
In particular, the methods of the present invention may be performed under non-stress conditions or under conditions of mild drought to give plants having increased yield relative to 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 signalling pathways and cellular responses, such as the production of stress proteins, up-regulation of anti-oxidants, accumulation of compatible solutes and growth arrest. The term “non-stress” conditions as used herein are those environmental conditions that allow optimal growth of plants. Persons skilled in the art are aware of normal soil conditions and climatic conditions for a given location.
An increase in yield and/or growth rate occurs whether the plant is under non-stress conditions or whether the plant is exposed to various stresses compared to control plants. Plants typically respond to exposure to stress by growing more slowly. In conditions of severe stress, the plant may even stop growing altogether. Mild stress on the other hand is defined herein as being any stress to which a plant is exposed which does not result in the plant ceasing to grow altogether without the capacity to resume growth. Due to advances in agricultural practices (irrigation, fertilization, pesticide treatments) severe stresses are not often encountered in cultivated crop plants. As a consequence, the compromised growth induced by mild stress is often an undesirable feature for agriculture. Mild stresses are the everyday biotic and/or abiotic (environmental) stresses to which a plant is exposed. Abiotic stresses may be due to drought or excess water, anaerobic stress, salt stress, chemical toxicity, oxidative stress and hot, cold or freezing temperatures. 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. Biotic stresses are typically those stresses caused by pathogens, such as bacteria, viruses, fungi and insects.
Performance of the methods of the invention gives in one embodiment plants grown under non-stress conditions or under mild stress conditions having increased yield-related traits, relative to control plants grown under comparable conditions. Therefore, according to the present invention, there is provided a method for increasing yield-related traits in plants grown under non-stress conditions or under mild stress conditions, which method comprises increasing expression in a plant of a nucleic acid sequence encoding a GRF polypeptide.
Performance of the methods according to the present invention results in plants grown under abiotic stress conditions having increased yield-related traits relative to control plants grown under comparable stress conditions. 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 signalling 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 should not be seen as a limitation to drought stress, but more as a screen to indicate the involvement of GRF polypeptides as defined above, in increasing yield-related traits relative to control plants grown in comparable stress conditions, in abiotic stresses in general.
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 stress caused by one or more of: NaCl, KCl, LiCl, MgCl2, CaCl2, amongst others.
Performance of the methods of the invention gives plants having increased yield-related traits, under abiotic stress conditions relative to control plants grown in comparable stress conditions. Therefore, according to the present invention, there is provided a method for increasing yield-related traits, in plants grown under abiotic stress conditions, which method comprises increasing expression in a plant of a nucleic acid sequence encoding a GRF polypeptide. According to one aspect of the invention, the abiotic stress is an osmotic stress, selected from one or more of the following: water stress, salt stress, oxidative stress and ionic stress.
Another example of abiotic environmental stress is the reduced availability of one or more nutrients that need to be assimilated by the plants for growth and development. Because of the strong influence of nutrition utilization efficiency on plant yield and product quality, a huge amount of fertilizer is poured onto fields to optimize plant growth and quality. Productivity of plants ordinarily is limited by three primary nutrients, phosphorous, potassium and nitrogen, which is usually the rate-limiting element in plant growth of these three. Therefore the major nutritional element required for plant growth is nitrogen (N). It is a constituent of numerous important compounds found in living cells, including amino acids, proteins (enzymes), nucleic acids, and chlorophyll. 1.5% to 2% of plant dry matter is nitrogen and approximately 16% of total plant protein. Thus, nitrogen availability is a major limiting factor for crop plant growth and production (Frink et al. (1999) Proc Natl Acad Sci USA 96(4): 1175-1180), and has as well a major impact on protein accumulation and amino acid composition. Therefore, of great interest are crop plants with increased yield-related traits, when grown under nitrogen-limiting conditions.
Performance of the methods of the invention gives plants grown under conditions of reduced nutrient availability, particularly under conditions of reduced nitrogen availablity, having increased yield-related traits relative to control plants grown under comparable conditions. Therefore, according to the present invention, there is provided a method for increasing yield-related traits in plants grown under conditions of reduced nutrient availablity, preferably reduced nitrogen availability, which method comprises increasing expression in a plant of a nucleic acid sequence encoding a GRF polypeptide. Reduced nutrient availability may result from a deficiency or excess of nutrients such as nitrogen, phosphates and other phosphorous-containing compounds, potassium, calcium, cadmium, magnesium, manganese, iron and boron, amongst others. Preferably, reduced nutrient availablity is reduced nitrogen availability.
Performance of the methods of the invention gives in one embodiment plants grown under non-stress conditions or under mild drought conditions increased yield relative to control plants grown under comparable conditions. Therefore, according to the present invention, there is provided a method for increasing yield in plants grown under non-stress conditions or under mild drought conditions, which method comprises modulating expression in a plant of a nucleic acid encoding a RAA1-like polypeptide.
Performance of the methods of the invention gives in one embodiment plants grown under conditions of nutrient deficiency, particularly under conditions of nitrogen deficiency, increased yield relative to control plants grown under comparable conditions. Therefore, according to the present invention, there is provided a method for increasing yield in plants grown under conditions of nutrient deficiency, which method comprises modulating expression in a plant of a nucleic acid encoding a RAA1-like polypeptide. Nutrient deficiency may result from a lack of nutrients such as nitrogen, phosphates and other phosphorous-containing compounds, potassium, calcium, cadmium, magnesium, manganese, iron and boron, amongst others.
Performance of the methods of the invention gives plants grown under abiotic stress conditions such as mild to severe drought conditions increased yield relative to control plants grown under comparable conditions. Therefore, according to the present invention, there is provided a method for increasing yield in plants grown under abiotic stress conditions such as mild to severe drought conditions, which method comprises increasing expression in a plant of a nucleic acid encoding a SYR polypeptide. The term “severe drought conditions” or “severe drought stress” as used herein are those drought conditions that cause a yield reduction of 50% or more in the control plants, compared to the yield of control plants grown under non-stress conditions.
Performance of the methods of the invention gives in one embodiment plants grown under conditions of nutrient deficiency, particularly under conditions of nitrogen deficiency, increased yield relative to control plants grown under comparable conditions. Therefore, according to the present invention, there is provided a method for increasing yield in plants grown under conditions of nutrient deficiency, which method comprises increasing expression in a plant of a nucleic acid encoding a SYR polypeptide. Nutrient deficiency may result from a lack of nutrients such as nitrogen, phosphates and other phosphorous-containing compounds, potassium, calcium, cadmium, magnesium, manganese, iron and boron, amongst others.
Performance of the methods of the invention gives plants grown under non-stress conditions or under mild drought conditions increased yield relative to control plants grown under comparable conditions. Therefore, according to the present invention, there is provided a method for increasing yield in plants grown under non-stress conditions or under mild drought conditions, which method comprises modulating expression in a plant of a nucleic acid encoding an ARKL polypeptide.
Performance of the methods of the invention gives in one embodiment plants grown under conditions of nutrient deficiency, particularly under conditions of nitrogen deficiency, increased yield relative to control plants grown under comparable conditions. Therefore, according to the present invention, there is provided a method for increasing yield in plants grown under conditions of nutrient deficiency, which method comprises modulating expression in a plant of a nucleic acid encoding an ARKL polypeptide. Nutrient deficiency may result from a lack of nutrients such as nitrogen, phosphates and other phosphorous-containing compounds, potassium, calcium, cadmium, magnesium, manganese, iron and boron, amongst others.
Performance of the methods of the invention gives plants grown under non-stress conditions or under mild drought conditions increased yield relative to control plants grown under comparable conditions. Therefore, according to the present invention, there is provided a method for increasing yield in plants grown under non-stress conditions or under mild drought conditions, which method comprises modulating expression in a plant of a nucleic acid encoding a YTP polypeptide.
Performance of the methods of the invention gives plants grown under conditions of nutrient deficiency, particularly under conditions of nitrogen deficiency, increased yield relative to control plants grown under comparable conditions. Therefore, according to the present invention, there is provided a method for increasing yield in plants grown under conditions of nutrient deficiency, which method comprises modulating expression in a plant of a nucleic acid encoding a YTP polypeptide. Nutrient deficiency may result from a lack of nutrients such as nitrogen, phosphates and other phosphorous-containing compounds, potassium, calcium, cadmium, magnesium, manganese, iron and boron, amongst others.
The present invention encompasses plants or parts thereof (including seeds) or cells thereof obtainable by the methods according to the present invention. The plants or parts thereof or cells thereof comprise a nucleic acid transgene encoding a polypeptide selected from the group consisting of: GRF polypeptide, RAA1-like polypeptide, SYR polypeptide, ARKL polypeptide, and YTP polypeptide respectively as defined above.
The invention also provides genetic constructs and vectors to facilitate introduction and/or increased expression in plants of nucleic acid sequences encoding a polypeptide selected from the group consisting of: GRF polypeptide, RAA1-like polypeptide, SYR polypeptide, ARKL polypeptide, and YTP polypeptide respectively. The gene constructs may be inserted into vectors, which may be commercially available, suitable for transforming into plants and for expression of the gene of interest in the transformed cells. The invention also provides use of a gene construct as defined herein in the methods of the invention.
More specifically, the present invention provides in one embodiment a construct comprising:

    
    
        (a) a nucleic acid sequence encoding a GRF polypeptide as defined above;
        (b) one or more control sequences capable of increasing expression of the nucleic acid sequence of (a); and optionally
        (c) a transcription termination sequence.
    
    


Preferably, the nucleic acid sequence encoding a GRF polypeptide is as defined above. The term “control sequence” and “termination sequence” are as defined herein.
Preferably, one of the control sequences of a construct is a constitutive promoter isolated from a plant genome. An example of a plant constitutive promoter is a GOS2 promoter, preferably a rice GOS2 promoter, more preferably a GOS2 promoter as represented by SEQ ID NO: 117.
More specifically, the present invention provides in one embodiment a construct comprising:

    
    
        (d) a nucleic acid encoding a RAA1-like polypeptide as defined above;
        (e) one or more control sequences capable of driving expression of the nucleic acid sequence of (a); and optionally
        (f) a transcription termination sequence.
    
    


Preferably, the nucleic acid encoding a RAA1-like polypeptide is as defined above. The term “control sequence” and “termination sequence” are as defined herein.
More specifically, the present invention provides in one embodiment a construct comprising:

    
    
        1. a nucleic acid encoding a SYR polypeptide as defined above;
        2. one or more control sequences capable of driving expression of the nucleic acid sequence of (a); and optionally
        3. a transcription termination sequence.
    
    


Preferably, the nucleic acid encoding a SYR polypeptide is as defined above. The term “control sequence” and “termination sequence” are as defined herein.
More specifically, the present invention provides in one embodiment a construct comprising:

    
    
        1. a nucleic acid encoding an ARKL polypeptide as defined above;
        2. one or more control sequences capable of driving expression of the nucleic acid sequence of (a); and optionally
        3. a transcription termination sequence.
    
    


Preferably, the nucleic acid encoding an ARKL polypeptide is as defined above. The term “control sequence” and “termination sequence” are as defined herein.
More specifically, the present invention provides in one embodiment a construct comprising:

    
    
        1. a nucleic acid encoding a YTP polypeptide as defined above;
        2. one or more control sequences capable of driving expression of the nucleic acid sequence of (a); and optionally
        3. a transcription termination sequence.
    
    


Preferably, the nucleic acid encoding a YTP polypeptide is as defined above. The term “control sequence” and “termination sequence” are as defined herein.
Plants are transformed with a vector comprising any of the nucleic acid sequences described above. The skilled artisan is well aware of the genetic elements that must be present on the vector in order to successfully transform, select and propagate host cells containing the sequence of interest. The sequence of interest is operably linked to one or more control sequences (at least to a promoter).
Advantageously, any type of promoter, whether natural or synthetic, may be used to increase expression of the nucleic acid sequence. A constitutive promoter is particularly useful in the methods, preferably a constitutive promoter isolated from a plant genome. The plant constitutive promoter drives expression of a coding sequence at a level that is in all instances below that obtained under the control of a 35S CaMV viral promoter.
Other organ-specific promoters, for example for preferred expression in leaves, stems, tubers, meristems, seeds (embryo and/or endosperm), are useful in performing the methods of the invention. See the “Definitions” section herein for definitions of the various promoter types.
Preferably in one embodiment the constitutive promoter is also a ubiquitous promoter. See the “Definitions” section herein for definitions of the various promoter types.
It should be clear that the applicability of the present invention is not restricted to a nucleic acid sequence encoding the GRF polypeptide, as represented by SEQ ID NO: 1, nor is the applicability of the invention restricted to expression of a GRF polypeptide-encoding nucleic acid sequence when driven by a constitutive promoter.
It should be clear that the applicability of the present invention is not restricted to the RAA1-like polypeptide-encoding nucleic acid represented by SEQ ID NO: 120, nor is the applicability of the invention restricted to expression of a RAA1-like polypeptide-encoding nucleic acid when driven by a constitutive promoter.
The constitutive promoter is preferably a GOS2 promoter, preferably a GOS2 promoter from rice. Further preferably the constitutive promoter is represented by a nucleic acid sequence substantially similar to SEQ ID NO: 124, most preferably the constitutive promoter is as represented by SEQ ID NO: 124 or SEQ ID NO: 211. According to another preferred feature of the invention, the constitutive promoter is a High Mobility Group Protein (HMGP) promoter, preferably a HMGP promoter from rice, more preferably substantially similar to SEQ ID NO: 125, most preferably identical to SEQ ID NO: 125. See Table 2 in the “Definitions” section herein for further examples of constitutive promoters.
It should be clear that the applicability of the present invention is not restricted to the SYR polypeptide-encoding nucleic acid represented by SEQ ID NO: 168, nor is the applicability of the invention restricted to expression of a SYR polypeptide-encoding nucleic acid when driven by a constitutive promoter.
The constitutive promoter is preferably a GOS2 promoter, preferably a GOS2 promoter from rice. Further preferably the constitutive promoter is represented by a nucleic acid sequence substantially similar to SEQ ID NO: 172 or SEQ ID NO: 211, most preferably the constitutive promoter is as represented by SEQ ID NO: 172 or SEQ ID NO: 211. See Table 2 in the “Definitions” section herein for further examples of useful constitutive promoters.
It should be clear that the applicability of the present invention is not restricted to the ARKL polypeptide-encoding nucleic acid represented by SEQ ID NO: 212, nor is the applicability of the invention restricted to expression of an ARKL polypeptide-encoding nucleic acid when driven by a constitutive promoter, or when driven by a root-specific promoter.
The constitutive promoter is preferably a GOS2 promoter, preferably a GOS2 promoter from rice. Further preferably the constitutive promoter is represented by a nucleic acid sequence substantially similar to SEQ ID NO: 406, most preferably the constitutive promoter is as represented by SEQ ID NO: 406 or SEQ ID NO: 211. See Table 2 in the “Definitions” section herein for further examples of constitutive promoters.
It should be clear that the applicability of the present invention is not restricted to the YTP polypeptide-encoding nucleic acid represented by SEQ ID NO: 1, nor is the applicability of the invention restricted to expression of a YTP polypeptide-encoding nucleic acid when driven by a constitutive promoter.
The constitutive promoter is preferably a GOS2 promoter, preferably a GOS2 promoter from rice. Further preferably the constitutive promoter is represented by a nucleic acid sequence substantially similar to SEQ ID NO: 548, most preferably the constitutive promoter is as represented by SEQ ID NO: 548 or SEQ ID NO: 211. See Table 2 in the “Definitions” section herein for further examples of constitutive promoters.
Optionally, one or more terminator sequences may be used in the construct introduced into a plant. Additional regulatory elements may include transcriptional as well as translational increasers. Those skilled in the art will be aware of terminator and increaser sequences that may be suitable for use in performing the invention. An intron sequence may also be added to the 5′ untranslated region (UTR) or in the coding sequence to increase the amount of the mature message that accumulates in the cytosol, as described in the definitions section. Other control sequences (besides promoter, increaser, silencer, intron sequences, 3′UTR and/or 5′UTR regions) may be protein and/or RNA stabilizing elements. Such sequences would be known or may readily be obtained by a person skilled in the art.
Optionally, one or more terminator sequences may be used in the construct introduced into a plant. Preferably, the construct comprises an expression cassette essentially similar or identical to SEQ ID NO: 166, comprising the GOS2 promoter, the nucleic acid encoding the RAA1-like polypeptide. In an alternative embodiment, the construct comprises an expression cassette essentially similar or identical to SEQ ID NO: 167, comprising the HMGP promoter, the nucleic acid encoding the RAA1-like polypeptide.
Optionally, one or more terminator sequences may be used in the construct introduced into a plant. Preferably, the construct comprises an expression cassette essentially similar or identical to SEQ ID NO: 407, comprising the GOS2 promoter, the nucleic acid encoding the Orysa_ARKL1 polypeptide and the T-zein+T-rubisco transcription terminator sequence.
Optionally, one or more terminator sequences may be used in the construct introduced into a plant. Preferably, the construct comprises an expression cassette essentially similar or identical to SEQ ID NO 549, comprising the GOS2 promoter, the nucleic acid encoding the YTP polypeptide and the T-zein+T-rubisco transcription terminator sequence.
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. An intron sequence may also be added to the 5′ untranslated region (UTR) or in the coding sequence to increase the amount of the mature message that accumulates in the cytosol, as described in the definitions section. Other control sequences (besides promoter, enhancer, silencer, intron sequences, 3′UTR and/or 5′UTR regions) may be protein and/or RNA stabilizing elements. Such sequences would be known or may readily be obtained by a person skilled in the art.
The genetic constructs of the invention may further include an origin of replication sequence that is required for maintenance and/or replication in a specific cell type. One example is when a genetic construct is required to be maintained in a bacterial cell as an episomal genetic element (e.g. plasmid or cosmid molecule). Preferred origins of replication include, but are not limited to, the f1-on and colE1.
For the detection of the successful transfer of the nucleic acid sequences as used in the methods of the invention and/or selection of transgenic plants comprising these nucleic acid sequences, it is advantageous to use marker genes (or reporter genes). Therefore, the genetic construct may optionally comprise a selectable marker gene. Selectable markers are described in more detail in the “definitions” section herein.
The marker genes may be removed or excised from the transgenic cell once they are no longer needed. Techniques for marker removal are known in the art, useful techniques are described above in the definitions section.
It is known that upon stable or transient integration of nucleic acid sequences into plant cells, only a minority of the cells takes up the foreign DNA and, if desired, integrates it into its genome, depending on the expression vector used and the transfection technique used. To identify and select these integrants, a gene coding for a selectable marker (such as the ones described above) is usually introduced into the host cells together with the gene of interest. These markers can for example be used in mutants in which these genes are not functional by, for example, deletion by conventional methods. Furthermore, nucleic acid sequence molecules encoding a selectable marker can be introduced into a host cell on the same vector that comprises the sequence encoding the polypeptides of the invention or used in the methods of the invention, or else in a separate vector. Cells which have been stably transfected with the introduced nucleic acid sequence can be identified for example by selection (for example, cells which have integrated the selectable marker survive whereas the other cells die). The marker genes may be removed or excised from the transgenic cell once they are no longer needed. Techniques for marker gene removal are known in the art, useful techniques are described above in the definitions section.
The invention also provides a method for the production of transgenic plants having increased yield-related traits relative to control plants, comprising introduction and expression in a plant of any nucleic acid sequence encoding a polypeptide selected from the group consisting of: GRF polypeptide, RAA1-like polypeptide, SYR polypeptide, ARKL polypeptide, and YTP polypeptide respectively as defined hereinabove.
More specifically, the present invention provides in one embodiment a method for the production of transgenic plants having increased yield-related traits relative to control plants, which method comprises:

    
    
        (i) introducing and expressing in a plant, plant part, or plant cell a nucleic acid sequence encoding a GRF polypeptide, under the control of plant constitutive promoter; and
        (ii) cultivating the plant cell, plant part or plant under conditions promoting plant growth and development.
    
    


The nucleic acid sequence of (i) may be any of the nucleic acid sequences capable of encoding a GRF polypeptide as defined herein.
More specifically, the present invention provides in one embodiment a method for the production of transgenic plants having increased enhanced yield-related traits, particularly increased biomass and/or seed yield, which method comprises:

    
    
        i) introducing and expressing in a plant or plant cell a RAA1-like polypeptide-encoding nucleic acid; and
        ii) cultivating the plant cell under conditions promoting plant growth and development.
    
    


The nucleic acid of (i) may be any of the nucleic acids capable of encoding a RAA1-like polypeptide as defined herein.
More specifically, the present invention provides in one embodiment a method for the production of transgenic plants having increased enhanced yield-related traits, particularly increased (seed) yield and/or increased biomass, which method comprises:

    
    
        (i) introducing and expressing in a plant or plant cell a SYR polypeptide-encoding nucleic acid; and
        (ii) cultivating the plant cell under conditions promoting plant growth and development.
    
    


The nucleic acid of (i) may be any of the nucleic acids capable of encoding a SYR polypeptide as defined herein.
More specifically, the present invention provides in one embodiment a method for the production of transgenic plants having increased enhanced yield-related traits, particularly increased (seed) yield, which method comprises:
(i) introducing and expressing in a plant or plant cell an ARKL polypeptide-encoding nucleic acid; and
(ii) cultivating the plant cell under conditions promoting plant growth and development.
The nucleic acid of (i) may be any of the nucleic acids capable of encoding an ARKL polypeptide as defined herein.
More specifically, the present invention provides in one embodiment a method for the production of transgenic plants having increased enhanced yield-related traits, particularly increased (seed) yield, which method comprises:

    
    
        i) introducing and expressing in a plant or plant cell a YTP polypeptide-encoding nucleic acid; and
        ii) cultivating the plant cell under conditions promoting plant growth and development.
    
    


The nucleic acid of (i) may be any of the nucleic acids capable of encoding a YTP polypeptide as defined herein.
The nucleic acid sequence 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 sequence is preferably introduced into a plant by transformation. The term “transformation” is described in more detail in the “definitions” section herein.
The genetically modified plant cells can be regenerated via all methods with which the skilled worker is familiar. Suitable methods can be found in the abovementioned publications by S. D. Kung and R. Wu, Potrykus or Höfgen and Willmitzer.
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. To select transformed plants, the plant material obtained in the transformation is, as a rule, subjected to selective conditions so that transformed plants can be distinguished from untransformed plants. For example, the seeds obtained in the above-described manner can be planted and, after an initial growing period, subjected to a suitable selection by spraying. A further possibility consists in growing the seeds, if appropriate after sterilization, on agar plates using a suitable selection agent so that only the transformed seeds can grow into plants. Alternatively, the transformed plants are screened for the presence of a selectable marker such as the ones described above.
Following DNA transfer and regeneration, putatively transformed plants may also 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).
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 sequence encoding a polypeptide selected from the group consisting of: GRF polypeptide, RAA1-like polypeptide, SYR polypeptide, ARKL polypeptide, and YTP polypeptide respectively as defined hereinabove, opereably linked to a plant constitutive promoter. Preferred host cells according to the invention are plant cells. Host plants for the nucleic acid sequences or the vector used in the method according to the invention, the expression cassette or construct or vector are, in principle, advantageously all plants, which are capable of synthesizing the polypeptides used in the inventive method.
The methods of the invention are advantageously applicable to any plant. 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. According to a preferred embodiment of the present invention, the plant is a crop plant. Examples of crop plants include soybean, sunflower, canola, alfalfa, rapeseed, cotton, tomato, potato and tobacco. Further preferably, the plant is a monocotyledonous plant. Examples of monocotyledonous plants include sugarcane. More preferably the plant is a cereal. Examples of cereals include rice, maize, wheat, barley, millet, rye, triticale, sorghum, emmer, spelt, secale, einkorn, teff, milo and oats.
The invention also extends to harvestable parts of a plant comprising an isolated nucleic acid sequence encoding a polypeptide selected from the group consisting of: GRF polypeptide, RAA1-like polypeptide, SYR polypeptide, ARKL polypeptide, and YTP polypeptide respectively (as defined hereinabove) operably linked to a plant constitutive promoter, 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.
According to a preferred feature of the invention, the modulated expression is increased expression. Methods for increasing expression of nucleic acids or genes, or gene products, are well documented in the art and examples are provided in the definitions section.
As mentioned above, a preferred method for increasing expression of a nucleic acid sequence encoding a polypeptide selected from the group consisting of: GRF polypeptide, RAA1-like polypeptide, SYR polypeptide, ARKL polypeptide, and YTP polypeptide respectively is by introducing and expressing in a plant a nucleic acid sequence encoding a polypeptide selected from the group consisting of: GRF polypeptide, RAA1-like polypeptide, SYR polypeptide, ARKL polypeptide, and YTP polypeptide respectively; however the effects of performing the method, i.e. increasing yield-related traits, may also be achieved using other well known techniques, including but not limited to T-DNA activation tagging, TILLING, homologous recombination. A description of these techniques is provided in the definitions section.
The present invention also encompasses in one embodiment use of nucleic acid sequences encoding GRF polypeptides as described herein and use of these GRF polypeptides in increasing any of the aforementioned yield-related traits in plants, under normal growth conditions, under abiotic stress growth (preferably osmotic stress growth conditions) conditions, and under growth conditions of reduced nutrient availability, preferably under conditions of reduced nitrogen availability.
The present invention also encompasses in one embodiment use of nucleic acids encoding RAA1-like polypeptides as described herein and use of these RAA1-like polypeptides in enhancing any of the aforementioned yield-related traits in plants.
The present invention also encompasses in one embodiment use of nucleic acids encoding SYR polypeptides as described herein and use of these SYR polypeptides in enhancing any of the aforementioned yield-related traits in plants when grown under abiotic stress conditions.
The present invention also encompasses in one embodiment use of nucleic acids encoding ARKL polypeptides as described herein and use of these ARKL polypeptides in enhancing any of the aforementioned yield-related traits in plants.
The present invention also encompasses in one embodiment use of nucleic acids encoding YTP polypeptides as described herein and use of these YTP polypeptides in enhancing any of the aforementioned yield-related traits in plants.
Nucleic acid sequences encoding a polypeptide selected from the group consisting of: GRF polypeptide, RAA1-like polypeptide, SYR polypeptide, ARKL polypeptide, and YTP polypeptide respectively, described herein, or the polypeptides of the invention themselves, may find use in breeding programmes in which a DNA marker is identified that may be genetically linked to a GRF polypeptide-encoding gene. The genes/nucleic acid sequences, or the GRF polypeptide, RAA1-like polypeptide, SYR polypeptide, ARKL polypeptide, and YTP polypeptide respectively, themselves 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-related traits, as defined hereinabove in the methods of the invention.
Allelic variants of a gene/nucleic acid sequence encoding a polypeptide selected from the group consisting of: GRF polypeptide, RAA1-like polypeptide, SYR polypeptide, ARKL polypeptide, and YTP polypeptide respectively 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-related traits. 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.
Nucleic acid sequences encoding polypeptide selected from the group consisting of: GRF polypeptide, RAA1-like polypeptide, SYR polypeptide, ARKL polypeptide, and YTP polypeptide respectively 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 nucleic acid sequences encoding a polypeptide selected from the group consisting of: GRF polypeptide, RAA1-like polypeptide, SYR polypeptide, ARKL polypeptide, and YTP polypeptide respectively requires only a nucleic acid sequence of at least 15 nucleotides in length. The nucleic acid sequences encoding a polypeptide selected from the group consisting of: GRF polypeptide, RAA1-like polypeptide, SYR polypeptide, ARKL polypeptide, and YTP polypeptide respectively 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 nucleic acid sequences encoding a polypeptide selected from the group consisting of: GRF polypeptide, RAA1-like polypeptide, SYR polypeptide, ARKL polypeptide, and YTP polypeptide respectively. 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 acid sequences 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 nucleic acid sequence encoding a GRF polypeptide 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 sequence 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 sequence 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 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 sequence amplification-based methods for genetic and physical mapping may be carried out using the nucleic acid sequences. 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 sequence Res. 18:3671), Radiation Hybrid Mapping (Walter et al. (1997) Nat. Genet. 7:22-28) and Happy Mapping (Dear and Cook (1989) Nucleic acid sequence Res. 17:6795-6807). For these methods, the sequence of a nucleic acid sequence 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 one embodiment in plants having increased yield-related traits, as described hereinbefore. These traits may also be combined with other economically advantageous traits, such as further yield-increasing traits, tolerance to abiotic and biotic stresses, tolerance to herbicides, insectides, traits modifying various architectural features and/or biochemical and/or physiological features.
The methods according to the present invention result in one embodiment in plants having enhanced yield-related traits, as described hereinbefore. These traits 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.
Methods for gene stacking in transgenic plants are well known in the art (see for example, a review by Halpin (2005) Plant Biotech J (3): 141-155. Gene stacking can proceed by interative steps, where two or more transgenes can be sequentially introduced into a plant by crossing a plant containing one transgene with individuals harbouring other transgenes or, alternatively, by re-transforming (or super-transforming) a plant containing one transgene with new genes. One limitation of the iterative procedure is that the transgenes are not linked and will be located at different random loci in the plant genome. The consequence is that the two loci can segregate apart in subsequent generations, which has consequences for breeding programs.
Alternatively, gene stacking can occur via co-transformation, which is faster and can be used in a whole range of transformation techniques. When using Agrobacterium transformation for example, the transgenes (at least two) can be present in a number of conformations:

    
    
        (i) the coding sequences are fused to form a single polypeptide when translated, and placed under the control of a single promoter;
        (ii) the coding sequences are sequentially placed downstream of a single promoter, separated by nucleic acid signals that influence mRNA synthesis (internal ribosome entry sites IRES, 2A stuttering signals, etc.), or polypeptide synthesis (polyproteins separated by protease substrate sites, etc.);
        (iii) the coding sequences are independently driven by separate promoters, and the promoter-coding sequence combinations are located within the same T-DNA;
        (iv) the coding sequences are independently driven by separate promoters, and the promoter-coding sequence combinations are located in different T-DNAs on the same plasmid;
        (v) the coding sequences are independently driven by separate promoters, and the promoter-coding sequence combinations are located in different T-DNAs on different plasmids hosted in the same or in separate Agrobacterium strains.
    
    


In another embodiment, the present invention provides a method for increasing yield-related traits in plants relative to control plants, comprising increasing expression in a plant of a nucleic acid sequence encoding a GRF polypeptide and modulating expression in the same plant of a nucleic acid sequence encoding a second polypeptide
Surprisingly, it has now been found that increasing expression in a plant of a nucleic acid sequence encoding a GRF polypeptide gives plants having increased yield-related traits relative to control plants. According to a first embodiment, the present invention provides a method for increasing yield-related traits in plants relative to control plants, comprising increasing expression in a plant of a nucleic acid sequence encoding a GRF polypeptide.
In one embodiment the invention relates to subject matter summarized as follows:

    1. A method for increasing yield-related traits in plants relative to control plants, comprising increasing expression in a plant of a nucleic acid sequence encoding a Growth-Regulating Factor (GRF) polypeptide, which GRF polypeptide comprises; (i) a domain having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more amino acid sequence identity to a QLQ domain as represented by SEQ ID NO: 115; and (ii) a domain having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more amino acid sequence identity to a WRC domain as represented by SEQ ID NO: 116, and optionally selecting for plants having increased yield-related traits.
    2. Method according to item 1, wherein said GRF polypeptide comprises: (i) a QLQ domain with an InterPro accession IPR014978 (PFAM accession PF08880); (ii) a WRC domain with an InterPro accession IPR014977 (PFAM accession PF08879); and (iii) an Effector of Transcription (ET) domain comprising three Cys and one His residues in a conserved spacing (CX9CX10CX2H).
    3. Method according to item 1 or 2, wherein said GRF polypeptide has in increasing order of preference at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more amino acid sequence identity to the GRF polypeptide as represented by SEQ ID NO: 2 or to any of the polypeptide sequences given in Table A herein.
    4. Method according to any preceding item, wherein said nucleic acid sequence encoding a GRF polypeptide is represented by any one of the nucleic acid sequence SEQ ID NOs given in Table A or a portion thereof, or a sequence capable of hybridising with any one of the nucleic acid sequences SEQ ID NOs given in Table A.
    5. Method according to any preceding item, wherein said nucleic acid sequence encodes an orthologue or paralogue of any of the polypeptide sequence SEQ ID NOs given in Table A.
    6. Method according to any preceding item, wherein said increased expression is effected by any one or more of: T-DNA activation tagging, TILLING, or homologous recombination.
    7. Method according to any preceding item, wherein said increased expression is effected by introducing and expressing in a plant a nucleic acid sequence encoding a GRF polypeptide.
    8. Method according to any preceding item, wherein said increased yield-related trait is one or more of: (i) increased early vigour; (ii) increased aboveground biomass; (iii) increased total seed yield per plant; (iv) increased seed filling rate; (v) increased harvest index; or (vi) increased thousand kernel weight (TKW).
    9. Method according to any preceding item, wherein said nucleic acid sequence is operably linked to a constitutive promoter, preferably to a plant constitutive promoter, more preferably to a GOS2 promoter, most preferably to a GOS2 promoter from rice as represented by SEQ ID NO: 117.
    10. Method according to any preceding item, wherein said nucleic acid sequence encoding a GRF polypeptide is of plant origin, preferably from a dicotyledonous plant, further preferably from the family Brassicaceae, most preferably from Arabidopsis thaliana. 
    11. Plants, parts thereof (including seeds), or plant cells obtainable by a method according to any preceding item, wherein said plant, part or cell thereof comprises an isolated nucleic acid transgene encoding a GRF polypeptide operably linked to a plant constitutive promoter.
    12. Construct comprising:
    
        1. A nucleic acid sequence encoding a GRF polypeptide as defined in any one of items 1 to 5;
        2. one or more control sequences capable of driving expression of the nucleic acid sequence of (a); and optionally
        3. a transcription termination sequence.
    
    
    13. Construct according to item 12, wherein said control sequence is a plant constitutive promoter, preferably a GOS2 promoter, more preferably a GOS2 promoter as represented by SEQ ID NO: 117.
    14. Use of a construct according to items 12 or 13 in a method for making plants having increased yield-related traits relative to control plants, which increased yield-related traits are one or more of: (i) increased early vigour; (ii) increased aboveground biomass; (iii) increased total seed yield per plant; (iv) increased seed filling rate; (v) increased harvest index; or (vi) increased thousand kernel weight (TKW).
    15. Plant, plant part or plant cell transformed with a construct according to item 12 or 13.
    16. Method for the production of transgenic plants having increased yield-related traits relative to control plants, comprising:
    
        (i) introducing and expressing in a plant, plant part, or plant cell, a nucleic acid sequence encoding a GRF polypeptide as defined in any one of items 1 to 5, under the control of plant constitutive promoter; and
        (ii) cultivating the plant cell, plant part, or plant under conditions promoting plant growth and development.
    
    
    17. Transgenic plant having increased yield-related traits relative to control plants, resulting from increased expression of a nucleic acid sequence encoding a GRF polypeptide as defined in any one of items 1 to 5, operably linked to a plant constitutive promoter, or a transgenic plant cell or transgenic plant part derived from said transgenic plant.
    18. Transgenic plant according to item 11, 15 or 17, wherein said plant is a crop plant or a monocot or a cereal, such as rice, maize, wheat, barley, millet, rye, triticale, sorghum and oats, or a transgenic plant cell derived from said transgenic plant.
    19. Harvestable parts comprising an isolated nucleic acid sequence encoding a GRF polypeptide of a plant according to item 18, wherein said harvestable parts are preferably seeds.
    20. Products derived from a plant according to item 18 and/or from harvestable parts of a plant according to item 19.
    21. Use of a nucleic acid sequence encoding a GRF polypeptide as defined in any one of items 1 to 5 in increasing yield-related traits, comprising one or more of: (i) increased early vigour; (ii) increased aboveground biomass; (iii) increased total seed yield per plant; (iv) increased seed filling rate; (v) increased harvest index; or (vi) increased thousand kernel weight (TKW).


In one embodiment the invention relates to subject matter summarized as follows:

    22. A method for enhancing yield-related traits in plants relative to control plants, comprising modulating expression in a plant of a nucleic acid encoding a RAA1-like polypeptide, wherein said RAA1-like polypeptide comprises two or more of the following motifs:









(SEQ ID NO: 162)








(i)motif 1:
GVW(V/L)F,


 







(SEQ ID NO: 163)








(ii)motif 2:
LGW(E/S)RY(Y/F),


 







(SEQ ID NO: 164)








(iii) motif 3:
(D/H)L(L/I)S(I/V/L)P(R/K/A)(S/D)F,


 







(SEQ ID NO: 165)








(iv)motif 4:
(H/Y)(F/M)YD(V/I)VVK(N/T)(R/P),






    23. Method according to item 22, wherein said RAA1-like polypeptide furthermore has a MW between 10 and 21 KDa and a pl above 8.5.
    24. Method according to item 22 or 23, wherein said modulated expression is effected by introducing and expressing in a plant a nucleic acid encoding a RAA1-like polypeptide.
    25. Method according to any preceding item, wherein said nucleic acid encoding a RAA1-like polypeptide encodes any one of the proteins listed in Table A or is a portion of such a nucleic acid, or a nucleic acid capable of hybridising with such a nucleic acid.
    26. Method according to any preceding item, wherein said nucleic acid sequence encodes an orthologue or paralogue of any of the proteins given in Table A.
    27. Method according to any preceding item, wherein said enhanced yield-related traits comprise increased yield, preferably increased biomass and/or increased seed yield relative to control plants.
    28. Method according to any one of items 22 to 27, wherein said enhanced yield-related traits are obtained under non-stress conditions.
    29. Method according to any one of items 22 to 27, wherein said enhanced yield-related traits are obtained under conditions of nitrogen deficiency.
    30. Method according to any one of items 24 to 29, wherein said nucleic acid is operably linked to a constitutive promoter
    31. Method according to item 30, wherein said constitutive promoter is a GOS2 promoter or an HMGP promoter, preferably a GOS2 promoter or HMGP promoter from rice.
    32. Method according to any preceding item, wherein said nucleic acid encoding a RAA1 polypeptide is of plant origin, preferably from a dicotyledonous plant, further preferably from the family Poaceae, more preferably from the genus Oryza, most preferably from Oryza sativa. 
    33. Plant or part thereof, including seeds, obtainable by a method according to any preceding item, wherein said plant or part thereof comprises a recombinant nucleic acid encoding a RAA1-like polypeptide.
    34. Construct comprising:
    
        (i) nucleic acid encoding a RAA1-like polypeptide;
        (ii) one or more control sequences capable of driving expression of the nucleic acid sequence of (a); and optionally
        (iii) a transcription termination sequence.
    
    
    35. Construct according to item 34, wherein one of said control sequences is a constitutive promoter.
    36. Construct according to item 35, wherein said constitutive promoter is a GOS2 promoter or an HMGP promoter, preferably a GOS2 promoter or an HMGP promoter from rice.
    37. Use of a construct according to any of items 34 to 36 in a method for making plants having increased yield, particularly increased biomass and/or increased seed yield relative to control plants.
    38. Plant, plant part or plant cell transformed with a construct according to any of items 34 to 36.
    39. Method for the production of a transgenic plant having increased yield, particularly increased biomass and/or increased seed yield relative to control plants, comprising:
    
        (i) introducing and expressing in a plant a nucleic acid encoding a RAA1-like polypeptide; and
        (ii) cultivating the plant cell under conditions promoting plant growth and development.
    
    
    40. Transgenic plant having increased yield, particularly increased biomass and/or increased seed yield, relative to control plants, resulting from modulated expression of a nucleic acid encoding a RAA1-like polypeptide, or a transgenic plant cell derived from said transgenic plant.
    41. Transgenic plant according to item 33, 38 or 39, or a transgenic plant cell derived thereof, wherein said plant is a crop plant or a monocot or a cereal, such as rice, maize, wheat, barley, millet, rye, triticale, sorghum and oats.
    42. Harvestable parts of a plant according to item 41, wherein said harvestable parts are preferably root biomass and/or seeds.
    43. Products derived from a plant according to item 41 and/or from harvestable parts of a plant according to 42.
    44. Use of a nucleic acid encoding a RAA1-like polypeptide in increasing yield, particularly in increasing seed yield and/or root biomass in plants, relative to control plants.


In one embodiment the invention relates to subject matter summarized as follows:

    45. A method for increasing abiotic stress resistance in plants relative to control plants, comprising modulating expression in a plant of a nucleic acid encoding a SYR polypeptide, which SYR polypeptide comprises a leucine rich domain, preceded by the conserved tripeptide motif 5 (one of SEQ ID NO: 173, 174, 175 or 176)) and followed by the conserved motif 6 (SEQ ID NO: 177), wherein said increased abiotic stress resistance is increased nutrient uptake efficiency and/or increased drought stress tolerance, relative to control plants.
    46. Method according to item 45, wherein said SYR polypeptide has, in increasing order of preference, at least 27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more sequence identity to the SYR polypeptide represented by SEQ ID NO: 169.
    47. Method according to item 45 or 46, wherein said nucleic acid encoding a SYR polypeptide is represented by any one of the nucleic acid SEQ ID NOs given in Table A or a portion thereof, or a sequence capable of hybridising with any one of the nucleic acids SEQ ID NOs given in Table A.
    48. Method according to any of items 45 to 47, wherein said nucleic acid sequence encodes an orthologue or paralogue of any of the SEQ ID NOs given in Table A.
    49. Method according to any preceding item, wherein said SYR protein furthermore comprises the conserved motif 7 (SEQ ID NO: 178).
    50. Method according to any preceding item, wherein said nutrient uptake efficiency results in increased seed yield and/or increased biomass.
    51. Method of item 50, wherein said increased seed yield comprises at least increased total weight of seeds, Thousand Kernel Weight and/or increased number of filled seeds.
    52. Method of item 50, wherein said increased biomass is increased shoot biomass and/or increased root biomass.
    53. Method according to any preceding item, wherein said increased nutrient uptake efficiency occurs under mild drought conditions.
    54. Method according to any preceding item, wherein said increased drought stress tolerance results in increased seed yield.
    55. Method of item 54, wherein said increased seed yield comprises at least increased total weight of seeds, fillrate and/or Harvest Index.
    56. Method according to any preceding item, wherein said modulated expression is effected by introducing and expressing in a plant a nucleic acid encoding a SYR polypeptide.
    57. Method according to item 56, wherein said nucleic acid is operably linked to a constitutive promoter, preferably to a GOS2 promoter.
    58. Method according to any preceding item, wherein said nucleic acid encoding a SYR polypeptide is of plant origin, preferably from a monocotyledonous plant, further preferably from the family Poaceae, more preferably from the genus Oryza, most preferably from Oryza sativa. 
    59. Use of a construct in a method for making plants having increased abiotic stress resistance, said construct comprising
    
        (a) nucleic acid encoding a SYR polypeptide as defined in any one of items 45 to 49;
        (b) one or more control sequences capable of driving expression of the nucleic acid sequence of (a); and optionally
        (c) a transcription termination sequence,

and wherein one of said control sequences is a constitutive promoter, preferably a GOS2 promoter and wherein said increased abiotic stress resistance is increased nutrient uptake efficiency and/or increased drought stress tolerance, relative to control plants.

    
    
    60. Use of a nucleic acid encoding a SYR polypeptide in a method for increasing abiotic stress resistance in plants relative to control plants, wherein said increased abiotic stress resistance is increased nutrient uptake efficiency and/or increased drought stress tolerance, relative to control plants.
    61. Use according to item 60, wherein said increased nutrient uptake efficiency results in increased seed yield and/or increased biomass.
    62. Use according to item 60, wherein said increased drought stress tolerance results in increased seed yield.


In one embodiment the invention relates to subject matter summarized as follows:

    63. A method for enhancing yield-related traits in plants relative to control plants, comprising modulating expression in a plant of a nucleic acid encoding an ARKL polypeptide.
    64. Method according to item 63, wherein said ARKL polypeptide comprises one or more of the following domains:
    
        (i) A ZfC3H2C3 domain as represented by SEQ ID NO: 400 or a domain having in increasing order of preference at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more sequence identity to one or more of the ZfC3H2C3 domains as represented by SEQ ID NO: 95 to SEQ ID NO. 351; and
        (II) A DAR1 domain having in increasing order of preference at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more sequence identity to one or more of the PfamB2828 domains as represented by SEQ ID NO: 352 to SEQ ID NO. 398.
    
    
    65. Method according to item 63 and 64, wherein said ARKL polypeptide comprises one or more of the following:
    
        (i) A ZfC3H2C3 domain as represented by SEQ ID NO: 401;
        (ii) A Motif 8 as represented by SEQ ID NO: 399;
    
    
    66. Method according to item 63 to 65, wherein said modulated expression is effected by introducing and expressing in a plant a nucleic acid encoding an ARKL polypeptide.
    67. Method according to any preceding item, wherein said nucleic acid encoding an ARKL polypeptide encodes any one of the proteins listed in Table A or is a portion of such a nucleic acid, or a nucleic acid capable of hybridising with such a nucleic acid.
    68. Method according to any preceding item, wherein said nucleic acid sequence encodes an orthologue or paralogue of any of the proteins given in Table A.
    69. Method according to any preceding item, wherein said enhanced yield-related traits comprise increased yield, preferably increased seed yield relative to control plants.
    70. Method according to preceding item, wherein said enhanced yield-related traits are obtained under non-stress conditions.
    71. Method according to preceding item, wherein said enhanced yield-related traits are obtained under conditions of drought stress.
    72. Method according to any preceding item, wherein said nucleic acid is operably linked to a constitutive promoter, preferably to a GOS2 promoter, most preferably to a GOS2 promoter from rice.
    73. Method according to any preceding item, wherein said nucleic acid encoding an ARKL polypeptide is of plant origin, preferably from a monocotyledonous plant, further preferably from the family Poaceae, more preferably from the genus Oryza, most preferably from Oryza sativa. 
    74. Plant or part thereof, including seeds, obtainable by a method according to any preceding item, wherein said plant or part thereof comprises a recombinant nucleic acid encoding an ARKL polypeptide.
    75. Construct comprising:
    
        (i) nucleic acid encoding an ARKL polypeptide as defined in items 63 to 65;
        (ii) one or more control sequences capable of driving expression of the nucleic acid sequence of (a); and optionally
        (iii) a transcription termination sequence.
    
    
    76. Construct according to item 75, wherein one of said control sequences is a constitutive promoter, preferably a GOS2 promoter, most preferably a GOS2 promoter from rice.
    77. Use of a construct according to item 75 or 76 in a method for making plants having enhanced yield-related traits preferably increased yield, more preferably increased seed yield relative to control plants.
    78. Plant, plant part or plant cell transformed with a construct according to item 75 or 76.
    79. Method for the production of a transgenic plant having increased yield, particularly increased seed yield relative to control plants, comprising:
    
        (i) introducing and expressing in a plant a nucleic acid encoding an ARKL polypeptide as defined in item 63 to 65; and
        (ii) cultivating the plant cell under conditions promoting plant growth and development.
    
    
    80. Transgenic plant having increased yield, particularly increased seed yield, relative to control plants, resulting from increased expression of a nucleic acid encoding an ARKL polypeptide as defined in item 63 to 65, or a transgenic plant cell derived from said transgenic plant.
    81. Transgenic plant according to item 74, 78 or 80, or a transgenic plant cell derived thereof, wherein said plant is a crop plant or a monocot or a cereal, such as rice, maize, wheat, barley, millet, rye, triticale, sorghum and oats.
    82. Harvestable parts of a plant according to item 81, wherein said harvestable parts are preferably shoot biomass and/or seeds.
    83. Products derived from a plant according to item 81 and/or from harvestable parts of a plant according to item 82.
    84. Use of a nucleic acid encoding an ARKL polypeptide enhancing yield-related traits preferably increased yield, more preferably increased seed yield relative to control plants.


In one embodiment the invention relates to subject matter summarized as follows:

    85. A method for improving yield-related traits in plants relative to control plants, comprising modulating expression in a plant of a nucleic acid encoding a YTP polypeptide comprising
    
        (i) at least one transmembrane domain and
        (ii) at least a portion of a DUF221 domain
    
    
    86. Method according to item 85, wherein said portion has in increasing order of preference at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity any one of the domains represented by SEQ ID NO: 518 to SEQ ID NO: 544.
    87. Method according to item 85 or 86, wherein said YTP said nucleic acid encodes a polypeptide having in increasing order of preference at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity amino acids to any one of the polypeptides of Table A.
    88. Method according to any preceding item, wherein said YTP polypeptide further comprises Motif 9 (SEQ ID NO: 545).
    89. Method according to any preceding item, wherein said modulated expression is effected by introducing and expressing in a plant a nucleic acid encoding a YTP polypeptide.
    90. Method according to any preceding item, wherein said nucleic acid encoding a YTP polypeptide encodes any one of the proteins listed in Table A or is a portion of such a nucleic acid, or a nucleic acid capable of hybridising with such a nucleic acid.
    91. Method according to any preceding item, wherein said nucleic acid sequence encodes an orthologue or paralogue of any of the proteins given in Table A or is a portion of such a nucleic acid, or a nucleic acid capable of hybridising with such a nucleic acid.
    92. Method according to any preceding item, wherein said enhanced yield-related traits comprise increased yield, preferably increased seed yield relative to control plants.
    93. Method according to any one of items 85 to 92, wherein said enhanced yield-related traits are obtained under non-stress conditions.
    94. Method according to any one of items 89 to 93, wherein said nucleic acid is operably linked to a constitutive promoter, preferably to a GOS2 promoter, most preferably to a GOS2 promoter from rice.
    95. Method according to any preceding item, wherein said nucleic acid encoding a YTP polypeptide is of plant origin, preferably from a dicotyledonous plant, further preferably from the family Poaceae, more preferably from the genus Oryza, most preferably from Oryza sativa. 
    96. Plant or part thereof, including seeds, obtainable by a method according to any preceding item, wherein said plant or part thereof comprises a recombinant nucleic acid encoding a YTP polypeptide.
    97. Construct comprising:
    
        (i) nucleic acid encoding a YTP polypeptide as defined in items 85 to 88
        (ii) one or more control sequences capable of driving expression of the nucleic acid sequence of (a); and optionally
        (iii) a transcription termination sequence.
    
    
    98. Construct according to item 97, wherein one of said control sequences is a constitutive promoter, preferably a GOS2 promoter, most preferably a GOS2 promoter from rice.
    99. Use of a construct according to item 97 or 98 in a method for making plants having increased yield, particularly increased seed yield relative to control plants.
    100. Plant, plant part or plant cell transformed with a construct according to item 97 or 98.
    101. Method for the production of a transgenic plant having increased yield, particularly increased seed yield relative to control plants, comprising:
    
        (i) introducing and expressing in a plant a nucleic acid encoding a YTP polypeptide as defined in item 85 to 88; and
        (ii) cultivating the plant cell under conditions promoting plant growth and development.
    
    
    102. Transgenic plant having increased yield, particularly increased seed yield, relative to control plants, resulting from modulated expression of a nucleic acid encoding a YTP polypeptide as defined in item 85 to 88 or a transgenic plant cell derived from said transgenic plant.
    103. Transgenic plant according to item 96, 100 or 102, or a transgenic plant cell derived thereof, wherein said plant is a crop plant or a monocot or a cereal, such as rice, maize, wheat, barley, millet, rye, triticale, sorghum and oats.
    104. Harvestable parts of a plant according to item 103, wherein said harvestable parts are preferably shoot biomass and/or seeds.
    105. Products derived from a plant according to item 103 and/or from harvestable parts of a plant according to 104.
    106. Use of a nucleic acid encoding a YTP polypeptide in increasing yield, particularly in increasing seed yield and/or shoot biomass in plants, relative to control plants.





DESCRIPTION OF FIGURES
The present invention will now be described with reference to the following figures in which:
FIG. 1 represents a cartoon of a GRF polypeptide as represented by SEQ ID NO: 2, which comprises the following features: (i) a QLQ domain with an InterPro accession IPR014978 (PFAM accession PF08880); (ii) a WRC domain with an InterPro accession IPR014977 (PFAM accession PF08879); and (iii) an Effector of Transcription (ET) domain comprising three Cys and one His residues in a conserved spacing (CX9CX10CX2H), located in the WRC domain.
FIG. 2 shows an AlignX (from Vector NTI 10.3, Invitrogen Corporation) multiple sequence alignment of the QLQ domain of GRF polypeptides from Table A (as represented by SEQ ID NO: 115 for SEQ ID NO: 2). The conserved QLQ amino acid residues are located on the top of the multiple alignment. Two other very conserved residues (boxed in black) are E (Glu) and P (Pro).
FIG. 3 shows an AlignX (from Vector NTI 10.3, Invitrogen Corporation) multiple sequence alignment of the WRC domain of GRF polypeptides from Table A (as represented by SEQ ID NO: 116 for SEQ ID NO: 2). The conserved WRC amino acid residues are in bold in the consensus sequence. The three Cys and one His residues in a conserved spacing (CX9CX10CX2H), designated as the Effector of Transcription (ET) domain, are boxed vertically across the alignment, and also identified at the bottom of the alignment. The putative nuclear localisation signal (NLS) comprised in the WRC domain, is double-underlined.
FIG. 4 shows the binary vector for increased expression in Oryza sativa of a nucleic acid sequence encoding a GRF polypeptide under the control of a GOS2 promoter (pGOS2) from rice.
FIG. 5 details examples of sequences useful in performing the methods according to the present invention.
FIG. 6 represents the sequence of a rice RAA1-like protein (SEQ ID NO: 121) with the conserved signature sequences indicated in bold underlined.
FIG. 7 shows a multiple alignment of various RAA1-like proteins. NP_001046787 corresponds to Q0E1D7, NP_001052368 corresponds to Q0JEF5, AAR97604 corresponds to Q6RIBO, NP_001042631 corresponds to Q9LGE3, NP_001045304 corresponds to Q8LR63, NP_974763 corresponds to Q9LXB5, NP_197868 corresponds to Q23624, NP_194866 corresponds to Q5Q0B3, NP_001060595 corresponds to Q8H475.
FIG. 8 shows a phylogenetic tree of RAA1-like polypeptides (Ge et al., 2004). OsRAA1 corresponds to SEQ ID NO: 121.
FIG. 9 represents the binary vector for increased expression in Oryza sativa of a RAA1-like-encoding nucleic acid under the control of a rice GOS2 promoter (pGOS2).
FIG. 10 details examples of sequences useful in performing the methods according to the present invention.
FIG. 11 gives an overview of the conserved motifs present in SEQ ID NO: 169. The leucine rich domain is underlined, the conserved motifs 5, 6 and 7 are indicated in bold and the sequence in italics represents the putative N-glycosylation site with the putative protein kinase C phosphorylation site.
FIG. 12 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. 11, the various domains and conserved motifs in SEQ ID NO: 171 can be easily identified in the other SYR proteins.
FIG. 13 shows binary vector pGOS2::SYR for transformation and expression in Oryza sativa of an Oryza sativa SYR nucleic acid under the control of a rice GOS2 promoter.
FIG. 14 details examples of sequences useful in performing the methods according to the present invention, or useful in isolating such sequences. Sequences may result from public EST assemblies, with lesser quality sequencing. As a consequence, a few nucleic acid substitutions may be expected. Both 5′ and 3′ UTRs may also be used for the performing the methods of the invention. SEQ ID NO: 193 represents the ARGOS protein sequence (GenBank accession AY305869).
FIG. 15 represents the amino acid sequence of SEQ ID NO: 213. Conserved domains pfamB2828 and ZfC3H2C3 (pfam00097) are highlighted in bold and underlined characters respectively. The highly conserved Motif 8 is indicated by an underlined dotted line. The most highly conserved amino acid residues in ARKL polypeptides are boxed. The conserved amino metal ligand positions (numbers) and zinc (Zn2+) coordinating amino acid pairs are illustrated.
FIG. 16 represents a multiple alignment of selected ARKL polypeptides. Highly conserved amino acid residues are indicated in the consensus sequence. As shown in the Figure the sequence of the C-terminus of ARKL polypeptides is more highly conserved than the N-terminus.
FIG. 17 shows a phylogenetic tree of ARKL polypeptides based on an alignment of the RING finger (pfam00097) domain comprised in ARKL polypeptides as represented by SEQ ID NO: 306-314, 316-318, 322, 323, 402 (SEQ ID NO: 402 comprises the pfam00097 (RING zinc finger) domain present in the Akadia polypeptide of Mus musculus and SEQ ID NO: 403 represents the pfam00097 domain as present in the Goliath polypeptide of Mus musculus. Abbreviations used: Os: Oryza sativa (Orysa); Hv: Hordeum vulgare (Horvu); Gm: Glycine max (Glyma); Zm: Zea mays (Zeama); Musmu: Mus musculus. 
FIG. 18 represents the binary vector for increased expression of OS_ARKL1 nucleic acid as represented by SEQ ID NO: 212 under the control of a rice GOS2 promoter (pGOS2).
FIG. 19 details examples of sequences useful in performing the methods according to the present invention.
FIG. 20 represents the sequence of YTP1 (SEQ ID NO: 409). Transmembrane domains are boxed. A part of a DUF221 domain of 124 amino acid residues is highlighted in bold. Motif 8 is underlined. Invariable residues in Motif 8 are indicated with a bigger sized letter type. The first and third loops are predicted to be located on the outside of the membrane; the second loop to the inside.
FIG. 21 shows phylogenetic tree of a selection of YTP polypeptides.
FIG. 22 represents multiple alignment of selected YTP polypeptides. A consensus sequence as represented by SEQ ID NO: 544 is given. Conserved amino acids residues are indicated in the consensus sequence; blanks represent regions of low conservation.
FIG. 23 represents the binary vector for increased expression in Oryza sativa of a YTP1-encoding nucleic acid under the control of a rice GOS2 promoter (pGOS2).
FIG. 24 details examples of sequences useful in performing the methods according to the present invention.



EXAMPLES
The present invention will now be described with reference to the following examples, which are by way of illustration alone. The following examples are not intended to completely define or 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. Standard materials and methods for plant molecular work are described in Plant Molecular Biology Labfax (1993) by R. D. D. Croy, published by BIOS Scientific Publications Ltd (UK) and Blackwell Scientific Publications (UK).
Example 1
Identification of Sequences Related to the Nucleic Acid Sequence Used in the Methods of the Invention
Sequences (full length cDNA, ESTs or genomic) related to the nucleic acid sequence used in the methods of the present invention 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 is used to find regions of local similarity between sequences by comparing nucleic acid sequence or polypeptide sequences to sequence databases and by calculating the statistical significance of matches. For example, the polypeptide encoded by the nucleic acid sequence of the present invention 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 reflect 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 sequence (or polypeptide) sequences over a particular length. In some instances, the default parameters may be adjusted to modify the stringency of the search. For example the E-value may be increased to show less stringent matches. This way, short nearly exact matches may be identified.
Table A provides a list of nucleic acid sequences related to the nucleic acid sequence used in the methods of the present invention.







TABLE A1







Examples of GRF polypeptide sequences, and encoding nucleic acid sequences:















Database



Source
Nucleic acid
Polypeptide
accession


Name
organism
SEQ ID NO:
SEQ ID NO:
number














Arath_GRF_At3G13960.1
Arabidopsis
1
2
AT3G13960.1



thaliana


Arath_GRF_At2G06200.1
Arabidopsis
3
4
At2G06200.1



thaliana


Arath_GRF_At2G22840.1
Arabidopsis
5
6
At2G22840.1



thaliana


Arath_GRF_At2G36400.1
Arabidopsis
7
8
At2G36400.1



thaliana


Arath_GRF_At2G45480.1
Arabidopsis
9
10
At2G45480.1



thaliana


Arath_GRF_At3G52910.1
Arabidopsis
11
12
At3G52910.1



thaliana


Arath_GRF_At4G24150.1
Arabidopsis
13
14
At4G24150.1



thaliana


Arath_GRF_At4G37740.1
Arabidopsis
15
16
At4G37740.1



thaliana


Arath_GRF_At5G53660.1
Arabidopsis
17
18
At5G53660.1



thaliana


Aqufo_GRF
Aquilegia
19
20
DT756681.1



formosa x


DR946716.1



Aquilegia



pubescens


Brana_GRF
Brassica
21
22
CN730217.1



napus


ES922527


Horvu_GRF
Hordeum
23
24
AK250947.1



vulgare


Lyces_GRF
Lycopersicon
25
26
BT013977.1



esculentum


Medtr_GRF
Medicago
27
28
AC144645.17



truncatula


Medtr_GRF like
Medicago
29
30
AC174350.4



truncatula


Orysa_GRF_Os02g47280.2
Oryza sativa
31
32
Os02g47280.2


Orysa_GRF_Os02g53690.1
Oryza sativa
33
34
Os02g53690.1


Orysa_GRF_Os03g51970.1
Oryza sativa
35
36
Os03g51970.1


Orysa_GRF_Os04g48510.1
Oryza sativa
37
38
Os04g48510.1


Orysa_GRF_Os04g51190.1
Oryza sativa
39
40
Os04g51190.1


Orysa_GRF_Os06g02560.1
Oryza sativa
41
42
Os06g02560.1


Orysa_GRF_Os11g35030.1
Oryza sativa
43
44
Os11g35030.1


Orysa_GRF_Os12g29980.1
Oryza sativa
45
46
Os12g29980.1


Oyrsa_GRF_Os03g47140.1
Oryza sativa
47
48
Os03g47140.1


Orysa_GRF_gi_115447910_ref_NM_001054270.1
Oryza sativa
49
50
NM_001054270.1


Orysa_GRF_gi_115460325_ref_NM_001060298.1
Oryza sativa
51
52
NM_001060298.1


Orysa_GRF_gi_115471984_ref_NM_001066126.1
Oryza sativa
53
54
NM_001066126.1


Poptr_GRF_lcl_scaff_XIV.39
Populus
55
56
lcl_scaff_XIV.39



tremuloides


Poptr_GRF_lcl_scaff_II.1070
Populus
57
58
lcl_scaff_II.1070



tremuloides


Poptr_GRF_lcl_scaff_I.1018
Populus
59
60
lcl_scaff_I.1018



tremuloides


Poptr_GRF_lcl_scaff_28.10
Populus
61
62
lcl_scaff_28.10



tremuloides


Poptr_GRF_lcl_scaff_I.995
Populus
63
64
lcl_scaff_I.995



tremuloides


Poptr_GRF_lcl_scaff_III.741
Populus
65
66
lcl_scaff_III.741



tremuloides


Poptr_GRF_lcl_scaff_VII.1274
Populus
67
68
lcl_scaff_VII.1274



tremuloides


Poptr_GRF_lcl_scaff_XII.277
Populus
69
70
lcl_scaff_XII.277



tremuloides


Poptr_GRF_lcl_scaff_XIII.769
Populus
71
72
lcl_scaff_XIII.769



tremuloides


Poptr_GRF_lcl_scaff_XIV.174
Populus
73
74
lcl_scaff_XIV.174



tremuloides


Poptr_GRF_lcl_scaff_XIV.51
Populus
75
76
lcl_scaff_XIV.51



tremuloides


Poptr_GRF_lcl_scaff_XIX.480
Populus
77
78
lcl_scaff_XIX.480



tremuloides


Poptr_GRF_lcl_scaff_28.309
Populus
79
80
lcl_scaff_28.309



tremuloides


Poptr_GRF_lcl_scaff_I.688
Populus
81
82
lcl_scaff_I.688



tremuloides


Sacof_GRF
Saccharum
83
84
CA084837.1



officinarum


CA238919.1






CA122516.1


Vitvi_GRF
Vitis vinifera
85
86
AM468035


Zeama_GRF10_gi_146008494_gb_EF515849.1
Zea mays
87
88
EF515849.1


Zeama_GRF11_gi_146008515_gb_EF515850.1
Zea mays
89
90
EF515850.1


Zeama_GRF12_gi_146008534_gb_EF515851.1
Zea mays
91
92
EF515851.1


Zeama_GRF13_gi_146008539_gb_EF515852.1
Zea mays
93
94
EF515852.1


Zeama_GRF14_gi_146008560_gb_EF515853.1
Zea mays
95
96
EF515853.1


Zeama_GRF1_gi_146008330_gb_EF515840.1
Zea mays
97
98
EF515840.1


Zeama_GRF2_gi_146008352_gb_EF515841.1
Zea mays
99
100
EF515841.1


Zeama_GRF3_gi_146008368_gb_EF515842.1
Zea mays
101
102
EF515842.1


Zeama_GRF4_gi_146008393_gb_EF515843.1
Zea mays
103
104
EF515843.1


Zeama_GRF5_gi_146008412_gb_EF515844.1
Zea mays
105
106
EF515844.1


Zeama_GRF6_gi_146008429_gb_EF515845.1
Zea mays
107
108
EF515845.1


Zeama_GRF7_gi_146008440_gb_EF515846.1
Zea mays
109
110
EF515846.1


Zeama_GRF8_gi_146008461_gb_EF515847.1
Zea mays
111
112
EF515847.1


Zeama_GRF9_gi_146008475_gb_EF515848.1
Zea mays
113
114
EF515848.1
















TABLE A2







Examples of RAA1-like polypeptides:












Nucleic acid




Plant Source
SEQ ID NO:
Protein SEQ ID NO:







Q9LGE3
SEQ ID NO: 120
SEQ ID NO: 121



Q8H475
SEQ ID NO: 126
SEQ ID NO: 127



A3BNA1
SEQ ID NO: 128
SEQ ID NO: 129



O23624
SEQ ID NO: 130
SEQ ID NO: 131



Q8LR63
SEQ ID NO: 132
SEQ ID NO: 133



Q9LXB5
SEQ ID NO: 134
SEQ ID NO: 135



Q5Q0B3
SEQ ID NO: 136
SEQ ID NO: 137



Q7XX25
SEQ ID NO: 138
SEQ ID NO: 139



A2WN18
SEQ ID NO: 140
SEQ ID NO: 141



O24340
SEQ ID NO: 142
SEQ ID NO: 143



A2X4J6
SEQ ID NO: 144
SEQ ID NO: 145



Q0E1D7
SEQ ID NO: 146
SEQ ID NO: 147



O49587
SEQ ID NO: 148
SEQ ID NO: 149



A2XRE0
SEQ ID NO: 150
SEQ ID NO: 151



Q6RIB0
SEQ ID NO: 152
SEQ ID NO: 153



Q9LXB6
SEQ ID NO: 154
SEQ ID NO: 155



Q0JEF5
SEQ ID NO: 156
SEQ ID NO: 157



NP1050091
SEQ ID NO: 158
SEQ ID NO: 159



A5BZJ2
SEQ ID NO: 160
SEQ ID NO: 161










In addition to the publicly available nucleic acid sequences available at NCBI, proprietary sequence databases are also searched following the same procedure as described herein above.
Table A3 provides a list of nucleic acid and protein sequences related to the nucleic acid sequence as represented by SEQ ID NO: 168 and the protein sequence represented by SEQ ID NO: 169.







TABLE A3







Nucleic acid sequences related to the nucleic acid sequence


(SEQ ID NO: 168) useful in the methods of the present invention,


and the corresponding deduced polypeptides.














Poly-peptide
Nucleic acid
Database



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





OsSYR
Oryza sativa
169
168
/
Full length or







partial


rice SYR
Oryza sativa
179
194
XP_472637
Full length


homologue 1







rice SYR
Oryza sativa
180

AP008218
Full length


homologue 2







corn SYR
Zea mays
181
195
AY110705
partial


homologue







wheat SYR
Triticum aestivum
182

/
Full length


homologue







barley SYR
Hordeum vulgare
183
203
CB871444
Full length


homologue







sugar cane SYR
Saccharum
184
204
CA165713
partial


homologue 1
officinarum






sugar cane SYR
Saccharum
185
205
CA242805
Full length


homologue 2
officinarum






sorghum SYR
Sorghum bicolor
186
206
CX611532
Full length


homologue







AtSYR homologue 1
Arabidopsis
187
207
NM_115853
Full length



thaliana






AtSYR homologue 2
Arabidopsis
188
208
NM_180078
Full length



thaliana






grape SYR
Vitis vinifera
189
196
CF404276
Full length


homologue







Citrus SYR
Citrus reticulata
190
197
CF830612
partial


homologue







tomato SYR
Lycopersicon
191
199
AI774560
Full length


homologue 1
esculentum






tomato SYR
Lycopersicon
192
198
BG125370
Full length


homologue 2
esculentum






Argos
Arabidopsis
193
209
AY305869
Full length



thaliana
















TABLE A4







Examples of ARKL nucleic acids and the respectively encoded


polypeptides.













Protein




Nucleic acid
SEQ ID


Description*
Plant Source
SEQ ID NO:
NO:













Orysa_ARKL1
Oryza sativa
212
213


Orysa_ARKL2
Oryza sativa
214
215


Orysa_ARKL3
Oryza sativa
216
217


Orysa_ARKL4
Oryza sativa
218
219


Orysa_ARKL5
Oryza sativa
220
221


Orysa_ARKL6
Oryza sativa
222
223


Orysa_ARKL7
Oryza sativa
224
225


Orysa_ARKL8
Oryza sativa
226
227


Orysa_ARKL9
Oryza sativa
228
229


Zeama_ARKL1
Zea mays
230
231


Zeama_ARKL2
Zea mays
232
233


Horvu_ARKL1
Hordeum vulgare
234
235


Horvu_ARKL2
Hordeum vulgare
236
237


Horvu_ARKL3
Hordeum vulgare
238
239


Lyces_ARKL1
Lycopersicum esculentum
240
241


Lyces_ARKL2
Lycopersicum esculentum
242
243


Lyces_ARKL3
Lycopersicum esculentum
244
245


Glyma_ARKL1
Glycine max
246
247


Glyma_ARKL2
Glycine max
248
249


Zinel_ARKL1
Zinnia elegans
250
251


Lotja_ARKL1
Lotus japonicus
252
253


Arath_ARKL1
Arabidopsis thaliana
254
255


Arath_ARKL2
Arabidopsis thaliana
256
257


Arath_ARKL3
Arabidopsis thaliana
258
259


Arath_ARKL4
Arabidopsis thaliana
260
261


Arath_ARKL5
Arabidopsis thaliana
262
263


Arath_ARKL6
Arabidopsis thaliana
264
265


Arath_ARKL7
Arabidopsis thaliana
266
267


Arath_ARKL8
Arabidopsis thaliana
268
269


Arath_ARKL9
Arabidopsis thaliana
270
271


Arath_ARKL10
Arabidopsis thaliana
272
273


Arath_ARKL11
Arabidopsis thaliana
274
275


Arath_ARKL12
Arabidopsis thaliana
276
277


Poptr_ARKL1
Populus trichocarpa
278
279


Poptr_ARKL2
Populus trichocarpa
280
281


Poptr_ARKL3
Populus trichocarpa
282
283


Poptr_ARKL4
Populus trichocarpa
284
285


Poptr_ARKL5
Populus trichocarpa
286
287


Poptr_ARKL6
Populus trichocarpa
288
289


Poptr_ARKL7
Populus trichocarpa
290
291


Poptr_ARKL8
Populus trichocarpa
292
293


Poptr_ARKL9
Populus trichocarpa
294
295


Poptr_ARKL10
Populus trichocarpa
296
297


Medtr_ARKL1
Medicago truncatula
298
299


Medtr_ARKL2
Medicago truncatula
300
301


Medtr_ARKL3
Medicago truncatula
302
303


Medtr_ARKL4
Medicago truncatula
304
305





*Orysa: Oryza sativa; Zeama: Zea mays; Horvu: Hordeum vulgare; Lyces: Lycopersicum esculentum; Glyma: Glycine max; Zinel: Zinnia elegans; Lotja: Lotus japonicus; Arath: Arabidopsis thaliana; Poptr: Populus thricocarpa; Medtr: Medicago truncatula.






Table A5 provides a list of nucleic acid sequences related to the nucleic acid sequence used in the methods of the present invention.







TABLE A5







Examples of YTP nucleic acids and polypeptides:














Nucleic Acid
Protein


Name
Name Alias
Source Organism
SEQ ID NO
SEQ ID NO





YTP1
YTP1_PARTIAL
Oryza sativa
408
409


YTP2
Os01g0534900 (768)
Oryza sativa
410
411


YTP3
Os01g0950900 (701)
Oryza sativa
412
413


YTP4
Os03g0137400 (792)
Oryza sativa
414
415


YTP5
Os03g0673800 (777)
Oryza sativa
416
417


YTP6
Os03g0726300 (743)
Oryza sativa
418
419


YTP7
Os05g0393800 (767)
Oryza sativa
420
421


YTP8
Os05g0594700 (766)
Oryza sativa
422
423


YTP9
Os07g0150100 (731)
Oryza sativa
424
425


YTP10
Os10g0579100 (810)
Oryza sativa
426
427


YTP11
Os12g0582800 (695)
Oryza sativa
428
429


YTP12
Os12g0633600 (763)
Oryza sativa
430
431


YTP13
AT1G10090 (762)
Arabidopsis thaliana
432
433


YTP14
AT1G11960 (375)
Arabidopsis thaliana
434
435


YTP15
AT1G30360 (724)
Arabidopsis thaliana
436
437


YTP16
AT1G58520 (657)
Arabidopsis thaliana
438
439


YTP17
AT1G62320 (769)
Arabidopsis thaliana
440
441


YTP18
AT1G69450 (711)
Arabidopsis thaliana
442
443


YTP19
AT3G01100 (596)
Arabidopsis thaliana
444
445


YTP20
AT3G21620 (756)
Arabidopsis thaliana
446
447


YTP21
AT3G54510 (617)
Arabidopsis thaliana
448
449


YTP22
AT4G02900 (806)
Arabidopsis thaliana
450
451


YTP23
AT4G04340 (772)
Arabidopsis thaliana
452
453


YTP24
AT4G15430 (761)
Arabidopsis thaliana
454
455


YTP25
AT4G22120 (771)
Arabidopsis thaliana
456
457


YTP26
AT4G35870 (817)
Arabidopsis thaliana
458
459


YTP27
AQGI.2hit1partialAquilegia
Aquilegia species
460
461



(PGI) (707)


YTP28
lcl_175_Medicago (712)
Medicago truncatula
462
463


YTP29
lcl_21269_Medicago (790)
Medicago truncatula
464
465


YTP30
lcl_24278_Medicago (766)
Medicago truncatula
466
467


YTP31
lcl_3723_Medicago (461)
Medicago truncatula
468
469


YTP32
lcl_scaff_1405.2 (301)
Populus trichocarpa
470
471


YTP33
lcl_scaff_1405.3 (276)
Populus trichocarpa
472
473


YTP34
lcl_scaff_166.26 (775)
Populus trichocarpa
474
475


YTP35
lcl_scaff_166.27 (774)
Populus trichocarpa
476
477


YTP36
lcl_scaff_29.271 (831)
Populus trichocarpa
478
479


YTP37
lcl_scaff_I.2570 (724)
Populus trichocarpa
480
481


YTP38
lcl_scaff_II.1056 (706)
Populus trichocarpa
482
483


YTP39
lcl_scaff_II.2075 (767)
Populus trichocarpa
484
485


YTP40
lcl_scaff_III.1644 (726)
Populus trichocarpa
486
487


YTP41
lcl_scaff_III.729 (516)
Populus trichocarpa
488
489


YTP42
lcl_scaff_IV.1089 (436)
Populus trichocarpa
490
491


YTP43
lcl_scaff_VIII.848 (714)
Populus trichocarpa
492
493


YTP44
lcl_scaff_XI.92 (546)
Populus trichocarpa
494
495


YTP45
lcl_scaff_XI.94 (708)
Populus trichocarpa
496
497


YTP46
lcl_scaff_XIV.1036 (846)
Populus trichocarpa
498
499


YTP47
Triae_TA80116_4565 (535)
Triticum aestivum
500
501


YTP48
volvox2_104236
Volvox carteri
502
503


YTP49
VOLVOX_95919
Volvox carteri
504
505


YTP50
chlamy-174910 (1129)
Chlamydomonas reinhardtii
506
507


YTP51
chlamy-194774 (1429)
Chlamydomonas reinhardtii
508
509


YTP52
ref_NP_014557.1_(991)
Schizosaccharomyces pombe
510
511


YTP53
ref_NP_592939.1_(871)
Ashbya gossypii
512
513


YTP54
ref_NP_984890.1_(875)
Kluyveromyces lactis
514
515


YTP55
ref_XP_452699.1_(967)
Saccharomyces cerevisiae
516
517









In some instances, related sequences have tentatively been assembled and publicly disclosed by research institutions, such as The Institute for Genomic Research (TIGR). The Eukaryotic Gene Orthologs (EGO) database may be used to identify such related sequences, either by keyword search or by using the BLAST algorithm with the nucleic acid sequence or polypeptide sequence of interest. On other instances, special nucleic acid sequence databases have been created for particular organisms, such as by the Joint Genome Institute, for example for poplar and Ostreococcus tauri. 
Example 2
a) Alignment of GRF Polypeptide Sequences
Mutliple sequence alignment of all the GRF polypeptide sequences in Table A was performed using the AlignX algorithm (from Vector NTI 10.3, Invitrogen Corporation). Results of the alignment for the QLQ domain of GRF polypeptides from Table A (as represented by SEQ ID NO: 115 for SEQ ID NO: 2) are shown in FIG. 2 of the present application. The conserved QLQ amino acid residues are located on the top of the multiple alignment. Two other very conserved residues (boxed in black) are E (Glu) and P (Pro). Results of the alignment for the WRC domain of the GRF polypeptides from Table A (as represented by SEQ ID NO: 116 for SEQ ID NO: 2) are shown in FIG. 3 of the present application. The conserved WRC amino acid residues are in bold in the consensus sequence. The three Cys and one His residues in a conserved spacing (CX9CX10CX2H), designated as the Effector of Transcription (ET) domain, are boxed vertically across the alignment, and also identified at the bottom of the alignment.
b) Alignment of RAA1-like Polypeptide Sequences
Alignment of polypeptide sequences was performed using the AlignX programme from the Vector NTI (Invitrogen) which is based on the popular Clustal W algorithm of progressive alignment (Thompson et al. (1997) Nucleic Acids Res 25:4876-4882; Chenna et al. (2003). Nucleic Acids Res 31:3497-3500). 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). Minor manual editing was done to further optimise the alignment. Sequence conservation among RAA1-like polypeptides is essentially throughout the whole sequence with the exception of a Gly and/or Ser rich region in the N-terminal half of the protein. The RAA1-like polypeptides are aligned in FIG. 7.
c) Alignment of SYR_Polypeptide Sequences
AlignX (Vector NTI, Invitrogen) is based on the popular Clustal algorithm of progressive alignment (Thompson et al. (1997) Nucleic Acids Res 25:4876-4882; Chenna 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. 12. The leucine rich repeat and the conserved motifs can be easily discriminated in the various sequences.
d) Alignment of ARKL Polypeptide Sequences
Alignment of polypeptide sequences was performed using the AlignX programme from the Vector NTI (Invitrogen) which is based on the popular Clustal W algorithm of progressive alignment (Thompson et al. (1997) Nucleic Acids Res 25:4876-4882; Chenna et al. (2003). Nucleic Acids Res 31:3497-3500). 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). Minor manual editing may be done to further optimise the alignment. Sequence conservation among ARKL polypeptides is essentially in the C-terminal along the DAR1 and RING-H2 domain of the polypeptides, the N-terminal domain usually being more variable in sequence length and composition. The ARKL polypeptides are aligned in FIG. 16.
A phylogenetic tree of ARKL polypeptides (FIG. 17) was constructed using a neighbour-joining clustering algorithm as provided in the AlignX programme from the Vector NTI (Invitrogen).
e) Alignment of YTP Polypeptide Sequences
Alignment of polypeptide sequences was performed using the AlignX programme from the Vector NTI (Invitrogen) which is based on the popular Clustal W algorithm of progressive alignment (Thompson et al. (1997) Nucleic Acids Res 25:4876-4882; Chenna et al. (2003). Nucleic Acids Res 31:3497-3500). 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). Minor manual editing may be done to further optimise the alignment. Sequence conservation among YTP polypeptides is higher in the C-terminus of the protein of the polypeptides along the DUF221 domain. The N-terminal domain is usually more variable in sequence length and composition. The YTP polypeptides are aligned in FIG. 22. Highly conserved amino acid residues are indicated in the consensus sequence (see FIG. 22).
A phylogenetic tree of YTP polypeptides (FIG. 21) was constructed using a neighbour-joining clustering algorithm as provided in the AlignX programme from the Vector NTI (Invitrogen). As shown in FIG. 21, Group 1 comprises the YTP polypeptides clustering with SEQ ID NO: 409 (YTP1 in FIG. 21).
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).







TABLE B1





MatGAT results for global similarity and identity over the


full length of the polypeptide sequences related to GRF.






























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





 1. Aqufo_GRF
  
31
22
25
23
38
22
19
22
23
39
31
21
46


 2. Arath_GRF_
43
  
18
23
20
28
18
17
18
21
27
26
21
32


AT2G06200.1
















 3. Arath_GRF_
36
25
 
26
19
22
22
23
57
21
21
24
27
22


AT2G22840.1
















 4. Arath_GRF_
43
31
38
 
23
27
48
23
26
26
24
26
47
25


AT2G36400.1
















 5. Arath_GRF_
38
30
33
39
 
21
21
16
17
23
22
23
21
24


AT2G45480.1
















 6. Arath_GRF_
53
38
34
44
34
 
23
18
21
22
83
29
22
45


AT3G13960.1
















 7. Arath_GRF_
34
26
40
56
36
36
  
20
24
22
23
22
31
22


AT3G52910.1
















 8. Arath_GRF_
31
25
38
36
32
30
35
 
23
25
19
21
25
18


AT4G24150.1
















 9. Arath_GRF_
35
24
72
38
33
31
40
39
  
21
23
23
26
23


AT4G37740.1
















10. Arath_GRF_
37
30
35
40
35
37
34
36
33
 
24
27
25
23


AT5G53660.1
















11. Brana_GRF
54
39
33
41
35
90
33
33
34
39
  
28
21
47


12. Horvu_GRF
49
34
35
42
39
44
35
32
35
41
47
  
25
25


13. Lyces_GRF
42
30
38
64
37
41
43
38
36
41
40
42
  
24


14. Medtr_GRF
61
44
34
38
36
65
34
31
36
38
63
44
40
  


15. Medtr_GRF\like
37
27
32
33
46
37
33
31
34
37
37
37
34
36


16. Orysa_GRF_NM_
27
37
23
31
25
24
23
24
24
28
25
29
32
27


001054270.1
















17. Orysa_GRF_NM_
53
35
36
44
35
46
35
32
35
42
46
78
41
48


001060298.1
















18. Orysa_GRF_NM_
27
38
24
29
28
29
23
26
25
26
28
30
31
29


001066126.1
















19. Orysa_GRF_
51
38
36
47
36
46
36
35
35
39
49
73
44
47


Os02g47280.2
















20. Orysa_GRF_
57
38
36
43
36
52
33
32
34
36
52
49
40
52


Os02g53690.1
















21. Orysa_GRF_
40
31
49
40
39
40
37
29
45
40
40
38
38
40


Os03g51970.1
















22. Orysa_GRF_
29
41
26
30
28
26
24
27
26
31
28
31
33
30


Os04g48510.1
















23. Orysa_GRF_
52
35
35
44
34
45
36
32
34
41
46
79
41
44


Os04g51190.1
















24. Orysa_GRF_
52
38
32
38
37
41
33
29
33
40
45
56
40
46


Os06g02560.1
















25. Orysa_GRF_
38
32
43
37
36
38
35
34
40
38
38
38
39
35


Os11g35030.1
















26. Orysa_GRF_
39
33
46
40
37
40
35
37
43
39
40
41
40
38


Os12g29980.1
















27. Oyrsa_GRF_
37
30
39
40
40
37
35
34
36
40
42
39
38
32


Os03g47140.1
















28. Poptr_GRF_lcl_scaff_
67
43
34
40
39
52
37
31
34
37
55
53
42
60


28.10
















29. Poptr_GRF_lcl_scaff_
40
32
36
65
33
38
46
33
35
42
38
42
59
41


28.309
















30. Poptr_GRF_lcl_scaff_
62
43
32
39
35
58
35
29
32
42
59
45
39
71


I.1018
















31. Poptr_GRF_lcl_scaff_
32
25
39
36
32
31
37
46
38
35
32
35
38
33


I.688
















32. Poptr_GRF_lcl_scaff_
26
34
22
27
24
24
20
22
22
28
25
26
28
28


I.995
















33. Poptr_GRF_lcl_scaff_
31
24
59
36
32
33
39
35
54
34
31
34
33
32


II.1070
















34. Poptr_GRF_lcl_scaff_
52
38
34
38
36
45
32
31
33
36
45
53
38
48


III.741
















35. Poptr_GRF_lcl_scaff_
38
25
57
37
34
35
41
37
58
37
34
35
36
34


VII.1274
















36. Poptr_GRF_lcl_scaff_
34
25
40
37
33
33
37
42
44
38
33
33
34
32


XII.277
















37. Poptr_GRF_lcl_scaff_
57
42
32
42
38
46
32
30
34
34
46
53
39
53


XIII.769
















38. Poptr_GRF_lcl_scaff_
33
25
36
35
43
35
39
34
36
35
35
35
35
35


XIV.174
















39. Poptr_GRF_lcl_scaff_
34
22
59
36
33
32
38
34
55
35
30
34
36
32


XIV.39
















40. Poptr_GRF_lcl_scaff_
37
27
60
41
35
35
42
40
54
37
36
37
36
37


XIV.51
















41. Poptr_GRF_lcl_scaff_
54
42
32
40
35
44
31
28
32
36
47
51
38
49


XIX.480
















42. Sacof_GRF
37
28
41
39
37
39
37
35
39
36
41
37
40
35


43. Vitvi_GRF
70
43
35
41
35
56
33
32
33
37
58
48
39
69


44. Zeama_GRF10_
26
36
23
29
27
27
23
26
25
26
26
31
32
26


EF515849.1
















45. Zeama_GRF11_
50
41
29
41
33
42
28
25
30
35
42
44
35
45


EF515850.1
















46. Zeama_GRF12_
44
38
31
40
32
41
30
30
30
39
44
46
38
42


EF515851.1
















47. Zeama_GRF13_
37
29
39
40
37
40
36
37
39
36
38
40
37
35


EF515852.1
















48. Zeama_GRF14_
49
36
33
45
36
43
35
30
33
42
42
53
42
43


EF515853.1
















49. Zeama_GRF1_
50
35
38
47
37
47
36
34
34
39
45
67
41
43


EF515840.1
















50. Zeama_GRF2_
42
35
38
41
36
41
30
31
37
45
41
40
38
41


EF515841.1
















51. Zeama_GRF3_
51
36
33
41
38
49
34
27
31
36
49
45
40
46


EF515842.1
















52. Zeama_GRF4_
24
36
24
30
27
28
21
25
26
25
28
31
31
26


EF515843.1
















53. Zeama_GRF5_
50
35
35
42
35
42
34
32
34
40
43
75
40
41


EF515844.1
















54. Zeama_GRF6_
50
36
35
40
35
44
33
30
36
39
45
76
40
42


EF515845.1
















55. Zeama_GRF7_
48
41
31
39
34
44
29
27
31
37
45
42
35
47


EF515846.1
















56. Zeama_GRF8_
38
29
39
38
36
38
34
37
40
37
38
37
39
35


EF515847.1
















57. Zeama_GRF9_
57
42
31
37
31
49
32
29
31
32
50
45
40
52


EF515848.1
























15
16
17
18
19
20
21
22
23
24
25
26
27





 1. Aqufo_GRF
23
18
34
15
33
41
29
18
34
35
23
23
23


 2. Arath_GRF_
19
21
25
21
26
28
22
21
25
27
20
22
20


AT2G06200.1















 3. Arath_GRF_
20
16
24
15
26
23
32
21
24
22
30
31
28


AT2G22840.1















 4. Arath_GRF_
20
24
28
19
28
25
25
23
28
27
24
25
25


AT2G36400.1















 5. Arath_GRF_
29
16
23
16
21
22
23
17
22
24
18
22
21


AT2G45480.1















 6. Arath_GRF_
21
16
29
16
29
35
26
17
28
27
23
25
22


AT3G13960.1















 7. Arath_GRF_
19
17
22
15
23
21
23
18
22
20
23
22
22


AT3G52910.1















 8. Arath_GRF_
16
17
21
18
23
19
17
18
22
20
22
23
20


AT4G24150.1















 9. Arath_GRF_
20
18
23
17
24
21
27
19
24
24
29
29
26


AT4G37740.1















10. Arath_GRF_
23
19
24
14
25
22
22
18
24
25
23
24
25


AT5G53660.1















11. Brana_GRF
21
16
29
15
31
35
26
18
29
28
21
24
24


12. Horvu_GRF
23
21
68
20
62
29
24
22
70
42
25
28
25


13. Lyces_GRF
21
25
25
18
27
22
24
25
25
25
23
26
24


14. Medtr_GRF
22
17
31
14
31
38
26
17
28
30
22
24
22


15. Medtr_GRF\like
  
16
22
20
24
20
20
18
22
21
24
23
22


16. Orysa_GRF_NM_
24
  
22
35
22
18
16
66
21
20
21
21
19


001054270.1















17. Orysa_GRF_NM_
36
30
  
20
70
33
24
23
98
42
25
27
26


001060298.1















18. Orysa_GRF_NM_
29
46
32
  
20
14
16
34
20
14
20
18
18


001066126.1















19. Orysa_GRF_
37
30
78
29
 
34
25
24
70
41
25
29
24


Os02g47280.2















20. Orysa_GRF_
33
26
49
26
50
 
27
20
33
34
23
25
22


Os02g53690.1















21. Orysa_GRF_
38
23
38
24
40
40
 
18
24
25
28
38
25


Os03g51970.1















22. Orysa_GRF_
28
71
32
47
32
29
24
  
23
22
23
22
19


Os04g48510.1















23. Orysa_GRF_
35
29
98
31
78
50
37
32
 
42
24
27
26


Os04g51190.1















24. Orysa_GRF_
34
28
54
24
55
47
35
32
54
  
24
25
23


Os06g02560.1















25. Orysa_GRF_
37
27
37
29
40
39
45
29
39
36
 
37
28


Os11g35030.1















26. Orysa_GRF_
39
27
41
28
42
37
55
29
41
37
54
  
28


Os12g29980.1















27. Oyrsa_GRF_
38
26
36
28
37
35
44
27
40
34
43
48
 


Os03g47140.1















28. Poptr_GRF_lcl_scaff_
37
24
55
27
53
55
40
27
55
48
38
38
38


28.10















29. Poptr_GRF_lcl_scaff_
33
30
40
32
41
39
34
33
41
42
39
40
39


28.309















30. Poptr_GRF_lcl_scaff_
33
27
50
28
48
52
40
31
48
48
38
37
35


I.1018















31. Poptr_GRF_lcl_scaff_
33
22
34
26
36
33
36
24
34
33
34
37
37


I.688















32. Poptr_GRF_lcl_scaff_
22
51
26
42
27
27
21
50
26
28
25
24
24


I.995















33. Poptr_GRF_lcl_scaff_
33
21
33
24
34
33
50
24
33
31
40
46
39


II.1070















34. Poptr_GRF_lcl_scaff_
38
28
53
27
50
43
38
32
53
58
38
40
36


III.741















35. Poptr_GRF_lcl_scaff_
33
22
36
25
36
34
52
25
37
32
41
46
41


VII.1274















36. Poptr_GRF_lcl_scaff_
33
22
32
24
35
32
37
22
33
31
36
37
35


XII.277















37. Poptr_GRF_lcl_scaff_
35
31
57
26
57
48
37
35
56
57
38
39
38


XIII.769















38. Poptr_GRF_lcl_scaff_
42
23
31
25
36
34
37
25
32
34
33
35
35


XIV.174















39. Poptr_GRF_lcl_scaff_
32
21
32
24
33
33
47
22
32
30
38
42
38


XIV.39















40. Poptr_GRF_lcl_scaff_
37
22
36
23
35
40
57
24
38
36
43
52
40


XIV.51















41. Poptr_GRF_lcl_scaff_
32
33
54
28
52
47
35
34
53
53
35
37
35


XIX.480















42. Sacof_GRF
37
27
37
28
38
35
43
30
40
35
45
46
82


43. Vitvi_GRF
34
26
51
24
50
58
40
29
51
50
38
40
36


44. Zeama_GRF10_
30
44
32
81
32
22
24
44
32
28
30
28
29


EF515849.1















45. Zeama_GRF11_
33
31
46
30
45
53
35
35
46
47
35
36
33


EF515850.1















46. Zeama_GRF12_
31
32
46
33
45
40
34
32
45
67
36
35
34


EF515851.1















47. Zeama_GRF13_
38
26
39
29
41
35
44
28
40
36
43
47
78


EF515852.1















48. Zeama_GRF14_
34
28
55
27
54
47
40
30
54
77
39
36
39


EF515853.1















49. Zeama_GRF1_
38
29
74
29
79
51
42
30
74
50
40
41
43


EF515840.1















50. Zeama_GRF2_
39
29
43
31
40
40
46
33
43
42
66
54
42


EF515841.1















51. Zeama_GRF3_
36
27
48
25
50
80
38
29
48
46
37
39
33


EF515842.1















52. Zeama_GRF4_
28
45
31
80
32
27
24
44
32
26
30
27
29


EF515843.1















53. Zeama_GRF5_
36
31
80
31
72
48
38
32
80
54
38
41
39


EF515844.1















54. Zeama_GRF6_
37
30
80
32
71
46
38
31
81
51
38
39
36


EF515845.1















55. Zeama_GRF7_
32
32
46
31
45
54
35
34
45
48
34
36
35


EF515846.1















56. Zeama_GRF8_
38
27
37
30
38
35
43
28
39
34
46
44
79


EF515847.1















57. Zeama_GRF9_
35
31
45
27
45
73
40
31
45
46
35
39
33


EF515848.1


























28
29
30
31
32
33
34
35
36
37
38
39
40
41
42





 1. Aqufo_GRF
54
23
47
21
18
20
34
25
21
44
23
21
24
43
22


 2. Arath_GRF_
34
22
32
16
24
16
27
17
15
30
19
15
18
30
20


AT2G06200.1

















 3. Arath_GRF_
22
26
22
24
17
42
22
40
27
21
20
44
39
21
29


AT2G22840.1

















 4. Arath_GRF_
23
51
27
26
20
25
25
27
23
27
22
25
27
27
27


AT2G36400.1

















 5. Arath_GRF_
22
19
23
18
17
17
23
21
19
24
28
20
21
23
19


AT2G45480.1

















 6. Arath_GRF_
36
21
41
20
17
22
27
23
21
31
22
22
25
30
24


AT3G13960.1

















 7. Arath_GRF_
24
37
22
22
14
23
22
22
21
23
23
22
25
21
22


AT3G52910.1

















 8. Arath_GRF_
19
22
20
32
15
21
19
20
25
20
17
20
22
18
21


AT4G24150.1

















 9. Arath_GRF_
21
24
23
24
17
38
21
42
27
23
19
42
35
23
27


AT4G37740.1

















10. Arath_GRF_
22
28
26
24
18
22
22
25
27
23
21
23
24
23
25


AT5G53660.1

















11. Brana_GRF
37
21
44
19
17
20
30
22
21
31
23
21
24
31
24


12. Horvu_GRF
32
25
30
21
21
23
35
23
23
39
21
22
26
38
24


13. Lyces_GRF
22
45
26
25
21
23
24
25
23
25
22
25
24
25
24


14. Medtr_GRF
43
24
56
22
18
22
29
22
21
36
23
21
23
35
23


15. Medtr_GRF\like
22
19
20
21
16
19
25
20
21
23
29
19
22
22
21


16. Orysa_GRF_NM_
16
23
18
18
38
16
19
16
16
21
18
16
17
21
20


001054270.1

















17. Orysa_GRF_NM_
36
25
32
22
20
22
36
25
24
43
19
22
25
39
25


001060298.1

















18. Orysa_GRF_NM_
12
19
15
18
29
16
16
15
16
14
16
16
14
15
19


001066126.1

















19. Orysa_GRF_
33
27
32
23
20
23
35
25
25
44
23
23
23
42
25


Os02g47280.2

















20. Orysa_GRF_
41
23
38
19
19
22
30
23
20
35
23
21
25
35
21


Os02g53690.1

















21. Orysa_GRF_
27
21
28
22
15
37
25
38
24
25
22
34
43
26
26


Os03g51970.1

















22. Orysa_GRF_
18
24
19
18
38
18
22
17
17
23
20
16
18
21
22


Os04g48510.1

















23. Orysa_GRF_
36
24
31
22
21
22
36
25
24
42
22
22
24
39
26


Os04g51190.1

















24. Orysa_GRF_
36
27
30
21
21
22
39
24
21
42
24
22
26
41
22


Os06g02560.1

















25. Orysa_GRF_
21
24
25
25
19
29
23
29
23
23
20
27
29
22
29


Os11g35030.1

















26. Orysa_GRF_
24
26
26
22
17
32
26
34
24
27
24
31
37
27
26


Os12g29980.1

















27. Oyrsa_GRF_
22
26
25
23
18
27
23
27
23
26
21
27
25
24
71


Os03g47140.1

















28. Poptr_GRF_lcl_scaff_
 
25
44
21
19
23
35
24
22
41
22
21
25
39
22


28.10

















29. Poptr_GRF_lcl_scaff_
42
  
26
24
20
24
24
24
22
26
21
23
24
25
25


28.309

















30. Poptr_GRFscaff_
59
39
 
22
20
19
32
23
22
36
21
21
23
36
23


I.1018

















31. Poptr_GRFscaff_
36
33
31
  
16
25
20
25
29
20
21
24
24
21
25


I.688

















32. Poptr_GRFscaff_
25
29
28
21
 
14
19
16
13
22
17
14
15
21
18


I.995

















33. Poptr_GRFscaff_
34
32
31
39
19
 
22
47
28
22
21
50
75
21
27


II.1070

















34. Poptr_GRFscaff_
51
41
50
32
28
31
  
23
20
47
22
20
25
44
24


III.741

















35. Poptr_GRFscaff_
35
35
33
39
20
62
34
 
28
23
23
74
50
21
26


VII.1274

















36. Poptr_GRFscaff_
35
34
33
46
18
43
31
41
 
22
22
28
27
22
25


XII.277

















37. Poptr_GRFscaff_
54
41
54
29
31
30
63
32
31
 
23
22
25
81
26


XIII.769

















38. Poptr_GRFscaff_
34
35
35
37
23
37
33
39
39
33
 
23
24
25
20


XIV.174

















39. Poptr_GRFscaff_
33
31
30
36
19
68
30
78
40
30
37
 
45
21
25


XIV.39

















40. Poptr_GRFscaff_
38
34
34
41
20
79
35
66
45
34
41
61
 
22
26


XIV.51

















41. Poptr_GRFscaff_
52
39
53
30
31
30
59
31
29
88
31
30
32
 
22


XIX.480

















42. Sacof_GRF
38
39
35
38
24
40
40
40
38
37
33
37
42
35
 


43. Vitvi_GRF
65
40
70
31
27
30
51
34
32
54
34
31
37
53
36


44. Zeama_GRF10_
25
34
25
24
41
23
25
23
24
28
24
22
24
26
28


EF515849

















45. Zeama_GRF11_
49
38
46
29
29
28
46
31
29
50
30
27
31
48
34


EF515850

















46. Zeama_GRF12_
45
39
45
30
32
27
50
31
30
52
32
28
33
48
36


EF515851

















47. Zeama_GRF13_
37
37
34
37
23
41
38
39
38
37
34
38
42
35
90


EF515852

















48. Zeama_GRF14_
50
41
45
35
28
32
52
33
32
52
35
31
36
48
38


EF515853

















49. Zeama_GRF1_
49
42
45
37
25
37
46
38
37
52
36
34
41
50
42


EF515840

















50. Zeama_GRF2_
40
36
43
33
29
39
41
40
35
42
33
38
41
38
40


EF515841

















51. Zeama_GRF3_
53
39
49
33
25
30
41
33
31
45
34
29
33
43
35


EF515842

















52. Zeama_GRF4_
25
32
28
26
41
24
27
23
24
28
25
23
24
30
27


EF515843

















53. Zeama_GRF5_
52
41
45
34
26
33
49
35
33
52
34
33
35
52
38


EF515844.1

















54. Zeama_GRF6_
51
41
43
33
26
33
51
35
33
53
33
32
36
51
37


EF515845.1

















55. Zeama_GRF7_
47
36
49
31
30
28
48
31
29
49
29
27
32
47
35


EF515846.1

















56. Zeama_GRF8_
38
38
36
37
23
39
37
40
37
35
33
38
41
35
94


EF515847.1

















57. Zeama_GRF9_
52
39
52
30
27
31
45
32
29
47
32
28
36
45
35


EF515848.1






43
44
45
46
47
48
49
50
51
52
53
54
55
56
57





 1. Aqufo_GRF
57
15
34
28
23
33
34
25
37
13
32
32
33
22
38


 2. Arath_GRF_
32
20
29
27
20
26
24
23
27
20
27
25
28
20
30


AT2G06200.1

















 3. Arath_GRF_
23
16
21
22
29
23
25
29
21
15
25
24
21
30
22


AT2G22840.1

















 4. Arath_GRF_
24
18
26
30
25
29
31
27
26
19
26
25
24
25
24


AT2G36400.1

















 5. Arath_GRF_
23
17
21
21
18
25
22
21
21
17
21
20
22
19
19


AT2G45480.1

















 6. Arath_GRF_
40
15
29
26
23
28
29
25
33
15
28
29
31
23
33


AT3G13960.1

















 7. Arath_GRF_
21
14
19
21
22
22
24
22
21
14
21
20
20
23
20


AT3G52910.1

















 8. Arath_GRF_
19
19
17
22
22
21
22
20
17
17
22
22
18
22
18


AT4G24150.1

















 9. Arath_GRF_
22
15
20
21
29
23
24
28
20
16
23
22
21
28
20


AT4G37740.1

















10. Arath_GRF_
22
14
22
24
24
25
24
24
21
13
26
23
22
24
21


AT5G53660.1

















11. Brana_GRF
41
13
29
27
25
26
30
24
34
13
28
28
30
23
34


12. Horvu_GRF
31
20
31
35
25
38
54
26
30
19
63
64
30
23
29


13. Lyces_GRF
20
20
23
27
25
25
23
25
23
19
25
24
23
24
24


14. Medtr_GRF
53
12
30
26
22
27
29
25
33
13
28
27
32
22
35


15. Medtr_GRF\like
21
19
20
21
21
22
22
24
22
20
25
25
20
21
23


16. Orysa_GRF_NM_
17
36
21
23
21
20
23
22
18
34
23
21
22
20
20


001054270.1

















17. Orysa_GRF_NM_
34
19
34
37
26
41
63
28
35
18
69
68
35
26
32


001060298.1

















18. Orysa_GRF_NM_
15
73
17
20
19
15
19
19
15
73
19
20
20
18
19


001066126.1

















19. Orysa_GRF_
34
20
32
35
28
41
72
26
32
20
61
59
31
25
31


Os02g47280.2

















20. Orysa_GRF_
42
15
43
30
22
32
36
26
69
16
33
33
43
21
64


Os02g53690.1

















21. Orysa_GRF_
27
14
25
25
26
27
27
30
26
14
25
25
27
26
28


Os03g51970.1

















22. Orysa_GRF_
19
34
21
23
21
23
24
26
19
32
23
21
22
20
20


Os04g48510.1

















23. Orysa_GRF_
34
19
34
36
26
41
62
28
34
19
71
69
35
26
32


Os04g51190.1

















24. Orysa_GRF_
33
15
35
57
24
68
39
27
34
14
42
41
36
23
33


Os06g02560.1

















25. Orysa_GRF_
23
18
23
23
29
25
24
55
22
18
24
24
22
29
21


Os11g35030.1

















26. Orysa_GRF_
27
16
25
24
28
24
27
42
25
16
28
26
25
26
25


Os12g29980.1

















27. Oyrsa_GRF_
22
15
24
24
65
25
26
28
22
17
25
25
23
67
22


Os03g47140.1

















28. Poptr_GRF_lcl_scaff_
52
14
35
28
25
35
32
25
37
14
33
31
33
23
38


28.10

















29. Poptr_GRF_lcl_scaff_
25
19
24
27
25
25
25
23
23
19
25
24
24
25
24


28.309

















30. Poptr_GRFscaff_
56
14
34
31
24
31
33
29
36
14
30
30
34
24
37


I.1018

















31. Poptr_GRFscaff_
21
16
20
21
25
23
25
22
20
16
25
21
21
24
19


I.688

















32. Poptr_GRFscaff_
19
30
21
23
17
20
19
21
18
29
20
20
22
17
18


I.995

















33. Poptr_GRFscaff_
21
15
20
20
27
22
24
28
20
16
23
22
19
25
20


II.1070

















34. Poptr_GRFscaff_
33
15
30
34
25
36
32
25
29
16
35
36
30
24
32


III.741

















35. Poptr_GRFscaff_
25
15
22
22
25
23
26
31
21
16
25
25
22
25
21


VII.1274

















36. Poptr_GRFscaff_
22
17
19
20
24
22
23
22
20
16
24
24
19
25
18


XII.277

















37. Poptr_GRFscaff_
41
13
33
35
25
40
39
26
33
13
41
40
32
25
34


XIII.769

















38. Poptr_GRFscaff_
22
18
20
23
21
23
23
21
21
18
22
23
19
19
21


XIV.174

















39. Poptr_GRFscaff_
21
14
19
21
25
22
23
28
19
14
24
23
19
26
19


XIV.39

















40. Poptr_GRFscaff_
24
16
23
23
25
24
26
30
24
14
25
25
22
25
23


XIV.51

















41. Poptr_GRFscaff_
39
15
30
33
24
37
37
26
32
17
40
40
32
22
32


XIX.480

















42. Sacof_GRF
22
18
22
25
86
24
25
30
22
17
27
26
23
91
21


43. Vitvi_GRF
 
14
34
29
22
33
33
26
40
13
31
32
36
23
42


44. Zeama_GRF10_
27
 
17
21
17
14
19
18
15
86
19
19
19
18
16


EF515849

















45. Zeama_GRF11_
46
28
  
32
23
34
33
26
41
18
33
31
75
22
41


EF515850

















46. Zeama_GRF12_
45
33
46
 
27
61
33
24
29
20
34
34
32
24
31


EF515851

















47. Zeama_GRF13_
35
26
35
35
 
24
26
29
22
18
27
26
23
86
22


EF515852

















48. Zeama_GRF14_
48
25
45
67
38
 
38
26
33
15
40
39
34
24
34


EF515853

















49. Zeama_GRF1_
48
30
46
43
42
52
  
29
35
18
57
57
32
25
34


EF515840

















50. Zeama_GRF2_
42
30
39
36
41
42
42
  
23
19
28
26
27
30
24


EF515841

















51. Zeama_GRF3_
54
24
52
42
33
50
52
38
  
15
33
34
41
22
72


EF515842

















52. Zeama_GRF4_
28
90
31
33
28
26
31
33
25
 
19
20
20
18
16


EF515843

















53. Zeama_GRF5_
47
32
46
44
38
53
68
43
47
31
  
87
33
27
31


EF515844.1

















54. Zeama_GRF6_
46
31
44
43
38
53
71
38
47
31
90
 
33
25
32


EF515845.1

















55. Zeama_GRF7_
51
32
83
46
35
47
44
38
52
31
47
43
 
23
40


EF515846.1

















56. Zeama_GRF8_
37
27
33
34
91
38
40
43
36
29
39
35
34
 
21


EF515847.1

















57. Zeama_GRF9_
59
25
54
43
35
48
44
39
79
27
45
44
54
33
 


EF515848.1









The percentage identity between the full length polypeptide sequences useful in performing the methods of the invention can be as low as 15% amino acid identity compared to SEQ ID NO: 2.
The percentage identity can be substantially increased if the identity calculation is performed between the QLQ domain SEQ ID NO: 2 (as represented by SEQ ID NO: 115 comprised in SEQ ID NO: 2; QLQ domain of the GRF polypeptides of Table A represented in FIG. 2) and the QLQ domains of the polypeptides useful in performing the invention. Similarly, the percentage identity can be substantially increased if the identity calculation is performed between the WRC domain SEQ ID NO: 2 (as represented by SEQ ID NO: 116 comprised in SEQ ID NO: 2; WRC domain of the GRF polypeptides of Table A represented in FIG. 3) and the WRC domains of the polypeptides useful in performing the invention. Percentage identity over the QLQ domain amongst the polypeptide sequences useful in performing the methods of the invention ranges between 25% and 99% amino acid identity, and percentage identity over the WRC domain amongst the polypeptide sequences useful in performing the methods of the invention ranges between 60% and 99% amino acid identity. As can also be observed in FIG. 3, the WRC domain is better conserved than the QLQ domain amongst the different GRF polypeptides, as shown in FIG. 2.
The percentages in amino acid acid identity between the QLQ domains, and the percentage identity between the WRC domains are significantly higher than the percentage amino acid identity calculated between the full length GRF polypeptide sequences.
Results of the software analysis related to RAA1-like polypeptide are shown in Table B2 for the global similarity and identity over the full length of the polypeptide sequences. Percentage similarity is given above the diagonal and percentage identity is given below the diagonal (bold face).
The percentage identity between the RAA1-like polypeptide sequences useful in performing the methods of the invention can be as low as 31% amino acid identity compared to SEQ ID NO: 121, leaving Q9LXB6 (SEQ ID NO: 155) out of consideration.







TABLE B2





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


polypeptide sequences. SEQ ID NO: 121 is represented by Q9LGE3.

























A2WN18
A2XRE0
Q6RIB0
A3BNA1
Q9LGE3
Q5Q0B3
Q0E1D7
O24340
Q8H475





A2WN18

63.6
73.4
70.6
100
46.4
67.2
74.5
68.8


A2XRE0
50

72
59.3
63.6
44.8
81.1
66.1
57.6


Q6RIB0
59.6
57.6

74.3
72.5
47.5
68
74.5
72.6


A3BNA1
55.3
48.8
52.8

70.6
39.8
60.7
64.5
95.3


Q9LGE3
98.2
50.8
58.7
56.1

46.4
67.2
73.6
68.8


Q5Q0B3
39.8
32.4
37
32.1
39.8

48.6
55.8
39.2


Q0E1D7
54.9
76.2
58.2
46.4
55.7
37.4

70.5
59.8


O24340
58
45
60.9
46.1
58.9
44.5
53.2

66.4


Q8H475
53
47.1
51.4
95.3
53.9
31.9
45.6
45.7



Q0JEF5
50
99.2
57.6
48.8
50.8
32.4
76.2
45
47.1


O49587
58.1
46.6
54
45.7
58.1
68.5
53.4
64.8
45.4


A2X4J6
54.9
76.2
58.2
46.4
55.7
37.9
99.2
51.6
45.6


Q7XX25
51.4
91.5
59.5
49.1
52.3
32.6
71.3
45.6
48.7


Q8LR63
58.3
47
52
45.5
59.1
39.2
50.7
54.7
45.5


Q9LXB5
58.9
46.7
56.2
45.3
58.9
46.2
52
79.6
44.9


Q9LXB6
8.5
7.3
7.6
6.7
8.5
8.3
8.7
11.1
6.8


O23624
58
45
59.1
47
58
45.6
52.4
92.7
44.8



















Q0JEF5
O49587
A2X4J6
Q7XX25
Q8LR63
Q9LXB5
Q9LXB6
O23624





A2WN18
63.6
67.7
67.2
67
70.1
72.3
10.4
73.6


A2XRE0
100
65.3
81.1
91.5
64.6
67.8
10.4
65.3


Q6RIB0
72
69.4
68
76.9
65.4
71.4
10
74.5


A3BNA1
59.3
58.9
60.7
63
59.1
62.5
9.2
65.5


Q9LGE3
63.6
67.7
67.2
67
70.1
72.3
10.4
72.7


Q5Q0B3
44.8
68.5
48.6
43.6
49.7
55.2
13.9
54.7


Q0E1D7
81.1
71
100
76.2
69.3
66.4
11.2
68.9


O24340
66.1
81.5
68
68.2
70.9
90.2
12.6
98.2


Q8H475
57.6
58.1
59.8
63
58.3
62.5
9.3
65.5


Q0JEF5

65.3
81.1
91.5
64.6
67.8
10.4
65.3


O49587
46.6

71
63.7
70.9
80.6
12.4
79.8


A2X4J6
76.2
54.1

76.2
69.3
67.2
11.3
68.9


Q7XX25
91.5
47.6
71.3

64.6
69.6
9.7
67.3


Q8LR63
47
56.6
50.7
48.8

70.1
11.3
70.1


Q9LXB5
46.7
67.2
52
47.4
54.7

13.8
89.3


Q9LXB6
7.2
10
8.7
6.6
8.7
13.6

12.3


O23624
45
66.4
52.4
45.6
53.9
79.6
11.1









The percentage identity between the polypeptide sequences related to SYR polypeptides useful in performing the methods of the invention can be as low as 27% amino acid identity compared to SEQ ID NO: 169.







TABLE B3







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

























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



























 1. SEQID 169

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. SEQID 179
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. SEQID 180
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. SEQID 181
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. SEQID 182
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. SEQID 183
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. SEQID 184
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. SEQID 185
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. SEQID 186
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. SEQID 187
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. SEQID 188
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. SEQID 189
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. SEQID 190
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. SEQID 191
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. SEQID 192
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. SEQID 201
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. SEQID 202
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









The percentage identity between the ARKL polypeptide sequences useful in performing the methods of the invention can be as low as 10% amino acid identity compared to SEQ ID NO: 213 (Orysa_ARKL1).







TABLE B4







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























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

























1. Orysa

25.6
19.2
22.9
26.4
21.5
22.6
21.3
16.6
19.6
25.1
20.4
64.7
18.0
18.8


ARKL1

















2. Orysa
34.8

23.7
21.2
23.2
22.0
20.9
13.9
10.0
12.8
62.0
24.6
24.7
22.3
14.8


ARKL3

















3. Orysa
27.6
37.8

23.0
17.9
22.3
21.8
14.0
9.6
12.0
25.1
27.3
19.1
27.8
11.0


ARKL4

















4. Orysa
38.1
36.2
35.1

20.6
45.4
23.6
15.3
11.1
13.7
20.9
23.5
23.1
22.0
14.6


ARKL5

















5. Orysa
41.4
31.0
25.7
33.9

21.0
22.4
21.6
24.2
18.6
22.9
19.5
25.1
16.6
19.8


ARKL6

















6. Orysa
33.4
35.5
34.6
57.0
30.6

23.7
15.3
11.0
13.5
23.3
24.2
22.1
21.3
14.6


ARKL7

















7. Orysa
32.6
35.0
32.7
42.7
33.6
38.6

14.6
11.6
14.2
20.6
24.0
21.8
20.7
11.9


ARKL8

















8. Orysa
35.1
24.3
21.7
24.6
32.1
25.0
25.6

15.5
15.3
15.5
13.4
20.1
11.2
20.1


ARKL9

















9. Zeama
23.4
14.0
12.3
16.6
28.0
15.8
15.8
22.3

43.2
10.1
8.4
15.5
7.3
17.1


ARKL1

















10. Zeama
25.3
16.9
14.4
16.8
26.1
17.3
19.4
23.0
64.2

11.9
17.3
18.2
11.5
19.0


ARKL2

















11. Horvu
35.7
73.4
38.6
38.1
33.0
38.3
35.2
23.5
14.3
15.8

24.0
24.9
23.7
13.9


ARKL1

















12. Horvu
29.6
38.9
42.9
37.2
28.8
36.6
37.7
21.7
13.0
18.8
37.8

20.3
32.5
12.3


ARKL2

















13. Horvu
76.5
34.3
27.1
35.1
40.1
32.5
31.2
32.6
22.5
23.5
35.1
28.6

17.1
19.3


ARKL3

















14. Glyma
26.4
35.6
43.6
33.3
23.4
33.9
31.4
18.8
10.8
13.6
35.8
46.9
25.8

11.3


ARKL1

















15. Glyma
31.6
21.6
19.0
23.8
29.9
22.0
23.0
32.5
26.0
30.5
20.4
19.1
31.0
16.8



ARKL2

















16. Musmu
18.0
26.1
28.6
22.9
17.4
25.4
23.9
15.2
7.1
8.4
26.1
29.8
18.8
32.6
14.2


ARKL3
















TABLE B5-1





MatGAT results for global similarity and identity over the DUF221 domain or


domain fragment in a selection of YTP polypeptides from Table A.





















YTP13_DUF221
YTP16_DUF221
YTP5_DUF221
YTP12_DUF221
YTP6_DUF221





YTP13_DUF221

81.1
59.3
58.8
56.2


YTP16_DUF221
90.3

63.0
63.0
58.8


YTP5_DUF221
76.9
76.5

84.0
60.4


YTP12_DUF221
77.7
78.2
91.5

60.2


YTP6_DUF221
74.8
74.5
77.2
78.4



YTP18_DUF221
60.5
59.8
63.9
63.2
60.8


YTP11_DUF221
61.8
62.0
63.1
64.8
64.6


YTP19_DUF221
49.4
50.7
52.2
54.4
51.5


YTP21_DUF221
59.0
58.1
60.7
63.4
59.3


YTP3_DUF221
55.5
56.9
56.3
58.7
57.5


YTP15_DUF221
48.8
49.3
50.0
52.9
51.7


YTP9_DUF221
50.4
49.2
52.3
54.9
51.3


YTP26_DUF221
36.1
34.8
35.4
37.0
34.4


YTP4_DUF221
41.5
40.4
38.5
38.7
37.1


YTP1_DUF221
16.1
14.7
16.0
19.7
17.5






YTP18_DUF221
YTP11_DUF221
YTP19_DUF221
YTP21_DUF221
YTP3_DUF221





YTP13_DUF221
43.6
44.1
34.1
39.0
36.2


YTP16_DUF221
42.6
45.5
34.5
37.6
37.7


YTP5_DUF221
45.3
48.5
36.1
40.7
38.7


YTP12_DUF221
43.3
49.8
37.9
40.5
38.3


YTP6_DUF221
41.8
45.1
34.8
36.4
36.8


YTP18_DUF221

55.0
44.8
37.6
36.1


YTP11_DUF221
71.4

41.1
37.6
37.3


YTP19_DUF221
59.3
56.4

30.6
28.4


YTP21_DUF221
54.0
56.4
47.0

61.9


YTP3_DUF221
54.2
56.7
46.7
77.6



YTP15_DUF221
47.6
49.0
38.0
49.0
49.3


YTP9_DUF221
49.2
49.9
37.6
50.4
48.0


YTP26_DUF221
38.9
38.7
33.9
36.3
36.1


YTP4_DUF221
40.4
40.0
33.6
38.5
38.2


YTP1_DUF221
14.0
15.6
18.2
15.2
15.9






YTP ♯899 UF221
YTP9_DUF221
YTP26_DUF221
YTP4_DUF221
YTP1_DUF221





YTP13_DUF221
29.4
29.3
20.1
20.6
9.6


YTP16_DUF221
30.3
30.3
18.6
19.1
9.0


YTP5_DUF221
29.2
30.9
17.4
19.3
9.0


YTP12_DUF221
32.2
32.8
17.7
18.9
11.2


YTP6_DUF221
30.2
27.6
17.4
19.1
9.2


YTP18_DUF221
26.4
28.4
19.8
21.4
7.3


YTP11_DUF221
27.8
29.1
19.4
20.3
9.0


YTP19_DUF221
21.3
21.4
18.6
16.2
11.1


YTP21_DUF221
28.2
30.8
17.5
17.9
9.7


YTP3_DUF221
28.1
28.3
17.4
18.0
8.9


YTP15_DUF221

59.8
18.5
17.9
16.8


YTP9_DUF221
77.5

20.8
19.7
29.3


YTP26_DUF221
35.7
38.7

68.6
9.2


YTP4_DUF221
37.4
37.8
83.6

7.0


YTP1_DUF221
23.0
30.4
15.5
13.8









Table B5-2 shows the SEQ ID NO: corresponding to the sequences used in Table B5-1.







TABLE B5-2







DUF221 domains.










Description
SEQ ID NO







YTP13_DUF221
530



YTP16_DUF221
533



YTP5_DUF221
522



YTP12_DUF221
529



YTP6_DUF221
523



YTP18_DUF221
525



YTP11_DUF221
528



YTP19_DUF221
536



YTP21_DUF221
538



YTP3_DUF221
520



YTP15_DUF221
532



YTP9_DUF221
526



YTP26_DUF221
543



YTP4_DUF221
521



YTP1_DUF221
518










Example 4
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, ProDom 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: 2 are presented in Table C.







TABLE C1







InterPro scan results of the polypeptide sequence as represented


by SEQ ID NO: 2











Integrated




InterPro accession
database
Integrated database
Integrated database


number and name
name
accession number
accession name





IPR014977
PFAM
PF08879
WRC


WRC domain


IPR014978
PFAM
PF08880
QLQ


QLQ domain









The results of the InterPro scan of the polypeptide sequence as represented by SEQ ID NO: 213 are presented in Table C1.







TABLE C2-1







InterPro scan results (major accession numbers) of the polypeptide sequence


as represented by SEQ ID NO: 213.




















Amino acid









coordinates





Description



on the




InterPRO
accession
Method
Database
Accession
Query



Query
accession
interpro
search
searched
in database
sequence
evalue





Orysa_ARKL1
IPR0013083
Zinc finger,
Gene3D
Gene3D
G3DSA:
315-365
1.5e−10




RING/FYVE/PHD-


3.30.40.10






type







Orysa_ARKL1
IPR001841
Zinc Finger, Ring-
HMM
Smart
SM00184
319-359
7.4e−07




type
Smart






Orysa_ARKL1
no IPR
RING FINGER
HMM
Panther
PTHR22766
316-365
9.6e−12



accession
PROTEIN 24-
Panther








RELATED







Orysa_ARKL1
IPR001841
Zinc Finger, Ring-
HMM
Pfam
PF00097
319-359
4.9e−09




type
Pfam






Orysa_ARKL1
no IPR
NA*
HMM
Pfam
PF2828
266-296




accession

Pfam









Table C2 gives the SEQ ID NO: comprising the conserved RING domain (ZfC3HC4) and DAR1 (PfamB2828) in the ARKL polypeptides of Table A.







TABLE C2-2







RING and DAR1 domains in ARKL polypeptides













SEQ



Domain
Reference Protein
ID NO:







ZfC3HC4_Orysa_ARKL1
Orysa_ARKL1
306



ZfC3HC4_Orysa_ARKL3
Orysa_ARKL3
307



ZfC3HC4_Orysa_ARKL4
Orysa_ARKL4
308



ZfC3HC4_Orysa_ARKL5
Orysa_ARKL5
309



ZfC3HC4_Orysa_ARKL6
Orysa_ARKL6
310



ZfC3HC4_Orysa_ARKL7
Orysa_ARKL7
311



ZfC3HC4_Orysa_ARKL8
Orysa_ARKL8
312



ZfC3HC4_Orysa_ARKL9
Orysa_ARKL9
313



ZfC3HC4_Zeama_ARKL1
Zeama_ARKL1
314



ZfC3HC4_Zeama_ARKL2
Zeama_ARKL2
315



ZfC3HC4_Horvu_ARKL1
Horvu_ARKL1
316



ZfC3HC4_Horvu_ARKL2
Horvu_ARKL2
317



ZfC3HC4_Horvu_ARKL3
Horvu_ARKL3
318



ZfC3HC4_Lyces_ARKL1
Lyces_ARKL1
319



ZfC3HC4_Lyces_ARKL2
Lyces_ARKL2
320



ZfC3HC4_Lyces_ARKL3
Lyces_ARKL3
321



ZfC3HC4_Glyma_ARKL1
Glyma_ARKL1
322



ZfC3HC4_Glyma_ARKL2
Glyma_ARKL2
323



ZfC3HC4_Zinel_ARKL1
Zinel_ARKL1
324



ZfC3HC4_Lotja_ARKL1
Lotja_ARKL1
325



ZfC3HC4_Arath_ARKL1
Arath_ARKL1
326



ZfC3HC4_Arath_ARKL2
Arath_ARKL2
327



ZfC3HC4_Arath_ARKL3
Arath_ARKL3
328



ZfC3HC4_Arath_ARKL4
Arath_ARKL4
329



ZfC3HC4_Arath_ARKL5
Arath_ARKL5
330



ZfC3HC4_Arath_ARKL6
Arath_ARKL6
331



ZfC3HC4_Arath_ARKL7
Arath_ARKL7
332



ZfC3HC4_Arath_ARKL8
Arath_ARKL8
333



ZfC3HC4_Arath_ARKL9
Arath_ARKL9
334



ZfC3HC4_Arath_ARKL10
Arath_ARKL10
335



ZfC3HC4_Arath_ARKL11
Arath_ARKL11
336



ZfC3HC4_Arath_ARKL12
Arath_ARKL12
337



ZfC3HC4_Poptr_ARKL1
Poptr_ARKL1
338



ZfC3HC4_Poptr_ARKL2
Poptr_ARKL2
339



ZfC3HC4_Poptr_ARKL3
Poptr_ARKL3
340



ZfC3HC4_Poptr_ARKL4
Poptr_ARKL4
341



ZfC3HC4_Poptr_ARKL5
Poptr_ARKL5
342



ZfC3HC4_Poptr_ARKL6
Poptr_ARKL6
343



ZfC3HC4_Poptr_ARKL7
Poptr_ARKL7
344



ZfC3HC4_Poptr_ARKL8
Poptr_ARKL8
345



ZfC3HC4_Poptr_ARKL9
Poptr_ARKL9
346



ZfC3HC4_Poptr_ARKL10
Poptr_ARKL10
347



ZfC3HC4_Medtr_ARKL1
Medtr_ARKL1
348



ZfC3HC4_Medtr_ARKL2
Medtr_ARKL2
349



ZfC3HC4_Medtr_ARKL3
Medtr_ARKL3
350



ZfC3HC4_Medtr_ARKL4
Medtr_ARKL4
351



PfamB2828_Orysa_ARKL1
Orysa_ARKL1
352



PfamB2828_Orysa_ARKL2
Orysa_ARKL2
353



PfamB2828_Orysa_ARKL3
Orysa_ARKL3
354



PfamB2828_Orysa_ARKL4
Orysa_ARKL4
355



PfamB2828_Orysa_ARKL5
Orysa_ARKL5
356



PfamB2828_Orysa_ARKL6
Orysa_ARKL6
357



PfamB2828_Orysa_ARKL7
Orysa_ARKL7
358



PfamB2828_Orysa_ARKL8
Orysa_ARKL8
359



PfamB2828_Orysa_ARKL9
Orysa_ARKL9
360



PfamB2828_Zeama_ARKL1
Zeama_ARKL1
361



PfamB2828_Zeama_ARKL2
Zeama_ARKL2
362



PfamB2828_Horvu_ARKL1
Horvu_ARKL1
363



PfamB2828_Horvu_ARKL2
Horvu_ARKL2
364



PfamB2828_Horvu_ARKL3
Horvu_ARKL3
365



PfamB2828_Lyces_ARKL1
Lyces_ARKL1
366



PfamB2828_Lyces_ARKL2
Lyces_ARKL2
367



PfamB2828_Lyces_ARKL3
Lyces_ARKL3
368



PfamB2828_Glyma_ARKL1
Glyma_ARKL1
369



PfamB2828_Glyma_ARKL2
Glyma_ARKL2
370



PfamB2828_Zinel_ARKL1
Zinel_ARKL1
371



PfamB2828_Lotja_ARKL1
Lotja_ARKL1
372



PfamB2828_Arath_ARKL1
Arath_ARKL1
373



PfamB2828_Arath_ARKL2
Arath_ARKL2
374



PfamB2828_Arath_ARKL3
Arath_ARKL3
375



PfamB2828_Arath_ARKL4
Arath_ARKL4
376



PfamB2828_Arath_ARKL5
Arath_ARKL5
377



PfamB2828_Arath_ARKL6
Arath_ARKL6
378



PfamB2828_Arath_ARKL7
Arath_ARKL7
379



PfamB2828_Arath_ARKL8
Arath_ARKL8
380



PfamB2828_Arath_ARKL9
Arath_ARKL9
381



PfamB2828_Arath_ARKL10
Arath_ARKL10
382



PfamB2828_Arath_ARKL11
Arath_ARKL11
383



PfamB2828_Arath_ARKL12
Arath_ARKL12
384



PfamB2828_Poptr_ARKL1
Poptr_ARKL1
385



PfamB2828_Poptr_ARKL2
Poptr_ARKL2
386



PfamB2828_Poptr_ARKL3
Poptr_ARKL3
387



PfamB2828_Poptr_ARKL4
Poptr_ARKL4
388



PfamB2828_Poptr_ARKL5
Poptr_ARKL5
389



PfamB2828_Poptr_ARKL6
Poptr_ARKL6
390



PfamB2828_Poptr_ARKL7
Poptr_ARKL7
391



PfamB2828_Poptr_ARKL8
Poptr_ARKL8
392



PfamB2828_Poptr_ARKL9
Poptr_ARKL9
393



PfamB2828_Poptr_ARKL10
Poptr_ARKL10
394



PfamB2828_Medtr_ARKL1
Medtr_ARKL1
395



PfamB2828_Medtr_ARKL2
Medtr_ARKL2
396



PfamB2828_Medtr_ARKL3
Medtr_ARKL3
397



PfamB2828_Medtr_ARKL4
Medtr_ARKL4
398










The results of the Pfam search of the polypeptide sequence as represented by SEQ ID NO: 409 are presented in Table C3-1 (Trusted matches) and Table C2 (Matches to Pfam-B).







TABLE C3-1:







Trusted matches from a Pfam search using SEQ ID NO: 409


as query sequence. Trusted matches have a higher than the


gathering threshold for the specific domain in Pfam. DUF221


is a Pfam-A domain with accession number PF02714.













Domain
Start
End
Bits
Evalue
Alignment
Mode





DUF221
305
411
44.10
2.3e−12
Align
fs
















TABLE C3-2







Matches to Pfam-B












Domain
Start
End
Alignment
















Pfam-B_1332
1
110
Align



Pfam-B_4698
144
233
Align



Pfam-B_131006
234
304
Align










Example 5
Subcellular Localisation Prediction of the Polypeptide Sequences Useful in Performing the Methods of the Invention
Experimental methods for protein localization range from immunolocalization to tagging of proteins using green fluorescent protein (GFP) or beta-glucuronidase (GUS). For example, a GRF polypeptide fused to a GUS reporter gene was used to transform transiently onion epidermal cells (van der Knapp et al. (2000) Plant Phys 122: 695-704). The nucleus was identified as the subcellular compartment of the GRF polypeptide. Such methods to identify subcellular compartmentalisation of GRF polypeptides are well known in the art.
A predicted nuclear localisation signal (NLS) was found by multiple sequence alignment, followed by eye inspection, in the WRC domain (CRRTDGKKWRC) (found within SEQ ID NO: 116) of the GRF polypeptide of Table A. An NLS is one or more short sequences of positively charged lysines or arginines.
Computational prediction of protein localisation from sequence data was performed. Among algorithms well known to a person skilled in the art are available at the ExPASy Proteomics tools hosted by the Swiss Institute for Bioinformatics, for example, PSort, TargetP, ChloroP, LocTree, Predotar, LipoP, MITOPROT, PATS, PTS1, SignalP and others.
LOCtree is an algorithm that can predict the subcellular localization and DNA-binding propensity of non-membrane proteins in non-plant and plant eukaryotes as well as prokaryotes. LOCtree classifies eukaryotic animal proteins into one of five subcellular classes, while plant proteins are classified into one of six classes and prokaryotic proteins are classified into one of three classes. Table D below shows the output of LOCtree using the polypeptide sequence information of SEQ ID NO: 2. High confidence predictions have reliability index values greater than 5.







TABLE D







Output of LOCtree using the polypeptide sequence information of


SEQ ID NO: 2.












Intermediate localization



Predicted
Reliability
prediction (output of different
Reliability


Localization
index
SVMs in hierarchical tree)
index





DNA binding
6
Not secreted, Nuclear,
8, 6, 9




DNA-binding









The predicted subcellular compartment of the GRF polypeptide as represented by SEQ ID NO: 2 using the LOCTree algorithm is the nucleus.
Example 6
Assay Related to the Polypeptide Sequences Useful in Performing the Methods of the Invention
GRF polypeptides useful in the methods of the present invention (at least in their native form) typically, but not necessarily, have transcriptional regulatory activity and capacity to interact with other proteins. DNA-binding activity and protein-protein interactions may readily be determined in vitro or in vivo using techniques well known in the art (for example in Current Protocols in Molecular Biology, Volumes 1 and 2, Ausubel et al. (1994), Current Protocols). GRF polypeptides are capable of transcriptional activation of reporter genes in yeast cells (Kim & Kende (2004) Proc Natl Acad Sci 101(36): 13374-13379). GRF polypeptides are also capable of interacting with GRF-interacting factor polypeptides (GIF1 to GIF3; also called SYT1 to SYT3) in vivo in yeast cells, using a yeast two-hybrid protein-protein interaction assay (Kim & Kende, supra). In vitro binding assays are also used to show that GRF polypeptides and GIF (also called SYT) polypeptides are interacting partners (Kim & Kende, supra). The experiments described in this publication are useful in characterizing GRF polypeptides, and are well known in the art.
Example 7
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: 121 are presented Table E1. 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: 121 is likely to be the cytoplasm, no transit peptide (SignalP) or nuclear localisation signal (PredictNLS) is predicted.







TABLE E1





TargetP 1.1 analysis of the polypeptide sequence as represented


by SEQ ID NO: 121


















Length (AA)
109



Chloroplastic transit peptide
0.098



Mitochondrial transit peptide
0.404



Secretory pathway signal
0.025



peptide



Other subcellular targeting
0.450



Predicted Location
/



Reliability class
5



Predicted transit peptide length
/










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;
        TMHMM, hosted on the server of the Technical University of Denmark
    
    


The results of TargetP 1.1 analysis of the polypeptide sequence as represented by SEQ ID NO: 169 are presented Table E2. 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: 169 may be the mitochondrion; however it should be noted that the reliability class is 5 (i.e. the lowest reliability class).







TABLE E2





TargetP 1.1 analysis of the polypeptide sequence as represented


by SEQ ID NO: 169


















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 are identified by the TMHMM program, hosted on the server of the Center for Biological Sequence Analysis, Technical University of Denmark. The probability that the N-terminus is located inside is 0.997. Further details on the orientation are given in Table F:







TABLE F







results of TMHMM 2.0












begin-end




Orientation
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 8
Functional Assay for the ARKL Polypeptides
The ubiquitination assay is carried out essentially as described by Stone et al. 2005. GST labeled ARKL protein is incubated at 30 C and pH 7.5 with yeast E1, purifed E2 At UBCC8, and ubiquitin (Sigma). The reaction is stopped and analyzed by SDS-PAGE electrophoresis followed by western blotting using ubiquitin antibodies.
Zinc chelating experiments are done by incubating TPEN-treated bead bound GST-ARKL protein with ZnC12.
Example 9
Transmembrane Topology Prediction of the Polypeptide Sequences Useful in Performing the Methods of the Invention
TMHMM V 2.O algorithm (Krogh et al. 2001 J Mol Biol, 305, 567-580) was used to predict transmembrane helices in SEQ ID NO: 409.
As shown below there are 4 predicted transmembrane helices. The position of the amino acide residues for the helices is also indicated. The loops between the transmembrane helices are predicted to be located at the inside of the membrane for loops between residues 28-85 and 172-373 and outside for loop between residues 109-151.















# Sequence Number of predicted TMHs: 4



# Sequence Exp number of AAs in TMHs: 89.26923



# Sequence Exp number, first 60 AAs: 22.14249



# Sequence Total prob of N-in: 0.04519



# Sequence POSSIBLE N-term signal sequence




















start end (amino acid coordinate)




















SequenceTMHMM2.0
outside
1
4



SequenceTMHMM2.0
TMhelix
5
27



SequenceTMHMM2.0
inside
28
85



SequenceTMHMM2.0
TMhelix
86
108



SequenceTMHMM2.0
outside
109
151



SequenceTMHMM2.0
TMhelix
152
171



SequenceTMHMM2.0
inside
172
373



SequenceTMHMM2.0
TMhelix
374
396



SequenceTMHMM2.0
outside
397
428










Example 10
a) Cloning of Nucleic Acid Sequence as Represented by SEQ ID NO: 1
Unless otherwise stated, recombinant DNA techniques are performed according to standard protocols described in (Sambrook (2001) Molecular Cloning: a laboratory manual, 3rd Edition Cold Spring Harbor Laboratory Press, CSH, New York) or in Volumes 1 and 2 of Ausubel et al. (1994), Current Protocols in Molecular Biology, Current Protocols. Standard materials and methods for plant molecular work are described in Plant Molecular Biology Labfax (1993) by R. D. D. Croy, published by BIOS Scientific Publications Ltd (UK) and Blackwell Scientific Publications (UK).
The Arabidopsis thaliana cDNA encoding the GRF polypeptide sequence as represented by SEQ ID NO: 2 was amplified by PCR using as template an Arabidopsis cDNA bank synthesized from mRNA extracted from mixed plant tissues. The following primers, which include the AttB sites for Gateway recombination, were used for PCR amplification:







1) Prm 10010 (SEQ ID NO: 118, sense):


5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTTAAACAATGATGAGTCTA


 


AGTGGAAGTAG-3′


 


2) Prm 10011 (SEQ ID NO: 119, reverse,


complementary):


5′-GGGGACCACTTTGTACAAGAAAGCTGGGTAGCTCTACTTAATTAGCT


 


ACCAG-3′






PCR was performed using Hifi Taq DNA polymerase in standard conditions. A PCR fragment of the expected length (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 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.
b) Cloning of the Nucleic Acid Sequence Coding for RAA1-Like Polypeptides Used in the Methods of the Invention
The nucleic acid sequence used in the methods of the invention was amplified by PCR using as template a custom-made Oryza sativa seedlings cDNA library (in pCMV Sport 6.0; Invitrogen, Paisley, UK). PCR was performed using Hifi Taq DNA polymerase in standard conditions, using 200 ng of template in a 50 μl PCR mix. The primers used were prm09129 (SEQ ID NO: 122; sense, start codon in bold):







5′ ggggacaagtttgtacaaaaaagcaggcttaaacaatgtcaggggtt


 


tgggtg 3′






and prm09988 (SEQ ID NO: 123; reverse, complementary):








5′ ggggaccactttgtacaagaaagctgggttgtcgcataggtcaattt


 


agg 3′,






which include the AttB sites for Gateway recombination. The amplified PCR fragment was 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”, pRAA1-like. Plasmid pDONR201 was purchased from Invitrogen, as part of the Gateway® technology.

c) Gene Cloning of the Nucleic Acid Sequence Coding for SYR Polypeptides
DNA manipulation: unless otherwise stated, recombinant DNA techniques are performed according to standard protocols described in (Sambrook (2001) Molecular Cloning: a laboratory manual, 3rd Edition, Cold Spring Harbor Laboratory Press, CSH, New York) or in Volumes 1 and 2 of Ausubel et al. (1994), Current Protocols in Molecular Biology, Current Protocols. Standard materials and methods for plant molecular work are described in Plant Molecular Biology Labfax (1993) by R. D. D. Croy, published by BIOS Scientific Publications Ltd (UK) and Blackwell Scientific Publications (UK).
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: 170; sense, start codon in bold, AttB1 site in italic:







5′-ggggacaagtttgtacaaaaaagcaggcttaaacaatggaaggtgta


 


ggtgctagg-3′)






and prm08171 (SEQ ID NO: 171; reverse, complementary, AttB2 site in italic:








5′-ggggaccactttgtacaagaaagctgggtcaaaaacaaaaataaatt


 


cccc-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.

d) Cloning of the Nucleic Acid Sequence Coding for ARKL Polypeptides Used in the Methods of the Invention
The nucleic acid sequence used in the methods of the invention was amplified by PCR using as template a custom-made Oryza sativa seedlings and panicles cDNA library (in pCMV Sport 6.0; Invitrogen, Paisley, UK). PCR was performed using Hifi Taq DNA polymerase in standard conditions, using 200 ng of template in a 50 μl PCR mix. The primers used were prm04873(SEQ ID NO: 404; sense, start codon in bold):







5′-ggggacaagtttgtacaaaaaagcaggcttaaacaatggatgatcac


 


atgggaaga-3′






and prm04874 (SEQ ID NO: 405; reverse, complementary):








5′-ggggaccactttgtacaagaaagctgggttttggtttctgaagaagc


 


acc-3′,






which include the AttB sites for Gateway recombination. The amplified PCR fragment was 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”, pARKL. Plasmid pDONR201 was purchased from Invitrogen, as part of the Gateway® technology.

e) Cloning of the Nucleic Acid Coding for YTP Polypeptides Used in the Methods of the Invention
The nucleic acid sequence used in the methods of the invention was amplified by PCR using as template a custom-made Oryza sativa seedlings cDNA library (in pCMV Sport 6.0; Invitrogen, Paisley, UK). PCR was performed using Hifi Taq DNA polymerase in standard conditions, using 200 ng of template in a 50 μl PCR mix. The primers used were (SEQ ID NO: 546: sense, start codon in bold):







5′-ggggacaagtttgtacaaaaaagcaggcttaaacaatggacaccgcg


 


tcgt-3′






and (SEQ ID NO: 547; reverse, complementary):








5′-ggggaccactttgtacaagaaagctgggtcagcacttgcattagatg


 


gat-3′,






which include the AttB sites for Gateway recombination. The amplified PCR fragment was 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”, pENTR-YTP1. Plasmid pDONR201 was purchased from Invitrogen, as part of the Gateway® technology.

Example 11
a) Expression Vector Construction Using the Nucleic Acid Sequence as Represented by SEQ ID NO: 1
The entry clone comprising SEQ ID NO: 1 was subsequently used in an LR reaction with a destination vector 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 nucleic acid sequence of interest already cloned in the entry clone. A rice GOS2 promoter (SEQ ID NO: 117) for constitutive expression was located upstream of this Gateway cassette.
After the LR recombination step, the resulting expression vector pGOS2::GRF (FIG. 4) was transformed into Agrobacterium strain LBA4044 according to methods well known in the art.
b) Expression Vector Construction Using the Nucleic Acid Sequence as Represented by SEQ ID NO: 120
The entry clone comprising SEQ ID NO: 120 was then used in an LR reaction with a destination vector 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 nucleic acid sequence of interest already cloned in the entry clone. A rice GOS2 promoter (SEQ ID NO: 124) for constitutive expression was located upstream of this Gateway cassette. In an alternative embodiment, a rice HMGP promoter (SEQ ID NO: 125) for constitutive expression was located upstream of the Gateway cassette.
After the LR recombination step, the resulting expression vector pGOS2::RAA1-like (FIG. 9), or pHMGP::RAA1-like, was transformed into Agrobacterium strain LBA4044 according to methods well known in the art.
c) Expression Vector Construction Using the Nucleic Acid Sequence Coding for SYR Polypeptides
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: 211) 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: 200 or SEQ ID NO: 210) 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. 13) were transformed into Agrobacterium strain LBA4044 and subsequently to Oryza sativa plants.
d) Expression Vector Construction Using the Nucleic Acid Sequence as Represented by SEQ ID NO: 212
The entry clone comprising SEQ ID NO: 212 was then used in an LR reaction with a destination vector 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 nucleic acid sequence of interest already cloned in the entry clone. A rice GOS2 promoter (SEQ ID NO: 406) for root specific expression was located upstream of this Gateway cassette.
After the LR recombination step, the resulting expression vector pGOS2::ARKL (FIG. 18) was transformed into Agrobacterium strain LBA4044 according to methods well known in the art.
e) Expression Vector Construction Using the Nucleic Acid Sequence as Represented by SEQ ID NO: 408
The entry clone comprising SEQ ID NO: 408 was then used in an LR reaction with a destination vector 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 nucleic acid sequence of interest already cloned in the entry clone. A rice GOS2 promoter (SEQ ID NO: 548) for root specific expression was located upstream of this Gateway cassette.
After the LR recombination step, the resulting expression vector pGOS2::YTP1 (FIG. 23) was transformed into Agrobacterium strain LBA4044 according to methods well known in the art.
Example 12
Plant Transformation
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-cultivation (to boost cell division activity).
Agrobacterium strain LBA4404 containing each individual expression vector was used independently for co-cultivation. 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 T0 rice transformants were generated for each 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 Hodges1996, Chan et al. 1993, Hiei et al. 1994).
Example 13
Phenotypic Evaluation Procedure
13.1-1 Evaluation Setup General
Approximately 35 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. Six events, of which the T1 progeny segregated 3:1 for presence/absence of the transgene, were retained. For each of these events, approximately 10 T1 seedlings containing the transgene (hetero- and homo-zygotes) and approximately 10 T1 seedlings lacking the transgene (nullizygotes) were selected by monitoring visual marker expression. The transgenic plants and the corresponding nullizygotes were grown side-by-side at random positions. Greenhouse conditions were of shorts days (12 hours light), 28° C. in the light and 22° C. in the dark, and a relative humidity of 70%.
13.1-2 Evaluation Set-Up for Plants Transformed with SYR Under the Control of the Rice GOS2 Promoter or the HMGP 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. 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%.
General Setup
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.
Four 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. 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.
13.2 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 to verify for an overall effect of the gene, also known as a global gene effect. The threshold for significance for a true global gene effect was set at a 5% probability level for the F-test. A significant F-test value points to a gene effect, meaning that it is not only the mere presence or position of the gene that is causing the differences in phenotype.
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.
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.
13.3 Parameters Measured
Biomass-Related Parameter Measurement (General Method)
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 above ground area is the area measured at the time point at which the plant had reached its maximal leafy biomass. The early vigour is the plant (seedling) aboveground area three weeks post-germination. Increase in root biomass is expressed as an increase in total root biomass (measured as maximum biomass of roots observed during the lifespan of a plant); or as an increase in the root/shoot index (measured as the ratio between root mass and shoot mass in the period of active growth of root and shoot).
Early vigour was determined by counting the total number of pixels from aboveground plant parts discriminated from the background. This value was averaged for the pictures taken on the same time point from different angles and was converted to a physical surface value expressed in square mm by calibration. The results described below are for plants three weeks post-germination.
Seed-Related Parameter Measurements (General Method)
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 weight per plant was measured by weighing all filled husks harvested from one 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 weight per plant 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).
Nitrogen Use Efficiency Screen (for Plants Transformed with SYR)
Rice plants from T2 seeds were grown in potting soil under normal conditions except for the nutrient solution. The pots were watered from transplantation to maturation with a specific nutrient solution containing reduced N nitrogen (N) content, usually between 7 to 8 times less. The rest of the cultivation (plant maturation, seed harvest) was the same as for plants not grown under abiotic stress. Growth and yield parameters are recorded as detailed for growth under normal conditions.
Drought Stress Screen (for Plants Transformed with SYR)
Rice plants from T1, T2 or further generations 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 re-transferred 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. Growth and yield parameters were recorded as detailed for growth under normal conditions. The applied drought conditions were “severe drought conditions” as defined above.
Example 14
Results of the Phenotypic Evaluation of the Transgenic Rice Plants Expressing the Nucleic Acid Sequence Encoding a GRF Polypeptide as Represented by SEQ ID NO: 2
The results of the evaluation of transgenic rice plants expressing the nucleic acid sequence encoding a GRF polypeptide as represented by SEQ ID NO: 2, under the control of the GOS2 promoter for constitutive expression, and grown under normal growth conditions, are presented below.
There was a significant increase in the early vigor, in the aboveground biomass, in the total seed yield per plant, in the seed filling rate, in the harvest index, and in the thousand kernel weight (TKW) of the transgenic plants compared to corresponding nullizygotes (controls), as shown in Table G.







TABLE G







Results of the evaluation of transgenic rice plants expressing the


nucleic acid sequence encoding a GRF polypeptide as represented


by SEQ ID NO: 2, under the control of the GOS2 promoter for


constitutive expression.











0verall average % increase



Trait
in 6 events in the T1 generation














Aboveground biomass
2%



Early vigor
13%



Total seed yield per plant
12%



Seed filling rate
5%



Harvest index
11%



TKW
11%










Example 15
a) Results of the Phenotypic Evaluation of the Transgenic Rice Plants Expressing Nucleic Acid Sequences Encoding Other GRF Polypeptides
Transgenic rice plants were generated, independently expressing the nucleic acid sequences encoding other GRF polypeptides, as shown in the Table H below, under the control of the GOS2 promoter for constitutive expression.
There was an increase in the Thousand Kernel Weight (TKW) of the seeds of transgenic plants compared to corresponding nullizygotes (controls), for the three constructs. This increase was less pronounced than for the seeds of transgenic plants expressing the nucleic acid sequence coding for the GRF polypeptide as represented by SEQ ID NO: 2.







TABLE H







Other GRF nucleic acid and polypeptide sequences tested in


transgenic rice plants, under the control of the GOS2 promoter


for constitutive expression.











Polypeptide


GRF polypeptide tested
Nucleic acid SEQ ID NO
SEQ ID NO





AT4G37740
SEQ ID NO: 15
SEQ ID NO: 16


AT2G36400
SEQ ID NO: 7
SEQ ID NO: 8


AT2G22840
SEQ ID NO: 5
SEQ ID NO: 6









b) Results of the Phenotypic Evaluation of the Transgenic Plants Expressing an RAM-like Nucleic Acid
The results of the evaluation of transgenic rice plants expressing an RAA1-like nucleic acid under control of a constitutive promoter (whether GOS2 or HMGP) under non-stress conditions were as follows: an increase of at least 2% was observed for Thousand Kernel Weight and an increase of more than 5% was observed for at least one of the following parameters: root/shoot index, total root biomass, flowers per panicle. Also under conditions of reduced nitrogen availability, an increase was observed in one or more of: root biomass, height, and greenness index.
c1) Measurement of Yield-Related Parameters for pGOS2::SYR Transformants Grown Under Conditions of Nutrient Deficiency
Upon analysis of the seeds as described above, the inventors found that plants transformed with the pGOS2::SYR gene construct and grown under nutrient deficiency stress, had a higher seed yield, expressed as number of filled seeds (increase of more than 5%), total weight of seeds (increase of more than 5%) and TKW (increase of more than 2.5%), compared to plants lacking the SYR transgene. There was also observed an increase in shoot biomass (more than 5%) and root biomass (several lines more than 5%).
c2) Measurement of Yield-Related Parameters for pGOS2::SYR Transformants Grown Under Conditions of Severe Drought Stress
Upon analysis of the seeds as described above, the inventors found that plants transformed with the pGOS2::SYR gene construct and grown under severe drought stress, had a higher seed yield, expressed as total weight of seeds (increase of more than 5%), fill rate (increase of more than 5%) and Harvest Index (increase of more than 5%), compared to plants lacking the SYR transgene.
d) Results of the Phenotypic Evaluation of the Transgenic Plants Expressing the Orysa_ARKL1 Nucleic Acid
The results of the evaluation of transgenic rice plants expressing the Orysa_ARKL1 nucleic acid under non-stress conditions are presented below. An increase of at least 5% was observed for emergence vigour (early vigour), total seed yield, number of filled seeds, thousand kernel weight and harvest index, and of 3% for thousand kernel weight.







TABLE I







Phenotypic evaluation results.











% Increase in transgenic/control



Trait
plants under non-stress conditions














Total seed yield
11



Number of filled
8



seeds



TKW
3



Early vigour
11



Harvest index
6










The transgenic rice plants expressing the Orysa_ARKL1 nucleic acid were also evaluated under drought stress conditions as described above. The same parameters (Increase in seed yield, number of filled seeds, early vigour and harvest index) were also increased in transgenic plants versus the corresponding control plant, though to a lower degree.
e) Results of the Phenotypic Evaluation of the Transgenic Plants Expressing an YTP1 Nucleic Acid
The results of the evaluation of transgenic rice plants expressing an YTP1 nucleic acid under non-stress conditions are presented below. An increase of at least 5% was observed for total seed yield, seed filling rate, number of flowers per panicle, harvest index, and 2% for thousand kernel weight
The results of the evaluation of transgenic rice plants expressing an YTP1 nucleic acid under non-stress conditions are presented hereunder. An increase was observed for total seed weight, number of filled seeds, fill rate, harvest index and thousand-kernel weight (Table J).







TABLE J







Phenotypic evaluation results.











% increase in the transgenic with



Yield related trait
respect to the control plants














Total seed weight
10



Seed filling rate
6



Flowers per panicle
7



Harvest index
12



Thousand kernel weight
2










Example 16
Examples of Transformation of Other Crops
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 (MS0) 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 Am 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.
Cotton Transformation
Cotton (Gossypium hirsutum L.) transformation is performed using Agrobacterium tumefaciens, on hypocotyls explants. The commercial cultivars such as Coker 130 or Coker 312 (SeedCo, Lubbock, Tex.) are standard varieties used for transformation, but other varieties can also be used. The seeds are surface sterilized and germinated in the dark. Hypocotyl explants are cut from the germinated seedlings to lengths of about 1-1.5 centimeter. The hypotocyl explant is submersed in the Agrobacterium tumefaciens inoculum containing the expression vector, for 5 minutes then co-cultivated for about 48 hours on MS+1.8 mg/l KNO3+2% glucose at 24° C., in the dark. The explants are transferred the same medium containing appropriate bacterial and plant selectable markers (renewed several times), until embryogenic calli is seen. The calli are separated and subcultured until somatic embryos appear. Plantlets derived from the somatic embryos are matured on rooting medium until roots develop. The rooted shoots are transplanted to potting 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.
Example 17
Examples of Abiotic Stress Screens
Drought Screen
Plants from a selected number of events are grown in potting soil under normal conditions until they approached the heading stage. They are then transferred to a “dry” section where irrigation is withheld. Humidity probes are inserted in randomly chosen pots to monitor the soil water content (SWC). When SWC go below certain thresholds, the plants are automatically re-watered continuously until a normal level is reached again. The plants are then re-transferred to normal conditions. The rest of the cultivation (plant maturation, seed harvest) is the same as for plants not grown under abiotic stress conditions. Growth and yield parameters are recorded as detailed for growth under normal conditions.
Salt Stress Screen
Plants are grown on a substrate made of coco fibers and argex (3 to 1 ratio). A normal nutrient solution is used during the first two weeks after transplanting the plantlets in the greenhouse. After the first two weeks, 25 mM of salt (NaCl) is added to the nutrient solution, until the plants were harvested. Growth and yield parameters are recorded as detailed for growth under normal conditions.
Reduced Nutrient (Nitrogen) Availability Screen
Plants from six events (T2 seeds) are grown in potting soil under normal conditions except for the nutrient solution. The pots are watered from transplantation to maturation with a specific nutrient solution containing reduced N nitrogen (N) content, usually between 7 to 8 times less. The rest of the cultivation (plant maturation, seed harvest) is the same as for plants not grown under abiotic stress. Growth and yield parameters are recorded as detailed for growth under normal conditions.

## Claims

1. A method for increasing seed yield-related traits in a plant relative to a control plant, comprising introducing and expressing in a plant a nucleic acid encoding a Growth-Regulating Factor (GRF) polypeptide, and selecting for a plant having increased seed yield-related traits on the basis of said plant showing increased seed yield-related traits relative to a control plant, wherein said seed yield-related traits comprise increased total seed yield per plant.

2. The method of claim 1, wherein said GRF polypeptide comprises:
(i) a domain having at least 80% or more amino acid sequence identity to a QLQ domain comprising the amino acid sequence of SEQ ID NO: 115; and
(ii) a domain having at least 80% or more amino acid sequence identity to a WRC domain comprising the amino acid sequence of SEQ ID NO: 116,
and has at least 80% or more amino acid sequence identity to the amino acid sequence of SEQ ID NO: 2.

3. The method of claim 1, wherein said GRF polypeptide has at least 90% or more amino acid sequence identity to the amino acid sequence of SEQ ID NO: 2.

4. The method of claim 1, wherein said nucleic acid encoding a GRF polypeptide comprises:
(a) the nucleotide sequence of SEQ ID NO: 1;
(b) a nucleotide sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 2; or
(c) a nucleotide sequence capable of hybridizing with the nucleotide sequence of (a) or (b) under stringency hybridization conditions comprising hybridization at 50° C. in 4×SSC or at 40° C. in 6×SSC and 50% formamide, followed by washing at 50° C. in 2×SSC.

5. The method of claim 1, wherein said increased seed yield-related traits further comprise one or more of: (i) increased seed filling rate; (ii) increased harvest index; or (iii) increased thousand kernel weight (TKW).

6. The method of claim 1, wherein said nucleic acid is operably linked to a constitutive promoter, a plant constitutive promoter, a GOS2 promoter, or a GOS2 promoter from rice comprising the nucleotide sequence of SEQ ID NO: 117.

7. A plant produced by the method of claim 1, or a plant part, plant cell, seed or progeny of said plant, wherein said plant, or said plant part, plant cell, seed or progeny, comprises a transgene comprising said nucleic acid encoding a Growth-Regulating Factor (GRF) polypeptide operably linked to a constitutive promoter isolated from a plant genome, and wherein said plant is a monocotyledonous plant.

8. A method for making a plant having an increased yield-related trait relative to a control plant, comprising introducing into a plant cell, plant, or part thereof a construct comprising a nucleic acid sequence encoding a GRF polypeptide operably linked to one or more control sequences, and selecting for a plant having increased yield-related traits on the basis of said plant showing increased yield-related traits relative to a control plant, wherein the increased yield-related trait is one or more of: (i) increased early vigour; (ii) increased biomass; (iii) increased total seed yield per plant; (iv) increased seed filling rate; (v) increased harvest index; (vi) increased thousand kernel weight (TKW), (vii) increased abiotic stress resistance, or (viii) increased nutrient uptake efficiency.

9. A method for the production of a transgenic plant having increased seed yield-related traits relative to a control plant, comprising:
(i) introducing and expressing in a plant, plant part, or plant cell, a nucleic acid encoding a GRF polypeptide under control of a plant constitutive promoter;
(ii) cultivating the plant cell, plant part, or plant under conditions promoting plant growth and development; and
(iii) selecting for a transgenic plant having increased seed yield-related traits on the basis of said transgenic plant showing increased seed yield-related traits relative to a control plant,
wherein said seed yield-related traits comprise increased total seed yield per plant.

10. A transgenic plant having increased seed yield-related traits relative to a control plant resulting from increased expression of a nucleic acid encoding a GRF polypeptide operably linked to a plant constitutive promoter, or a transgenic plant cell or transgenic plant part derived from said transgenic plant, wherein said seed yield-related traits comprise increased total seed yield per plant, and wherein said plant is a monocotyledonous plant.

11. The transgenic plant of claim 10, wherein said plant is a crop plant, a monocot, a cereal, rice, maize, wheat, barley, millet, rye, triticale, sorghum, or oats, or a transgenic plant cell derived from said transgenic plant.

12. Harvestable parts, including seed, of the transgenic plant of claim 10, wherein said harvestable parts comprise a transgene comprising the nucleic acid encoding a GRF polypeptide.

13. The method of claim 9, wherein said GRF polypeptide has at least 95% or more amino acid sequence identity to the amino acid sequence of SEQ ID NO: 2.

14. The method of claim 9, wherein the plant is a crop plant, a monocot, or a cereal, or wherein the plant is rice, maize, wheat, barley, millet, rye, triticale, sorghum, or oat.

15. The plant of claim 7, wherein the plant is a crop plant or a cereal, or wherein the plant is rice, maize, wheat, barley, millet, rye, triticale, sorghum, or oat.

16. The method of claim 8, wherein said GRF polypeptide has at least 95% or more amino acid sequence identity to the amino acid sequence of SEQ ID NO: 2.

17. The method of claim 8, wherein said nucleic acid encoding a GRF polypeptide comprises:
(a) the nucleotide sequence of SEQ ID NO: 1;
(b) a nucleotide sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 2; or
(c) a nucleotide sequence capable of hybridizing with the nucleotide sequence of (a) or (b) under stringency hybridization conditions comprising hybridization at 50° C. in 4×SSC or at 40° C. in 6×SSC and 50% formamide, followed by washing at 50° C. in 2×SSC.

18. The method of claim 8, wherein one of the control sequences is a plant constitutive promoter, a GOS2 promoter, or a GOS2 promoter comprising the nucleotide sequence of SEQ ID NO: 117.

19. The method of claim 9, wherein said GRF polypeptide has at least 95% or more amino acid sequence identity to the amino acid sequence of SEQ ID NO: 2.

20. The method of claim 9, wherein said nucleic acid encoding a GRF polypeptide comprises:
(a) the nucleotide sequence of SEQ ID NO: 1;
(b) a nucleotide sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 2; or
(c) a nucleotide sequence capable of hybridizing with the nucleotide sequence of (a) or (b) under stringency hybridization conditions comprising hybridization at 50° C. in 4×SSC or at 40° C. in 6×SSC and 50% formamide, followed by washing at 50° C. in 2×SSC.

21. The method of claim 9, wherein said nucleic acid is operably linked to a constitutive promoter, a plant constitutive promoter, a GOS2 promoter, or a GOS2 promoter from rice comprising the nucleotide sequence of SEQ ID NO: 117.