# Patent

## Title
Plants having enhanced yield-related traits and a method for making the same

## Abstract

The present invention relates generally to the field of molecular biology and concerns a method for enhancing yield related traits by modulating expression in a plant of a nucleic acid encoding a eRF1 polypeptide, a SCAMP-like (secretory carrier membrane proteins) polypeptide, a PLATZ (plant AT-rich sequence- and zinc binding protein) polypeptide, a PLST-like polypeptide or a Glomalin (HSP60, chaperonin CNP60) polypeptide. The present invention also concerns plants having modulated expression of a nucleic acid encoding said polypeptides, which plants have enhanced yield-related traits relative to corresponding wild type plants or other 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/EP2010/058129, filed Jun. 10, 2010; which claims benefit of European Application No. 09163277.8, filed Jun. 19, 2009; European Application No. 09163257.0, filed Jun. 19, 2009; European Application No. 09163287.7, filed Jun. 19, 2009; U.S. Provisional Application No. 61/223,431, filed Jul. 7, 2009; U.S. Provisional Application Ser. No. 61/223,429, filed Jul. 7, 2009; U.S. Provisional Application Ser. No. 61/223,389, filed Jul. 7, 2009; European Application No. 09165779.1, filed Jul. 17, 2009; U.S. Provisional Application Ser. No. 61/226,307, filed Jul. 17, 2009; U.S. Provisional Application Ser. No. 61/227,448, filed Jul. 22, 2009; European Application No. 09166083.7, filed Jul. 22, 2009; U.S. Provisional Application Ser. No. 61/227,803, filed Jul. 23, 2009; and European Application No. 09166200.7, filed Jul. 23, 2009.


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 filed containing the Sequence Listing is Revised _Sequence _List _13987 _00160 _US. The size of the text file is 1,182 KB and the text file was created on Feb. 1, 2012.
The present invention relates generally to the field of molecular biology and concerns a method for enhancing yield related traits by modulating expression in a plant of a nucleic acid encoding an eRF1 polypeptide. The present invention also concerns plants having modulated expression of a nucleic acid encoding this eRF1 polypeptide, which plants have enhanced yield-related traits relative to corresponding wild type plants or other control plants. The invention also provides constructs useful in the methods of the invention.
The present invention relates generally to the field of molecular biology and concerns a method for enhancing various economically important yield-related traits in plants. More specifically, the present invention concerns a method for enhancing yield-related traits in plants by modulating expression in a plant of a nucleic acid encoding an SCAMP-like (secretory carrier membrane proteins) polypeptide. The present invention also concerns plants having modulated expression of a nucleic acid encoding an SCAMP-like polypeptide, which plants have enhanced yield-related traits relative to control plants. The invention also provides hitherto unknown SCAMP-like-encoding nucleic acids, and constructs comprising the same, useful in performing the methods of the invention.
The present invention relates generally to the field of molecular biology and concerns a method for enhancing various yield-related traits in plants by modulating expression in a plastid of a plant of a nucleic acid encoding a fibrillin polypeptide. The present invention also concerns plants having modulated expression in a plastid of a plant of a nucleic acid encoding a fibrillin, which plants have enhanced yield-related traits relative to corresponding wild type plants or other control plants. The invention also provides constructs useful in the methods of the invention.
The present invention relates generally to the field of molecular biology and concerns a method for improving various plant growth characteristics by modulating expression in a plant of a nucleic acid encoding a PLATZ (plant AT-rich sequence- and zinc binding protein) polypeptide. The present invention also concerns plants having modulated expression of a nucleic acid encoding a PLATZ polypeptide, which plants have improved growth characteristics relative to corresponding wild type plants or other control plants. The invention also provides constructs useful in the methods of the invention.
The present invention relates generally to the field of molecular biology and concerns a method for enhancing yield related traits by modulating expression in a plant of a nucleic acid encoding a PLST-like polypeptide. The present invention also concerns plants having modulated expression of a nucleic acid encoding a PLST-like polypeptide, which plants have enhanced yield-related traits relative to corresponding wild type plants or other control plants. The invention also provides constructs useful in the methods of the invention.
The present invention relates generally to the field of molecular biology and concerns a method for enhancing yield-related traits in plants by modulating expression in a plant of a nucleic acid encoding a Glomalin (HSP60, chaperonin CNP60) polypeptide. The present invention also concerns plants having modulated expression of a nucleic acid encoding a Glomalin polypeptide, which plants have enhanced yield-related traits relative to corresponding wild type plants or other 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 enhanced yield characteristics. 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 abovementioned 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 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.
A further important trait is that of improved abiotic stress tolerance. Abiotic stress is a primary cause of crop loss worldwide, reducing average yields for most major crop plants by more than 50% (Wang et al., Planta (2003) 218: 1-14). Abiotic stresses may be caused by drought, salinity, extremes of temperature, chemical toxicity, excess or deficiency of nutrients (macroelements and/or microelements), radiation and oxidative stress. The ability to improve plant tolerance to abiotic stress, namely to drought, 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 enhanced 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.
It has now been found that various yield related traits may be enhanced in plants by modulating expression in a plant of a nucleic acid encoding an eRF1 protein-like in a plant.
It has also now been found that various growth characteristics may be improved in plants by modulating expression in a plant of a nucleic acid encoding a SCAMP-like in a plant.
It has also now been found that various yield-related traits may be improved in plants by modulating expression of a nucleic acid encoding a fibrillin polypeptide in a plant plastid.
It has also now been found that various growth characteristics may be improved in plants by modulating expression in a plant of a nucleic acid encoding a PLATZ (plant AT-rich sequence- and zinc binding protein) in a plant.
It has also now been found that various yield related traits may be enhanced in plants by modulating expression in a plant of a nucleic acid encoding a PLST-like protein in a plant.
It has also 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 Glomalin (HSP60, chaperonin CNP60) polypeptide in a plant.
BACKGROUND
1. SCAMP-Like Polypeptides
Considerable evidence for endocytosis in plants has accumulated during recent years (Samaj et al., 2004; Plant Physiol. 135: 1150-1161). Some of the components of the clathrin-based internalization machinery have been identified and data for the uptake of cell surface receptor-ligand complexes is accumulating (Russinova et al. 2004, Plant Cell 16: 3216-3229). Recently it has been hypothesized that plant SCAMP proteins might play a role in mediating endocytosis in plant cells (Lam et al. 2007; The Plant Cell, Vol. 19: 296-319). SCAMP proteins were Initially identified as secretory vesicle components in mammalian exocrine glands and later found to be ubiquitous proteins in eukaryotes (Fernandez-Chacon and Sudhof, 2000; J. Neurosci. 20: 7941-7950). SCAMPs were found in both the trans-Golgi and the endosomal recycling compartment, and they appear to be concentrated within the motile population of early and recycling endosomes (Castle and Castle, 2005) J. Cell Sci. 118: 3769-3780. Plant SCAMP homologs have been found amongst others in rice (Oryza sativa), Arabidopsis, and pea (Pisum sativum) and are thought to be present in many other plant species (Fernandez-Chacon and Sudhof, 2000). In plants SCAMPs have been localized at the plasma memebrane and the and mobile cytosolic organelles (Lam et al. 2007).
2. Fibrillin Polypeptides
The most prominent proteins in plastoglobulins (PGs) are fibrillins. Fibrillins are plastid-associated lipid-binding proteins that are ubiquitous in plants and cyanobacteria. They have been primarily characterized from chromoplasts of tomato and pepper fruits and are known to accumulate during abiotic stress in plastids e.g., inflicted by high light, cold, and drought, and also during pathogen infection. The family of fibrillin-like proteins contain a hydrophobic domain that associates with or anchors within lipids. Fibrillins associate with stromal lamellae of thylakoids and fibrillic carotenoid-containing structures of chromoplasts. A model for the fibrillic structures predicts a layer of fibrillin shielding polar lipids and carotenoids. Furthermore, fibrillin is known to accumulate during high-light conditions, and fibrillin affects photosynthetic efficiency (see Yang et al., Proc Natl Acad Sci USA. 2006 April 11; 103(15): 6061-6066). Evidence is also available for the association of these proteins to various lipid globules under non-stressed conditions preventing plastoglobule coalescence (see CAB Abstracts, Simkin et al., Recent Research Developments in Biochemistry, 2004).
The Arabidopsis genome has 13 fibrillin genes that are all predicted to encode plastid localized proteins (Laizet et al., 2004). Rey et al., (Plant J. 2000 March; 21(5):483-94) disclose transgenic Nicotiana tabacum plants over-expressing fibrillin using a constitutive promoter. No growth difference between wild-type plants and transgenic plants was noticed under low light conditions, however transgenic plants were reported to exhibit a longer main stem, enhanced development of lateral stems and accelerated floral development under higher light intensities.
3. PLATZ Polypeptides
PLATZ proteins form a plant specific family of DNA-binding proteins. So far, only one member has been described in more detail (PLATZ1, Nagano et al, Nucl. Acids Res. 29, 4097-4105, 2001). Sequence comparison between PLATZ1 and other putative PLATZ proteins revealed the presence of two Zn-binding domains with conserved cysteine and histidine residues. DNA-binding activity required the presence of Zn. PLATZ1 was shown to bind A/T-rich regions in a non-specific way, and was able to induce expression of the GTPase pra2 and plastocyanin petE genes (Nagano et al., 2001). Though DNA-binding proteins are implicated in DNA replication and in regulation of gene expression, a precise characterisation of the role of PLATZ proteins is still lacking.
4. Glomalin Polypeptides
Glomalin was first identified as a high molecular mass glycoprotein produced by the arbuscular mycorrhizal fungi (like Glomus sp). It is secreted into the environment and the sugar moiety was postulated to play a role in sequestering of Cu and Zn in the soil. Gadkar and Rillig (FEMS Microbiol Lett. 263, 93-101, 2006) have shown that the glomalin of Glomus intraradices is a protein of 590 amino acids with three N-terminal glycosylation sites and a string of GGM motifs at the C-terminal end. The genomic sequence had three introns of 67, 76 and 131 bp length. The protein had homology to heat shock protein 60 (hsp 60); a plant homologue of hsp60 reportedly plays a role in acclimating photosynthesis to heat stress, possibly by protecting Rubisco activase from thermal denaturation (Salvucci M., E., J Exp Bot. 2008; 59(7):1923-33). However, the precise role of glomalin orthologues in plant biology remains to be elucidated.
SUMMARY
1. eRF1 Polypeptides
Surprisingly, it has now been found that modulating expression of a nucleic acid encoding an eRF1 polypeptide gives plants having enhanced yield-related traits, in particular enhanced yield relative to control plants.
According one embodiment, there is provided a method for enhanced yield related traits of a plant relative to control plants, comprising modulating expression of a nucleic acid encoding an eRF1 polypeptide in a plant.
2. SCAMP-Like Polypeptides
Surprisingly, it has now been found that modulating expression of a nucleic acid encoding a SCAMP-like polypeptide gives plants having enhanced yield-related traits relative to control plants.
According one embodiment, there is provided a method for enhancing yield-related traits of a plant relative to control plants, comprising modulating expression of a nucleic acid encoding a SCAMP-like polypeptide in a plant.
3. Fibrillin Polypeptides
Surprisingly, it has now been found that modulating expression in a plastid of a plant of a nucleic acid encoding a fibrillin polypeptide gives plants having enhanced yield-related traits relative to control plants.
According one embodiment, there is provided a method for enhancing yield-related traits relative to control plants, comprising modulating expression in a plant plastid of a nucleic acid encoding a fibrillin polypeptide.
4. PLATZ Polypeptides
Surprisingly, it has now been found that modulating expression of a nucleic acid encoding a PLATZ 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 PLATZ polypeptide in a plant.
5. PLST-Like Polypeptides
Surprisingly, it has now been found that modulating expression of a nucleic acid encoding a PLST-like polypeptide gives plants having enhanced yield-related traits, in particular enhanced yield relative to control plants.
According one embodiment, there is provided a method for enhanced yield related traits of a plant relative to control plants, comprising modulating expression of a nucleic acid encoding a PLST-like polypeptide in a plant.
6. Glomalin Polypeptides
Surprisingly, it has now been found that modulating expression of a nucleic acid encoding a Glomalin polypeptide gives plants having enhanced yield-related traits, in particular increased seed yield relative to control plants.
According one embodiment, there is provided 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 Glomalin polypeptide.
DEFINITIONS
The following definitions will be used throughout the present specification.
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.
Homologue(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 and may range from 1 to 10 amino acids; 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










Residue
Conservative Substitutions







Ala
Ser



Arg
Lys



Asn
Gln; His



Asp
Glu



Gln
Asn



Cys
Ser



Glu
Asp



Gly
Pro



His
Asn; Gln



Ile
Leu, Val



Leu
Ile; Val



Lys
Arg; Gln



Met
Leu; Ile



Phe
Met; Leu; Tyr



Ser
Thr; Gly



Thr
Ser; Val



Trp
Tyr



Tyr
Trp; Phe



Val
Ile; Leu










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).
Orthologue(s)/Paralogue(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, Motif/Consensus Sequence/Signature
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.
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).
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)). Domains or motifs may also be identified using routine techniques, such as by sequence alignment.
Methods for the alignment of sequences for comparison are well known in the art, such methods include GAP, BESTFIT, BLAST, FASTA and TFASTA. GAP uses the algorithm of Needleman and Wunsch ((1970) J Mol Biol 48: 443-453) to find the 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., BMC Bioinformatics. 2003 Jul. 10; 4:29. 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 or amino acid 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).
Reciprocal BLAST
Typically, this involves a first BLAST involving BLASTing a query sequence (for example using any of the sequences listed in Table A2, A3, A4, A5 or A6 of the Examples section) 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. 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.
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.
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 acids are in solution. The hybridisation process can also occur with one of the complementary nucleic acids immobilised to a matrix such as magnetic beads, Sepharose beads or any other resin. The hybridisation process can furthermore occur with one of the complementary nucleic acids immobilised to a solid support such as a nitro-cellulose or nylon membrane or immobilised by e.g. photolithography to, for example, a siliceous glass support (the latter known as nucleic acid arrays or microarrays or as nucleic acid chips). In order to allow hybridisation to occur, the nucleic acid molecules are generally thermally or chemically denatured to melt a double strand into two single strands and/or to remove hairpins or other secondary structures from single stranded nucleic acids.
The 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 acids 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 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 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-RNAs hybrids:
    
        For <20 nucleotides: Tm=2 (In)
        For 20-35 nucleotides: Tm=22+1.46 (In)
    
    
    a or for other monovalent cation, but only accurate in the 0.01-0.4 M range.
    b only accurate for % GC in the 30% to 75% range.
    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 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 acids 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.
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.
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 acids 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).
Construct
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-ori 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 acids, 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.
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 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, enhancers and silencers) which alter gene expression in response to developmental and/or external stimuli, or in a tissue-specific manner. Also included within the term is a transcriptional regulatory sequence of a classical prokaryotic gene, in which case it may include a −35 box sequence and/or −10 box transcriptional regulatory sequences. The term “regulatory element” also encompasses a synthetic fusion molecule or derivative that confers, activates or enhances expression of a nucleic acid molecule in a cell, tissue or organ.
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” can also originate 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 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 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 lower level than a strong promoter, in particular at a level that is in all instances below that obtained when 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 constitutive promoters








Gene Source
Reference





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


HMGP
WO 2004/070039


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


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


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



WO 2004/065596


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


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


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


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


histone


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


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


Rubisco small
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


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



7831-7846


V-ATPase
WO 01/14572


Super promoter
WO 95/14098


G-box proteins
WO 94/12015









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





RCc3
Plant Mol Biol. 1995 January; 27(2): 237-48


Arabidopsis PHT1
Kovama et al., 2005; Mudge et al. (2002,



Plant J. 31: 341)


Medicago phosphate
Xiao et al., 2006


transporter


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


root-expressible genes
Tingey et al., EMBO J. 6: 1, 1987.


tobacco auxin-
Van der Zaal et al., Plant Mol. Biol. 16,


inducible gene
983, 1991.


β-tubulin
Oppenheimer, et al., Gene 63: 87, 1988.


tobacco root-
Conkling, et al., Plant Physiol. 93: 1203, 1990.


specific genes


B. napus G1-3b gene
U.S. Pat. No. 5,401,836


SbPRP1
Suzuki et al., Plant Mol. Biol. 21: 109-119,



1993.


LRX1
Baumberger et al. 2001, Genes & Dev. 15: 1128


BTG-26 Brassica
US 20050044585


napus


LeAMT1 (tomato)
Lauter et al. (1996, PNAS 3: 8139)


The LeNRT1-1
Lauter et al. (1996, PNAS 3: 8139)


(tomato)


class I patatin
Liu et al., Plant Mol. Biol. 153: 386-395, 1991.


gene (potato)


KDC1 (Daucus
Downey et al. (2000, J. Biol. Chem. 275: 39420)


carota)


TobRB7 gene
W Song (1997) PhD Thesis, North Carolina State



University, Raleigh, NC USA


OsRAB5a (rice)
Wang et al. 2002, Plant Sci. 163: 273


ALF5 (Arabidopsis)
Diener et al. (2001, Plant Cell 13: 1625)


NRT2; 1Np (N.
Quesada et al. (1997, Plant Mol. Biol. 34: 265)


plumbaginifolia)









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. The seed specific promoter may be endosperm/aleurone/embryo specific. Examples of seed-specific promoters (endosperm/aleurone/embryo specific) are shown in Table 2c to Table 2f 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
Mol Gen Genet 216: 81-90, 1989; NAR 17: 461-2, 1989


glutenin-1


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 pyrophos-
Trans Res 6: 157-68, 1997


phorylase


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
unpublished


ITR1 (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
















TABLE 2d







examples of endosperm-specific promoters








Gene source
Reference





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



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


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


wheat LMW
Colot et al. (1989) Mol Gen Genet 216: 81-90,


and HMW
Anderson et al. (1989) NAR 17: 461-2


glutenin-1


wheat SPA
Albani et al. (1997) Plant Cell 9: 171-184


wheat gliadins
Rafalski et al. (1984) EMBO 3: 1409-15


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


promoter


barley B1, C, D,
Cho et al. (1999) Theor Appl Genet 98: 1253-62;


hordein
Muller et al. (1993) Plant J 4: 343-55;



Sorenson et al. (1996) Mol Gen Genet 250: 750-60


barley DOF
Mena et al, (1998) Plant J 116(1): 53-62


blz2
Onate et al. (1999) J Biol Chem 274(14): 9175-82


synthetic promoter
Vicente-Carbajosa et al. (1998) Plant J 13: 629-640


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


NRP33


rice globulin
Wu et al. (1998) Plant Cell Physiol 39(8) 885-889


Glb-1


rice globulin
Nakase et al. (1997) Plant Molec Biol 33: 513-522


REB/OHP-1


rice ADP-glucose
Russell et al. (1997) Trans Res 6: 157-68


pyrophosphorylase


maize ESR
Opsahl-Ferstad et al. (1997) Plant J 12: 235-46


gene family


sorghum kafirin
DeRose et al. (1996) Plant Mol Biol 32: 1029-35
















TABLE 2e







Examples of embryo specific promoters:








Gene source
Reference





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



1996


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


PRO0151
WO 2004/070039


PRO0175
WO 2004/070039


PRO005
WO 2004/070039


PRO0095
WO 2004/070039
















TABLE 2f







Examples of aleurone-specific promoters:








Gene source
Reference





α-amylase
Lanahan et al, Plant Cell 4: 203-211, 1992;


(Amy32b)
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 2g below.







TABLE 2g







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 specific
WO 2004/070039


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 green meristem-specific promoters which may be used to perform the methods of the invention are shown in Table 2h below.







TABLE 2h







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. Sci.



stage to seedling stage
USA, 93: 8117-8122


Rice metallothionein
Meristem specific
BAD87835.1


WAK1 & WAK 2
Shoot and root apical
Wagner & Kohorn



meristems, and in ex-
(2001) Plant Cell



panding leaves and sepals
13(2): 303-318









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.
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 construct of the invention. These marker genes enable the identification of a successful transfer of the nucleic acid 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 acids 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 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 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 acids have been introduced successfully, the process according to the invention for introducing the nucleic acids 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 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 (known as the Ac/Ds technology). The transformants can be crossed with a transposase source or the transformants are transformed with a nucleic acid 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 acids 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 acids to be expressed homologously or heterologously. However, as mentioned, transgenic also means that, while the nucleic acids 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 acids according to the invention at an unnatural locus in the genome, i.e. homologous or, preferably, heterologous expression of the nucleic acids takes place. Preferred transgenic plants are mentioned herein.
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 enhancement of gene expression is typically greatest when placed near the 5′ end of the transcription unit. Use of the maize introns Adh1-S intron 1, 2, and 6, the Bronze-1 intron are known in the art. For general information see: The Maize Handbook, Chapter 116, Freeling and Walbot, Eds., Springer, N.Y. (1994).
Decreased Expression
Reference herein to “decreased expression” 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 encoding the protein of interest (target gene), or from any nucleic acid 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 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., Dev. Cell 8, 517-527, 2005). Convenient tools for design and generation of amiRNAs and their precursors are also available to the public (Schwab et al., Plant Cell 18, 1121-1133, 2006).
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 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.
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 acids 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).
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 above-mentioned 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).
T-DNA Activation Tagging
T-DNA activation tagging (Hayashi et al. Science (1992) 1350-1353), involves insertion of T-DNA, usually containing a promoter (may also be a translation enhancer or an intron), in the genomic region of the gene of interest or 10 kb up- or 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 acids 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 CR, 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 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 Related Traits
Yield related traits comprise one or more of early flowering time; yield, biomass, seed yield, early vigour, greenness index, increased growth rate, improved agronomic traits such as improved Water Use Efficiency (WUE), Nitrogen Use Efficiency (NUE), etc.
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 and “plant yield” are used interchangeably herein may relate to vegetative biomass such as root and/or shoot biomass, to reproductive organs, and/or to propagules such as seeds of that plant.
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 square meter, 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 square meter, number of panicles per plant, panicle length, number of spikelets per panicle, number of flowers (florets) per panicle, 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. In rice, submergence tolerance may also result in increased yield.
Early Flowering Time
Plants having an “early flowering time” as used herein are plants which start to flower earlier than control plants. Hence this term refers to plants that show an earlier start of flowering. Flowering time of plants can be assessed by counting the number of days (“time to flower”) between sowing and the emergence of a first inflorescence. The “flowering time” of a plant can for instane be determined using the method as described in WO 2007/093444.
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.
Increased Growth Rate
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 speed of germination, 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 enhanced vigour. The increase in growth rate may alter the harvest cycle of a plant allowing plants to be sown later and/or harvested sooner than would otherwise be possible (a similar effect may be obtained with earlier flowering time). 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 square meter (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.
Stress Resistance
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. Mild stress in the sense of the invention leads to a reduction in the growth of the stressed plants of less than 40%, 35%, 30% or 25%, more preferably less than 20% or 15% 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.
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. Plants with optimal growth conditions, (grown under non-stress conditions) typically yield in increasing order of preference at least 97%, 95%, 92%, 90%, 87%, 85%, 83%, 80%, 77% or 75% of the average production of such plant in a given environment. Average production may be calculated on harvest and/or season basis. Persons skilled in the art are aware of average yield productions of a crop.
Nutrient deficiency may result from a lack of nutrients such as nitrogen, phosphates and other phosphorous-containing compounds, potassium, calcium, magnesium, manganese, iron and boron, amongst others.
The term salt stress is not restricted to common salt (NaCl), but may be any one or more of: NaCl, KCl, LiCl, MgCl2, CaCl2, amongst others.
Increase/Improve/Enhance
The terms “increase”, “improve” or “enhance” are interchangeable and shall mean in the sense of the application at least a 3%, 4%, 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 square meter;
        b) increased number of flowers per plant;
        c) increased number of seeds and/or 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, i.e. biomass of aboveground plant parts; and
        f) 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 seed yield may also manifest itself as an increase in seed area and/or seed length and/or seed width and/or seed perimeter.
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.
Biomass
The term “biomass” as used herein is intended to refer to the total weight of a plant. Within the definition of biomass, a distinction may be made between the biomass of one or more parts of a plant, which may include:

    
    
        aboveground (harvestable) parts such as but not limited to shoot biomass, seed biomass, leaf biomass, etc. and/or
        (harvestable) parts below ground, such as but not limited to root biomass, etc., and/or
        vegetative biomass such as root biomass, shoot biomass, etc., and/or
        reproductive organs, and/or
        propagules such as seed.

Marker Assisted Breeding

    
    


Such breeding programmes sometimes require introduction of allelic variation by mutagenic treatment of the plants, using for example EMS mutagenesis; alternatively, the programme may start with a collection of allelic variants of so called “natural” origin caused unintentionally. Identification of allelic variants then takes place, for example, by PCR. This is followed by a step for selection of superior allelic variants of the sequence in question and which give increased yield. Selection is typically carried out by monitoring growth performance of plants containing different allelic variants of the sequence in question. Growth performance may be monitored in a greenhouse or in the field. Further optional steps include crossing plants in which the superior allelic variant was identified with another plant. This could be used, for example, to make a combination of interesting phenotypic features.
Use as Probes in (Gene Mapping)
Use of nucleic acids encoding the protein of interest for genetically and physically mapping the genes requires only a nucleic acid sequence of at least 15 nucleotides in length. These nucleic acids 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 acids encoding the protein of interest. The resulting banding patterns may then be subjected to genetic analyses using computer programs such as MapMaker (Lander et al. (1987) Genomics 1: 174-181) in order to construct a genetic map. In addition, the nucleic acids may be used to probe Southern blots containing restriction endonuclease-treated genomic DNAs of a set of individuals representing parent and progeny of a defined genetic cross. Segregation of the DNA polymorphisms is noted and used to calculate the position of the nucleic acid encoding the protein of interest in the genetic map previously obtained using this population (Botstein et al. (1980) Am. J. Hum. Genet. 32:314-331).
The production and use of plant gene-derived probes for use in genetic mapping is described in Bernatzky and Tanksley (1986) Plant Mol. Biol. Reporter 4: 37-41. Numerous publications describe genetic mapping of specific cDNA clones using the methodology outlined above or variations thereof. For example, F2 intercross populations, backcross populations, randomly mated populations, near isogenic lines, and other sets of individuals may be used for mapping. Such methodologies are well known to those skilled in the art.
The nucleic acid probes may also be used for physical mapping (i.e., placement of sequences on physical maps; see Hoheisel et al. In: Non-mammalian Genomic Analysis: A Practical Guide, Academic press 1996, pp. 319-346, and references cited therein).
In another embodiment, the nucleic acid probes may be used in direct fluorescence in situ hybridisation (FISH) mapping (Trask (1991) Trends Genet. 7:149-154). Although current methods of FISH mapping favour use of large clones (several kb to several hundred kb; see Laan et al. (1995) Genome Res. 5:13-20), improvements in sensitivity may allow performance of FISH mapping using shorter probes.
A variety of nucleic acid amplification-based methods for genetic and physical mapping may be carried out using the nucleic acids. Examples include allele-specific amplification (Kazazian (1989) J. Lab. Clin. Med 11:95-96), polymorphism of PCR-amplified fragments (CAPS; Sheffield et al. (1993) Genomics 16:325-332), allele-specific ligation (Landegren et al. (1988) Science 241:1077-1080), nucleotide extension reactions (Sokolov (1990) Nucleic Acid Res. 18:3671), Radiation Hybrid Mapping (Walter et al. (1997) Nat. Genet. 7:22-28) and Happy Mapping (Dear and Cook (1989) Nucleic Acid Res. 17:6795-6807). For these methods, the sequence of a nucleic acid is used to design and produce primer pairs for use in the amplification reaction or in primer extension reactions. The design of such primers is well known to those skilled in the art. In methods employing PCR-based genetic mapping, it may be necessary to identify DNA sequence differences between the parents of the mapping cross in the region corresponding to the instant nucleic acid sequence. This, however, is generally not necessary for mapping methods.
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 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 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 elata, 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, Eragrostis tef, 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., Tripsacum dactyloides, Triticosecale rimpaui, Triticum spp. (e.g. Triticum aestivum, Triticum durum, Triticum turgidum, Triticum hybernum, Triticum macha, Triticum sativum, Triticum monococcum 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.
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.
DETAILED DESCRIPTION OF THE INVENTION
Surprisingly, it has now been found that modulating expression in a plant of a nucleic acid encoding an eRF1 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 eRF1 polypeptide and optionally selecting for plants having enhanced yield-related traits.
The invention also provides hitherto unknown eRF1-encoding nucleic acids and eRF1 polypeptides.
According to a further embodiment of the present invention, there is therefore provided an isolated nucleic acid molecule selected from:

    
    
        (i) a nucleic acid represented by any one of the following nucleic acid sequences: G.max_GM06MC33657_sm55b10@32878 having SEQ ID NO: 15; H.vulgare_c64960768hv270303@2598 having SEQ ID NO: 17;
        (ii) the complement of a nucleic acid represented by said sequences: G.max_GM06MC33657_sm55b10@32878 having SEQ ID NO: 15; H.vulgare_c64960768hv270303@2598 having SEQ ID NO: 17;
        (iii) a nucleic acid encoding the polypeptide as represented by any one G.max_GM06MC33657_sm55b10@32878 having SEQ ID NO: 16; H.vulgare_c64960768hv270303@2598 having SEQ ID NO: 18 preferably as a result of the degeneracy of the genetic code, said isolated nucleic acid can be derived from a polypeptide sequence as represented by any one of SEQ ID NO: 16 and 18 and further preferably confers enhanced yield-related traits relative to control plants;
        (iv) a nucleic acid having, 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% sequence identity with any of the nucleic acid sequences of Table A1 and further preferably conferring enhanced yield-related traits relative to control plants;
        (v) a nucleic acid molecule which hybridizes with a nucleic acid molecule of (i) to (iv) under stringent hybridization conditions and preferably confers enhanced yield-related traits relative to control plants;
        (vi) a nucleic acid encoding a eRF1 polypeptide having, in increasing order of preference, at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence represented by any one of SEQ ID NO: 16 and 18 and any of the other amino acid sequences in Table A1 and preferably conferring enhanced yield-related traits relative to control plants.
    
    


According to a further embodiment of the present invention, there is also provided an isolated polypeptide selected from:

    
    
        (i) an amino acid sequence represented by any one of SEQ ID NO: 16 and 18;
        (ii) an amino acid sequence having, in increasing order of preference, at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence represented by any one of SEQ ID NO: 16 and 18 and any of the other amino acid sequences in Table A1 and preferably conferring enhanced yield-related traits relative to control plants.
        (iii) derivatives of any of the amino acid sequences given in (i) or (ii) above.
    
    


Furthermore, it has now surprisingly been found that modulating expression in a plant of a nucleic acid encoding a SCAMP-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 SCAMP-like polypeptide and optionally selecting for plants having enhanced yield-related traits.
The invention also provides hitherto unknown SCAMP-like-encoding nucleic acids and polypeptides.
According to a further embodiment of the present invention, there is therefore provided an isolated nucleic acid molecule selected from:

    
    
        (i) a nucleic acid represented by any one of SEQ ID NO: 100, 102, 104, 106, 180, 182, 184, 186, 188, 190 and 192;
        (ii) the complement of a nucleic acid represented by any one of (i) SEQ ID NO: 100, 102, 104, 106, 108, 182, 184, 186, 188, 190 and 192;
        (iii) a nucleic acid encoding the polypeptide as represented by any one of SEQ ID NO: 101, 103, 105, 107, 181, 183, 185, 187, 189, 191 and 193; preferably as a result of the degeneracy of the genetic code, said isolated nucleic acid can be derived from a polypeptide sequence as represented by any one of SEQ ID NO: 101, 103, 105, 107, 181, 183, 185, 187, 189, 191 and 193 and further preferably confers enhanced yield-related traits relative to control plants;
        (iv) a nucleic acid having, 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% sequence identity with any of the nucleic acid sequences of Table A2 and further preferably conferring enhanced yield-related traits relative to control plants;
        (v) a nucleic acid molecule which hybridizes with a nucleic acid molecule of (i) to (iv) under stringent hybridization conditions and preferably confers enhanced yield-related traits relative to control plants;
        (vi) a nucleic acid encoding a polypeptide having, in increasing order of preference, at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence represented by any one of SEQ ID NO: 101, 103, 105, 107, 181, 183, 185, 187, 189, 191 and 193 and any of the other amino acid sequences in Table A2 and preferably conferring enhanced yield-related traits relative to control plants.
    
    


According to a further embodiment of the present invention, there is also provided an isolated polypeptide selected from:

    
    
        (i) an amino acid sequence represented by any one of SEQ ID NO: 101, 103, 105, 107, 181, 183, 185, 187, 189, 191 and 193;
        (ii) an amino acid sequence having, in increasing order of preference, at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence represented by any one of SEQ ID NO: 101, 103, 105, 107, 181, 183, 185, 187, 189, 191 and 193 and any of the other amino acid sequences in Table A2 and preferably conferring enhanced yield-related traits relative to control plants.
        (iii) derivatives of any of the amino acid sequences given in (i) or (ii) above.
    
    


Furthermore, it has now surprisingly been found that modulating expression in a plant plastid of a nucleic acid encoding a fibrillin 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 plastid of a nucleic acid encoding a fibrillin polypeptide and optionally selecting for plants having enhanced yield-related traits.
According to a further embodiment of the present invention, there is therefore provided an isolated nucleic acid molecule selected from:

    
    
        (i) a nucleic acid represented by any one of the following nucleic acid sequences: B.napus_BN06MC20042_46499279@19975 having SEQ ID NO: 206; G.max_GMO6MC19234_59694709@18873 having SEQ ID NO: 220;
        (ii) the complement of a nucleic acid represented by said sequences B.napus_BN06MC20042_46499279@19975 having SEQ ID NO: 206; G.max_GM06MC19234_59694709@18873 having SEQ ID NO 220;
        (iii) a nucleic acid encoding the polypeptide as represented by any one SEQ ID NO: 207; SEQ ID NO 221 preferably as a result of the degeneracy of the genetic code, said isolated nucleic acid can be derived from a polypeptide sequence as represented by any one of SEQ ID NO: 207 and 221 and further preferably confers enhanced yield-related traits relative to control plants;
        (iv) a nucleic acid having, 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% sequence identity with any of the nucleic acid sequences of Table A3 and further preferably conferring enhanced yield-related traits relative to control plants;
        (v) a nucleic acid molecule which hybridizes with a nucleic acid molecule of (i) to (iv) under stringent hybridization conditions and preferably confers enhanced yield-related traits relative to control plants;
        (vi) a nucleic acid encoding a fibrillin polypeptide having, in increasing order of preference, at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence represented by any one of SEQ ID NO: 207, and 221 and any of the other amino acid sequences in Table A3 and preferably conferring enhanced yield-related traits relative to control plants.
    
    


According to a further embodiment of the present invention, there is also provided an isolated polypeptide selected from:

    
    
        (i) an amino acid sequence represented by any one of SEQ ID NO: 207 and 221;
        (ii) an amino acid sequence having, in increasing order of preference, at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence represented by any one of SEQ ID NO: 207 and 221 and any of the other amino acid sequences in Table A3 and preferably conferring enhanced yield-related traits relative to control plants.
        (iii) derivatives of any of the amino acid sequences given in (i) or (ii) above.
    
    


Furthermore, it has now surprisingly been found that modulating expression in a plant of a nucleic acid encoding a PLATZ 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 PLATZ polypeptide and optionally selecting for plants having enhanced yield-related traits.
The invention also provides hitherto unknown PLATZ-encoding nucleic acids and PLATZ polypeptides.
According to a further embodiment of the present invention, there is therefore provided an isolated nucleic acid molecule selected from:

    
    
        (i) a nucleic acid represented by SEQ ID NO: 354;
        (ii) the complement of a nucleic acid represented by SEQ ID NO: 354;
        (iii) a nucleic acid encoding a PLATZ polypeptide having, in increasing order of preference, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the amino acid sequence represented by SEQ ID NO: 355, and having in increasing order of preference at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to one or more of the motifs as defined herein.
    
    


According to a further embodiment of the present invention, there is also provided an isolated polypeptide selected from:

    
    
        (i) an amino acid sequence represented by SEQ ID NO: 355;
        (ii) an amino acid sequence having, in increasing order of preference, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the amino acid sequence represented by SEQ ID NO: 355, and having in increasing order of preference at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to one or more of the motifs as defined herein;
        (iii) derivatives of any of the amino acid sequences given in (i) or (ii) above.
    
    


Furthermore, it has now surprisingly been found that modulating expression in a plant of a nucleic acid encoding a PLST-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 PLST-like polypeptide and optionally selecting for plants having enhanced yield-related traits.
The invention also provides hitherto unknown PLST-like-encoding nucleic acids and PLST-like polypeptides.
According to a further embodiment of the present invention, there is therefore provided an isolated nucleic acid molecule selected from:

    
    
        (i) a nucleic acid represented by any one of the following nucleic acid sequences: having SEQ ID NO: 414; SEQ ID NO: 426; SEQ ID NO: 428; SEQ ID NO: 434; SEQ ID NO: 438;
        (ii) the complement of a nucleic acid represented by said sequences having SEQ ID NO: 414; SEQ ID NO: 426; SEQ ID NO: 428; SEQ ID NO: 434; SEQ ID NO: 438;
        (iii) a nucleic acid encoding the polypeptide as represented by any one having SEQ ID NO: 415; SEQ ID NO: 427; SEQ ID NO: 429; SEQ ID NO: 435; SEQ ID NO: 439 preferably as a result of the degeneracy of the genetic code, said isolated nucleic acid can be derived from a polypeptide sequence as represented by any one of SEQ ID NO: 415; SEQ ID NO: 427; SEQ ID NO: 429; SEQ ID NO: 435; SEQ ID NO: 439: and further preferably confers enhanced yield-related traits relative to control plants;
        (iv) a nucleic acid having, 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% sequence identity with any of the nucleic acid sequences of Table A5 and further preferably conferring enhanced yield-related traits relative to control plants;
        (v) a nucleic acid molecule which hybridizes with a nucleic acid molecule of (i) to (iv) under stringent hybridization conditions and preferably confers enhanced yield-related traits relative to control plants;
        (vi) a nucleic acid encoding a PLST-like polypeptide having, in increasing order of preference, at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence represented by any one of SEQ ID NO: 415; SEQ ID NO: 427; SEQ ID NO: 429; SEQ ID NO: 435; SEQ ID NO: 439: and any of the other amino acid sequences in Table A5 and preferably comprising the motifs 19 to 21 and 44 to 60 and the PF domain, conferring enhanced yield-related traits relative to control plants.
    
    


According to a further embodiment of the present invention, there is also provided an isolated polypeptide selected from:

    
    
        (i) an amino acid sequence represented by any one of SEQ ID NO: 415; SEQ ID NO: 427; SEQ ID NO: 429; SEQ ID NO: 435; SEQ ID NO: 439;
        (ii) an amino acid sequence having, in increasing order of preference, at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence represented by any one of SEQ ID NO: 415; SEQ ID NO: 427; SEQ ID NO: 429; SEQ ID NO: 435; SEQ ID NO: 439; and any of the other amino acid sequences in Table A5 and preferably comprising the motifs 19 to 21 and 44 to 60 and the PF domain, conferring enhanced yield-related traits relative to control plants.
        (iii) derivatives of any of the amino acid sequences given in (i) or (ii) above.
    
    


Furthermore, it has now surprisingly been found that modulating expression in a plant of a nucleic acid encoding a Glomalin 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 Glomalin polypeptide and optionally selecting for plants having enhanced yield-related traits.
The invention also provides hitherto unknown Glomalin-encoding nucleic acids and Glomalin polypeptides.
According to a further embodiment of the present invention, there is therefore provided an isolated nucleic acid molecule selected from:

    
    
        (i) a nucleic acid represented by any of SEQ ID NO: 568, SEQ ID NO: 569, or SEQ ID NO: 570;
        (ii) the complement of a nucleic acid represented by any of SEQ ID NO: 568, SEQ ID NO: 569, or SEQ ID NO: 570;
        (iii) a nucleic acid encoding a Glomalin polypeptide having in increasing order of preference at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence represented by any of SEQ ID NO: 592, SEQ ID NO: 593, or SEQ ID NO: 594, and additionally or alternatively comprising one or more motifs having in increasing order of preference two, one or no sequence mismatch compared to any of the Motifs 31 to 43 (SEQ ID NO: 596 to SEQ ID NO: 608), and further preferably conferring enhanced yield-related traits relative to control plants.
        (iv) a nucleic acid molecule which hybridizes with a nucleic acid molecule of (i) to (iii) under high stringency hybridization conditions and preferably confers enhanced yield-related traits relative to control plants.
    
    


According to a further embodiment of the present invention, there is also provided an isolated polypeptide selected from:

    
    
        (i) an amino acid sequence represented by any of SEQ ID NO: 592, SEQ ID NO: 593, or SEQ ID NO: 594;
        (ii) an amino acid sequence having, in increasing order of preference, at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence represented by any of SEQ ID NO: 592, SEQ ID NO: 593, or SEQ ID NO: 594, and additionally or alternatively comprising one or more motifs having in increasing order of preference two, one or no sequence mismatch compared to any of the motifs 41 to 43 (SEQ ID NO: 596 to SEQ ID NO: 608), and further preferably conferring enhanced yield-related traits relative to control plants;
        (iii) derivatives of any of the amino acid sequences given in (i) or (ii) above.
    
    


A preferred method for modulating, and preferably increasing, expression of a nucleic acid encoding an eRF1 polypeptideis by introducing and expressing in a plant a nucleic acid encoding an eRF1 polypeptide. Another preferred method for modulating, preferably increasing, expression of a nucleic acid encoding a SCAMP-like polypeptide is by introducing and expressing in a plant a nucleic acid encoding a SCAMP-like polypeptide. Yet another preferred method for modulating, preferably increasing, expression of a nucleic acid encoding a fibrillin polypeptide is by introducing and expressing in a plant a nucleic acid encoding a fibrillin polypeptide. Another preferred method for modulating, preferably increasing, expression of a nucleic acid encoding a PLATZ polypeptide is by introducing and expressing in a plant a nucleic acid encoding a PLATZ polypeptide. Still another preferred method for modulating, preferably increasing, expression of a nucleic acid encoding a PLST-like polypeptide is by introducing and expressing in a plant a nucleic acid encoding a PLST-like polypeptide. Another preferred method for modulating, preferably increasing, expression of a nucleic acid encoding a Glomalin polypeptide is by introducing and expressing in a plant a nucleic acid encoding a Glomalin polypeptide.
In one embodiment, a “protein useful in the methods of the invention” is taken to mean an eRF1 polypeptide as defined herein. In another embodiment, a “nucleic acid useful in the methods of the invention” is taken to mean a nucleic acid capable of encoding such an eRF1 polypeptide. A nucleic acid to be introduced into a plant, and therefore useful in performing the methods of the invention, is in such embodiment any nucleic acid encoding the type of protein which will now be described, hereafter also named “eRF1 nucleic acid” or “eRF1 gene”.
The “eRF1 polypeptide” as defined herein refers to any polypeptide comprising at least tree consensus domains—eRF1 domain 1, eRF1 domain 2 and eRF1 domain 3, with PFam accession numbers respectively PF03463, PF03464 and PF03465.
Preferably, the eRF1 domain 1 of an eRF1 polypeptide has at least, in increasing order of preference, 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% or more sequence identity to the sequence located between amino acid 6 and 140 of SEQ ID NO 2.
Preferably, the eRF1 domain 2 of an eRF1 polypeptide has at least, in increasing order of preference, 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% or more sequence identity to the sequence located between amino acid 144 and 278 of SEQ ID NO 2. Preferably, the eRF1 domain 3 of an eRF1 polypeptide has at least, in increasing order of preference, 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% or more sequence identity to the sequence located between amino acid 281 and 418 of SEQ ID NO 2.
Further preferable the eRF1 polypeptide of the present invention comprises one or more of the following peptides: GGQ, NIKS and [GA][IMLV]LR[YW] having SEQ ID NO: 73, 74 and 75 respectively.
Alternatively, the eRF1 polypeptide useful in the methods of the invention comprises one or more sequence motifs having at least, in increasing order of preference 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% or more sequence identity to anyone of the following motifs:
The amino acids as indicated herein in brackets represent a possible replacement by any other amino acid.








Motif 1:



(SEQ ID NO: 76)



FGTLSGNTREVLHKF[TS]VDLPKKHGRGGQSALRFARLRMEKRHNYVRK[TV]AE;



 


Motif 2:


(SEQ ID NO: 77)



YN[KR]VPPNGLVLY[TC]GT[IV]VT[ED][DE]GKEKKV[TN]IDFEPF[KR]PIN[AT]SLYLCD



 


NKFHTE;


 


Motif 3:


(SEQ ID NO: 78)



ARGNGTSMISLI[MI]PP[RK]DQ[IV]SRVTKML[GA]DE[YF]GTASNIKSRVNR[QL]SV



 


L[GS]AIT.






Motifs 1 to 3 are typically found in any eRF1 polypeptide.
In another preferred embodiment of the present invention the eRF1 polypeptide of the invention may comprise any one or more sequence motifs having at least, in increasing order of preference 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% or more sequence identity to anyone of the following motifs:








Motif 4:



(SEQ ID NO: 79)



F[TS]VDLPKKHGRGGQSALRFARLR[EM]EKRHNYVRKVAE[VL]A[VT]QNFITND



 


[KR][PV]NV;


 


Motif 5:


(SEQ ID NO: 80)



Y[NT][KR]VPPNGLV[VLI]YCG[TD][IV][ILM]T[ED][ED]GKE[KR]K[VM][NT]ID[FI]E



 


PFKPINTSLYLCDNKFHTE;


 


Motif 6:


(SEQ ID NO: 81)



ARGNGTSMISL[IV][IM]PPK[DG]Q[IV]S[RL]V[QA]KML[AT][DE]EYGTASNIKSRVN



 


R[LQ]SVL[SG]AIT.






Motifs 4, 5 and 6 correspond to a consensus sequences which represent conserved protein regions in an eRF1 polypeptide of non-Streptophyta origin.
Most preferably, the eRF1 polypeptide of the invention may comprise any one or more of the following sequence motifs having at least, in increasing order of preference 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% or more sequence identity to anyone of the following motifs:







Motif 7:


(SEQ ID NO: 82)


VDLPKKHGRGGQSALRFARLRMEKRHNYVRKTAELATQF[YF]INPATS


 


QPNV


 


Motif 8:


(SEQ ID NO: 83)


YNKVPPNGLVLYTGTIVT[ED]DGKEKKVTIDFEPF[KR]PINASLYLC


 


DNKFHTE


 


Motif 9:


(SEQ ID NO: 84)


TSMISLIMPPRDQ[VI]SRVTKMLGDE[FY]GTASNIKSRVNRQSVLGA


 


ITSAQQR






Motifs 7, 8 and 9 correspond to a consensus sequences which represent conserved protein regions in an eRF1 polypeptide of Streptophyta cluster to which Arabidopsis belongs.
It is understood that Motif 1, 2, 3, 4, 5, 6, 7, 8 and 9 as referred herein represent the consensus sequence of the homologous motifs as present in a specific eRF1 polypeptide, preferably in any eRF1 polypeptide of Table A1, more preferably in SEQ ID NO: 2. Motifs as defined herein are not limited to their respective sequence but they encompass the homologous motifs as present in any eRF1.
Methods to identify homologous motif to Motifs 1 to 9 in a polypeptide useful in the methods of the present invention are well known in the art. For example the polypeptide may be compared to the motif by aligning their respective amino acid sequence to identify regions with similar sequence using an algorithm such as Blast (Altschul et al. (1990) J Mol Biol 215: 403-10).
Alternatively, the homologue of eRF1 polypeptide 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 any of the polypeptides of Table A1, preferably by the SEQ ID NO: 2, provided that the homolog polypeptide comprises the conserved motifs 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 and preferably with sequences of mature proteins (i.e. without taking into account secretion signals or transit peptides). Compared to overall sequence identity, the sequence identity will generally be higher when only conserved domains or motifs are considered. For local alignments, the Smith-Waterman algorithm is particularly useful (Smith T F, Waterman M S (1981) J. Mol. Biol 147(1); 195-7).
Preferably, the sequences of eRF1 polypeptides, when used in the construction of a phylogenetic tree, such as the one depicted in FIG. 1, clusters with the group of eRF1 polypeptides comprising the amino acid sequences represented by SEQ ID NO: 2.
In another embodiment, a “protein/polypeptide useful in the methods of the invention” is taken to mean a SCAMP-like polypeptide as defined herein. In another embodiment a “nucleic acid useful in the methods of the invention” is taken to mean a nucleic acid capable of encoding such a SCAMP-like polypeptide. A nucleic acid to be introduced into a plant, and therefore useful in performing the methods of the invention, is in such embodiment any nucleic acid encoding the type of protein which will now be described, hereafter also named “SCAMP-like nucleic acid” or “SCAMP-like gene”.
A “SCAMP-like polypeptide” as defined herein refers to any polypeptide comprising a SCAMP domain (HMM PFam PF04144).
Preferably the SCAMP domain of a SCAMP-like polypeptide has in increasing order of preference at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid of the SCAMP domain present in any of the polypeptides of Table A2, preferably to the SCAMP domain represented by the sequence located between amino acids 91 and 265 of SEQ ID NO: 89.
More preferably and in addition to the SCAMP domain, the SCAMP-like polypeptide useful in the methods of the invention has one or more, preferably and at least 4, or 3, or 2, or 1 transmembrane domain regions (TMRs).
Transmembrane domain regions (TMRs) in a polypeptide and methods to identify the same are well known in the art. Examples of such methods are further provided in the Example section.
More preferably the TMR of a SCAMP-like polypeptide useful in the methods of the invention has in increasing order of preference at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid of one or more of the TMRs present in any of the polypeptides of Table A2, preferably of SEQ ID NO: 89.
The TMRs as present in SEQ ID NO: 89 are represented by the following sequences:














N-ter-

C-ter-

SEQ ID


minal
transmembrane region
minal
length
NO:







121
AFTTLLGLVGCLLWNIVAVTVAW
143
23
194


 


151
IWLLSIIYFLAGVPGAYVLWYRP
173
23
195


 


186
FGAFFFFYVFHIAFCGFAAVAPP
208
23
196


 


227
TTNAAVGIMYFIGAGFFCIETLL
249
23
197









The N-terminal and C-terminal columns indicate the amino acid coordinates of the transmembrane domain in SEQ ID NO: 89
Further more preferably the SCAMP-like polypeptide useful in the methods of the invention comprises at least 2, 1, 0, 3, 4 or 5 repeats or the tripeptide NPF (SEQ ID NO: 198). This tripeptide has been associated with the biological role of SCAMP proteins to recruit by binding EH-proteins during endocytosis. The NPF tripeptide in a SCAMP polypeptide are preferably located in increasing order of preference at the N-terminus, the C-terminus and at both the N- and the C-terminus.
Typically and even more preferably the SCAMP-like polypeptides of the invention share a common domain structure composed of a cytoplasmic N-terminal domain with multiple NPF repeats, four highly conserved transmembranes regions (TMRs), and a short cytoplasmic C-terminal tail.
The N-terminal tail of a preferred SCAMP-like polypeptide of the invention has in increasing order of preference at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid of the N-terminal tail as present in any of the polypeptides of Table A2, preferably of SEQ ID NO: 89 and represented by MARHDPNPFADEEINPFANHTSVPPASNSYLKPLPPEPYDRGATVDIPLDSGNDLRAKEM ELQAKENELKRKEQELKRREDAIARTGVVIEEKNWPEFFPLIHHDIPNEIPIHLQKIQYV (SEQ ID NO: 199).
The C-terminal tail of a preferred SCAMP-like polypeptide of the invention has in increasing order of preference at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid of the C-terminal tail as present in any of the polypeptides of Table A2, preferably of SEQ ID NO: 89 and represented by NIWVIQQVYAYFRGSGKAAEMKREA TKSTLMRAL (SEQ ID NO: 200).
Typically polypeptides comprise an E peptide. An E peptide refers to a conserved amino acid motif, having in increasing order of preference at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% overall sequence identity to the amino acid represented LWYRPLYRAFRTDSA (SEQ ID NO: 201) or LWYRPLYNAMRTESA (SEQ ID NO: 202).
Alternatively, a SCAMP-like nucleic acid of the invention is any nucleic acid encoding a homologue of any of the polypeptides of Table A2, preferably a paralogue or an orthologue thereof. Preferably such homologue has an equivalent (or similar) biological function, for example recruiting EH-proteins during endocytosis processes occurring at cell separation, for example during fruit abscission and dehiscence.
Alternatively, the homologue of a SCAMP-like protein useful in the methods of the invention has in increasing order of preference at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% overall sequence identity to the amino acid represented by any of the polypeptides of Table A2, preferably to SEQ ID NO: 89.
The overall sequence identity is determined using a global alignment algorithm, such as the Needleman Wunsch algorithm in the program GAP (GCG Wisconsin Package, Accelrys), preferably with default parameters and preferably with sequences of mature proteins (i.e. without taking into account secretion signals or transit peptides). Compared to overall sequence identity, the sequence identity will generally be higher when only conserved domains or motifs are considered. For local alignments, the Smith-Waterman algorithm is particularly useful (Smith T F, Waterman M S (1981) J. Mol. Biol 147(1); 195-7).
In another embodiment, a “protein useful in the methods of the invention” is taken to mean a fibrillin polypeptide as defined herein. In another embodiment, a “nucleic acid useful in the methods of the invention” is taken to mean a nucleic acid capable of encoding such a fibrillin polypeptide. A nucleic acid to be introduced into a plant, and therefore useful in performing the methods of the invention, in such embodiment is any nucleic acid encoding the type of protein which will now be described, hereafter also named “fibrillin nucleic acid” or “fibrillin gene”.
A “fibrillin polypeptide” as defined herein refers to any polypeptide comprising the following

    
    
        (i) a PAP fibrillin domain as represented by PFAM Accession number PF04755; and
        (ii) a C-terminal domain represented by KFECQNESRGGLVRNVIKWSVPRLLEEN EGATLIVTARFSSVSARNIYLKFEEIGLQNINISDDLQAVIAPAILPRSFLSLQILQF IRSFKARVPVTSPERHSVGGLYYLSYLDKNMLLGRAVGGGGVFIFTRAHTL (SEQ ID NO: 253) which may contain between 0 and 5 gaps representing between 1 and 15 residues, or a domain having in increasing order of preference at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% overall sequence identity to (SEQ ID NO: 253); and optionally
        (iii) a transit peptide within the N-terminal region of the polypeptide.
    
    


SEQ ID NO: 253 represents the amino acid sequence of the C-terminal domain as it appears in the fibrillin represented by SEQ ID NO: 205.
According to a preferred feature of the present invention, the PAP fibrillin domain is represented by: ENRKYELLNIIQDTQRGLVTTADQRSTIEEAMVVVEGFDAGKEIDLSKL DG TWQYTSAPDVLILFESAARLPFFQVGQIFQ (SEQ ID NO: 252) which may contain between 0 and 5 gaps representing between 1 and 15 residues, or a domain having in increasing order of preference at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% overall sequence identity to SEQ ID NO: 252. SEQ ID NO: 252 represents the amino acid sequence of the PAP fibrillin domain as it appears in the fibrillin represented by SEQ ID NO: 205.
A further feature of fibrillin poypeptides is the presence of one or more of the following domains:

    Domain X:NIYLQF[EQ]E[IA]S[VL]Q[ND]INISE[EQ]LQAL[IL]APA[IL]LPRSFL[SN]LQILQ[FA][LI][RK][TS]F[KR]AQ[VI]P (SEQ ID NO: 254);
    Domain Y: YYL[ST]YLD[RN][ND]MLLGR[AS]VGGGGV (SEQ ID NO: 255);
    Domain Z: [PA][IL]DL[AS]KLDGTWRLQYTSA[SP]DV (SEQ ID NO: 256); or a domain having in increasing order of preference at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% overall sequence identity to any one or more of Domains X, Y and Z.


Fibrillin poylpeptides typically have in increasing order of preference at least 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%, 99% or more overall sequence identity to the amino acid represented by SEQ ID NO: 205. The overall sequence identity is determined using a global alignment algorithm, such as the Needleman Wunsch algorithm in the program GAP (GCG Wisconsin Package, Accelrys), preferably with default parameters and preferably with sequences of mature proteins (i.e. without taking into account secretion signals or transit peptides). Compared to overall sequence identity, the sequence identity will generally be higher when only conserved domains or motifs are considered. For local alignments, the Smith-Waterman algorithm is particularly useful (Smith T F, Waterman M S (1981) J. Mol. Biol 147(1); 195-7).
Preferably, the polypeptide sequence which when used in the construction of a phylogenetic tree, such as the one depicted in FIG. 6, clusters with the group of fibrillin polypeptides comprising the amino acid sequence represented by SEQ ID NO: 205 rather than with any other group.
In another embodiment, a “protein useful in the methods of the invention” is taken to mean a PLATZ polypeptide as defined herein. In another embodiment, a “nucleic acid useful in the methods of the invention” is taken to mean a nucleic acid capable of encoding such a PLATZ polypeptide. A nucleic acid to be introduced into a plant, and therefore useful in performing the methods of the invention, is in such embodiment any nucleic acid encoding the type of protein which will now be described, hereafter also named “PLATZ nucleic acid” or “PLATZ gene”.
A “PLATZ polypeptide” as defined herein refers to any polypeptide comprising a PLATZ domain (Pfam accession PF04640; InterPro accession IPR006734); and comprising an N-terminal zinc binding region with the consensus sequence C-x(2)-H-x(11)-C-x(2)-C-x(4-5)-C-x(2)-C-x(3-7)-H-x(2)-H (SEQ ID NO: 262) and a zinc binding region with the consensus sequence C-x(2)-C-x(10-11)-C-x(3)-C (SEQ ID NO: 263) in the central region, as described in Nagano et al. (2001). In other words, a “PLATZ polypeptide” as defined herein refers to any polypeptide comprising a PLATZ domain (Pfam accession PF04640; InterPro accession IPR006734); and comprising an N-terminal zinc binding region with a consensus sequence as given by any of the following sequences: CxxHxxxxxxxxxxxCxxCxxxxCxxCxxxHxxH (SEQ ID NO: 645); CxxHxxxxxxxxxxx CxxCxxxxCxxCxxxxHxxH (SEQ ID NO: 646); CxxHxxxxxxxxxxxCxxCxxxxCxxCxxxxxHxxH (SEQ ID NO: 647); CxxHxxxxxxxxxxxCxxCxxxxCxxCxxxxxxHxxH (SEQ ID NO: 648); CxxHxxxxxxxxxxxCxxCxxxxCxxCxxxxxxxHxxH (SEQ ID NO: 649); CxxHxxxxxxxxxxxCxx CxxxxxCxxCxxxHxxH (SEQ ID NO: 650); CxxHxxxxxxxxxxxCxxCxxxxxCxxCxxxxHxxH (SEQ ID NO: 651); CxxHxxxxxxxxxxxCxxCxxxxxCxxCxxxxxHxxH (SEQ ID NO: 652); CxxHxxxxxxxxxxxCxxCxxxxxCxxCxxxxxxHxxH (SEQ ID NO: 653); CxxHxxxxxxxxxxx CxxCxxxxxCxxCxxxxxxxHxxH (SEQ ID NO: 654); and a zinc binding region with the consensus sequence CxxCxxxxxxxxxxCxxxC (SEQ ID NO: 655) or CxxCxxxxxxxxxxx CxxxC (SEQ ID NO: 656) in the central region, as described in Nagano et al. (2001).
Preferably, the PLATZ protein useful in the methods of the present invention comprises one or more of the following motifs (defined by MEME; Bailey and Elkan, Proceedings of the Second International Conference on Intelligent Systems for Molecular Biology, pp. 28-36, AAAI Press, Menlo Park, Calif., 1994, Bailey et al., Nucleic Acids Research, 34, W369-W373, 2006):

    Motif 10 (SEQ ID NO: 264): [VI]QTY[TVI]INSA[KR]V[VI]FL[NK][QE]RPQ[SP]R
    Motif 11 (SEQ ID NO: 265): HRSHR[LV][LI]Q[VI]RR[YS][VS]YHDV[VI]R[LV]x[DE][LI][QE]KL[IL]D[CI]
    Motif 12 (SEQ ID NO: 266): [KR]NE[KC]N[IV]FCLDC


More preferably, the PLATZ proteins comprise one or more of the following motifs, as defined by MEME:

    Motif 13 (SEQ ID NO: 267): HRSH[RP]LLQVRRYVYHDV[VI]RLEDL[EQ]KLIDCS
    Motif 14 (SEQ ID NO: 268): FKG[SL]GN[SY]CT[TS]CDR[SI]LQEP[FY][HR][FHY]CS[LV] [SG]CKV
    Motif 15 (SEQ ID NO: 269): AWLE[AG]L[LY][TA][EDQ]KFFV[GA]C[SP]xHEx[AR][KR]KNE KN[IV][FC]C[LV]DC[CS][AT]SIC[PQ]HC


Most preferably, the PLATZ proteins comprise one or more of the following motifs, as defined by MEME:

    Motif 16 (SEQ ID NO: 270): RRYVYHDVVRLEDL[EQ]KLIDCS[NS]VQ[AS]YTINS[AS]KV VF[IL]KKRPQNRQFKG[SA]GN
    Motif 17 (SEQ ID NO: 271): PAWLEALY[TA][QE]KFF[VA][GA]CS[YF]HE[HNT]AKKNEKN [IV]CCLDCC[TI]SICPHC[VL]P[SA]HR[FV]HR
    Motif 18 (SEQ ID NO: 272): CTSCDRSLQEP[FY][IF]HCSL[GD]CKV[DE][FY][VI]L[KR][HQ] [YK]KDLS[PA]YLR[PTR]C[KN][TS]L[QT]L[GS]PDF[FL]IP


Furthermore preferably, the PLATZ polypeptide comprises in increasing order of preference, at least 2 or at least 3 of the motifs listed above.
Alternatively or additionally, the homologue of a PLATZ 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: 261, provided that the homologous protein comprises one or more of the conserved motifs 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 and preferably with sequences of mature proteins (i.e. without taking into account secretion signals or transit peptides). Compared to overall sequence identity, the sequence identity will generally be higher when only conserved domains or motifs are considered. Preferably the motifs in a PLATZ polypeptide have, in increasing order of preference, at least 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% sequence identity to the motifs represented by SEQ ID NO: 264 to SEQ ID NO: 272 (Motifs 10 to 18).
Preferably, the polypeptide sequence which when used in the construction of a phylogenetic tree, such as the one depicted in FIG. 12, clusters with the group of PLATZ-A polypeptides, more preferably with the group of PLATZ-A1 proteins, most preferably with the group of PLATZ-A1-α proteins, comprising the amino acid sequence represented by SEQ ID NO: 261 rather than with any other group of PLATZ proteins.
In another embodiment, a “protein useful in the methods of the invention” is taken to mean a PLST-like polypeptide as defined herein. In another embodiment, a “nucleic acid useful in the methods of the invention” is taken to mean a nucleic acid capable of encoding such a PLST-like polypeptide. A nucleic acid to be introduced into a plant, and therefore useful in performing the methods of the invention, is in such embodiment any nucleic acid encoding the type of protein which will now be described, hereafter also named “PLST-like nucleic acid” or “PLST-like gene”.
The “PLST-like polypeptide” as defined herein refers to any polypeptide comprising at least a PLST-like consensus domain—with PFam accession number PF02298.
Preferably, the PLST-like domain of a PLST-like polypeptide has at least, in increasing order of preference, 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% or more sequence identity to the sequence located between amino acid 38 and 124 of SEQ ID NO 411.
Alternatively, the PLST-like polypeptide useful in the methods of the invention comprises one or more sequence motifs having at least, in increasing order of preference 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% or more sequence identity to anyone of the following motifs:

    Motif 19: [DH]SV[LI]QV[TS]KE[DA][YF][DK]SCNT[SK][NSD]P (SEQ ID NO: 530);
    Motif 20: [FHY]YF[IT]SGV[PK][GD][HN]C (SEQ ID NO: 531);
    Motif 21: Y[NT][QK]WA[ESK][KS]NRF[KQ][IV]GD[ST][LI][VL]F[KL]YP (SEQ ID NO: 532);
    Motif 44: [DHN][SND][VLM]xx[VL]xxxx[FYHN]xxCxxTx[NSD] (SEQ ID NO: 615);
    Motif 45: [GD]xx[FY][FYI]x[SGC][GAIT] (SEQ ID NO: 616); and preferably said x in position 3 is selected from the following amino acids: F, H, Y, V, and A.










Motif 46:



(SEQ ID NO: 617)



[GV][GDAE]xxxWx[VITLA]xxxxxxW[ASPV]xxxx[FL]xx[GDNS]Dxxxxx[FY];



 


Motif 47:


(SEQ ID NO: 618)



[GV][GDAE]xxxWx[VITLA]xxxxxxxW[ASPV]xxxx[FL]xx[GDNS]Dxxxxx[FY];



 


Motif 48:


(SEQ ID NO: 619)



[GV][GDAE]xxxWx[VITLA]xxxxxxxxW[ASPV]xxxx[FL]xx[GDNS]Dxxxxx[FY];



 


Motif 49:


(SEQ ID NO: 620)



[GV][GDAE[xxxWx[VITLA]xxxxxxxxxW[ASPV]xxxx[FL]xx[GDNS]Dxxxxx[FY];



 


Motif 50:


(SEQ ID NO: 621)



[GV][GDAE]xxxWx[VITLA]xxxxxxxxxxW[ASPV]xxxx[FL]xx[GDNS]Dxxxxx[FY];



 


Motif 51


(SEQ ID NO: 622)



[GV][GDAE]xxxWx[VITLA]xxxxxxxxxxxW[ASPV]xxxx[FL]xx[GDNS]Dxxxxx[FY];



 


Motif 52:


(SEQ ID NO: 623)



[GV][GDAE]xxxWx[VITLA]xxxxxxxxxxxxW[ASPV]xxxx[FL]xx[GDNS]Dxxxxx[FY];



 


Motif 53:


(SEQ ID NO: 624)



[GV][GDAE]xxxxWx[VITLA]xxxxxxW[ASPV]xxxx[FL]xx[GDNS]Dxxxxx[FY];



 


Motif 54:


(SEQ ID NO: 625)



[GV][GDAE]xxxxWx[VITLA]xxxxxxxW[ASPV]xxxx[FL]xx[GDNS]Dxxxxx[FY];



 


Motif 55:


(SEQ ID NO: 626)



[GV][GDAE]xxxxWx[VITLA]xxxxxxxxW[ASPV]xxxx[FL]xx[GDNS]Dxxxxx[FY];



 


Motif 56:


(SEQ ID NO: 627)



[GV][GDAE]xxxxWx[VITLA]xxxxxxxxxW[ASPV]xxxx[FL]xx[GDNS]Dxxxxx[FY];



 


Motif 57:


(SEQ ID NO: 628)



[GV][GDAE]xxxxWx[VITLA]xxxxxxxxxxW[ASPV]xxxx[FL]xx[GDNS]Dxxxxx[FY];



 


Motif 58


(SEQ ID NO: 629)



[GV][GDAE]xxxxWx[VITLA]xxxxxxxxxxxW[ASPV]xxxx[FL]xx[GDNS]Dxxxxx[FY];



 


Motif 59


(SEQ ID NO: 630)



[GV][GDAE]xxxxWx[VITLA]xxxxxxxxxxxxW[ASPV]xxxx[FL]xx[GDNS]Dxxxxx[FY].







Alternatively, or in combination therewith, in another embodiment, a PLST-like polypeptide useful in methods of the invention comprises a sequence motifs having at least, in increasing order of preference 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% or more sequence identity to following motif:









Motif 60:



(SEQ ID NO: 631)



[FHY][YF][FY][TAI]S[GAD]xx[GD][HRN]C.






These motifs are consensus sequences and will not always be identical in PLST-like sequences. It is noted that the amino acids herein indicated in square brackets represent alternative amino acids for a particular position. In the herein given sequences the “x” can be any distinct amino acid.
In another preferred embodiment of the present invention the PLST-like polypeptide of the invention may comprise any one or more sequence motifs having at least, in increasing order of preference 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% or more sequence identity to anyone of the following motifs:








Motif 22:



(SEQ ID NO: 533)



[DN]GN[TS][LVK][FV][KN][LF][DT]R[SP]GP[FY]YF[IT]SG[VA][KP][GD][HN]CEK



 


[GN][QE]K;


 


Motif 23:


(SEQ ID NO: 534)



[YL]N[QK]WA[EK][KS][NH]RF[KQ][IV]GD[ST]L[LV]F[LK]Y[PD];



 


Motif 24:


(SEQ ID NO: 535)



[KQ]DSV[LI]QVTKE[DA]YKSCNT[SK][DSN]PI;



 


Motif 61:


(SEQ ID NO: 632)



[DNT][GDE][NDKH][TS][LVKM][FVYI]x[LIF]xxxGx[FYHV][YF][FYI][ITVA]S[GAD]xxxxxC;



 


Motif 62:


(SEQ ID NO: 633)



[DNT][GDE][NDKH][TS][LVKM][FVYI]x[LIF]xxxGx[FYHV][YF][FYI][ITVA]S[GAD]xxxxxxC;



 


Motif 63:


(SEQ ID NO: 667)



[DNT][GDE][NDKH][TS][LVKM][FVYI]x[LIF]xxxGx[FYHV][YF][FYI][ITVA]S[GAD]xxxxC;



 


Motif 64:


(SEQ ID NO: 634)



[NSDT]xW[ASPV]xxx[RSNT][FLV]x[VILT][GN]Dx[LIV]x[FLW]x[YF];



 


Motif 65:


(SEQ ID NO: 635)



S[VALM][LIM]x[VL]xxxx[YF]xxC[NTKD][SKTDG]xx[PAHY].







Motifs 22, 23 and 24, and motifs 61 to 65 correspond to consensus sequences, which represent conserved protein regions in a polypeptide of a group comprising the PLST-like, P_Class (to which Arabidopsis thaliana and P. trichocarpa belong), and the NDL.
Most preferably, the PLST-like polypeptide of the invention may comprise any one or more sequence motifs having at least, in increasing order of preference 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% or more sequence identity to anyone of the following motifs:








Motif 25:



(SEQ ID NO: 536)



DSVI[QV]VT[EKA][EQ]S[YF][KN][SK]CNL[KST]DPIL[YF][MS]N[ND]GN[ST]



 


[LV]FN [LI][TD][RS]PGL[FY]YF[TI]SG[VA][PS]GHC[EQ][KR];


 


Motif 26:


(SEQ ID NO: 537)



P[PT]SA[DN]P[DQ][VL]YTKW[AS][KS][NS][HN][RN]FK[IL]GD[ST][LI]LFLYP



 


Motif 27:


(SEQ ID NO: 538)



XVS[CS]Y[QE][YF]KVG[DG]LD[AGS]W;



 


Motif 66:


(SEQ ID NO: 636)



DS[VALM][LVIM][QVE][VL][STA]xxx[FY]xxC[NDQ]x[KST]xP[LVIQH][LATF]



 


[YFTKS][FSLM]x[ND][GE][NDK][ST]x[FYI]x[LIF][TDSE];


 


Motif 67:


(SEQ ID NO: 637)



[YF]xxW[APV][KSG]xxx[FLV][KART][LIV][GN]D[SAT][LI]xFxY;



 


Motif 68:


(SEQ ID NO: 638)



[YF]x[VA]G[DAGE]xx[GAS]Wx[VAI]P.







Motifs 25, 26 and 27 and motifs 66 to 68 correspond to a consensus sequence, which represents the most conserved protein regions in polypeptides belonging to PLST-like and P_Class group polypeptide to which Arabidopsis thaliana belongs.
In a most preferred embodiment of the present invention the PLST-like polypeptide of the invention may comprise any one or more sequence motifs having at least, in increasing order of preference 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% or more sequence identity to anyone of the following motifs:








Motif 28:



(SEQ ID NO: 539)



HN[FL]K[IL]GDSLLFLYPPSQDSVIQVTA[QE][SAN][YF][KN]SC[ND]L[KS]DPILY



 


MN[DN]GNSLFN[IL]T;


 


Motif 29:


(SEQ ID NO: 540)



GDFYFTSG[AVE]PGHC[EQ]K[SK]QKLH[IV];



 


Motif 30:


(SEQ ID NO: 541)



VSCYQYKVGDLD[AS]WGIPTSA[NK];



 


Motif 69:


(SEQ ID NO: 639)



[FLV]x[LIV]GD[SA][LIV][LFW]FLY[PL]PS[QE]DS[LMAV][LIV]Q[VL][TA]x2[ASN][FY];



 


Motif 70:


(SEQ ID NO: 640)



C[NDQ]X[SKT][DNS]P[LVI][LAT]X[MFL]X[ND]GN[ST][LAV][NK][LFI][ST];



 


Motif 71:


(SEQ ID NO: 641)



Gxx[FHY][YF][FY][TAI]S[GAD]xxG[HR]Cx[KR]x[QS][KR][LAI];



 


Motif 72:


(SEQ ID NO: 642)



[YF][KQ]VG[DAGN]L[DQN][AS]W[GAN][VAI]P[TIPS];



 


Motif 73:


(SEQ ID NO: 643)



GD[SA][LIV][LFW]FLY[PL]PS[QE];



 


Motif 74:


(SEQ ID NO: 644)



LY[PL]PS[QE].







It is understood that Motif determined by MEME (Timothy L. Bailey and Charles Elkan, “Fitting a mixture model by expectation maximization to discover motifs in biopolymers”, Proceedings of the Second International Conference on Intelligent Systems for Molecular Biology, pp. 28-36, AAAI Press, Menlo Park, Calif., 1994) 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30 as referred herein represent the consensus sequence of motifs as present in a PLST-like polypeptide, preferably in a PLST-like polypeptide of Table A5, more preferably in SEQ ID NO: 411. Motifs as defined herein are not limited to their respective sequence but they encompass the homologous motifs as present in any PLST-like.
It is understood that Motif determined by MEME (Timothy L. Bailey and Charles Elkan, “Fitting a mixture model by expectation maximization to discover motifs in biopolymers”, Proceedings of the Second International Conference on Intelligent Systems for Molecular Biology, pp. 28-36, AAAI Press, Menlo Park, Calif., 1994) 44 to 74 as referred herein represent the consensus sequence of motifs as present in a PLST-like polypeptide, preferably in a PLST-like polypeptide of Table A5, more preferably in SEQ ID NO: 411. Motifs as defined herein are not limited to their respective sequence but they encompass the homologous motifs as present in any PLST-like
Methods to identify homologous motif to Motifs 19 to 30 and/or Motifs 44 to 74 in a polypeptide useful in the methods of the present invention are well known in the art. For example the polypeptide may be compared to the motif by aligning their respective amino acid sequence to identify regions with similar sequence using an algorithm such as Blast (Altschul et al. (1990) J. Mol. Biol. 215: 403-10).
Alternatively, the homologue of PLST-like polypeptide 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 any of the polypeptides of Table A5, preferably by the SEQ ID NO: 411, provided that the homolog polypeptide comprises the conserved motifs 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 and preferably with sequences of mature proteins (i.e. without taking into account secretion signals or transit peptides). Compared to overall sequence identity, the sequence identity will generally be higher when only conserved domains or motifs are considered. For local alignments, the Smith-Waterman algorithm is particularly useful (Smith T F, Waterman M S (1981) J. Mol. Biol. 147(1); 195-7).
Preferably, the sequences of PLST-like polypeptides, when used in the construction of a phylogenetic tree, such as the one depicted in FIG. 15, clusters with the group of PLST-like polypeptides comprising the amino acid sequences represented by SEQ ID NO: 411.
In another embodiment, a “protein useful in the methods of the invention” is taken to mean a Glomalin polypeptide as defined herein. In another embodiment, a “nucleic acid useful in the methods of the invention” is taken to mean a nucleic acid capable of encoding such a Glomalin polypeptide. A nucleic acid to be introduced into a plant, and therefore useful in performing the methods of the invention, is in such embodiment any nucleic acid encoding the type of protein which will now be described, hereafter also named “Glomalin nucleic acid” or “Glomalin gene”.
A “Glomalin polypeptide” as defined herein refers to any heat shock protein 60 (HSP60 polypeptide) comprising a Cpn60_TCP1 domain (Pfam accession PF00118) and further comprising Motifs 31, 32 and 33, or a motif having not more than 2 mismatches to Motifs 31 to 33.

    Motif 31 (SEQ ID NO: 596): DDT(I/V/L)(I/V)L(D/H)G(G/A/L)G(N/D/E)K(A/T/K/Q)X(I/L)(E/D) (E/D), wherein X is any amino acid, preferably one of I, A, S, G, L, Q, T
    Motif 32 (SEQ ID NO: 597): (T/A)(S/A/N/E)N(F/E/D)(D/G)(E/Q)(K/R)(V/I/R)GV(Q/E)
    Motif 33 (SEQ ID NO: 598): (E/Q)X(N/D)X(N/S/D)(I/L/F/M)G(Y/F/C)DAA(K/R)(A/G/D) (E/K/T)Y(V/L)(D/H)M, wherein X in position 2 is any amino acid, preferably one of A, T, S, D, E, X in position 4 is any amino acid, preferably one of L, P, T, C


Additionally or alternatively, the Glomalin polypeptide comprises one or more of the Motifs 34 to 43.

    Motif 34 (SEQ ID NO: 599): L(Q/R/K)GV(E/S/N)(Q/E/D)(V/L)A(D/E)AV(K/Q)VTMGPKGR (T/H/N)V(V/I)(I/L)EXS, wherein X is any amino acid, preferably one of Q, S, R, K, G, most preferably Q.
    Motif 35 (SEQ ID NO: 600): KDGVTVAK(A/S)(I/V)(E/S/K/N)F
    Motif 36 (SEQ ID NO: 601): (KQ)(N/S)(I/V)GA(S/E/D/N)LVK(S/Q)VA(S/N/E/D)(S/A)TN
    Motif 37 (SEQ ID NO: 602): TT(C/A)AT(V/I)L(T/A)(R/K/Q)AI(F/L)XEGCK(S/A)V(A/S)AG (M/V)(N/S)AMDLR, wherein X is any amino acid, preferably one of V, A, T, S, I
    Motif 38 (SEQ ID NO: 603): TISANG
    Motif 39 (SEQ ID NO: 604): GK(E/H/D)GVIT(V/I)XDG(K/N)T(L/M), wherein X is any amino acid, preferably one of S, A, Q, T, V
    Motif 40 (SEQ ID NO: 605): RGY(I/T)SPYF(V/I)T(N/D)
    Motif 41 (SEQ ID NO: 606): PLLI(V/I)(A/S)ED(V/L/I)(E/D)
    Motif 42 (SEQ ID NO: 607): K(A/S)PGFG(E/D)(N/S)R
    Motif 43 (SEQ ID NO: 608, located in the C-terminal end of the protein sequence): GMGG


More preferably, the Glomalin polypeptide comprises in increasing order of preference, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, or all 13 motifs. Furthermore, the glomalin preferably has no secretion signal.
Additionally or alternatively, the homologue of a Glomalin 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: 546, provided that the homologous protein comprises any one or more of the conserved motifs 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 and preferably with sequences of mature proteins (i.e. without taking into account secretion signals or transit peptides). Compared to overall sequence identity, the sequence identity will generally be higher when only conserved domains or motifs are considered. Preferably the motifs in a Glomalin polypeptide have, in increasing order of preference, at least 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% sequence identity to the motifs represented by SEQ ID NO: 596 to SEQ ID NO: 608 (Motifs 31 to 43).
Preferably, the polypeptide sequence which when used in the construction of a phylogenetic tree, such as the one depicted in FIG. 19, clusters with the group of Glomalin polypeptides comprising the amino acid sequence represented by SEQ ID NO: 546 rather than with any other group.
The terms “domain”, “signature” and “motif” are 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)). Domains or motifs may also be identified using routine techniques, such as by sequence alignment.
Concerning PLATZ polypeptides, an alignment of the PLATZ-A1-α polypeptides given in Table A4 herein is shown in FIG. 11. Such alignments are useful for identifying the most conserved domains or motifs between the PLATZ polypeptides as defined herein. One such domain is the PLATZ domain, indicated in bold italics in FIG. 10. Examples of such motifs are SEQ ID NO: 264 to SEQ ID NO: 272 marked with the respective motif numbers in FIG. 10.
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., BMC Bioinformatics. 2003 Jul. 10; 4:29. 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 or amino acid 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).
Concerning PLATZ polypeptides, example 3 describes in Table B3 the percentage identity between various PLATZ polypeptides, in particular the percentage identity between SEQ ID NO: 261 (indicated as Pt583639) and other PLATZ-A1-α polypeptides, which can be as low as 70%. Typically, the percentage identity within the group of PLATZ-A1-α polypeptides is 55% or higher.
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, TMHMM, and others.
Concerning PLATZ polypeptides, PLATZ proteins, as DNA-binding proteins likely have a nuclear localisation.
Furthermore, eRF1 polypeptide (at least in their native form) typically have a regulation of floral meristem activity. Tools and techniques for measuring floral meristem activity are well known in the art.
In addition, eRF1 polypeptide, when expressed in rice according to the methods of the present invention as outlined in the Examples section, give plants having enhanced yield related traits, in particular seed yield and also biomass.
Additionally, eRF1 polypeptide may display a preferred subcellular localization, typically one or more of nuclear, cytoplasmic, chloroplastic, or mitochondrial. 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, TMHMM, and others.
Furthermore, SCAMP-like polypeptides (at least in their native form) typically have EH-protein binding activity. Tools and techniques for measuring protein binding activity to EH-proteins are well known in the art.
In addition, SCAMP-like polypeptides, when expressed in rice according to the methods of the present invention as outlined in the Example section, give plants having increased yield related traits, in particular increase seed and/or biomass yield in plants growing under nitrogen deficiency growth conditions, such as those described in the Examples Section.
Additionally, SCAMP-like polypeptides may display a preferred subcellular localization, typically one or more of nuclear, cytoplasmic, chloroplastic, or mitochondrial. 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, TMHMM, and others. Preferably the SCAMP polypeptide of the invention is preferably attached to a membrane, preferably to a membrane of the endoplamic reticulum.
On another preferred embodiment of the invention, the SCAMP polypeptide of the invention is attached to a membrane. Methods to attach (also referred to as anchored) polypeptides to a membrane or to increase the proportion of a polypeptide in a cell attached to a membrane are well known in the art. For example, a GPI (glycosyl-phosphatidylinositol) (GPI) moiety motif may be linked typically, covalently linked, to in increasing order of preference the N-teminus, the C-terminus, the Central part of a SCAMP polypeptide (Bertozzi et al. Biochemistry. 2008 Jul. 8; 47(27):6991-7000). Alternatively, the SCAMP polypeptide may be enriched in the Proline and or Alanine reisidues. Such residues are typically modified by sugars on a cell and subsequently sequester in mebranes, predominantly the plasma membrane (Kjellbom P, Snogerup L, Stöhr C, Reuzeau C, McCabe P F, Pennell R I. Plant J. 1997 November; 12(5):1189-96).
Fibrillin polypeptides, when expressed in rice according to the methods of the present invention as outlined in the Examples section herein, give plants having increased yield related traits.
Fibrillin polypeptides are typically display a preferred subcellular localization in the chloroplast. 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 labour-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, TMHMM, and others.
Furthermore, PLATZ polypeptides (at least in their native form) typically have zinc-dependent DNA-binding activity. Tools and techniques for measuring DNA-binding activity are well known in the art (see for example Nagano et al., 2001). Further details are provided in The Examples section.
In addition, PLATZ polypeptides, when expressed in rice according to the methods of the present invention as outlined in the Examples section, give plants having increased yield related traits, in particular increased seed yield, such as e.g. increased total weight of seeds and/or increased fill rate and/or increased thousand kernel weight; and/or increased harvest index; and/or increased above-ground biomass (also referred to as areamax herein) and/or quick early development, etc.
In addition, PLST-like polypeptide, when expressed in rice according to the methods of the present invention as outlined in the Examples section, give plants having enhanced yield related traits, in particular seed yield and also biomass.
Additionally, PLST-like polypeptide may display a preferred subcellular localization, typically one or more of nuclear, cytoplasmic, chloroplastic, or mitochondrial. 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, TMHMM, and others.
Furthermore, Glomalin polypeptides (at least in their native form) typically rescue temperature sensitive mutants (Hemmingsen et al., Nature 333, 330-334, 1988, Salvucci, J. Exp. Bot. 59, 1923-1933, 2008).
In addition, Glomalin polypeptides, when expressed in rice according to the methods of the present invention as outlined in the Examples section, give plants having increased yield related traits, in particular increased harvest index.
Concerning eRF1 polypeptides, the present invention is illustrated by transforming plants with the nucleic acid sequence represented by SEQ ID NO: 1, encoding the 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 eRF1 polypeptide encoding nucleic acid or eRF1 polypeptide as defined herein.
Examples of nucleic acids encoding eRF1 polypeptide are given in Table A1 of the Examples section herein. Such nucleic acids are useful in performing the methods of the invention. The amino acid sequences given in Table A1 of the Examples section are example sequences of orthologues and paralogues of the eRF1 polypeptide represented by SEQ ID NO: 2, 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 A1 of the Examples section) 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. 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.
Concerning SCAMP-like polypeptides, the present invention is illustrated by transforming plants with the nucleic acid sequence represented by SEQ ID NO: 88, encoding the polypeptide sequence of SEQ ID NO: 89. However, performance of the invention is not restricted to these sequences; the methods of the invention may advantageously be performed using any SCAMP-like-encoding nucleic acid or SCAMP-LIKE polypeptide as defined herein.
Examples of nucleic acids encoding SCAMP-like polypeptides are given in Table A2 of the Examples section herein. Such nucleic acids are useful in performing the methods of the invention. The amino acid sequences given in Table A2 of the Examples section are example sequences of orthologues and paralogues of the SCAMP-like polypeptide represented by SEQ ID NO: 89, 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 A2 of the Examples section) 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: 88 or SEQ ID NO: 89, 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.
Concerning fibrillin polypeptides, the present invention is illustrated by transforming plants with the nucleic acid sequence represented by SEQ ID NO: 204, encoding the polypeptide sequence of SEQ ID NO: 205. However, performance of the invention is not restricted to these sequences; the methods of the invention may advantageously be performed using any fibrillin-encoding nucleic acid or fibrillin polypeptide as defined herein.
Examples of nucleic acids encoding fibrillin polypeptides are given in Table A3 of the Examples section herein. Such nucleic acids are useful in performing the methods of the invention. The amino acid sequences given in Table A3 of the Examples section are example sequences of orthologues and paralogues of the fibrillin polypeptide represented by SEQ ID NO: 205, 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 A3 of the Examples section) 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: 204 or SEQ ID NO: 205, the second BLAST would therefore be against tomato 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.
Concerning PLATZ polypeptides, the present invention is illustrated by transforming plants with the nucleic acid sequence represented by SEQ ID NO: 260, encoding the polypeptide sequence of SEQ ID NO: 261. However, performance of the invention is not restricted to these sequences; the methods of the invention may advantageously be performed using any PLATZ-encoding nucleic acid or PLATZ polypeptide as defined herein. For example, in another embodiment the present invention is illustrated by transforming plants with the nucleic acid sequence represented by SEQ ID NO: 356, encoding the polypeptide sequence of SEQ ID NO: 357.
Examples of nucleic acids encoding PLATZ polypeptides are given in Table A4 of the Examples section herein. Such nucleic acids are useful in performing the methods of the invention. The amino acid sequences given in Table A4 of the Examples section are example sequences of orthologues and paralogues of the PLATZ polypeptide represented by SEQ ID NO: 261, 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 A4 of the Examples section) 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: 260 or SEQ ID NO: 261, the second BLAST would therefore be against poplar 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.
Concerning PLST-like polypeptides, the present invention is illustrated by transforming plants with the nucleic acid sequence represented by SEQ ID NO: 410, encoding the polypeptide sequence of SEQ ID NO: 411. However, performance of the invention is not restricted to these sequences; the methods of the invention may advantageously be performed using any PLST-like polypeptide encoding nucleic acid or PLST-like polypeptide as defined herein.
Examples of nucleic acids encoding PLST-like polypeptide are given in Table A5 of the Examples section herein. Such nucleic acids are useful in performing the methods of the invention. The amino acid sequences given in Table A5 of the Examples section are example sequences of orthologues and paralogues of the PLST-like polypeptide represented by SEQ ID NO: 411, 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 A5 of the Examples section) 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. 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.
Concerning Glomalin polypeptides, the present invention is illustrated by transforming plants with the nucleic acid sequence represented by SEQ ID NO: 545, encoding the polypeptide sequence of SEQ ID NO: 546. However, performance of the invention is not restricted to these sequences; the methods of the invention may advantageously be performed using any Glomalin-encoding nucleic acid or Glomalin polypeptide as defined herein.
Examples of nucleic acids encoding Glomalin polypeptides are given in Table A6 of the Examples section herein. Such nucleic acids are useful in performing the methods of the invention. The amino acid sequences given in Table A6 of the Examples section are example sequences of orthologues and paralogues of the Glomalin polypeptide represented by SEQ ID NO: 546, 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 as described in the definitions section; where the query sequence is SEQ ID NO: 545 or SEQ ID NO: 546, the second BLAST (back-BLAST) would be against rice sequences.
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 acids encoding homologues and derivatives of any one of the amino acid sequences given in Table A1 to A6 of the Examples section, the terms “homologue” and “derivative” being as defined herein. Also useful in the methods of the invention are nucleic acids encoding homologues and derivatives of orthologues or paralogues of any one of the amino acid sequences given in Table A1 to A6 of the Examples section. 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. Further variants useful in practising the methods of the invention are variants in which codon usage is optimised or in which miRNA target sites are removed.
Further nucleic acid variants useful in practising the methods of the invention include portions of nucleic acids encoding an eRF1 polypeptide, or a SCAMP-like polypeptide, or a fibrillin polypeptide, or a PLATZ polypeptide, or a PLST-like polypeptide, or a Glomalin polypeptide, nucleic acids hybridising to nucleic acids encoding an eRF1 polypeptide, or a SCAMP-like polypeptide, or a fibrillin polypeptide, or a PLATZ polypeptide, or a PLST-like polypeptide, or a Glomalin polypeptide, splice variants of nucleic acids encoding an eRF1 polypeptide, or a SCAMP-like polypeptide, or a fibrillin polypeptide, or a PLATZ polypeptide, or a PLST-like polypeptide, or a Glomalin polypeptide, allelic variants of nucleic acids encoding an eRF1 polypeptide, or a SCAMP-like polypeptide, or a fibrillin polypeptide, or a PLATZ polypeptide, or a PLST-like polypeptide, or a Glomalin polypeptide, and variants of nucleic acids encoding an eRF1 polypeptide, or a SCAMP-like polypeptide, or a fibrillin polypeptide, or a PLATZ polypeptide, or a PLST-like polypeptide, or a Glomalin polypeptide, obtained by gene shuffling. The terms hybridising sequence, splice variant, allelic variant and gene shuffling are as described herein.
Nucleic acids encoding an eRF1 polypeptide, or a SCAMP-like polypeptide, or a fibrillin polypeptide, or a PLATZ polypeptide, or a PLST-like polypeptide, or a Glomalin polypeptide, need not be full-length nucleic acids, 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 enhancing 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 A1 to A6 of the Examples section, or a portion of a nucleic acid encoding an orthologue, paralogue or homologue of any of the amino acid sequences given in Table A1 to A6 of the Examples section.
A portion of a nucleic acid may be prepared, for example, by making one or more deletions to the nucleic acid. The portions may be used in isolated form or they may be fused to other coding (or non-coding) sequences in order to, for example, produce a protein that combines several activities. When fused to other coding sequences, the resultant polypeptide produced upon translation may be bigger than that predicted for the protein portion.
Concerning eRF1 polypeptides, portions useful in the methods of the invention, encode an eRF1 polypeptide as defined herein, and have substantially the same biological activity as the amino acid sequences given in Table A1 of the Examples section. Preferably, the portion is a portion of any one of the nucleic acids given in Table A1 of the Examples section, or is a portion of a nucleic acid encoding an orthologue or paralogue of any one of the amino acid sequences given in Table A1 of the Examples section. Preferably the portion is at least 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350 consecutive nucleotides in length, the consecutive nucleotides being of any one of the nucleic acid sequences given in Table A1 of the Examples section, or of a nucleic acid encoding an orthologue or paralogue of any one of the amino acid sequences given in Table A1 of the Examples section. Most preferably the portion is a portion of the nucleic acid of SEQ ID NO: 1. 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. 1, clusters with the group of eRF1 polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 rather than with any other group.
Concerning SCAMP-like polypeptides, portions useful in the methods of the invention, encode a SCAMP-like polypeptide as defined herein, and have substantially the same biological activity as the amino acid sequences given in Table A2 of the Examples section. Preferably, the portion is a portion of any one of the nucleic acids given in Table A2 of the Examples section, 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 the Examples section. Preferably the portion is at least 100, 200, 300, 400, 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 A2 of the Examples section, or of a nucleic acid encoding an orthologue or paralogue of any one of the amino acid sequences given in Table A2 of the Examples section. Most preferably the portion is a portion of the nucleic acid of SEQ ID NO: 88. Preferably, the portion encodes a fragment of an amino acid sequence comprising a SCAMP domain as defined herein.
Concerning fibrillin polypeptides, portions useful in the methods of the invention, encode a fibrillin polypeptide as defined herein, and have substantially the same biological activity as the amino acid sequences given in Table A3 of the Examples section. Preferably, the portion is a portion of any one of the nucleic acids given in Table A3 of the Examples section, 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 the Examples section. 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 A3 of the Examples section, or of a nucleic acid encoding an orthologue or paralogue of any one of the amino acid sequences given in Table A3 of the Examples section. Most preferably the portion is a portion of the nucleic acid of SEQ ID NO: 204. 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. 6, clusters with the group of fibrillin polypeptides comprising the amino acid sequence represented by SEQ ID NO: 205 rather than with any other group.
Concerning PLATZ polypeptides, portions useful in the methods of the invention, encode a PLATZ polypeptide as defined herein, and have substantially the same biological activity as the amino acid sequences given in Table A4 of the Examples section. Preferably, the portion is a portion of any one of the nucleic acids given in Table A4 of the Examples section, 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 the Examples section. Preferably the portion is at least 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100 consecutive nucleotides in length, the consecutive nucleotides being of any one of the nucleic acid sequences given in Table A4 of the Examples section, or of a nucleic acid encoding an orthologue or paralogue of any one of the amino acid sequences given in Table A4 of the Examples section. Most preferably the portion is a portion of the nucleic acid of SEQ ID NO: 260. Preferably, the portion encodes a fragment of an amino acid sequence which comprises a PLATZ domain and one or more of the motifs defined above, and which, when used in the construction of a phylogenetic tree, such as the one depicted in FIG. 12, clusters with the group of PLATZ-A polypeptides, more preferably with the group of PLATZ-A1 proteins, most preferably with the group of PLATZ-A1-α proteins, comprising the amino acid sequence represented by SEQ ID NO: 261 rather than with any other group of PLATZ proteins.
Concerning PLST-like polypeptides, portions useful in the methods of the invention, encode a PLST-like polypeptide as defined herein, and have substantially the same biological activity as the amino acid sequences given in Table A5 of the Examples section. Preferably, the portion is a portion of any one of the nucleic acids given in Table A5 of the Examples section, 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 the Examples section. Preferably the portion is at least 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700 consecutive nucleotides in length, the consecutive nucleotides being of any one of the nucleic acid sequences given in Table A5 of the Examples section, or of a nucleic acid encoding an orthologue or paralogue of any one of the amino acid sequences given in Table A5 of the Examples section. Most preferably the portion has one or more of motifs 1 to 12 and the PF domain and is a portion of the nucleic acid of SEQ ID NO: 410. 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. 19, clusters with the group of PLST-like polypeptide comprising the amino acid sequence represented by SEQ ID NO: 411 rather than with any other group.
Concerning Glomalin polypeptides, portions useful in the methods of the invention, encode a Glomalin polypeptide as defined herein, and have substantially the same biological activity as the amino acid sequences given in Table A6 of the Examples section. Preferably, the portion is a portion of any one of the nucleic acids given in Table A6 of the Examples section, or is a portion of a nucleic acid encoding an orthologue or paralogue of any one of the amino acid sequences given in Table A6 of the Examples section. Preferably the portion is at least 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900 consecutive nucleotides in length, the consecutive nucleotides being of any one of the nucleic acid sequences given in Table A6 of the Examples section, or of a nucleic acid encoding an orthologue or paralogue of any one of the amino acid sequences given in Table A6 of the Examples section. Most preferably the portion is a portion of the nucleic acid of SEQ ID NO: 545. Preferably, the portion encodes a fragment of an amino acid sequence which comprises a Cpn60_TCP1 domain (Pfam accession PF00118), or which when used in the construction of a phylogenetic tree, such as the one depicted in FIG. 19, clusters with the group of Glomalin polypeptides comprising the amino acid sequence represented by SEQ ID NO: 546 rather than with any other group.
Another nucleic acid variant useful in the methods of the invention is a nucleic acid capable of hybridising, under reduced stringency conditions, preferably under stringent conditions, with a nucleic acid encoding an eRF1 polypeptide, or a SCAMP-like polypeptide, or a fibrillin polypeptide, or a PLATZ polypeptide, or a PLST-like polypeptide, or a Glomalin polypeptide, as defined herein, or with a portion as defined herein.
According to the present invention, there is provided a method for enhancing yield-related traits in plants, comprising introducing and expressing in a plant a nucleic acid capable of hybridizing to any one of the nucleic acids given in Table A1 to A6 of the Examples section, or comprising introducing and expressing in a plant a nucleic acid capable of hybridising to a nucleic acid encoding an orthologue, paralogue or homologue of any of the nucleic acid sequences given in Table A1 to A6 of the Examples section.
Concerning eRF1 polypeptides, hybridising sequences useful in the methods of the invention encode an eRF1 polypeptide as defined herein, having substantially the same biological activity as the amino acid sequences given in Table A1 of the Examples section. Preferably, the hybridising sequence is capable of hybridising to the complement of any one of the nucleic acids given in Table A1 of the Examples section, or to a portion of any of these sequences, a portion being as defined above, or the hybridising sequence is capable of hybridising to the complement of a nucleic acid encoding an orthologue or paralogue of any one of the amino acid sequences given in Table A1 of the Examples section. Most preferably, the hybridising sequence is capable of hybridising to the complement of a nucleic acid as represented by SEQ ID NO: 1 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. 1, clusters with the group of eRF1 polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 rather than with any other group.
Concerning SCAMP-like polypeptides, hybridising sequences useful in the methods of the invention encode a SCAMP-like polypeptide as defined herein, having substantially the same biological activity as the amino acid sequences given in Table A2 of the Examples section. Preferably, the hybridising sequence is capable of hybridising to the complement of any one of the nucleic acids given in Table A2 of the Examples section, or to a portion of any of these sequences, a portion being as defined above, or the hybridising sequence is capable of hybridising to the complement of a nucleic acid encoding an orthologue or paralogue of any one of the amino acid sequences given in Table A2 of the Examples section. Most preferably, the hybridising sequence is capable of hybridising to the complement of a nucleic acid as represented by SEQ ID NO: 88 or to a portion thereof.
Preferably, the hybridising sequence encodes a polypeptide with an amino acid sequence comprising a SCAMP domain as defined herein.
Concerning fibrillin polypeptides, hybridising sequences useful in the methods of the invention encode a fibrillin polypeptide as defined herein, having substantially the same biological activity as the amino acid sequences given in Table A3 of the Examples section. Preferably, the hybridising sequence is capable of hybridising to the complement of any one of the nucleic acids given in Table A3 of the Examples section, or to a portion of any of these sequences, a portion being as defined above, or the hybridising sequence is capable of hybridising to the complement of a nucleic acid encoding an orthologue or paralogue of any one of the amino acid sequences given in Table A3 of the Examples section. Most preferably, the hybridising sequence is capable of hybridising to the complement of a nucleic acid as represented by SEQ ID NO: 204 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. 6, clusters with the group of fibrillin polypeptides comprising the amino acid sequence represented by SEQ ID NO: 205 rather than with any other group.
Concerning PLATZ polypeptides, hybridising sequences useful in the methods of the invention encode a PLATZ polypeptide as defined herein, having substantially the same biological activity as the amino acid sequences given in Table A4 of the Examples section. Preferably, the hybridising sequence is capable of hybridising to the complement of any one of the nucleic acids given in Table A4 of the Examples section, or to a portion of any of these sequences, a portion being as defined above, or the hybridising sequence is capable of hybridising to the complement of a nucleic acid encoding an orthologue or paralogue of any one of the amino acid sequences given in Table A4 of the Examples section. Most preferably, the hybridising sequence is capable of hybridising to the complement of a nucleic acid as represented by SEQ ID NO: 260 or to a portion thereof.
Preferably, the hybridising sequence encodes a polypeptide with an amino acid sequence which comprises a PLATZ domain and one or more of the motifs defined above, and which, when used in the construction of a phylogenetic tree, such as the one depicted in FIG. 12, clusters with the group of PLATZ-A polypeptides, more preferably with the group of PLATZ-A1 proteins, most preferably with the group of PLATZ-A1-α proteins, comprising the amino acid sequence represented by SEQ ID NO: 261 rather than with any other group of PLATZ proteins.
Concerning PLST-like polypeptides, hybridising sequences useful in the methods of the invention encode a PLST-like polypeptide as defined herein, having substantially the same biological activity as the amino acid sequences given in Table A5 of the Examples section. Preferably, the hybridising sequence is capable of hybridising to the complement of any one of the nucleic acids given in Table A5 of the Examples section, or to a portion of any of these sequences, a portion being as defined above, or the hybridising sequence is capable of hybridising to the complement of a nucleic acid encoding an orthologue or paralogue of any one of the amino acid sequences given in Table A5 of the Examples section. Most preferably, the hybridising sequence is capable of hybridising to the complement of a nucleic acid as represented by SEQ ID NO: 410 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. 15, has one or more of motifs 1 to 12 and the PF domain and clusters with the group of a PLST-like polypeptide comprising the amino acid sequence represented by SEQ ID NO: 411 rather than with any other group.
Concerning Glomalin polypeptides, hybridising sequences useful in the methods of the invention encode a Glomalin polypeptide as defined herein, having substantially the same biological activity as the amino acid sequences given in Table A6 of the Examples section. Preferably, the hybridising sequence is capable of hybridising to the complement of any one of the nucleic acids given in Table A6 of the Examples section, or to a portion of any of these sequences, a portion being as defined above, or the hybridising sequence is capable of hybridising to the complement of a nucleic acid encoding an orthologue or paralogue of any one of the amino acid sequences given in Table A6 of the Examples section. Most preferably, the hybridising sequence is capable of hybridising to the complement of a nucleic acid as represented by SEQ ID NO: 545 or to a portion thereof.
Preferably, the hybridising sequence encodes a polypeptide with an amino acid sequence which comprises a Cpn60_TCP1 domain (Pfam accession PF00118), or which when used in the construction of a phylogenetic tree, such as the one depicted in FIG. 19, clusters with the group of Glomalin polypeptides comprising the amino acid sequence represented by SEQ ID NO: 546 rather than with any other group.
Another nucleic acid variant useful in the methods of the invention is a splice variant encoding an eRF1 polypeptide, or a SCAMP-like polypeptide, or a fibrillin polypeptide, or a PLATZ polypeptide, or a PLST-like polypeptide, or a Glomalin polypeptide, as defined hereinabove, a splice variant being as defined herein.
According to the present invention, there is provided a method for enhancing yield-related traits in plants, comprising introducing and expressing in a plant a splice variant of any one of the nucleic acid sequences given in Table A1 to A6 of the Examples section, or a splice variant of a nucleic acid encoding an orthologue, paralogue or homologue of any of the amino acid sequences given in Table A1 to A6 of the Examples section.
Concerning eRF1 polypeptides, preferred splice variants are splice variants of a nucleic acid represented by SEQ ID NO: 1, or a splice variant of a nucleic acid encoding an orthologue or paralogue of SEQ ID NO: 2. 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. 1, clusters with the group of eRF1 polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 rather than with any other group.
Concerning SCAMP-like polypeptides, preferred splice variants are splice variants of a nucleic acid represented by SEQ ID NO: 88, or a splice variant of a nucleic acid encoding an orthologue or paralogue of SEQ ID NO: 89. Preferably, the amino acid sequence encoded by the splice variant comprises a SCAMP domain as defined herein.
Concerning fibrillin polypeptides, preferred splice variants are splice variants of a nucleic acid represented by SEQ ID NO: 204, or a splice variant of a nucleic acid encoding an orthologue or paralogue of SEQ ID NO: 205. 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. 6, clusters with the group of fibrillin polypeptides comprising the amino acid sequence represented by SEQ ID NO: 205 rather than with any other group.
Concerning PLATZ polypeptides, preferred splice variants are splice variants of a nucleic acid represented by SEQ ID NO: 260, or a splice variant of a nucleic acid encoding an orthologue or paralogue of SEQ ID NO: 261. Preferably, the amino acid sequence encoded by the splice variant which comprises a PLATZ domain and one or more of the motifs defined above, and which, when used in the construction of a phylogenetic tree, such as the one depicted in FIG. 12, clusters with the group of PLATZ-A polypeptides, more preferably with the group of PLATZ-A1 proteins, most preferably with the group of PLATZ-A1-α proteins, comprising the amino acid sequence represented by SEQ ID NO: 261 rather than with any other group of PLATZ proteins.
Concerning PLST-like polypeptides, preferred splice variants are splice variants of a nucleic acid represented by SEQ ID NO: 410, or a splice variant of a nucleic acid encoding an orthologue or paralogue of SEQ ID NO: 411. 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. 15, has one or more of motifs 19 to 30 and the PF domain and clusters with the group of a PLST-like polypeptide comprising the amino acid sequence represented by SEQ ID NO: 411 rather than with any other group.
Concerning Glomalin polypeptides, preferred splice variants are splice variants of a nucleic acid represented by SEQ ID NO: 545, or a splice variant of a nucleic acid encoding an orthologue or paralogue of SEQ ID NO: 546. Preferably, the amino acid sequence encoded by the splice variant comprises a Cpn60_TCP1 domain (Pfam accession PF00118), or when used in the construction of a phylogenetic tree, such as the one depicted in FIG. 19, clusters with the group of Glomalin polypeptides comprising the amino acid sequence represented by SEQ ID NO: 546 rather than with any other group.
Another nucleic acid variant useful in performing the methods of the invention is an allelic variant of a nucleic acid encoding an eRF1 polypeptide, or a SCAMP-like polypeptide, or a fibrillin polypeptide, or a PLATZ polypeptide, or a PLST-like polypeptide, or a Glomalin polypeptide, as defined hereinabove, an allelic variant being as defined herein.
According to the present invention, there is provided a method for enhancing yield-related traits in plants, comprising introducing and expressing in a plant an allelic variant of any one of the nucleic acids given in Table A1 to A6 of the Examples section, or comprising introducing and expressing in a plant an allelic variant of a nucleic acid encoding an orthologue, paralogue or homologue of any of the amino acid sequences given in Table A1 to A6 of the Examples section.
Concerning eRF1 polypeptides, the polypeptides encoded by allelic variants useful in the methods of the present invention have substantially the same biological activity as the eRF1 polypeptide of SEQ ID NO: 2 and any of the amino acids depicted in Table A1 of the Examples section. 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 encoding an orthologue or paralogue of SEQ ID NO: 2. 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. 1, clusters with the eRF1 polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 rather than with any other group.
Concerning SCAMP-like polypeptides, the polypeptides encoded by allelic variants useful in the methods of the present invention have substantially the same biological activity as the SCAMP-like polypeptide of SEQ ID NO: 89 and any of the amino acids depicted in Table A2 of the Examples section. 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: 88 or an allelic variant of a nucleic acid encoding an orthologue or paralogue of SEQ ID NO: 89. Preferably, the amino acid sequence encoded by the allelic variant comprises a SCAMP domain as defined herein.
Concerning fibrillin polypeptides, the polypeptides encoded by allelic variants useful in the methods of the present invention have substantially the same biological activity as the fibrillin polypeptide of SEQ ID NO: 205 and any of the amino acids depicted in Table A3 of the Examples section. 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: 204 or an allelic variant of a nucleic acid encoding an orthologue or paralogue of SEQ ID NO: 205. 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. 6, clusters with the fibrillin polypeptides comprising the amino acid sequence represented by SEQ ID NO: 205 rather than with any other group.
Concerning PLATZ polypeptides, the polypeptides encoded by allelic variants useful in the methods of the present invention have substantially the same biological activity as the PLATZ polypeptide of SEQ ID NO: 261 and any of the amino acids depicted in Table A4 of the Examples section. 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: 260 or an allelic variant of a nucleic acid encoding an orthologue or paralogue of SEQ ID NO: 261. Preferably, the amino acid sequence encoded by the allelic variant, which comprises a PLATZ domain and one or more of the motifs defined above, and which, when used in the construction of a phylogenetic tree, such as the one depicted in FIG. 12, clusters with the group of PLATZ-A polypeptides, more preferably with the group of PLATZ-A1 proteins, most preferably with the group of PLATZ-A1-α proteins, comprising the amino acid sequence represented by SEQ ID NO: 261 rather than with any other group of PLATZ proteins.
Concerning PLST-like polypeptides, the polypeptides encoded by allelic variants useful in the methods of the present invention have substantially the same biological activity as the PLST-like polypeptide of SEQ ID NO: 411 and any of the amino acids depicted in Table A5 of the Examples section. 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: 410 or an allelic variant of a nucleic acid encoding an orthologue or paralogue of SEQ ID NO: 411. 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. 14, has one or more of motifs 19 to 30 and the PF domain and clusters with the PLST-like polypeptide comprising the amino acid sequence represented by SEQ ID NO: 411 rather than with any other group.
Concerning Glomalin polypeptides, the polypeptides encoded by allelic variants useful in the methods of the present invention have substantially the same biological activity as the Glomalin polypeptide of SEQ ID NO: 546 and any of the amino acids depicted in Table A6 of the Examples section. 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: 545 or an allelic variant of a nucleic acid encoding an orthologue or paralogue of SEQ ID NO: 546. Preferably, the amino acid sequence encoded by the allelic variant comprises a Cpn60_TCP1 domain (Pfam accession PF00118), or when used in the construction of a phylogenetic tree, such as the one depicted in FIG. 19, clusters with the group of Glomalin polypeptides comprising the amino acid sequence represented by SEQ ID NO: 546 rather than with any other group.
Gene shuffling or directed evolution may also be used to generate variants of nucleic acids encoding an eRF1 polypeptide, or a SCAMP-like polypeptide, or a fibrillin polypeptide, or a PLATZ polypeptide, or a PLST-like polypeptide, or a Glomalin polypeptide, as defined above; the term “gene shuffling” being as defined herein.
According to the present invention, there is provided a method for enhancing yield-related traits in plants, comprising introducing and expressing in a plant a variant of any one of the nucleic acid sequences given in Table A1 to A6 of the Examples section, or comprising introducing and expressing in a plant a variant of a nucleic acid encoding an orthologue, paralogue or homologue of any of the amino acid sequences given in Table A1 to A6 of the Examples section, which variant nucleic acid is obtained by gene shuffling.
Concerning eRF1 polypeptides, preferably, 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. 1, clusters with the group of eRF1 polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 rather than with any other group.
Concerning SCAMP-like polypeptides, preferably, the amino acid sequence encoded by the variant nucleic acid obtained by gene shuffling comprises a SCAMP domain as defined herein.
Concerning fibrillin polypeptides, preferably, 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. 6, clusters with the group of fibrillin polypeptides comprising the amino acid sequence represented by SEQ ID NO: 205 rather than with any other group.
Concerning PLATZ polypeptides, preferably, the amino acid sequence encoded by the variant nucleic acid obtained by gene shuffling, which comprises a PLATZ domain and one or more of the motifs defined above, and which, when used in the construction of a phylogenetic tree, such as the one depicted in FIG. 12, clusters with the group of PLATZ-A polypeptides, more preferably with the group of PLATZ-A1 proteins, most preferably with the group of PLATZ-A1-α proteins, comprising the amino acid sequence represented by SEQ ID NO: 261 rather than with any other group of PLATZ proteins.
Concerning PLST-like polypeptides, preferably, 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. 15, clusters with the group of PLST-like polypeptide comprising the amino acid sequence represented by SEQ ID NO: 411 rather than with any other group.
Concerning Glomalin polypeptides, preferably, the amino acid sequence encoded by the variant nucleic acid obtained by gene shuffling comprises a Cpn60_TCP1 domain (Pfam accession PF00118), or when used in the construction of a phylogenetic tree, such as the one depicted in FIG. 19, clusters with the group of Glomalin polypeptides comprising the amino acid sequence represented by SEQ ID NO: 546 rather than with any other group.
Furthermore, nucleic acid 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 acids encoding eRF1 polypeptide 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 eRF1 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 SCAMP-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 SCAMP-LIKE polypeptide-encoding nucleic acid is from a plant, further preferably from a dicotyledonous plant, more preferably from the family Brasicaceae, most preferably the nucleic acid is from Arabidospis thaliana. 
Any of the aforementioned fibrillin polypeptide sequences may be targeted to a plastid. A preferred plastid is a chloroplast. Methods for targeting to plastids are well known in the art and include, but are not limited to, the use of transit peptides. Table 3 below shows examples of transit peptides suitable for targeting any fibrillin polypeptide to a plastid. The fibrillin polypeptide may not, in its natural form, be targeted to a plastid or may be targeted to a different plastid. Furthermore, the fibrillin may, in its natural form, be targeted to a plastid through a different transit peptide (for example, its natural transit peptide).







TABLE 3







Examples of transit peptide sequences useful in targeting


amino acids to plastids










NCBI Accession





Number/SEQ ID





NO
Source Organism
Protein Function
Transit Peptide Sequence





SEQ ID NO: 668
Chlamydomonas
Ferredoxin
MAMAMRSTFAARVGAKPAVR


P07839


GARPASRMSCMA


 


SEQ ID NO: 669
Chlamydomonas
Rubisco activase
MQVTMKSSAVSGQRVGGARV


AAR23425


ATRSVRRAQLQV


 


SEQ ID NO: 670
Arabidopsis thaliana
asp Amino
MASLMLSLGSTSLLPREINKDK


CAA56932

transferase
LKLGTSASNPFLKAKSFSRVT





MTVAVKPSR


 


SEQ ID NO: 671
Arabidopsis thaliana
Acyl carrier
MATQFSASVSLQTSCLATTRIS


CAA31991

protein1
FQKPALISNHGKTNLSFNLRR





SIPSRRLSVSC


 


SEQ ID NO: 672
Arabidopsis thaliana
Acyl carrier
MASIAASASISLQARPRQLAIA


CAB63798

protein2
ASQVKSFSNGRRSSLSFNLRQ





LPTRLTVSCAAKPETVDKVCA





VVRKQL


 


SEQ ID NO: 673
Arabidopsis thaliana
Acyl carrier
MASIATSASTSLQARPRQLVIG


CAB63799

protein3
AKQVKSFSYGSRSNLSFNLRQ





LPTRLTVYCAAKPETVDKVCA





VVRKQLSLKE









Nucleic acids encoding fibrillin 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 fibrillin polypeptide-encoding nucleic acid is from a plant, further preferably from a dicotyledonous plant, more preferably from the family Solanaceae, further preferably the nucleic acid is from the genus Lycopersicon, further preferably from the species Lycopersicum, most preferably from Lycopersicon esculentum. 
Nucleic acids encoding PLATZ 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 PLATZ polypeptide-encoding nucleic acid is from a plant, further preferably from a dicotyledonous plant, more preferably from the family Salicaceae, most preferably the nucleic acid is from Populus sp.
Nucleic acids encoding PLST-like polypeptide 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 PLST-like polypeptide-encoding nucleic acid is from a plant, further preferably from a dicocotyledonous plant, more preferably from the family Salicaceae, most preferably the nucleic acid is from Populus trichocarpa. 
Nucleic acids encoding Glomalin 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 Glomalin 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 plants having enhanced yield-related traits. In particular performance of the methods of the invention gives plants having enhanced 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.
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 particular, such harvestable parts are biomass and/or seeds, and performance of the methods of the invention results in plants having increased biomass and/or increased seed yield relative to the seed yield of 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 square meter, 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 square meter, 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, especially biomass and/or seed yield of plants, relative to control plants, which method comprises modulating expression in a plant and/or plant plastid of a nucleic acid encoding an eRF1 polypeptide, or a SCAMP-like polypeptide, or a fibrillin polypeptide, or a PLATZ polypeptide, or a PLST-like polypeptide, or a Glomalin polypeptide, as defined herein.
Since the transgenic plants according to the present invention have enhanced yield characteristics and/or yield, it is likely that these plants exhibit an increased growth rate (during at least part of their life cycle), relative to the growth rate of control plants at a corresponding stage in their life cycle.
The increased growth rate may be specific to one or more parts of a plant (including seeds), or may be throughout substantially the whole plant. 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 speed of germination, 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 enhanced vigour. The increase in growth rate may alter the harvest cycle of a plant allowing plants to be sown later and/or harvested sooner than would otherwise be possible (a similar effect may be obtained with earlier flowering time). 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 square meter (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 modulating expression in a plant of a nucleic acid encoding an eRF1 polypeptide as defined herein.
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. Mild stress in the sense of the invention leads to a reduction in the growth of the stressed plants of less than 40%, 35%, 30% or 25%, more preferably less than 20% or 15% 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. 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. The term non-stress conditions as used herein, encompasses the occasional or everyday mild stresses to which a plant is exposed, as defined herein, but does not encompass severe stresses.
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 enhanced yield characteristics 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. Plants with optimal growth conditions, (grown under non-stress conditions) typically yield at least 97%, 95%, 92%, 90%, 87%, 85%, 83%, 80%, 77% or 75% of the average production of such plant in a given environment. Average production may be calculated on harvest and/or season basis. Persons skilled in the art are aware of average yield productions of a crop.
Performance of the methods of the invention gives plants grown under non-stress conditions or under mild drought conditions enhanced yield characteristics and/or 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 and/or plant plastid of a nucleic acid encoding an eRF1 polypeptide, or a SCAMP-like polypeptide, or a fibrillin polypeptide, or a PLATZ polypeptide, or a PLST-like polypeptide, or a Glomalin polypeptide.
Performance of the methods of the invention gives plants grown under conditions of nutrient deficiency, particularly under conditions of nitrogen deficiency, enhanced yield characteristics 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 eRF1 polypeptide, or a SCAMP-like polypeptide, or a fibrillin polypeptide, or a PLST-like polypeptide, or a Glomalin polypeptide. Nutrient deficiency may result from a lack of nutrients such as nitrogen, phosphates and other phosphorous-containing compounds, potassium, calcium, magnesium, manganese, iron and boron, amongst others.
Concerning PLATZ polypeptides, 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. 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. Thus, 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 PLATZ polypeptide. Nutrient deficiency may result from a lack of nutrients such as nitrogen, phosphates and other phosphorous-containing compounds, potassium, calcium, magnesium, manganese, iron and boron, amongst others.
Performance of the methods of the invention gives plants grown under conditions of salt stress, enhanced yield characteristics 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 salt stress, which method comprises modulating expression in a plant of a nucleic acid encoding an eRF1 polypeptide, or a SCAMP-like polypeptide, or a fibrillin polypeptide, or a PLATZ polypeptide, or a PLST-like polypeptide, or a Glomalin polypeptide. The term salt stress is not restricted to common salt (NaCl), but may be any one or more of: NaCl, KCl, LiCl, MgCl2, CaCl2, amongst others.
The present invention encompasses plants or parts thereof (including seeds) obtainable by the methods according to the present invention. The plants or parts thereof comprise a nucleic acid transgene encoding an eRF1 polypeptide, or a SCAMP-like polypeptide, or a fibrillin polypeptide, or a PLATZ polypeptide, or a PLST-like polypeptide, or a Glomalin polypeptide, as defined above.
The invention also provides genetic constructs and vectors to facilitate introduction and/or expression in plants of nucleic acids encoding an eRF1 polypeptide, or a SCAMP-like polypeptide, or a fibrillin polypeptide, or a PLATZ polypeptide, or a PLST-like polypeptide, or a Glomalin polypeptide. The gene constructs may be inserted into vectors, which may be commercially available, suitable for transforming into plants and suitable for expression of the gene of interest in the transformed cells. The invention also provides use of a gene construct as defined herein in the methods of the invention.
More specifically, the present invention provides a construct comprising:

    
    
        (a) a nucleic acid encoding an eRF1 polypeptide, or a SCAMP-like polypeptide, or a fibrillin polypeptide, or a PLATZ polypeptide, or a PLST-like polypeptide, or a Glomalin polypeptide, as defined above;
        (b) one or more control sequences capable of driving expression of the nucleic acid sequence of (a); and optionally
        (c) a transcription termination sequence.
    
    


Preferably, the nucleic acid encoding an eRF1 polypeptide, or a SCAMP-like polypeptide, or a fibrillin polypeptide, or a PLATZ polypeptide, or a PLST-like polypeptide, or a Glomalin 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 acids 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 drive expression of the nucleic acid sequence, but preferably the promoter is of plant origin. A constitutive promoter is particularly useful in the methods. Preferably the constitutive promoter is also a ubiquitous promoter of medium strength. See the “Definitions” section herein for definitions of the various promoter types. Concerning eRF1 polypeptides and/or PLST-like polypeptides, also useful in the methods of the invention is a root-specific promoter.
Concerning Glomalin polypeptides, advantageously, any type of promoter, whether natural or synthetic, may be used to drive expression of the nucleic acid sequence, but preferably the promoter is of plant origin. See the “Definitions” section herein for definitions of the various promoter types. A root-specific promoter is particularly useful in the methods. The root-specific promoter is preferably an RCc3 promoter (Plant Mol Biol. 1995 January;27(2):237-48), more preferably the RCc3 promoter is from rice, further preferably the RCc3 promoter is represented by a nucleic acid sequence substantially similar to SEQ ID NO: 611, most preferably the promoter is as represented by SEQ ID NO: 611. Examples of other root-specific promoters which may also be used to perform the methods of the invention are shown in Table 2b in the “Definitions” section above.
Also useful in the methods of the invention is a constitutive promoter; preferably the constitutive promoter is a ubiquitous constitutive promoter of medium strength such as a GOS2 promoter, more preferably the promoter is the GOS2 promoter from rice. Further preferably the constitutive promoter is represented by a nucleic acid sequence substantially similar to SEQ ID NO: 612, most preferably the constitutive promoter is as represented by SEQ ID NO: 612. See the “Definitions” section herein for further examples of constitutive promoters.
Concerning eRF1 polypeptides, it should be clear that the applicability of the present invention is not restricted to the eRF1 polypeptide-encoding nucleic acid represented by SEQ ID NO: 1, nor is the applicability of the invention restricted to expression of an eRF1 polypeptide-encoding nucleic acid when driven by a constitutive promoter, or when driven by a root-specific promoter.
The constitutive promoter is preferably a medium strength promoter, more preferably selected from a plant derived promoter, such as a GOS2 promoter, more preferably is the promoter GOS2 promoter from rice. Further preferably the constitutive promoter is represented by a nucleic acid sequence substantially similar to SEQ ID NO: 85, most preferably the constitutive promoter is as represented by SEQ ID NO: 85. See 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. Preferably, the construct comprises an expression cassette comprising a GOS2 promoter, substantially similar to SEQ ID NO: 85, and the nucleic acid encoding the eRF1 polypeptide.
Concerning SCAMP-like polypeptides, it should be clear that the applicability of the present invention is not restricted to the SCAMP-like polypeptide-encoding nucleic acid represented by SEQ ID NO: 88, nor is the applicability of the invention restricted to expression of a SCAMP-like polypeptide-encoding nucleic acid when driven by a constitutive promoter.
The constitutive promoter is preferably a medium strength promoter, more preferably selected from a plant derived promoter, such as a GOS2 promoter, more preferably is the promoter GOS2 promoter from rice. Further preferably the constitutive promoter is represented by a nucleic acid sequence substantially similar to SEQ ID NO: 203, most preferably the constitutive promoter is as represented by SEQ ID NO: 203. See 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. Preferably, the construct comprises an expression cassette comprising a GOS2 promoter, substantially similar to SEQ ID NO: 203, and the nucleic acid encoding the SCAMP-LIKE polypeptide.
Concerning fibrillin polypeptides, it should be clear that the applicability of the present invention is not restricted to the fibrillin polypeptide-encoding nucleic acid represented by SEQ ID NO: 204, nor is the applicability of the invention restricted to expression of a fibrillin polypeptide-encoding nucleic acid when driven by a constitutive promoter.
The constitutive promoter is preferably a medium strength promoter, more preferably selected from a plant derived promoter, such as a GOS2 promoter, more preferably is the promoter GOS2 promoter from rice. Further preferably the constitutive promoter is represented by a nucleic acid sequence substantially similar to SEQ ID NO: 257, most preferably the constitutive promoter is as represented by SEQ ID NO: 257. See 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. Preferably, the construct comprises an expression cassette comprising a GOS2 promoter, substantially similar to SEQ ID NO: 257, and the nucleic acid encoding a fibrillin polypeptide.
Concerning PLATZ polypeptides, it should be clear that the applicability of the present invention is not restricted to the PLATZ polypeptide-encoding nucleic acid represented by SEQ ID NO: 260, nor is the applicability of the invention restricted to expression of a PLATZ polypeptide-encoding nucleic acid when driven by a constitutive promoter.
The constitutive promoter is preferably selected from a plant, such as a GOS2 promoter; more preferably the promoter is a GOS2 promoter from rice. Further preferably the constitutive promoter is represented by a nucleic acid sequence substantially similar to SEQ ID NO: 273, most preferably the constitutive promoter is as represented by SEQ ID NO: 273. See 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. Preferably, the construct comprises an expression cassette comprising a GOS2 promoter, substantially similar to SEQ ID NO: 273, and the nucleic acid encoding the PLATZ polypeptide.
Concerning PLST-like polypeptides, it should be clear that the applicability of the present invention is not restricted to the PLST-like polypeptide-encoding nucleic acid represented by SEQ ID NO: 410, nor is the applicability of the invention restricted to expression of a PLST-like polypeptide-encoding nucleic acid when driven by a constitutive promoter.
The constitutive promoter is preferably a medium strength promoter, more preferably selected from a plant derived promoter, such as a GOS2 promoter, more preferably is the promoter GOS2 promoter from rice. Further preferably the constitutive promoter is represented by a nucleic acid sequence substantially similar to SEQ ID NO: 542, most preferably the constitutive promoter is as represented by SEQ ID NO: 542. See 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. Preferably, the construct comprises an expression cassette comprising a GOS2 promoter, substantially similar to SEQ ID NO: 542, and the nucleic acid encoding the PLST-like polypeptide.
Concerning Glomalin polypeptides, it should be clear that the applicability of the present invention is not restricted to the Glomalin polypeptide-encoding nucleic acid represented by SEQ ID NO: 545, nor is the applicability of the invention restricted to expression of a Glomalin polypeptide-encoding nucleic acid when driven by a root-specific promoter, or when driven by a constitutive promoter.
Optionally, one or more terminator sequences may be used in the construct introduced into a plant. Preferably, the construct comprises an expression cassette comprising a RCc3 promoter, substantially similar to SEQ ID NO: 611, and the nucleic acid encoding the Glomalin polypeptide.
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.
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-ori 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 acids, 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.
The invention also provides a method for the production of transgenic plants having enhanced yield-related traits relative to control plants, comprising introduction and expression in a plant and/or a plant plastid of any nucleic acid encoding an eRF1 polypeptide, or a SCAMP-like polypeptide, or a fibrillin polypeptide, or a PLATZ polypeptide, or a PLST-like polypeptide, or a Glomalin polypeptide, as defined hereinabove.
More specifically, the present invention provides a method for the production of transgenic plants having enhanced yield-related traits, particularly increased seed yield and also biomass, which method comprises:

    
    
        (i) introducing and expressing in a plant or plant cell a nucleic acid encoding an eRF1 polypeptide, or a SCAMP-like polypeptide, or a fibrillin polypeptide, or a PLATZ polypeptide, or a PLST-like polypeptide, or a Glomalin polypeptide; 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 eRF1 polypeptide, or a SCAMP-like polypeptide, or a fibrillin polypeptide, or a PLATZ polypeptide, or a PLST-like polypeptide, or a Glomalin polypeptide, as defined herein.
The nucleic acid may be introduced directly into a plant cell or into the plant itself (including introduction into a tissue, organ or any other part of a plant). According to a preferred feature of the present invention, the nucleic acid is preferably introduced into a plant by transformation. The term “transformation” is 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 above-mentioned 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 encoding an eRF1 polypeptide, or a SCAMP-like polypeptide, or a fibrillin polypeptide, or a PLATZ polypeptide, or a PLST-like polypeptide, or a Glomalin polypeptide, as defined hereinabove. Preferred host cells according to the invention are plant cells. Host plants for the nucleic acids 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, linseed, 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 such as, but not limited to seeds, leaves, fruits, flowers, stems, roots, rhizomes, tubers and bulbs, which harvestable parts comprise a recombinant nucleic acid encoding an eRF1 polypeptide, or a SCAMP-like polypeptide, or a fibrillin polypeptide, or a PLATZ polypeptide, or a PLST-like polypeptide, or a Glomalin polypeptide. 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 modulating expression of a nucleic acid encoding an eRF1 polypeptide, or a SCAMP-like polypeptide, or a fibrillin polypeptide, or a PLATZ polypeptide, or a PLST-like polypeptide, or a Glomalin polypeptide, is by introducing and expressing in a plant a nucleic acid encoding an eRF1 polypeptide, or a SCAMP-like polypeptide, or a fibrillin polypeptide, or a PLATZ polypeptide, or a PLST-like polypeptide, or a Glomalin polypeptide; however the effects of performing the method, i.e. enhancing 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 use of nucleic acids encoding eRF1 polypeptide as described herein and use of these eRF1 polypeptides, or SCAMP-like polypeptides, or fibrillin polypeptides, or PLATZ polypeptides, or PLST-like polypeptides, or Glomalin polypeptides, in enhancing any of the aforementioned yield-related traits in plants.
Nucleic acids encoding an eRF1 polypeptide, or a SCAMP-like polypeptide, or a fibrillin polypeptide, or a PLATZ polypeptide, or a PLST-like polypeptide, or a Glomalin polypeptide, described herein, or the eRF1 polypeptides, or SCAMP-like polypeptides, or fibrillin polypeptides, or PLATZ polypeptides, or PLST-like polypeptides, or Glomalin polypeptides, themselves, may find use in breeding programmes in which a DNA marker is identified which may be genetically linked to a gene encoding an eRF1 polypeptide, or a SCAMP-like polypeptide, or a fibrillin polypeptide, or a PLATZ polypeptide, or a PLST-like polypeptide, or a Glomalin polypeptide. The nucleic acids/genes, or the eRF1 polypeptides, or SCAMP-like polypeptides, or fibrillin polypeptides, or PLATZ polypeptides, or PLST-like polypeptides, or Glomalin polypeptides, 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 enhanced yield-related traits as defined hereinabove in the methods of the invention.
Allelic variants of a nucleic acid/gene encoding an eRF1 polypeptide, or a SCAMP-like polypeptide, or a fibrillin polypeptide, or a PLATZ polypeptide, or a PLST-like polypeptide, or a Glomalin polypeptide, 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 enhanced yield characteristics. 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 acids encoding an eRF1 polypeptide, or a SCAMP-like polypeptide, or a fibrillin polypeptide, or a PLATZ polypeptide, or a PLST-like polypeptide, or a Glomalin polypeptide, may also be used as probes for genetically and physically mapping the genes that they are a part of, and as markers for traits linked to those genes. Such information may be useful in plant breeding in order to develop lines with desired phenotypes. Such use of nucleic acids encoding an eRF1 polypeptide, or a SCAMP-like polypeptide, or a fibrillin polypeptide, or a PLATZ polypeptide, or a PLST-like polypeptide, or a Glomalin polypeptide, requires only a nucleic acid sequence of at least 15 nucleotides in length. The nucleic acids encoding an eRF1 polypeptide, or a SCAMP-like polypeptide, or a fibrillin polypeptide, or a PLATZ polypeptide, or a PLST-like polypeptide, or a Glomalin polypeptide, 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 acids encoding an eRF1 polypeptide, or a SCAMP-like polypeptide, or a fibrillin polypeptide, or a PLATZ polypeptide, or a PLST-like polypeptide, or a Glomalin polypeptide. The resulting banding patterns may then be subjected to genetic analyses using computer programs such as MapMaker (Lander et al. (1987) Genomics 1: 174-181) in order to construct a genetic map. In addition, the nucleic acids may be used to probe Southern blots containing restriction endonuclease-treated genomic DNAs of a set of individuals representing parent and progeny of a defined genetic cross. Segregation of the DNA polymorphisms is noted and used to calculate the position of the nucleic acid encoding an eRF1 polypeptide, or a SCAMP-like polypeptide, or a fibrillin polypeptide, or a PLATZ polypeptide, or a PLST-like polypeptide, or a Glomalin 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 probes may also be used for physical mapping (i.e., placement of sequences on physical maps; see Hoheisel et al. In: Non-mammalian Genomic Analysis: A Practical Guide, Academic press 1996, pp. 319-346, and references cited therein).
In another embodiment, the nucleic acid probes may be used in direct fluorescence in situ hybridisation (FISH) mapping (Trask (1991) Trends Genet. 7:149-154). Although current methods of FISH mapping favour use of large clones (several kb to several hundred kb; see Laan et al. (1995) Genome Res. 5:13-20), improvements in sensitivity may allow performance of FISH mapping using shorter probes.
A variety of nucleic acid amplification-based methods for genetic and physical mapping may be carried out using the nucleic acids. Examples include allele-specific amplification (Kazazian (1989) J. Lab. Clin. Med 11:95-96), polymorphism of PCR-amplified fragments (CAPS; Sheffield et al. (1993) Genomics 16:325-332), allele-specific ligation (Landegren et al. (1988) Science 241:1077-1080), nucleotide extension reactions (Sokolov (1990) Nucleic Acid Res. 18:3671), Radiation Hybrid Mapping (Walter et al. (1997) Nat. Genet. 7:22-28) and Happy Mapping (Dear and Cook (1989) Nucleic Acid Res. 17:6795-6807). For these methods, the sequence of a nucleic acid is used to design and produce primer pairs for use in the amplification reaction or in primer extension reactions. The design of such primers is well known to those skilled in the art. In methods employing PCR-based genetic mapping, it may be necessary to identify DNA sequence differences between the parents of the mapping cross in the region corresponding to the instant nucleic acid sequence. This, however, is generally not necessary for mapping methods.
The methods according to the present invention result in plants having 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.
Items
1. eRF1 Polypeptides
In one aspect, the present invention is characterised by one or more of the following items:

    1. 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 eRF1 polypeptide, wherein said polypeptide comprises at least three consensus domains, eRF1 domain 1, eRF1 domain 2 and eRF1 domain 3, with PFam accession numbers respectively PF03463, PF03464 and PF03465.
    2. Method according to item 1, wherein the eRF1 domain 1 of an eRF1 polypeptide has at least, in increasing order of preference, 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% or more sequence identity to the sequence located between amino acid 6 and 140 of SEQ ID NO 2.
    3. Method according to item 1, wherein the eRF1 domain 2 of an eRF1 polypeptide has at least, in increasing order of preference, 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% or more sequence identity to the sequence located between amino acid 144 and 278 of SEQ ID NO 2.
    4. Method according to item 1, wherein the eRF1 domain 3 of an eRF1 polypeptide has at least, in increasing order of preference, 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% or more sequence identity to the sequence located between amino acid 281 and 418 of SEQ ID NO 2.
    5. Method according to any of the items 1 to 4 wherein the eRF1 polypeptide of the present invention comprises one or more of the following peptides: GGQ, NIKS and [GA][IMLV]LR[YW] having SEQ ID NO: 73, 74 and 75 respectively.
    6. Method according to item 1, wherein said eRF1 polypeptide may also comprise sequence motifs having at least, in increasing order of preference 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% or more sequence identity to anyone of the following motifs:









(i)


Motif 1:


(SEQ ID NO: 76)


FGTLSGNTREVLHKF[TS]VDLPKKHGRGGQSALRFARLRMEKRHNY


 


VRK[TV]AE,


 


(ii)


Motif 2:


(SEQ ID NO: 77)


YN[KR]VPPNGLVLY[TC]GT[IV]VT[ED][DE]GKEKKV[TN]IDFE


 


PF[KR]PIN[AT]SLYLCDNKFHTE,


 


(iii)


Motif 3:


(SEQ ID NO: 78)


ARGNGTSMISLI[MI]PP[RK]DQ[IV]SRVTKML[GA]DE[YF]GTAS


 


NI KSRVNR[QL]SVL[GS]AIT






    7. Method, according to item 1 or 6, wherein said eRF1 polypeptide may also comprise any one or more sequence motifs having at least, in increasing order of preference 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% or more sequence identity to anyone of the following motifs:









(i)


Motif 4:


(SEQ ID NO: 79)


[TS]VDLPKKHGRGGQSALRFARLR[EM]EKRHNYVRKVAE[VL]A[VT]


 


QNFITND[KR][PV]NV,


 


(ii)


Motif 5:


(SEQ ID NO: 80)


Y[NT][KR]VPPNGLV[VLI]YCG[TD][IV][ILM]T[ED][ED]GKE


 


[KR]K[VM][NT]ID[FI]EPFKPINTSLYLCDNKFHTE,


 


(iii)


Motif 6:


(SEQ ID NO: 81)


ARGNGTSMISL[IV][IM]PPK[DG]Q[IV]S[RL]V[QA]KML[AT]


 


[DE]EYGTASNIKSRVNR[LQ]SVL[SG]AIT






    8. Method, according to any of the items 6 to 7, wherein said eRF1 polypeptide may also comprise any one or more of the following sequence motifs having at least, in increasing order of preference 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% or more sequence identity to anyone of the following motifs:









(i)


Motif 7:


(SEQ ID NO: 82)


VDLPKKHGRGGQSALRFARLRMEKRHNYVRKTAELATQF[YF]INPAT


 


SQPNV,


 


(ii)


Motif 8:


(SEQ ID NO: 83)


YNKVPPNGLVLYTGTIVT[ED]DGKEKKVTIDFEPF[KR]PINASLYLC


 


DNKFHTE,


 


(iii)


Motif 9:


(SEQ ID NO: 84)


TSMISLIMPPRDQ[VI]SRVTKMLGDE[FY]GTASNIKSRVNRQSVLGA


 


ITSAQQR. 






    9. Method, according to any of the items 1 to 8, wherein the homologue of eRF1 polypeptide 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%, 7%, 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 any of the polypeptides of Table A1, preferably by the SEQ ID NO: 2.
    10. Method, according to any of the items 1 to 9, wherein said modulated expression is effected by introducing and expressing in a plant a nucleic acid encoding an eRF1 polypeptide as defined in any of the previous items.
    11. Method according to any one of items 1 to 10, wherein said nucleic acid encoding an eRF1 polypeptide encodes any one of the proteins listed in Table A1 or is a portion of such a nucleic acid, or a nucleic acid capable of hybridising with such a nucleic acid.
    12. Method according to any one of items 1 to 11, wherein said nucleic acid sequence encodes an orthologue or paralogue of any of the proteins given in Table A1.
    13. 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.
    14. Method according to any one of items 1 to 13, wherein said enhanced yield-related traits are obtained under non-stress conditions.
    15. Method according to any one of items 1 to 14, wherein said enhanced yield-related traits are obtained under conditions of drought stress, salt stress or nitrogen deficiency.
    16. Method according to any one of items 10 to 12, wherein said nucleic acid is operably linked to a constitutive promoter, preferably to a GOS2 promoter, most preferably to a GOS2 promoter from rice.
    17. Method according to any one of items 1 to 16, wherein said nucleic acid encoding an eRF1 polypeptide is of plant origin, preferably from a dicotyledonous plant, further preferably from the family Brassicaceae, more preferably from the genus Arabidopsis, most preferably from Arabidopsis thaliana. 
    18. Plant or part thereof, including seeds, obtainable by a method according to any one of items 1 to 17, wherein said plant or part thereof comprises a recombinant nucleic acid encoding an eRF1 polypeptide.
    19. Construct comprising:
    
        (i) nucleic acid encoding an eRF1 polypeptide as defined in items 1 to 9;
        (ii) one or more control sequences capable of driving expression of the nucleic acid sequence of (i); and optionally
        (iii) a transcription termination sequence.
    
    
    20. Construct according to item 19, wherein one of said control sequences is a constitutive promoter, preferably a GOS2 promoter, most preferably a GOS2 promoter from rice.
    21. Use of a construct according to items 19 or 20 in a method for making plants having increased yield, particularly increased biomass and/or increased seed yield relative to control plants.
    22. Plant, plant part or plant cell transformed with a construct according to items 19 or 20.
    23. 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 an eRF1 polypeptide as defined in items 1 to 9; and
        (ii) cultivating the plant cell under conditions promoting plant growth and development.
    
    
    24. 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 an eRF1 polypeptide as defined in items 1 to 9, or a transgenic plant cell derived from said transgenic plant.
    25. Transgenic plant according to item 18, 22 or 24, 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 emmer, spelt, secale, einkorn, teff, milo and oats.
    26. Harvestable parts of a plant according to item 25, wherein said harvestable parts are preferably shoot biomass and/or seeds.
    27. Products derived from a plant according to item 25 and/or from harvestable parts of a plant according to item 26.
    28. Use of a nucleic acid encoding an eRF1 polypeptide in enhancing yield characteristics, particularly in increasing seed yield and/or shoot biomass in plants, relative to control plants.
    29. An isolated nucleic acid molecule selected from:
    
        (i) a nucleic acid represented by any one of the following nucleic acid sequences: G.max_GM06MC33657_sm55b10@32878 having SEQ ID NO: 15; H.vulgare_c64960768hv270303@2598 having SEQ ID NO: 17;
        (ii) the complement of a nucleic acid represented by said sequences G.max_GM06MC33657_sm55b10@32878 having SEQ ID NO: 15; H.vulgare_c64960768hv270303@2598 having SEQ ID NO 17;
        (iii) a nucleic acid encoding the polypeptide as represented by any one SEQ ID NO: 16; SEQ ID NO 18 preferably as a result of the degeneracy of the genetic code, said isolated nucleic acid can be derived from a polypeptide sequence as represented by any one of SEQ ID NO: 16 and 18 and further preferably confers enhanced yield-related traits relative to control plants;
        (iv) a nucleic acid having, 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% sequence identity with any of the nucleic acid sequences of Table A1 and further preferably conferring enhanced yield-related traits relative to control plants;
        (v) a nucleic acid molecule which hybridizes with a nucleic acid molecule of (i) to (iv) under stringent hybridization conditions and preferably confers enhanced yield-related traits relative to control plants;
        (vi) a nucleic acid encoding a eRF1 polypeptide having, in increasing order of preference, at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence represented by any one of SEQ ID NO: 16, and 18 and any of the other amino acid sequences in Table A1 and preferably conferring enhanced yield-related traits relative to control plants.
    
    
    30. According to a further embodiment of the present invention, there is also provided an isolated polypeptide selected from:
    
        (i) an amino acid sequence represented by any one of SEQ ID NO: 16 and 18;
        (ii) an amino acid sequence having, in increasing order of preference, at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence represented by any one of SEQ ID NO: 16 and 18 and any of the other amino acid sequences in Table A1 and preferably conferring enhanced yield-related traits relative to control plants.
        (iii) derivatives of any of the amino acid sequences given in (i) or (ii) above.

2. SCAMP-Like Polypeptides

    
    


In another aspect, the invention is characterised by one or more of the following items:

    1. 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 SCAMP-like polypeptide, wherein said SCAMP-like polypeptide comprises a SCAMP domain.
    2. Method according to item 1, wherein said SCAMP domain has in increasing order of preference at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid of the SCAMP domain present in any of the polypeptides of Table A2, preferably to the SCAMP domain represented by the sequence located between amino acids 91 and 265 of SEQ ID NO: 89.
    3. Method according to item 1 or 2, wherein said modulated expression is effected by introducing and expressing in a plant a nucleic acid encoding a SCAMP-like polypeptide.
    4. Method according to any one of items 1 to 3, wherein said nucleic acid encoding a SCAMP-like polypeptide encodes any one of the proteins listed in Table A2 or is a portion of such a nucleic acid, or a nucleic acid capable of hybridising with such a nucleic acid.
    5. Method according to any one of items 1 to 4, wherein said nucleic acid sequence encodes an orthologue or paralogue of any of the proteins given in Table A2.
    6. 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.
    7. Method according to any one of items 1 to 6, wherein said enhanced yield-related traits are obtained under non-stress conditions.
    8. Method according to any one of items 1 to 6, wherein said enhanced yield-related traits are obtained under conditions of drought stress, salt stress or nitrogen deficiency.
    9. Method according to any one of items 3 to 8, wherein said nucleic acid is operably linked to a constitutive promoter, preferably to a GOS2 promoter, most preferably to a GOS2 promoter from rice.
    10. Method according to any one of items 1 to 9, wherein said nucleic acid encoding a LBD polypeptide is of plant origin, preferably from a dicotyledonous plant, further preferably from the family Brassicaceae, more preferably from the genus Arabidopsis, most preferably from Arabidopsis thaliana. 
    11. Plant or part thereof, including seeds, obtainable by a method according to any one of items 1 to 10, wherein said plant or part thereof comprises a recombinant nucleic acid encoding a SCAMP-like polypeptide.
    12. Construct comprising:
    
        (i) nucleic acid encoding a SCAMP-like polypeptide as defined in items 1 or 2;
        (ii) one or more control sequences capable of driving expression of the nucleic acid sequence of (i); and optionally
        (iii) a transcription termination sequence.
    
    
    13. Construct according to item 12, wherein one of said control sequences is a constitutive promoter, preferably a GOS2 promoter, most preferably a GOS2 promoter from rice.
    14. Use of a construct according to item 12 or 13 in a method for making plants having increased yield, particularly increased biomass and/or increased seed yield relative to control plants.
    15. Plant, plant part or plant cell transformed with a construct according to item 12 or 13.
    16. 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 SCAMP-like polypeptide as defined in item 1 or 2; and
        (ii) cultivating the plant cell under conditions promoting plant growth and development.
    
    
    17. 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 SCAMP-like polypeptide as defined in item 1 or 2, or a transgenic plant cell derived from said transgenic plant.
    18. Transgenic plant according to item 11, 15 or 17, 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 emmer, spelt, secale, einkorn, teff, milo and oats.
    19. Harvestable parts of a plant according to item 18, wherein said harvestable parts are preferably shoot biomass and/or 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 encoding a SCAMP-like polypeptide in increasing yield, particularly in increasing seed yield and/or shoot biomass in plants, relative to control plants.
    22. An isolated nucleic acid molecule selected from:
    
        (i) a nucleic acid represented by any one of SEQ ID NO: 100, 102, 104, 106, 180, 182, 184, 186, 188, 190 and 192;
        (ii) the complement of a nucleic acid represented by any one of (i) SEQ ID NO: 100, 102, 104, 106, 180, 182, 184, 186, 188, 190 and 192;
        (iii) a nucleic acid encoding the polypeptide as represented by any one of SEQ ID NO: 101, 103, 105, 107, 109, 183, 185, 187, 189, 191 and 193 preferably as a result of the degeneracy of the genetic code, said isolated nucleic acid can be derived from a polypeptide sequence as represented by any one of SEQ ID NO: 101, 103, 105, 107, 181, 183, 185, 187, 189, 191 and 193 and further preferably confers enhanced yield-related traits relative to control plants;
        (iv) a nucleic acid having, 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% sequence identity with any of the nucleic acid sequences of Table A2 and further preferably conferring enhanced yield-related traits relative to control plants;
        (v) a nucleic acid molecule which hybridizes with a nucleic acid molecule of (i) to (iv) under stringent hybridization conditions and preferably confers enhanced yield-related traits relative to control plants;
        (vi) a nucleic acid encoding a polypeptide having, in increasing order of preference, at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence represented by any one of SEQ ID NO: 101, 103, 105, 107, 181, 183, 185, 187, 189, 191 and 193 and any of the other amino acid sequences in Table A2 and preferably conferring enhanced yield-related traits relative to control plants.
    
    
    23. An isolated polypeptide selected from:
    
        (i) an amino acid sequence represented by any one of SEQ ID NO: 101, 103, 105, 107, 181, 183, 185, 187, 189, 191 and 193;
        (ii) an amino acid sequence having, in increasing order of preference, at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence represented by any one of SEQ ID NO: 101, 103, 105, 107, 181, 183, 185, 187, 189, 191 and 193 and any of the other amino acid sequences in Table A2 and preferably conferring enhanced yield-related traits relative to control plants.
        (iii) derivatives of any of the amino acid sequences given in (i) or (ii) above.

3. Fibrillin Polypeptides

    
    


In another aspect, the invention is characterised by one or more of the following items:

    1. 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 fibrillin polypeptide, comprising:
    
        (i) a PAP fibrillin domain as represented by PFAM Accession number PF04755; and
        (ii) a C-terminal domain represented by KFECQNESRGGLVRNVIKWSVPRLLE ENEGATLIVTARFSSVSARNIYLKFEEIGLQNINISDDLQAVIAPAILPRSFLSLQIL QFIRSFKARVPVTSPERHSVGGLYYLSYLDKNMLLGRAVGGGGVFIFTRAHTL (SEQ ID NO: 253) which may contain between 0 and 5 gaps representing between 1 and 15 residues, or a domain having in increasing order of preference at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% overall sequence identity to (SEQ ID NO: 253); and optionally
        (iii) a transit peptide within the N-terminal region of the polypeptide.
    
    
    2. Method according to item 1, wherein said PAP fibrillin domain is represented by: ENRKYELLNIIQDTQRGLVTTADQRSTIEEAMVVVEGFDAGKEIDLSKLDGTWQYTS APDVLILFESAARLPFFQVGQIFQ SEQ ID NO: 252 which may contain between 0 and 5 gaps representing between 1 and 15 residues, or a domain having in increasing order of preference at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% overall sequence identity to SEQ ID NO: 252.
    3. Method according to item 1 or 2, wherein said fibrillin polypeptide comprises one or more of the following one or more of the following domains:
    
        Domain X: NIYLQF[EQ]E[IA]S[VL]Q[ND]INISE[EQ]LQAL[IL]APA[IL]LPRSFL [SN]LQILQ[FA][LI][RK][TS]F[KR]AQ[VI]P;
        Domain Y: YYL[ST]YLD[RN][ND]MLLGR[AS]VGGGGV;
        Domain Z: [PA][IL]DL[AS]KLDGTWRLQYTSA[SP]DV; or
        a domain having in increasing order of preference at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% overall sequence identity to any one or more of Domains X, Y and Z.
    
    
    4. Method according to any one of items 1 to 3, wherein said modulated expression is effected by introducing and expressing in a plant a nucleic acid encoding a fibrillin polypeptide.
    5. Method according to any one of items 1 to 4, wherein said nucleic acid encoding a fibrillin polypeptide encodes any one of the proteins listed in Table A3 or is a portion of such a nucleic acid, or a nucleic acid capable of hybridising with such a nucleic acid.
    6. Method according to any one of items 1 to 5, wherein said nucleic acid sequence encodes an orthologue or paralogue of any of the proteins given in Table A3.
    7. Method according to any preceding item, wherein said enhanced yield-related traits comprise increased yield, preferably seed yield relative to control plants.
    8. Method according to any one of items 1 to 7, wherein said enhanced yield-related traits are obtained under non-stress conditions.
    9. Method according to any one of items 4 to 8, wherein said nucleic acid is operably linked to a constitutive promoter, preferably to a GOS2 promoter, most preferably to a GOS2 promoter from rice.
    10. Method according to any one of items 1 to 9, wherein said nucleic acid encoding a fibrillin polypeptide is of plant origin, preferably from a dicotyledonous plant, further preferably from the family more preferably from the family Solanaceae, further preferably the nucleic acid is from the genus Lycopersicon, further preferably from the species Lycopersicum, most preferably from Lycopersicon esculentum. 
    11. Plant or part thereof, including seeds, obtainable by a method according to any one of items 1 to 10, wherein said plant or part thereof comprises a recombinant nucleic acid encoding a fibrillin polypeptide.
    12. Construct comprising:
    
        (i) nucleic acid encoding a fibrillin polypeptide as defined in any one of items 1 to 3;
        (ii) one or more control sequences capable of driving expression of the nucleic acid sequence of (i); and optionally
        (iii) a transcription termination sequence.
    
    
    13. Construct according to item 12, wherein one of said control sequences is a constitutive promoter, preferably a GOS2 promoter, most preferably a GOS2 promoter from rice.
    14. Use of a construct according to item 12 or 13 in a method for making plants having increased yield, particularly increased seed yield relative to control plants.
    15. Plant, plant part or plant cell transformed with a construct according to item 12 or 13.
    16. 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 fibrillin polypeptide as defined in any one of items 1 to 3; and
        (ii) cultivating the plant cell under conditions promoting plant growth and development.
    
    
    17. Transgenic plant having increased yield, particularly increased seed yield, relative to control plants, resulting from modulated expression of a nucleic acid encoding a fibrillin polypeptide as defined in any one of items 1 to 3, or a transgenic plant cell derived from said transgenic plant.
    18. Transgenic plant according to item 11, 15 or 17, 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, emmer, spelt, secale, einkorn, teff, milo and oats.
    19. Harvestable parts 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 encoding a fibrillin polypeptide in increasing yield, particularly in increasing seed yield relative to control plants.
    22. An isolated nucleic acid molecule selected from:
    
        (i) a nucleic acid represented by any one of the following nucleic acid sequences: B.napus_BN06MC20042_46499279@19975 having SEQ ID NO: 206; G.max_GM06MC19234_59694709@18873 having SEQ ID NO: 220;
        (ii) the complement of a nucleic acid represented by said sequences B.napus_BN06MC20042_46499279@19975 having SEQ ID NO: 206; G.max_GM06MC19234_59694709@18873 having SEQ ID NO 220;
        (iii) a nucleic acid encoding the polypeptide as represented by any one SEQ ID NO: 207; SEQ ID NO 221 preferably as a result of the degeneracy of the genetic code, said isolated nucleic acid can be derived from a polypeptide sequence as represented by any one of SEQ ID NO: 207 and 221 and further preferably confers enhanced yield-related traits relative to control plants;
        (iv) a nucleic acid having, 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% sequence identity with any of the nucleic acid sequences of Table A3 and further preferably conferring enhanced yield-related traits relative to control plants;
        (v) a nucleic acid molecule which hybridizes with a nucleic acid molecule of (i) to (iv) under stringent hybridization conditions and preferably confers enhanced yield-related traits relative to control plants;
        (vi) a nucleic acid encoding a fibrillin polypeptide having, in increasing order of preference, at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence represented by any one of SEQ ID NO: 207, and 221 and any of the other amino acid sequences in Table A3 and preferably conferring enhanced yield-related traits relative to control plants.
    
    
    23. According to a further embodiment of the present invention, there is also provided an isolated polypeptide selected from:
    
        (i) an amino acid sequence represented by any one of SEQ ID NO: 207 and 221;
        (ii) an amino acid sequence having, in increasing order of preference, at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence represented by any one of SEQ ID NO: 207 and 221 and any of the other amino acid sequences in Table A3 and preferably conferring enhanced yield-related traits relative to control plants.
        (iii) derivatives of any of the amino acid sequences given in (i) or (ii) above.

4. PLATZ Polypeptides

    
    


In another aspect, the invention is characterised by one or more of the following items:

    1. 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 PLATZ polypeptide, wherein said PLATZ polypeptide comprises a PLATZ domain.
    2. Method according to item 1, wherein said PLATZ polypeptide comprises one or more of the motifs 10 to 18 (SEQ ID NO: 264 to SEQ ID NO: 272)
    3. Method according to item 1 or 2, wherein said modulated expression is effected by introducing and expressing in a plant a nucleic acid encoding a PLATZ polypeptide.
    4. Method according to any one of items 1 to 3, wherein said nucleic acid encoding a PLATZ polypeptide encodes any one of the proteins listed in Table A4 or is a portion of such a nucleic acid, or a nucleic acid capable of hybridising with such a nucleic acid.
    5. Method according to any one of items 1 to 4, wherein said nucleic acid sequence encodes an orthologue or paralogue of any of the proteins given in Table A4.
    6. 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.
    7. Method according to any one of items 1 to 6, wherein said enhanced yield-related traits are obtained under non-stress conditions.
    8. Method according to any one of items 3 to 7, wherein said nucleic acid is operably linked to a constitutive promoter, preferably to a GOS2 promoter, most preferably to a GOS2 promoter from rice.
    9. Method according to any one of items 1 to 8, wherein said nucleic acid encoding a PLATZ polypeptide is of plant origin, preferably from a dicotyledonous plant, further preferably from the family Salicaceae, more preferably from the genus Populus. 
    10. Plant or part thereof, including seeds, obtainable by a method according to any one of items 1 to 9, wherein said plant or part thereof comprises a recombinant nucleic acid encoding a PLATZ polypeptide.
    11. Construct comprising:
    
        (i) nucleic acid encoding a PLATZ polypeptide as defined in items 1 or 2;
        (ii) one or more control sequences capable of driving expression of the nucleic acid sequence of (i); and optionally
        (iii) a transcription termination sequence.
    
    
    12. Construct according to item 11, wherein one of said control sequences is a constitutive promoter, preferably a GOS2 promoter, most preferably a GOS2 promoter from rice.
    13. Use of a construct according to item 11 or 12 in a method for making plants having increased yield, particularly increased biomass and/or increased seed yield relative to control plants.
    14. Plant, plant part or plant cell transformed with a construct according to item 11 or 12.
    15. 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 PLATZ polypeptide as defined in item 1 or 2; and
        (ii) cultivating the plant cell under conditions promoting plant growth and development.
    
    
    16. 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 PLATZ polypeptide as defined in item 1 or 2, or a transgenic plant cell derived from said transgenic plant.
    17. Transgenic plant according to item 10, 14 or 16, 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 emmer, spelt, secale, einkorn, teff, milo and oats.
    18. Harvestable parts of a plant according to item 17, wherein said harvestable parts are preferably shoot biomass and/or seeds.
    19. Products derived from a plant according to item 17 and/or from harvestable parts of a plant according to item 19.
    20. Use of a nucleic acid encoding a PLATZ polypeptide in increasing yield, particularly in increasing seed yield and/or shoot biomass in plants, relative to control plants.
    21. An isolated nucleic acid molecule selected from:
    
        (i) a nucleic acid represented by SEQ ID NO: 354;
        (ii) the complement of a nucleic acid represented by SEQ ID NO: 354;
        (iii) a nucleic acid encoding a PLATZ polypeptide having, in increasing order of preference, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the amino acid sequence represented by SEQ ID NO: 355, and having in increasing order of preference at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to one or more of the motifs as defined hereabove.
    
    
    22. An isolated polypeptide selected from:
    
        (i) an amino acid sequence represented by SEQ ID NO: 355;
        (ii) an amino acid sequence having, in increasing order of preference, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the amino acid sequence represented by SEQ ID NO: 355, and having in increasing order of preference at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to one or more of the motifs as defined hereabove;
        (iii) derivatives of any of the amino acid sequences given in (i) or (ii) above.

5. PLST-Like Polypeptides

    
    


In another aspect, the invention is characterised by one or more of the following items:

    1. 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 PLST-like polypeptide, wherein said polypeptide comprising at least a PLST consensus domain with a PFam accession number PF02298.
    2. Method according to item 1, wherein the PLST domain of the PLST-like polypeptide has at least, in increasing order of preference, 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% or more sequence identity to the sequence located between amino acid 38 and 124 of SEQ ID NO 411.
    3. Method according to any of the items 1 or 2, wherein said PLST-like polypeptide may also comprise sequence motifs having at least, in increasing order of preference 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% or more sequence identity to anyone of the following motifs:











(i)



Motif 19:



(SEQ ID NO: 530)



[DH]SV[LI]QV[TS]KE[DA][YF][DK]SCNT[SK][NSD]P



 



(ii)



Motif 20:



(SEQ ID NO: 531)



[FHY]YF[IT]SGV[PK][GD][HN]C



 



(iii)



Motif 21:



(SEQ ID NO: 532)



Y[NT][QK]WA[ESK][KS]NRF[KQ][IV]GD[ST][LI][VL]F



 



[KL]YP






    4. Method, according to any of the items 1 to 3, wherein said PLST-like polypeptide may also comprise any one or more sequence motifs having at least, in increasing order of preference 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% or more sequence identity to anyone of the following motifs:









(i)


Motif 22:


(SEQ ID NO: 533)


[DN]GN[TS][LVK][FV][KN][LF][DT]R[SP]GP[FY]YF[IT] 


 


SG[VA][KP][GD][HN]CEK[GN][QE]K 


 


(ii)


Motif 23:


(SEQ ID NO: 534)


[YL]N[QK]WA[EK][KS][NH]RF[KQ][IV]GD[ST]L[LV]F[LK] 


 


Y[PD] 


 


(iii)


Motif 24:


(SEQ ID NO: 535)


[KQ]DSV[LI]QVTKE[DA]YKSCNT[SK][DSN]PI






    5. Method, according to any of the items 1 to 4, wherein said PLST-like polypeptide may also comprise any one or more of the following sequence motifs having at least, in increasing order of preference 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% or more sequence identity to anyone of the following motifs:









(i)


Motif 25:


(SEQ ID NO: 536)


DSVI[QV]VT[EKA][EQ]S[YF][KN][SK]CNL[KST]DPIL[YF]


 


[MS]N[ND]GN[ST][LV]FN[LI][TD][RS]PGL[FY]YF[TI]SG


 


[VA][PS]GHC[EQ][KR]


 


(ii)


Motif 26:


(SEQ ID NO: 537)


P[PT]SA[DN]P[DQ][VL]YTKW[AS][KS][NS][HN][RN]FK 


 


[IL]GD[ST][LI]LFLYP


 


(iii)


Motif 27:


(SEQ ID NO: 538)


XVS[CS]Y[QE][YF]KVG[DG]LD[AGS]W.






    6. Method, according to any of the items 1 to 5, wherein said PLST-like polypeptide may also comprise any one or more of the following sequence motifs having at least, in increasing order of preference 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% or more sequence identity to anyone of the following motifs:









(i)


Motif 28:


(SEQ ID NO: 539)


HN[FL]K[IL]GDSLLFLYPPSQDSVIQVTA[QE][SAN][YF][KN] 


 


SC[ND]L[KS]DPILYMN[DN]GNSLFN[IL]T 


 


(ii)


Motif 29:


(SEQ ID NO: 540)


GDFYFTSG[AVE]PGHC[EQ]K[SK]QKLH[IV]


 


(iii)


Motif 30:


(SEQ ID NO: 541)


VSCYQYKVGDLD[AS]WGIPTSA[NK].






    7. Method, according to any of the items 1 to 6, wherein the homologue of PLST-like polypeptide 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 any of the polypeptides of Table A5, preferably by the SEQ ID NO: 411.
    8. Method, according to any of the items 1 to 7, wherein said modulated expression is effected by introducing and expressing in a plant a nucleic acid encoding a PLST-like polypeptide as defined in any of the previous items.
    9. Method according to any one of items 1 to 8, wherein said nucleic acid encoding a PLST-like polypeptide encodes any one of the proteins listed in Table A5 or is a portion of such a nucleic acid, or a nucleic acid capable of hybridising with such a nucleic acid.
    10. Method according to any one of items 1 to 9, wherein said nucleic acid sequence encodes an orthologue or paralogue of any of the proteins given in Table A5.
    11. Method according to any preceding item, wherein said enhanced yield-related traits comprise increased yield, preferably increased seed yield relative to control plants.
    12. Method according to any one of items 1 to 11, wherein said enhanced yield-related traits are obtained under non-stress conditions.
    13. Method according to any one of items 1 to 11, wherein said enhanced yield-related traits are obtained under conditions of drought stress, salt stress or nitrogen deficiency.
    14. Method according to any one of items 8 to 10, wherein said nucleic acid is operably linked to a constitutive promoter, preferably to a GOS2 promoter, most preferably to a GOS2 promoter from rice.
    15. Method according to any one of items 1 to 14, wherein said nucleic acid encoding a PLST-like polypeptide is of plant origin.
    16. Method according to item 15 wherein said nucleic acid encoding a PLST-like polypeptide is from a dicotyledonous plant, further preferably from the family Salicaceae, most preferably the nucleic acid is from Populus trichocarpa.
    17. Plant or part thereof, including seeds, obtainable by a method according to any one of items 1 to 16, wherein said plant or part thereof comprises a recombinant nucleic acid encoding a PLST-like polypeptide.
    18. Construct comprising:
    
        (i) nucleic acid encoding a PLST-like polypeptide as defined in items 1 to 7;
        (ii) one or more control sequences capable of driving expression of the nucleic acid sequence of (a); and optionally
        (iii) a transcription termination sequence.
    
    
    19. Construct according to item 18, wherein one of said control sequences is a constitutive promoter, preferably a GOS2 promoter, most preferably a GOS2 promoter from rice.
    20. Use of a construct according to items 18 or 19 in a method for making plants having increased yield, particularly increased seed yield relative to control plants.
    21. Plant, plant part or plant cell transformed with a construct according to items 18 or 19.
    22. 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 PLST-like polypeptide as defined in items 1 to 7; and
        (ii) cultivating the plant cell under conditions promoting plant growth and development.
    
    
    23. 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 PLST-like polypeptide as defined in items 1 to 7, or a transgenic plant cell derived from said transgenic plant.
    24. Transgenic plant according to item 17, 21 or 23, 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 emmer, spelt, secale, einkorn, teff, milo and oats.
    25. Harvestable parts of a plant according to item 24, wherein said harvestable parts are preferably seeds.
    26. Products derived from a plant according to item 24 and/or from harvestable parts of a plant according to item 25.
    27. Use of a nucleic acid encoding a PLST-like polypeptide in enhancing yield characteristics, particularly in increasing seed yield in plants, relative to control plants.
    28. An isolated nucleic acid molecule selected from:
    
        (i) a nucleic acid represented by SEQ ID NO: 414; SEQ ID NO: 426; SEQ ID NO: 428; SEQ ID NO: 434; SEQ ID NO: 438;
        (ii) the complement of a nucleic acid represented by SEQ ID NO: 414; SEQ ID NO: 426; SEQ ID NO: 428; SEQ ID NO: 434; SEQ ID NO: 438;
        (iii) a nucleic acid encoding a PLST-like polypeptide as represented by any one of SEQ ID NO: 415; SEQ ID NO: 427; SEQ ID NO: 429; SEQ ID NO: 435; SEQ ID NO: 439, preferably as a result of the degeneracy of the genetic code, said isolated nucleic acid can be derived from a polypeptide sequence as represented by any one of said SEQ IDs and further preferably confers enhanced yield related traits relative to control plants;
        (iv) a nucleic acid having, in increasing order of preference, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity with any of the nucleic acid sequences of Table A5 and further preferably confers enhanced yield related traits relative to control plants;
        (v) a nucleic acid molecule which hybridizes with a nucleic acid molecule of (i) to (iv) under stringent hybridization conditions and preferably confers enhanced yield related traits relative to control plants;
        (vi) a nucleic acid encoding a PLST-like polypeptide having in increasing order of preference at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any one of SEQ ID NO: 415; SEQ ID NO: 427; SEQ ID NO: 429; SEQ ID NO: 435; SEQ ID NO: 439 and any of the other amino acid sequences in Table A5 and preferably confers enhanced yield related traits relative to control plants.
    
    
    29. According to a further embodiment of the present invention, there is also provided an isolated polypeptide selected from:
    
        (i) an amino acid sequence represented by SEQ ID NO: 415; SEQ ID NO: 427; SEQ ID NO: 429; SEQ ID NO: 435; SEQ ID NO: 439;
        (ii) an amino acid sequence having, in increasing order of preference, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the amino acid sequence represented by SEQ ID NO: Y, and having in increasing order of preference at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 415; SEQ ID NO: 427; SEQ ID NO: 429; SEQ ID NO: 435; SEQ ID NO: 439;
        (iii) derivatives of any of the amino acid sequences given in (i) or (ii) above.

6. Glomalin Polypeptides

    
    


In another aspect, the invention is characterised by one or more of the following items:

    1. 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 Glomalin polypeptide, wherein said Glomalin polypeptide comprises a Cpn60_TCP1 domain.
    2. Method according to item 1, wherein said Glomalin polypeptide comprises one or more of the motifs 31 to 43 (SEQ ID NO: 596 to SEQ ID NO: 608).
    3. Method according to item 1 or 2, wherein said modulated expression is effected by introducing and expressing in a plant a nucleic acid encoding a Glomalin polypeptide.
    4. Method according to any one of items 1 to 3, wherein said nucleic acid encoding a Glomalin polypeptide encodes any one of the proteins listed in Table A6 or is a portion of such a nucleic acid, or a nucleic acid capable of hybridising with such a nucleic acid.
    5. Method according to any one of items 1 to 4, wherein said nucleic acid sequence encodes an orthologue or paralogue of any of the proteins given in Table A6.
    6. Method according to any preceding item, wherein said enhanced yield-related traits comprise increased yield, preferably increased seed yield relative to control plants.
    7. Method according to any one of items 1 to 6, wherein said enhanced yield-related traits are obtained under non-stress conditions.
    8. Method according to any one of items 3 to 7, wherein said nucleic acid is operably linked to a root specific promoter, preferably to an RCc3 promoter, most preferably to an RCc3 promoter from rice.
    9. Method according to any one of items 1 to 8, wherein said nucleic acid encoding a Glomalin 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. 
    10. Plant or part thereof, including seeds, obtainable by a method according to any one of items 1 to 9, wherein said plant or part thereof comprises a recombinant nucleic acid encoding a Glomalin polypeptide.
    11. Construct comprising:
    
        (i) nucleic acid encoding a Glomalin polypeptide as defined in items 1 or 2;
        (ii) one or more control sequences capable of driving expression of the nucleic acid sequence of (a); and optionally
        (iii) a transcription termination sequence.
    
    
    12. Construct according to item 11, wherein one of said control sequences is a constitutive promoter, preferably an RCc3 promoter, most preferably an RCc3 promoter from rice.
    13. Use of a construct according to item 11 or 12 in a method for making plants having increased yield, particularly increased seed yield relative to control plants.
    14. Plant, plant part or plant cell transformed with a construct according to item 11 or 12.
    15. 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 Glomalin polypeptide as defined in item 1 or 2; and
        (ii) cultivating the plant cell under conditions promoting plant growth and development.
    
    
    16. Transgenic plant having increased yield, particularly increased seed yield, relative to control plants, resulting from modulated expression of a nucleic acid encoding a Glomalin polypeptide as defined in item 1 or 2, or a transgenic plant cell derived from said transgenic plant.
    17. Transgenic plant according to item 10, 14 or 16, 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 emmer, spelt, secale, einkorn, teff, milo and oats.
    18. Harvestable parts of a plant according to item 17, wherein said harvestable parts are preferably seeds.
    19. Products derived from a plant according to item 18 and/or from harvestable parts of a plant according to item 18.
    20. Use of a nucleic acid encoding a Glomalin polypeptide in increasing yield, particularly in increasing seed yield 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 the Phylogenetic tree of selected eRF1 proteins for the different clusters: Proteins of Streptophyta origins and non-Streptophyta origins are indicated. SPT=Streptophyta, CHL=chlorophyta, STR=Stramenopile, BAC=bacteria, FUNGI=fungi, HUM=human, PROT=protozoa. The alignment was generated using MAFFT (Katoh and Toh (2008) Briefings in Bioinformatics 9:286-298). A neighbour-joining tree was calculated using QuickTree (Howe et al. (2002), Bioinformatics 18(11): 1546-7), 100 bootstrap repetitions. The circular phylogram was drawn using Dendroscope (Huson et al. (2007), BMC Bioinformatics 8(1):460). Confidence for 100 bootstrap repetitions is indicated for major branching. Major branching position is indicated by circles.
FIG. 2 represents the binary vector used for increased expression in Oryza sativa of an eRF1-encoding nucleic acid under the control of a rice GOS2 promoter (pGOS2).
FIG. 3 represents a multiple alignment of SCAMP polypeptides. Sequences shown are: H.vulgare_TA39331_4513#1 (SEQ ID NO: 109); T.aestivum _TA81857_4565#1 (SEQ ID NO:177); Z.mays_ZM07MC31327_BFb0342A21@(SEQ ID NO: 181); Z.mays_ZM07MC32029_BFb0293C15@ (SEQ ID NO: 189); O.sativa_LOC_Os07g37740.1#1(SEQ ID NO: 137); O.sativa_LOC_Os05g42330.1#1(SEQ ID NO: 139); Z.mays_ZM07MC22858_BFb0220H23@ (SEQ ID NO: 183); H.vulgare_TA36210_4513#1(SEQ ID NO: 115); O.sativa_LOC Os01g57220.1#1(SEQ ID NO: 123); Z.mays_ZM07MC27067_BFb0182018@ (SEQ ID NO: 191); T.aestivum_TA51636_4565#(SEQ ID NO: 169); T.aestivum_CK163668#(SEQ ID NO: 179); T.aestivum_TA50955_4565#1(SEQ ID NO: 175); T.aestivum_TA72069_4565#1(SEQ ID NO: 167); A.thaliana_AT2G20840.1#1(SEQ ID NO: 97); B.napus_BN06MC09315_42883615@9SEQ ID NO: 103); P.trichocarpa_scaff_120.48#1(SEQ ID NO: 151); P.trichocarpa_scaff_XIII.1138# (SEQ ID NO: 153); A.thaliana_AT1G03550.1#1(SEQ ID NO: 89); S.lycopersicum_TA43976_4081#1(SEQ ID NO: 159); A.thaliana_AT1G61250.1#1(SEQ ID NO: 91); B.napus_BN06MC05708_42365297@5(SEQ ID NO: 101); A.thaliana_AT1G11180.1#1(SEQ ID NO: 99); M.truncatula_TA20357_3880#1(SEQ ID NO: 119); P.sativum_TA772_3888#1(SEQ ID NO: 147); G.max_GM06MC34782_sp08b05@3397(SEQ ID NO: 107); M.truncatula_TA21989_3880#1(SEQ ID NO: 117); P.trichocarpa_scaff_XI.291#1(SEQ ID NO: 149); S.lycopersicum_TA41016_4081#1(SEQ ID NO: 161); O.sativa_LOC_Os03g38590.2#1(SEQ ID NO: 125); O.sativa_LOC_Os03g38590.1#1(SEQ ID NO: 127); H.vulgare_TA44339_4513#1(SEQ ID NO: 113); Z.mays_ZM07MC25122_BFb0162CO2@ (SEQ ID NO: 185); Z.mays_ZM07MC20385_BFb0172E11@ (SEQ ID NO: 193); O.sativa_LOC_Os03g38600.1#1(SEQ ID NO: 131); P.patens_147248#1(SEQ ID NO: 141); P.patens_181545#1(SEQ ID NO: 143); P.patens_178454#1(SEQ ID NO: 145); A.cepa_TA5060_4679#1(SEQ ID NO: 95); M.truncatula_TA32267_3880#1(SEQ ID NO: 121); A.thaliana_AT1G32050.1#1 (SEQ ID NO: 93); B.napus_BN06MC16749_45336122@,1 (SEQ ID NO: 105); P.trichocarpa_scaff_III.723#1 (SEQ ID NO: 155); P.trichocarpa_scaff_29.268#1 (SEQ ID NO: 157); H.vulgare_TA38269_4513#1 (SEQ ID NO: 111); T.aestivum_TA75461_4565#1(SEQ ID NO: 173); T.aestivum_TA75459_4565#1 (SEQ ID NO: 165); Z.mays_ZM07MC20685_BFb0020D20@ (SEQ ID NO: 187); O.sativa_LOC_Os04g50890.1#1(SEQ ID NO: 127); O.sativa_LOC_Os02g47010.1#1(SEQ ID NO: 135); T.aestivum_DR738056#1(SEQ ID NO: 171); O.sativa_LOC_Os08g06440.1#1(SEQ ID NO: 133); and T.aestivum_TA95653_4565#1(SEQ ID NO: 163).
FIG. 4 represents the binary vector used for increased expression in Oryza sativa of a SCAMP-like-encoding nucleic acid under the control of a rice GOS2promoter (pGOS2).
FIG. 5 shows a multiple alignment of fibrillin polypeptide sequences which was performed using the AlignX (from Vector NTI 10.3, Invitrogen Corporation) multiple sequence alignment. The conserved PAP fibrillin PF04755 is marked by X under the consensus sequence. The conserved C-terminal domain is also marked by X under the consensus sequence. Sequences shown are: L.esculentum QC (SEQ ID NO: 205); AT2G46910.1 (SEQ ID NO: 225); B.napus_BN06MC20042 (SEQ ID NO: 207); P.patens_202760 (SEQ ID NO: 237); P.sitchensis_TA_14105 3332(SEQ ID NO: 239); O.sativa_AK241632 (SEQ ID NO: 233); S.bicolor_Sb01g017450.1 (SEQ ID NO: 243); Z.mays_TC447544 (SEQ ID NO: 251); C.solstitialis_TA2061_347529 (SEQ ID NO: 211); L.virosa_DW148855(SEQ ID NO: 227);
G,hirsutum_TC97719 (SEQ ID NO: 217); G.raimondii_TC7628(SEQ ID NO: 223); P.trichocarpa_552393 (SEQ ID NO: 241); M.domestica_TC4908(SEQ ID NO: 229); V.vinifera_GSVIVT00026214001 (SEQ ID NO: 249); G.max_Glyma07g00410.1 (SEQ ID NO: 219); G.max_GM06MC19234 (SEQ ID NO: 221); N.tabacum_TC21276 (SEQ ID NO: 231); T.pratense_TA1297_57577 (SEQ ID NO: 247); C.reinhardtii_190008 (SEQ ID NO: 209); O.taurii _36262 (SEQ ID NO: 235); S.moellendorffii_422148 (SEQ ID NO: 245); Chlorella_141300 (SEQ ID NO: 215); and C.vulgaris_102074 (SEQ ID NO: 213).
FIG. 6 shows a phylogenetic tree of fibrillin polypeptides constructed using a neighbour-joining clustering algorithm as provided in the AlignX programme from the Vector NTI (Invitrogen).
FIG. 7 taken from Ytterberg et al., 2006 (Plant Physiology, March 2006, Vol. 140, pp. 984-997) shows a schematic overview of proposed organization (A) and functional role of the plastoglobule (PG) and its proteome (B). PGs consist of a monolayer of lipids and sequester different hydrophilic small molecules, such as quinones and tocopherols. Structural proteins (fibrillins) and enzymes are attached to or embedded in the monolayer, but proteins lack transmembrane domains (A). Integration of PG functions in plastid metabolism (B).
FIG. 8 represents sequence logos for the three Conserved domain X, Y and Z as determined by MEME algorithm. Sequence logos are a graphical representation of an amino acid multiple sequence alignment, consisting of stacks of symbols, one stack for each position in the sequence. The overall height of the stack indicates the sequence conservation at that position, while the height of symbols within the stack indicates the relative frequency of each amino or nucleic acid at that position.
FIG. 9 represents the binary vector used for increased expression in Oryza sativaof a fibrillin-encoding nucleic acid under the control of a rice GOS2 promoter (pGOS2).
FIG. 10 represents the domain structure of SEQ ID NO: 261 with indication of the conserved motifs 10 to 18, and the PLATZ domain (bold italics).
FIG. 11 represents a multiple alignment of various PLATZ-A1-αpolypeptides using ClustalW with default settings for a slow alignment. Sequences shown are: Pt583639 (SEQ ID NO: 261); Pt779642 (SEQ ID NO: 347); Os02g09070.1 (SEQ ID NO: 345); Zm376 (SEQ ID NO: 355); Sb04g005680.1 (SEQ ID NO: 349); TaTC339412 (SEQ ID NO: 351); NtTC27363(SEQ ID NO: 343); VvT00005658001 (SEQ ID NO: 353); Gm13g23360.1 (SEQ ID NO: 337); Gm17g11470.1 (SEQ ID NO: 339); MtAC152347_6.5 (SEQ ID NO: 341); Gm09g12330.1 (SEQ ID NO: 335); AT2G12646.1 (SEQ ID NO: 333); and AsTA310_217475 (SEQ ID NO: 331).
FIG. 12 shows phylogenetic tree of PLATZ polypeptides, the alignment was generated using MAFFT (Katoh and Toh (2008) Briefings in Bioinformatics 9:286-298). A neighbour-joining tree was calculated using QuickTree (Howe et al. (2002), Bioinformatics 18(11): 1546-7), 100 bootstrap repetitions. The circular phylogram was drawn using Dendroscope (Huson et al. (2007), BMC Bioinformatics 8(1):460). Confidence for 100bootstrap repetitions is indicated for major branching. Major branching position is indicated by circles. SEQ ID NO: 261 is represented as P.trichocarpa_583639, in the clade PLATZ-A1-α.
FIG. 13 represents the binary vector used for increased expression in Oryza sativa of a PLATZ-encoding nucleic acid under the control of a rice GOS2 promoter (pGOS2)
FIG. 14 represents SEQ ID NO 411 with indication of PF 02298 domain and motifs 19 to 30.
FIG. 15 represents the Phylogenetic tree of selected PLST-like proteins for the different clusters: P-class=plastocyanin-like, NDL=nodulin-like, Z=others, PLST =PLST group.
The alignment was generated using MAFFT (Katoh and Toh (2008) Briefings in Bioinformatics 9:286-298). A neighbour-joining tree was calculated using QuickTree (Howe et al. (2002), Bioinformatics 18(11): 1546-7), 100 bootstrap repetitions. The circular phylogram was drawn using Dendroscope (Huson et al. (2007), BMC Bioinformatics 8(1):460). Confidence for 100 bootstrap repetitions is indicated for major branching. Major branching position is indicated by circles.
FIG. 16 represents the binary vector used for increased expression in Oryza sativaof a PLST-like-encoding nucleic acid under the control of a rice GOS2 promoter (pGOS2).
FIG. 17 represents the domain structure of SEQ ID NO: 546 with the conserved motifs 31 to 43 indicated and the Cpn60_TCP1 domain shown in bold.
FIG. 18 represents a multiple alignment of various Glomalin polypeptides. The asterisks indicate identical amino acids among the various protein sequences, colons represent highly conserved amino acid substitutions, and the dots represent less conserved amino acid substitution; on other positions there is no sequence conservation. These alignments can be used for defining further motifs, when using conserved amino acids. Sequences shown are: P.patens_226792 (SEQ ID NO: 580); P.patens_233067 (SEQ ID NO: 581); P.patens_163173(SEQ ID NO: 582); P.patens_56767 (SEQ ID NO: 583); S.lycopersicum_TC192865 (SEQ ID NO: 590); L.esculentum_gl _39 (SEQ ID NO: 591); S.lycopersicum_TC197855 (SEQ ID NO: 589); A.thaliana_AT3G23990 (SEQ ID NO: 574); A.thaliana_AT2G33210 (SEQ ID NO: 573); O.sativa_Os10g32550.1 (SEQ ID NO: 578); O.sativa_glomalin_SEQID2 (SEQ ID NO: 546); O.sativa_glomalin_39 (SEQ ID NO: 674); O.sativa_Os03g04970.1 (SEQ ID NO: 579); Z.mays_ZM07MC32795 (SEQ ID NO: 593); T.aestivum_c54647991 (SEQ ID NO: 592); A.cepa_CF435092 (SEQ ID NO: 571); P.trichocarpa_sc 1.447 (SEQ ID NO: 584); P.trichocarpa_sc_III.1436 (SEQ ID NO: 585);M.truncatula_AC161864_24 (SEQ ID NO: 575); M.truncatula_AC161864_3 (SEQ ID NO: 576); A.thaliana_AT3G13860.1 (SEQ ID NO: 572); P.trichocarpa_sc_44.102 (SEQ ID NO: 586); S.lycopersicum_TC204816 (SEQ ID NO: 588); O.sativa_Os05g46290 (SEQ ID NO: 577); and Z.mays_ZM07MC22894 (SEQ ID NO: 594).
FIG. 19 shows phylogenetic tree of Glomalin polypeptides. The sequence XM_002297581 from poplar represents the outgroup. The other sequences represent the cluster of the Glomalin sequences.
FIG. 20 represents the binary vector used for increased expression in Oryza sativa of a Glomalin-encoding nucleic acid under the control of a rice RCc3 promoter (pRC3)



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 tolimit 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 eRF1 sequences 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 or polypeptide sequences to sequence databases and by calculating the statistical significance of matches. For example, the polypeptide encoded by the nucleic acid used in 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 (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.
1.1. eRF1 Polypeptides
Table A1 provides a list of nucleic acid and polypeptide sequences related to SEQ ID NO: 1 and SEQ ID NO: 2.







TABLE A1







Examples of eRF1 nucleic acids and polypeptides:










Nucleic




acid
Polypeptide



SEQ
SEQ


Name
ID NO:
ID NO:












A.thaliana_AT1G12920.1
1
2


A.thaliana_AT3G26618.1
3
4


A.thaliana_AT5G47880.1
5
6


Aquilegia_sp_TC23338
7
8


C.sinensis_TC9326
9
10


C.solstitialis_TA682_347529
11
12


G.max_Glyma09g29600.1
13
14


G.max_GM06MC33657_sm55b10@32878
15
16


H.vulgare_c64960768hv270303@2598
17
18


M.truncatula_AC136505_1.4
19
20


O.sativa_LOC_Os01g71270.1
21
22


O.sativa_LOC_Os03g49580.1
23
24


P.glauca_TA15071_3330
25
26


P.patens_58108
27
28


P.trichocarpa_708902
29
30


P.trichocarpa_732023
31
32


S.lycopersicum_TC19702
33
34


S.tuberosum_TC166984
35
36


V.vinifera_GSVIVT0001464200
37
38


A.anophagefferens_32373
39
40


D.discoideum_XP_636638.1
41
42


P.falciparum_XP_001349629
43
44


D.melanogaster_NP_649210
45
46


H.sapiens_NP_004721.1
47
48


A.fumigatus_XP_754064.1
49
50


C.glabrata_XP_449070.1
51
52


D.hansenii_XP_457910.1
53
54


K.lactis_XP_452701.1
55
56


N.crassa_EAA28060.1
57
58


S.cerevisiae_XP_009701.1
59
60


Y.lipolyticaXP_504906.1
61
62


C.reinhardtii_182764
63
64


Chlorella_29482
65
66


O.RCC809_23895
67
68


L.braziliensis_XP_0015659401
69
70


T.brucei_XP_8283141
71
72









In some instances, related sequences have tentatively been assembled and publicly disclosed by research institutions, such as The Institute for Genomic Research (TIGR; beginning with TA). 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. Further, access to proprietary databases, has allowed the identification of novel nucleic acid and polypeptide sequences.
1.2. SCAMP-Like Polypeptides
Table A2 provides a list of nucleic acid and polypeptide sequences related to SEQ ID NO: 88 and SEQ ID NO: 89.







TABLE A2







Examples of SCAMP-like nucleic acids and polypeptides:










Nucleic




acid
Polypeptide



SEQ
SEQ


Name
ID NO:
ID NO:












A.thaliana_AT1G03550.1
88
89


A.thaliana_AT1G61250.1
90
91


A.thaliana_AT1G32050.1
92
93


A.cepa_TA5060_4679
94
95


A.thaliana_AT2G20840.1
96
97


A.thaliana_AT1G11180.1
98
99


B.napus_BN06MC05708_42365297@5693
100
101


B.napus_BN06MC09315_42883615@9289
102
103


B.napus_BN06MC16749_45336122@16695
104
105


G.max_GM06MC34782_sp08b05@33970
106
107


H.vulgare_TA39331_4513
108
109


H.vulgare_TA38269_4513
110
111


H.vulgare_TA44339_4513
112
113


H.vulgare_TA36210_4513
114
115


M.truncatula_TA21989_3880
116
117


M.truncatula_TA20357_3880
118
119


M.truncatula_TA32267_3880
120
121


O.sativa_LOC_Os01g57220.1
122
123


O.sativa_LOC_Os03g38590.2
124
125


O.sativa_LOC_Os04g50890.1
126
127


O.sativa_LOC_Os03g38590.1
128
129


O.sativa_LOC_Os03g38600.1
130
131


O.sativa_LOC_Os08g06440.1
132
133


O.sativa_LOC_Os02g47010.1
134
135


O.sativa_LOC_Os07g37740.1
136
137


O.sativa_LOC_Os05g42330.1
138
139


P.patens_147248
140
141


P.patens_181545
142
143


P.patens_178454
144
145


P.sativum_TA772_3888
146
147


P.trichocarpa_scaff_XI.291
148
149


P.trichocarpa_scaff_120.48
150
151


P.trichocarpa_scaff_XIII.1138
152
153


P.trichocarpa_scaff_III.723
154
155


P.trichocarpa_scaff_29.268
156
157


S.lycopersicum_TA43976_4081
158
159


S.lycopersicum_TA41016_4081
160
161


T.aestivum_TA95653_4565
162
163


T.aestivum_TA75459_4565
164
165


T.aestivum_TA72069_4565
166
167


T.aestivum_TA51636_4565
168
169


T.aestivum_DR738056
170
171


T.aestivum_TA75461_4565
172
173


T.aestivum_TA50955_4565
174
175


T.aestivum_TA81857_4565
176
177


T.aestivum_CK163668
178
179


Z.mays_ZM07MC31327_BFb0342A21@31234
180
181


Z.mays_ZM07MC22858_BFb0220H23@22794
182
183


Z.mays_ZM07MC25122_BFb0162C02@25049
184
185


Z.mays_ZM07MC20685_BFb0020D20@20631
186
187


Z.mays_ZM07MC32029_BFb0293C15@31934
188
189


Z.mays_ZM07MC27067_BFb0182O18@26987
190
191


Z.mays_ZM07MC20385_BFb0172E11@20333
192
193


A.thaliana_AT_x
663
664


A.thaliana_AT_y
665
666









In some instances, related sequences have tentatively been assembled and publicly disclosed by research institutions, such as The Institute for Genomic Research (TIGR; beginning with TA). 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. Further, access to proprietary databases, has allowed the identification of novel nucleic acid and polypeptide sequences.
1.3. Fibrillin Polypeptides
Table A3 provides a list of nucleic acid and polypeptide sequences related to SEQ ID NO: 204 and SEQ ID NO: 205.







TABLE A3







Examples of fibrillin nucleic acids and polypeptides:










Nucleic




acid
Polypeptide



SEQ
SEQ


Name
ID NO:
ID NO:





L.esculentum QC
204
205


B.napus_BN06MC20042_46499279@19975#1
206
207


C.reinhardtii_190008#1
208
209


C.solstitialis_TA2061_347529#1
210
211


C.vulgaris_102074#1
212
213


Chlorella_141300#1
214
215


G.hirsutum_TC97719#1
216
217


G.max_Glyma07g00410.1#1
218
219


G.max_GM06MC19234_59694709@18873#1
220
221


G.raimondii_TC7628#1
222
223


A.thaliana_AT2G46910.1#1
224
225


L.virosa_DW148855#1
226
227


M.domestica_TC4908#1
228
229


N.tabacum_TC21276#1
230
231


O.sativa_AK241632.1
232
233


O.taurii_36262#1
234
235


P.patens_202760#1
236
237


P.sitchensis_TA14105_3332#1
238
239


P.trichocarpa_552393#1
240
241


S.bicolor_Sb01g017450.1#1
242
243


S.moellendorffii_422148#1
244
245


T.pratense_TA1297_57577#1
246
247


V.vinifera_GSVIVT00026214001#1
248
249


Z.mays_TC447544#1
250
251









Research institutions, such as The Institute for Genomic Research (TIGR; beginning with TA), The Eukaryotic Gene Orthologs (EGO) database and The Joint Genome Institute may be used to identify further fibrillin sequences, either by a keyword search or by using the BLAST algorithm with the nucleic acid sequence or polypeptide sequence of interest.
1.4. PLATZ Polypeptides
Table A4 provides a list of nucleic acid and polypeptide sequences related to SEQ ID NO: 260 and SEQ ID NO: 261.







TABLE A4







Examples of PLATZ nucleic acids and polypeptides:












Nucleic





acid
Polypeptide




SEQ
SEQ



Name
ID NO:
ID NO:







P.trichocarpa_583639
260
261



A.thaliana_AT2G01818.1
276
277



A.thaliana_AT3G60670.1
278
279



Aquilegia_sp_TC23605
280
281



C.sinensis_TC11672
282
283



Chlorella_29200
284
285



Chlorella_50866
286
287



G.hirsutum_TC83646
288
289



G.hirsutum_TC86562
290
291



G.max_Glyma05g34370.1
292
293



G.max_Glyma07g27310.1
294
295



G.max_Glyma08g05270.1
296
297



G.max_Glyma11g03370.1
298
299



G.raimondii_TC4026
300
301



M.truncatula_AC142094_10.4
302
303



M.truncatula_AC195570_2.4
304
305



O.lucimarinus_29623
306
307



O.RCC809_43444
308
309



O.sativa_LOC_Os02g10000.1
310
311



O.sativa_LOC_Os03g12440.1
312
313



O.sativa_LOC_Os09g02790.1
314
315



P.trichocarpa_566415
316
317



P.trichocarpa_766209
318
319



S.bicolor_Sb01g033165.1
320
321



S.bicolor_Sb04g006330.1
322
323



S.moellendorffii_102589
324
325



V.vinifera_GSVIVT00007302001
326
327



V.vinifera_GSVIVT00026380001
328
329



A.stenosperma_TA310_217475
330
331



A.thaliana_AT2G12646.1
332
333



G.max_Glyma09g12330.1
334
335



G.max_Glyma13g23360.1
336
337



G.max_Glyma17g11470.1
338
339



M.truncatula_AC152347_6.5
340
341



N.tabacum_TC27363
342
343



O.sativa_LOC_Os02g09070.1
344
345



P.trichocarpa_779642
346
347



S.bicolor_Sb04g005680.1
348
349



T.aestivum_TC339412
350
351



V.vinifera_GSVIVT00005658001
352
353



Z.mays_376
354
355



A.thaliana_AT1G31040.1
356
357



Aquilegia_sp_TC28233
358
359



B.napus_TC69120
360
361



G.max_Glyma15g17040.1
362
363



I.nil_TC8897
364
365



O.sativa_LOC_Os02g07650.1
366
367



O.sativa_LOC_Os06g45540.1
368
369



P.sativum_AB045222
370
371



Pt_PLATZ4
372
373



S.bicolor_Sb04g004830.1
374
375



S.bicolor_Sb10g026620.1
376
377



S.officinarum_TC85346
378
379



V.vinifera_GSVIVT00030128001
380
381



Zea_mays_EU968977
382
383



C.japonica_AB254819
384
385



P.glauca_DR575106
386
387



P.glauca_TA21076_3330
388
389



P.pinaster_TA5209_71647
390
391



P.sitchensis_TA13012_3332
392
393



P.sitchensis_TA17699_3332
394
395



P.taeda_TA10616_3352
396
397



P.taeda_TA11415_3352
398
399



W.mirabilis_TA1807_3377
400
401



M.polymorpha_TA832_3197
402
403



P.patens_112207
404
405



P.patens_112256
406
407



S.moellendorffii_425306
408
409










In some instances, related sequences have tentatively been assembled and publicly disclosed by research institutions, such as The Institute for Genomic Research (TIGR; beginning with TA). 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. Further, access to proprietary databases, has allowed the identification of novel nucleic acid and polypeptide sequences.
1.5. PLST-Like Polypeptides
Table A5 provides a list of nucleic acid and polypeptide sequences related to SEQ ID NO: 410 and SEQ ID NO: 411.







TABLE A5







Examples of PLST-like nucleic acids and polypeptides:










Nucleic




acid
Polypeptide



SEQ
SEQ


Name
ID NO:
ID NO:





P.trichocarpa_scaff_I.715#1_PLST
410
411


A.thaliana_AT1G64640.1#1_PLST
412
413


B.napus_BN06MC03188_42122333@3181#1_PLST
414
415


C.canephora_TC195#1_PLST
416
417


G.hirsutum_DW500755#1_PLST
418
419


M.domestica_TC10680#1_PLST
420
421


V.vinifera_GSVIVT00000537001#1_PLST
422
423


G.max_Glyma13g10460.1#1_PLST
424
425


G.max_GM06MC08447_51315644@8367#1_PLST
426
427


T.erecta_SIN_01b-CS_Scarletade-7-M13.b1@1841#1_PLST
428
429


S.lycopersicum_TC196046#1_PLST
430
431


Triphysaria_sp_TC7525#1_PLST
432
433


H.vulgare_c62931576hv270303@6641#1_PLST
434
435


O.sativa_LOC_Os03g02400.1#1_PLST
436
437


Z.mays_ZM07MC02479_58582734@2470#1_PLST
438
439


P.glauca_TA13716_3330#1_PLST
440
441


A.thaliana_AT3G18590.1#1_P-class
442
443


N.tabacum_NP916758#1_P-class
444
445


G.max_Glyma05g07790.1#1_P-class
446
447


N.tabacum_TC30930#1_P-class
448
449


V.vinifera_GSVIVT00020596001#1_P-class
450
451


A.thaliana_AT1G79800.1#1_P-class
452
453


L.virosa_DW147584#1_P-Class
454
455


A.thaliana_AT2G23990.1#1_NDL
456
457


A.thaliana_AT2G25060.1#1_NDL
458
459


A.thaliana_AT4G31840.1#1_NDL
460
461


C.sativus_CK085664#1_NDL
462
463


C.sinensis_TC4954#1_NDL
464
465


P.trichocarpa_669166#1_NDL
466
467


L.japonicus_TC34883#1_NDL
468
469


P.persica_TC6359#1_NDL
470
471


F.vesca_TA9813_57918#1_NDL
472
473


M.truncatula_AC143339_7.5#1_NDL
474
475


S.henryi_TA1001_13258#1_NDL
476
477


M.crystallinum_TC7817#1_NDL
478
479


A.thaliana_AT3G20570.1#1_NDL
480
481


M.truncatula_AC202319_3.4#1_NDL
482
483


H.paradoxus_TA4880_73304#1_NDL
484
485


L.perennis_DW075689#1_NDL
486
487


S.tuberosum_TC170270#1_NDL
488
489


V.vinifera_GSVIVT00027380001#1_NDL
490
491


P.trichocarpa_571501#1_NDL
492
493


R.communis_TA4149_3988#1_NDL
494
495


M.crystallinum_TC10463#1_NDL
496
497


Z.officinale_TA6894_94328#1_NDL
498
499


O.sativa_LOC_Os01g57880.1#1_NDL
500
501


S.bicolor_Sb04g004360.1#1_NDL
502
503


C.clementina_TC6769#1_Oz
504
505


H.brasiliensis_TA329_3981#1_Oz
506
507


L.japonicus_TC35544#1_Oz
508
509


H.tuberosus_TA3846_4233#1_Oz
510
511


H.exilis_TA3895_400408#1_Oz
512
513


T.aestivum_TC309777#1_Oz
514
515


H.ciliaris_TA2829_73280#1_Oz
516
517


Triphysaria_sp_TC728#1_Oz
518
519


V.vinifera_GSVIVT00023717001#1_Oz
520
521


N.benthamiana_TC12249#1_Oz
522
523


C.sinensis_TC10549#1_Oz
524
525


M.polymorpha_BJ870068#1_Oz
526
527


B.vulgaris_BQ584719#1_Oz
528
529









In some instances, related sequences have tentatively been assembled and publicly disclosed by research institutions, such as The Institute for Genomic Research (TIGR; beginning with TA). 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. Further, access to proprietary databases, has allowed the identification of novel nucleic acid and polypeptide sequences.
1.6. Glomalin Polypeptides
Table A6 provides a list of nucleic acid and polypeptide sequences related to SEQ ID NO: 545 and SEQ ID NO: 546.







TABLE A6







Examples of Glomalin nucleic acids and polypeptides:










Nucleic




acid
Polypeptide



SEQ
SEQ


Name
ID NO:
ID NO:





O.sativa_glomalin
545
546


A.cepa_CF435092
547
571


A.thaliana_AT3G13860.1
548
572


A.thaliana_AT2G33210.1
549
573


A.thaliana_AT3G23990.1
550
574


M.truncatula_AC161864_24.5
551
575


M.truncatula_AC161864_3.5
552
576


O.sativa_LOC_Os05g46290.1
553
577


O.sativa_LOC_Os10g32550.1
554
578


O.sativa_LOC_Os03g04970.1
555
579


P.patens_226792
556
580


P.patens_233067
557
581


P.patens_163173
558
582


P.patens_56767
559
583


P.trichocarpa_scaff_I.447
560
584


P.trichocarpa_scaff_III.1436
561
585


P.trichocarpa_scaff_44.102
562
586


P.trichocarpa_scaff_II.2576
563
587


S.lycopersicum_TC204816
564
588


S.lycopersicum_TC197855
565
589


S.lycopersicum_TC192865
566
590


Lesculentum_gl_39
567
591


T.aestivum_c54647991@12535
568
592


Z.mays_ZM07MC32795_BFb0311C05@32697
569
593


Z.mays_ZM07MC22894_BFb0229L19@22830
570
594









Sequences have been tentatively assembled and publicly disclosed by research institutions, such as The Institute for Genomic Research (TIGR; beginning with TA). 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. Special nucleic acid sequence databases have been created for particular organisms, such as by the Joint Genome Institute. Furthermore, access to proprietary databases, has allowed the identification of novel nucleic acid and polypeptide sequences.
Example 2
Alignment of Sequences Related to the Polypeptide Sequences Used in the Methods of the Invention
2.1. eRF1 Polypeptides
Alignment of polypeptide sequences was performed using the ClustalW 2.0 algorithm of progressive alignment (Thompson et al. (1997) Nucleic Acids Res 25:4876-4882; Chenna et al. (2003). Nucleic Acids Res 31:3497-3500) with standard setting (slow alignment, similarity matrix: Gonnet (or Blosum 62 (if polypeptides are aligned), gap opening penalty 10, gap extension penalty: 0.2).
A phylogenetic tree of eRF1 polypeptide (FIG. 1) was constructed using a neighbour-joining clustering algorithm as provided in the AlignX programme from the Vector NTI (Invitrogen).
2.2. SCAMP-Like Polypeptides
Alignment of polypeptide sequences was performed using the Clustal W1.8 algorithm of progressive alignment (Thompson et al. (1997) Nucleic Acids Res 25:4876-4882; Chenna et al. (2003). Nucleic Acids Res 31:3497-3500) with the setting: gap opening penalty 10, gap extension penalty: 0.2). Minor manual editing was done to further optimise the alignment. The SCAMP-LIKE polypeptides are aligned in FIG. 3.
2.3. Fibrillin Polypeptides
Alignment of polypeptide sequences was performed using the AlignX programme from the Vector NTI (Invitrogen) with standard setting. Minor manual editing was done to further optimise the alignment. The fibrillin polypeptides are aligned in FIG. 5.
A phylogenetic tree of fibrillin polypeptides (FIG. 6) was constructed using a neighbour-joining clustering algorithm as provided in the AlignX programme from the Vector NTI (Invitrogen).
2.4. PLATZ Polypeptides
Alignment of polypeptide sequences was performed using the ClustalW 2.0 algorithm of progressive alignment (Thompson et al. (1997) Nucleic Acids Res 25:4876-4882; Chenna et al. (2003). Nucleic Acids Res 31:3497-3500) with standard setting (slow alignment, similarity matrix: Gonnet, gap opening penalty 10, gap extension penalty: 0.2). Minor manual editing was done to further optimise the alignment. The PLATZ polypeptides are aligned in FIG. 11.
This alignment can be used for determining conserved signature sequences of about 5 to 10 amino acids in length. Preferably the conserved regions of the proteins are used, recognisable by the asterisks (identical residues), the colons (highly conserved substitutions) and the dots (conserved substitutions).
A phylogenetic tree of PLATZ polypeptides (FIG. 12) was constructed using MAFFT (Katoh and Toh (2008) Briefings in Bioinformatics 9:286-298). A neighbour-joining tree was calculated using QuickTree (Howe et al. (2002), Bioinformatics 18(11): 1546-7), 100 bootstrap repetitions. The circular phylogram was drawn using Dendroscope (Huson et al. (2007), BMC Bioinformatics 8(1):460). Confidence for 100 bootstrap repetitions is indicated for major branching.
2.5. PLST-Like Polypeptides
Alignment of polypeptide sequences was performed using the ClustalW 2.0 algorithm of progressive alignment (Thompson et al. (1997) Nucleic Acids Res 25:4876-4882; Chenna et al. (2003). Nucleic Acids Res 31:3497-3500) with standard setting (slow alignment, similarity matrix: Gonnet (or Blosum 62 (if polypeptides are aligned), gap opening penalty 10, gap extension penalty: 0.2).
A phylogenetic tree of PLST-like polypeptide (FIG. 15) was constructed using a neighbour-joining clustering algorithm as provided in the AlignX programme from the Vector NTI (Invitrogen).
2.6. Glomalin Polypeptides
Alignment of polypeptide sequences was performed using the ClustalW 2.0 algorithm of progressive alignment (Thompson et al. (1997) Nucleic Acids Res 25:4876-4882; Chenna et al. (2003). Nucleic Acids Res 31:3497-3500) with standard setting (slow alignment, similarity matrix: Gonnet, gap opening penalty 10, gap extension penalty: 0.2). Minor manual editing was done to further optimise the alignment. The Glomalin polypeptides are aligned in FIG. 18.
A phylogenetic tree of Glomalin polypeptides (FIG. 19) was constructed using a neighbour-joining clustering algorithm as provided in the AlignX programme from the Vector NTI (Invitrogen).
Example 3
Calculation of Global Percentage Identity Between Polypeptide Sequences Useful in Performing the Methods of the Invention
3.1. eRF1 Polypeptides
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 B1 for the global similarity and identity over the full length of the polypeptide sequences. Percentage identity is given above the diagonal in bold and percentage similarity is given below the diagonal (normal face).
The percentage identity between the eRF1 polypeptide sequences useful in performing the methods of the invention can be as low as 49% amino acid identity compared to SEQ ID NO: 2.







TABLE B1





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


























1
2
3
4
5
6
7
8
9
10





 1. A.thaliana_AT1G12920.1

95.00
86.00
88.00
89.00
89.00
89.00
90.00
89.00
91.00


 2. A.thaliana_AT3G26618.1


86.00
90.00
91.00
90.00
90.00
93.00
90.00
92.00


 3. A.thaliana_AT5G47880.1



87.00
87.00
87.00
89.00
88.00
86.00
85.00


 4. Aquilegia_sp_TC23338




91.00
92.00
91.00
93.00
90.00
92.00


 5. C.sinensis_TC9326





94.00
92.00
96.00
92.00
92.00


 6. C.solstitialis_TA682_347529






93.00
94.00
92.00
91.00


 7. G.max_Glyma09g29600.1







93.00
91.00
91.00


 8. G.max_GM06MC33657_sm55b10








92.00
95.00


 9. H.vulgare_c64960768hv270303









89.00


10. M.truncatula_AC136505_11.4


11. O.sativa_LOC_Os01g71270.1


12. O.sativa_LOC_Os03g49580.1


13. P.glauca_TA15071_3330


14. P.patens_58108


15. P.trichocarpa_708902


16. P.trichocarpa_732023


17. S.lycopersicum_TC197021


18. S.tuberosum_TC166984


19. V.vinifera_GSVIVT00014642001






















11
12
13
14
15
16
17
18
19







 1. A.thaliana_AT1G12920.1
85.00
90.00
91.00
87.00
91.00
91.00
87.00
89.00
90.00



 2. A.thaliana_AT3G26618.1
85.00
91.00
93.00
88.00
91.00
93.00
88.00
90.00
91.00



 3. A.thaliana_AT5G47880.1
84.00
87.00
87.00
87.00
88.00
87.00
84.00
89.00
88.00



 4. Aquilegia_sp_TC23338
85.00
92.00
92.00
87.00
92.00
92.00
88.00
90.00
92.00



 5. C.sinensis_TC9326
87.00
95.00
95.00
89.00
92.00
95.00
89.00
90.00
94.00



 6. C.solstitialis_TA682_347529
86.00
93.00
94.00
89.00
93.00
94.00
89.00
91.00
94.00



 7. G.max_Glyma09g29600.1
86.00
92.00
93.00
89.00
94.00
93.00
88.00
92.00
94.00



 8. G.max_GM06MC33657_sm55b10
87.00
94.00
95.00
90.00
94.00
96.00
91.00
92.00
95.00



 9. H.vulgare_c64960768hv270303
87.00
95.00
92.00
87.00
91.00
92.00
87.00
90.00
90.00



10. M.truncatula_AC136505_11.4
86.00
91.00
93.00
88.00
92.00
94.00
89.00
90.00
93.00



11. O.sativa_LOC_Os01g71270.1

87.00
87.00
83.00
87.00
86.00
84.00
87.00
86.00



12. O.sativa_LOC_Os03g49580.1


93.00
89.00
93.00
93.00
89.00
92.00
92.00



13. P.glauca_TA15071_3330



91.00
93.00
95.00
90.00
91.00
95.00



14. P.patens_58108




89.00
90.00
86.00
88.00
89.00



15. P.trichocarpa_708902





94.00
89.00
92.00
94.00



16. P.trichocarpa_732023






90.00
91.00
96.00



17. S.lycopersicum_TC197021







87.00
90.00



18. S.tuberosum_TC166984








91.00



19. V.vinifera_GSVIVT00014642001










3.2. SCAMP-Like Polypeptides

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 typically used in the comparison are:

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

3.3. Fibrillin Polypeptides

    
    


Global percentages of similarity and identity between full length polypeptide sequences useful in performing the methods of the invention were determined using 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 B2 for the global similarity and identity over the full length of the polypeptide sequences. Percentage identity is given above the diagonal and percentage similarity is given below the diagonal.







TABLE B2





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































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





 1. L.esculentum_QC

61
61
31
61
30
28
62
62
62
61
61
61
89
53


 2. A.thaliana_AT2G46910.1
78

89
31
62
29
26
68
61
61
67
60
65
64
58


 3. B.napus_BN06MC20042_46499279
77
93

32
62
29
27
67
61
61
65
61
65
63
59


 4. C.reinhardtii_190008
50
51
50

32
30
29
34
30
30
33
31
30
32
32


 5. C.solstitialis_TA2061_347529
78
79
78
52

31
28
62
64
64
61
80
63
64
54


 6. C.vulgaris_102074
47
45
46
47
45

43
28
29
29
28
28
28
28
28


 7. Chlorella_141300
45
44
44
41
43
63

30
26
26
29
27
28
28
26


 8. G.hirsutum_TC97719
77
81
80
53
76
46
45

67
66
99
61
73
61
61


 9. G.max_Glyma07g00410.1
78
78
76
51
78
47
43
79

99
65
66
70
62
59


10. G.max_GM06MC19234_59694709
78
77
75
51
78
46
43
78
100

65
65
69
62
58


11. G.raimondii_TC7628
77
80
79
53
75
45
44
99
78
77

59
72
60
59


12. L.virosa_DW148855
77
77
77
52
88
43
42
75
79
79
74

64
63
52


13. M.domestica_TC4908
77
82
82
50
76
45
46
85
82
81
84
77

62
61


14. N.tabacum_TC21276
93
80
80
51
79
47
45
77
80
80
76
78
78

55


15. O.sativa_AK241632.1
71
71
73
50
71
41
42
72
73
72
71
71
75
72


16. O.taurii_36262
48
50
50
48
51
40
38
52
49
48
52
49
52
48
51


17. P.patens_202760
55
57
56
45
55
34
36
57
56
55
56
56
58
57
57


18. P.sitchensis_TA14105_3332
63
66
66
48
66
36
34
64
65
64
63
66
66
63
66


19. P.trichocarpa_552393
64
69
67
43
62
38
35
68
64
64
67
62
69
64
61


20. S.bicolor_Sb01g017450.1
70
73
72
54
72
42
42
75
72
72
74
71
74
73
87


21. S.moellendorffii_422148
63
63
66
49
65
51
49
62
65
64
61
63
64
67
60


22. T.pratense_TA1297_57577
78
79
76
48
74
42
41
77
82
82
76
77
79
79
73


23. V.vinifera_GSVIVT00026214001
76
78
78
49
76
47
43
80
77
77
79
75
80
77
72


24. Z.mays_TC447544
69
71
70
51
70
40
41
72
71
70
71
71
73
71
85


25. AT4G04020.1 Symbols: FIB FIB
37
40
41
41
37
31
28
40
38
37
40
39
41
37
41


(FIBRILLIN); structural molecule


chr4: 1932159-1933544


26. AT4G22240.1 Symbols: plastid-
39
45
42
40
40
31
29
43
40
39
43
43
43
40
42


lipid associated protein PAP,


putative chr4: 11766102-


27. AT3G23400.1 Symbols: plastid-
40
40
39
39
40
29
29
40
41
41
40
40
42
41
39


lipid associated protein PAP/


fibrillin family protein


28. AT2G35490.1 Symbols: plastid-
33
35
36
32
35
24
26
36
35
35
37
36
35
34
33


lipid associated protein PAP,


putative chr2: 14919388-


29. AT3G58010.1 Symbols: Identical
29
29
27
27
29
23
21
27
30
30
27
28
31
29
31


to Probable plastid-lipid-associated


protein 9,


30. AT2G42130.3 Symbols: Identical
31
28
28
31
29
24
26
29
27
29
28
29
31
31
29


to Probable plastid-lipid-associated


protein 13,






16
17
18
19
20
21
22
23
24
25
26
27
28
29
30





 1. L.esculentum_QC
33
38
48
51
54
47
60
62
52
20
19
21
19
15
16


 2. A.thaliana_AT2G46910.1
33
39
48
56
60
45
62
63
57
23
26
23
22
16
15


 3. B.napus_BN06MC20042_46499279
34
39
49
54
59
48
60
63
58
24
25
23
20
17
16


 4. C.reinhardtii_190008
34
30
31
29
35
35
28
30
32
23
22
23
17
14
19


 5. C.solstitialis_TA2061_347529
37
37
50
54
56
51
59
62
54
23
24
23
20
16
16


 6. C.vulgaris_102074
28
24
26
24
27
34
28
27
27
17
17
17
14
13
12


 7. Chlorella_141300
27
23
22
23
27
33
25
26
26
17
17
19
17
12
16


 8. G.hirsutum_TC97719
35
40
47
59
62
47
64
69
60
22
23
23
22
15
16


 9. G.max_Glyma07g00410.1
34
39
47
55
57
46
74
66
56
22
22
23
21
16
16


10. G.max_GM06MC19234_59694709
34
39
46
54
57
45
73
66
55
22
22
23
20
17
16


11. G.raimondii_TC7628
34
41
46
58
61
45
63
67
58
21
22
22
22
16
15


12. L.virosa_DW148855
34
38
50
52
55
49
61
62
53
22
24
23
21
15
16


13. M.domestica_TC4908
35
41
49
56
62
48
66
73
61
22
23
24
22
17
15


14. N.tabacum_TC21276
33
38
48
52
56
50
61
63
55
21
22
22
20
16
15


15. O.sativa_AK241632.1
34
42
50
49
80
46
57
59
79
25
23
23
21
18
17


16. O.taurii_36262

31
32
29
36
31
34
35
34
21
20
23
21
17
16


17. P.patens_202760
48

43
34
41
42
39
40
42
20
21
20
19
16
15


18. P.sitchensis_TA14105_3332
49
58

40
52
45
48
49
51
22
23
21
22
16
16


19. P.trichocarpa_552393
48
55
59

48
38
51
57
47
17
18
20
19
17
15


20. S.bicolor_Sb01g017450.1
52
56
64
61

48
56
59
90
22
24
25
21
18
16


21. S.moellendorffii_422148
47
57
58
50
60

44
47
46
22
23
20
18
15
14


22. T.pratense_TA1297_57577
51
56
66
66
75
62

60
54
20
21
24
20
15
16


23. V.vinifera_GSVIVT00026214001
51
54
65
67
72
63
75

57
21
24
24
19
18
16


24. Z.mays_TC447544
50
57
64
60
92
60
69
69

22
24
25
21
19
18


25. AT4G04020.1 Symbols: FIB FIB
39
39
42
37
39
37
40
38
40

75
22
39
19
20


(FIBRILLIN); structural molecule


chr4: 1932159-1933544


26. AT4G22240.1 Symbols: plastid-
40
40
41
38
43
39
40
42
43
83

23
38
16
18


lipid associated protein PAP,


putative chr4: 11766102-


27. AT3G23400.1 Symbols: plastid-
40
36
38
35
42
35
41
41
41
37
39

25
18
15


lipid associated protein PAP/


fibrillin family protein


28. AT2G35490.1 Symbols: plastid-
35
36
37
35
34
30
35
35
35
53
51
35

18
19


lipid associated protein PAP,


putative chr2: 14919388-


29. AT3G58010.1 Symbols: Identical
31
31
29
31
29
26
33
28
29
34
32
31
34

67


to Probable plastid-lipid-associated


protein 9,


30. AT2G42130.3 Symbols: Identical
31
32
29
31
28
26
33
29
30
37
32
30
35
79


to Probable plastid-lipid-associated


protein 13,









A MATGAT table for local alignment of a specific domain for example over the PAP fibrillin domain or over the C-terminal domain, or data on % identity/similarity between specific domains may also be performed.
3.4. PLATZ Polypeptides
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 B3 for the global similarity and identity over the full length of the polypeptide sequences. Percentage identity is given above the diagonal in bold and percentage similarity is given below the diagonal (normal face).
The percentage identity between the PLATZ-A1-α polypeptide sequences useful in performing the methods of the invention can be as low as 65% amino acid identity compared to SEQ ID NO: 261.







TABLE B3







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






















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

























 1. AsTA310_217475

55.4
67.1
65.0
66.7
62.7
55.6
57.2
65.2
64.8
57.4
56.7
62.9
57.1


 2. AT2G12646.1
65.6

66.3
68.3
68.3
65.8
63.7
62.1
72.3
70.7
60.9
62.2
65.9
61.4


 3. Gm09g12330.1
74.5
80.5

87.4
86.7
81.0
71.4
72.0
81.3
80.5
72.8
70.4
81.3
72.1


 4. Gm13g23360.1
72.5
82.8
90.3

98.0
86.5
74.6
70.0
82.1
81.7
70.9
68.1
83.7
69.8


 5. Gm17g11470.1
72.6
82.4
90.3
98.4

86.5
74.6
69.8
82.1
81.7
70.6
67.9
82.5
69.5


 6. MtAC152347_6.5
70.2
80.1
86.9
92.9
92.9

72.5
67.1
77.0
77.0
67.5
65.6
75.7
66.8


 7. NtTC27363
63.2
81.3
79.4
83.8
83.4
83.8

69.6
75.9
75.9
69.4
66.8
75.3
68.8


 8. Os02g09070.1
69.4
76.6
82.4
82.2
81.9
79.4
79.4

72.8
72.4
94.9
91.1
71.8
93.2


 9. Pt583639
70.8
85.5
88.1
88.7
88.7
86.5
84.6
83.1

93.4
71.4
70.3
82.4
70.7


10. Pt779642
70.0
84.0
86.4
87.9
87.9
86.1
84.6
81.9
96.3

71.0
71.1
83.3
70.3


11. Sb04g005680.1
68.6
77.0
83.7
83.0
82.7
80.2
80.6
97.0
82.3
81.5

90.7
73.1
95.4


12. TaTC339412
66.7
78.1
82.0
81.8
81.9
79.4
78.3
94.5
83.5
82.3
94.9

70.3
91.1


13. VvT00005658001
70.2
81.3
85.7
91.9
91.1
86.9
84.6
82.0
89.8
89.0
84.1
81.6

72.8


14. Zm376
66.7
77.0
82.8
82.2
82.3
79.8
79.4
95.4
82.3
81.5
96.6
95.4
82.9









3.5. PLST-Like Polypeptides

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 B4 for the global similarity and identity over the full length of the polypeptide sequences. Percentage identity is given above the diagonal in bold and percentage similarity is given below the diagonal (normal face).
The percentage identity between the PLST-like polypeptide sequences useful in performing the methods of the invention can be as low as 49% amino acid identity compared to SEQ ID NO: 411.







TABLE B4







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
























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



























 1. A.thaliana_AT1G64640.1#1

81
45
47
51
49
51
48
39
45
47
49
32
41
42
38


 2. B.napus_BN06MC03188_42122333


44
50
56
49
53
50
41
47
46
47
32
39
38
38


 3. C.canephora_TC195#1



57
59
61
55
53
45
48
57
50
30
36
39
41


 4. G.hirsutum_DW500755#1




68
67
65
53
46
51
56
56
35
36
38
41


 5. M.domestica_TC10680#1





71
67
61
45
52
57
57
33
37
38
40


 6. V.vinifera_GSVIVT00000537001#1






71
56
43
53
57
54
31
36
36
40


 7. P.trichocarpa_scaff_I.715#1







59
48
54
56
58
32
37
37
39


 8. G.max_Glyma13g10460.1#1








50
45
52
51
30
33
34
38


 9. G.max_GM06MC08447_51315644









39
40
45
24
27
28
30


10. T.erecta_SIN_01b-CS_Scarletade-7-M13.b1










47
49
32
32
31
37


11. S.lycopersicum_TC196046#1











53
30
37
36
39


12. Triphysaria_sp_TC7525#1












30
36
32
41


13. H.vulgare_c62931576hv270303













40
39
30


14. O.sativa_LOC_Os03g02400.1#1














66
38


15. Z.mays_ZM07MC02479_58582734















37


16. P.glauca_TA13716_3330#1









3.6. Glomalin Polypeptides

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. Percentage identity is given above the diagonal in bold and percentage similarity is given below the diagonal (normal face).
The sequence identity (in %) between the Glomalin polypeptide sequences useful in performing the methods of the invention is generally higher than 50% compared to SEQ ID NO: 546.







TABLE B5





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




























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





 1. S.lycopersicum_TC192865

77.1
76.8
76.4
75.4
69.3
26.8
67.4
65.9
70.1
83.6
87.4


 2. P.patens_226792
89.1

92.4
89.3
83.5
67.5
26.3
64.4
63.4
66.1
76.1
76.9


 3. P.patens_233067
89.1
97.9

89.3
84.2
66.4
25.5
64.7
63.7
65.8
76.1
76.9


 4. P.patens_163173
87.7
95.2
95.5

83.3
67.5
26.4
65.7
64.3
66.8
75.6
77.2


 5. P.patens_56767
87.6
92.9
92.8
91.4

66.0
25.3
64.8
64.3
66.5
74.0
75.0


 6. A.thaliana_AT3G13860.1
82.9
81.7
80.9
81.4
80.3

31.2
69.2
69.2
79.4
67.6
70.5


 7. P.trichocarpa_sc_44.102
33.2
32.4
32.2
31.7
30.7
36.2

27.2
26.3
30.6
26.3
27.2


 8. O.sativa_Os05g46290
84.3
82.3
82.6
82.6
81.9
85.2
34.3

88.9
71.3
66.2
68.2


 9. Z.mays_ZM07MC22894
83.8
81.0
81.2
81.0
81.4
85.2
32.9
95.4

71.4
65.4
68.1


10. S.lycopersicum_TC204816
84.1
82.4
82.1
82.4
81.9
89.5
35.4
86.4
85.9

68.7
72.3


11. A.thaliana_AT2G33210
91.6
87.7
88.0
86.2
85.6
81.9
32.1
82.2
81.0
81.4

86.0


12. A.thaliana_AT3G23990
94.6
89.4
89.1
88.6
87.8
83.7
33.4
85.4
84.1
84.9
92.5


13. L.esculentum_gl_39
99.8
88.9
89.0
87.6
87.4
82.7
33.2
84.2
83.6
84.3
91.5
94.5


14. S.lycopersicum_TC197855
95.2
89.1
88.3
87.1
86.7
83.9
33.5
84.9
83.4
84.8
91.6
95.8


15. A.cepa_CF435092
24.5
23.0
22.6
22.1
22.4
21.0
49.0
21.9
20.3
21.5
23.8
24.8


16. O.sativa_Os10g32550.1
85.5
81.5
82.0
80.1
80.1
76.3
30.8
77.8
76.7
77.8
83.8
86.8


17. O.sativa_glomalin_SEQID2
93.6
89.4
89.7
88.3
87.6
83.8
33.4
85.0
83.8
85.7
90.8
95.3


18. Z.mays_ZM07MC32795
93.1
89.3
89.0
87.7
87.1
83.3
33.3
84.2
82.8
85.1
90.8
94.5


19. O.sativa_Os03g04970.1
93.1
89.4
89.1
87.2
86.6
82.8
32.8
83.6
82.8
84.1
90.1
94.5


20. T.aestivum_c54647991
93.4
89.3
89.0
87.3
87.2
82.3
33.3
82.6
81.9
83.9
90.1
93.8


21. P.trichocarpa_sc_I.447
56.0
52.4
52.8
52.5
51.7
49.8
56.9
50.6
48.4
51.3
55.0
56.7


22. P.trichocarpa_sc_III.1436
90.5
84.8
85.2
83.8
82.8
81.3
32.0
81.1
80.2
80.8
88.2
91.7


23. M.truncatula_AC161864_24
94.6
89.1
89.5
88.7
88.3
84.3
33.4
85.5
84.0
85.5
92.1
96.0


24. M.truncatula_AC161864_3
94.1
88.9
89.0
88.4
87.9
83.2
33.0
84.3
82.9
84.2
92.0
95.5


25. P.trichocarpa_scaff_II.2576
93.4
88.9
87.9
87.7
86.4
83.7
33.2
84.7
83.1
84.2
89.9
93.9
























13
14
15
16
17
18
19
20
21
22
23
24
25





 1. S.lycopersicum_TC192865
99.8
88.6
22.1
76.7
84.8
83.3
83.4
83.2
50.6
81.7
85.3
83.6
84.7


 2. P.patens_226792
76.9
76.0
19.5
69.3
76.7
75.9
75.5
75.6
43.5
72.5
75.8
76.3
75.9


 3. P.patens_233067
76.6
75.6
18.9
69.4
76.8
75.5
75.0
75.0
43.2
73.3
76.6
76.6
75.1


 4. P.patens_163173
76.1
75.0
19.4
69.4
76.7
75.6
74.6
75.0
43.9
72.9
76.7
76.5
76.4


 5. P.patens_56767
75.3
73.9
18.9
68.1
75.1
73.5
72.9
73.9
43.3
71.5
75.6
74.6
74.1


 6. A.thaliana_AT3G13860.1
69.1
70.2
18.2
63.6
69.7
69.2
68.3
68.4
42.6
68.1
71.5
69.6
70.7


 7. P.trichocarpa_sc_44.102
26.8
27.2
38.6
25.6
27.4
27.4
27.2
27.1
47.2
25.6
27.7
27.1
26.3


 8. O.sativa_Os05g46290
67.2
66.6
18.6
62.2
68.7
68.4
67.8
67.9
40.1
66.3
67.8
65.8
68.9


 9. Z.mays_ZM07MC22894
65.8
65.3
17.2
62.2
68.4
67.5
67.0
68.0
38.4
65.7
67.6
65.4
67.6


10. S.lycopersicum_TC204816
70.1
72.5
18.8
66.3
73.2
71.8
70.5
70.0
44.1
69.5
72.2
70.3
70.0


11. A.thaliana_AT2G33210
83.4
84.1
21.7
75.7
83.2
82.8
82.4
81.2
49.2
80.9
85.0
83.6
82.4


12. A.thaliana_AT3G23990
87.2
89.1
22.3
79.5
87.9
85.9
85.8
84.6
51.7
84.4
89.4
87.7
85.8


13. L.esculentum_gl_39

88.4
22.1
76.5
84.6
83.1
83.2
83.1
50.6
81.5
85.1
83.4
84.5


14. S.lycopersicum_TC197855
95.0

22.5
78.9
87.3
85.8
85.3
84.2
51.7
84.0
87.6
85.2
85.6


15. A.cepa_CF435092
24.5
25.1

21.0
23.2
23.6
22.7
23.3
38.7
23.8
22.6
22.4
22.6


16. O.sativa_Os10g32550.1
85.3
85.8
22.7

90.4
86.6
85.9
83.0
47.3
77.3
79.8
77.2
78.1


17. O.sativa_glomalin_SEQID2
93.4
94.8
25.1
90.5

95.5
94.6
91.5
52.4
85.2
88.0
85.4
86.4


18. Z.mays_ZM07MC32795
92.9
93.9
25.3
88.5
97.4

93.1
90.3
51.4
85.2
88.0
85.1
85.8


19. O.sativa_Os03g04970.1
92.9
94.1
24.6
88.6
97.4
96.5

91.2
51.3
83.4
85.6
83.6
85.8


20. T.aestivum_c54647991
93.3
93.4
25.0
87.9
96.7
96.2
96.2

50.3
82.5
85.8
83.6
83.5


21. P.trichocarpa_sc_I.447
56.0
57.6
41.6
50.6
55.9
55.2
54.8
54.7

52.1
51.7
50.2
50.1


22. P.trichocarpa_sc_III.1436
90.3
92.1
25.6
82.0
90.6
90.5
89.4
89.2
56.0

85.9
83.0
82.6


23. M.truncatula_AC161864_24
94.5
95.8
24.7
86.6
95.6
94.4
93.8
93.9
57.1
92.2

94.8
88.2


24. M.truncatula_AC161864_3
94.0
94.6
24.7
86.0
94.6
93.9
93.9
93.4
56.6
91.1
98.1

86.3


25. P.trichocarpa_scaff_II.2576
93.3
94.3
25.0
84.9
93.6
92.5
93.1
92.2
55.5
89.6
95.1
94.4









Example 4
Identification of Domains Comprised in Polypeptide Sequences Useful in Performing the Methods of the Invention
4.1. eRF1 Polypeptides
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. Pfam is a large collection of multiple sequence alignments and hidden Markov models covering many common protein domains and families. Pfam is hosted at the Sanger Institute server in the United Kingdom. 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 C1.







TABLE C1







InterPro scan results (major accession numbers) of the


polypeptide sequence as represented by SEQ ID NO: 2.















Location






(amino acid


Interpro ID
Domain name
Domain ID
Short Name
coordinates)





IPR004403
Peptide chain release factor
PANTHER
eRF1
 1-436



eRF/aRF subunit 1
PTHR10113




TIGRFAMs
eRF1
 4-422




TIGR00108


IPR005140
eRF1 domain 1
PFAM
eRF1_1
 6-140




PF03463


IPR005141
eRF1 domain 2
PFAM
eRF1_2
144-278




PF03464


IPR005142
eRF1 domain 3
PFAM
eRF1_3
281-418




PF03465


unintegrated
unintegrated
GENE3D
—
279-436




G3DSA:3.30.1330.30




GENE3D
—
142-278




G3DSA:3.30.420.60




GENE3D
—
 27-131




G3DSA:3.30.960.10




PANTHER
—
 1-436




PTHR10113:SF1




SUPERFAMILY
—
142-277




SSF53137




SUPERFAMILY
—
278-423




SSF53135




SUPERFAMILY
—
 4-141




SSF55481









4.2. SCAMP-Like Polypeptides

Pfam is a large collection of multiple sequence alignments and hidden Markov models covering many common protein domains and families. Pfam is hosted at the Sanger Institute server in the United Kingdom.
The results of the Pfam scan for conserved HMM PFam domains of the polypeptide sequence as represented by SEQ ID NO: 89 are presented in Table C2.







TABLE C2







InterPro scan results (major accession numbers) of the


polypeptide sequence as represented by SEQ ID NO: 89.













Amino acid coordinates



Database
Accession number
on SEQ ID NO: 89







Pfam
PF044144
91-265










4.3. Fibrillin Polypeptides

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. Pfam is a large collection of multiple sequence alignments and hidden Markov models covering many common protein domains and families. Pfam is hosted at the Sanger Institute server in the United Kingdom. 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: 205 are presented in Table C3.







TABLE C3







InterPro scan results (major accession numbers) of the polypeptide


sequence as represented by SEQ ID NO: SEQ ID NO: 205.











InterPRO accesssion
Database
Entry name







InterPro PAP Fibrillin
PFAM PF04755
Pap fibrillin



Family IPR006843



No IPR integrated
SignalP
Signal Peptide










4.4. PLATZ Polypeptides

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. Pfam is a large collection of multiple sequence alignments and hidden Markov models covering many common protein domains and families. Pfam is hosted at the Sanger Institute server in the United Kingdom. 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: 261 are presented in Table C4.







TABLE C4







InterPro scan results (major accession numbers) of the


polypeptide sequence as represented by SEQ ID NO: 261.















Amino acid




Accession
Accession
coordinates on



Database
number
name
SEQ ID NO 261







HMMPfam
PF04640
PLATZ
21-131 (2e−67)










4.5. PLST-Like Polypeptides

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. Pfam is a large collection of multiple sequence alignments and hidden Markov models covering many common protein domains and families. Pfam is hosted at the Sanger Institute server in the United Kingdom. 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: 411 are presented in Table C5.







TABLE C5







InterPro scan results (major accession numbers) of the


polypeptide sequence as represented by SEQ ID NO: 411.















Location






(amino acid


Interpro ID
Domain ID
Domain ID
Short Name
coordinates)





IPR003245
PFAM
Plastocyanin-like
Cu_bind_like
38-124



PF02298


IPR008972
GENE3D
Cupredoxin
—
25-134



GSDA:2.60.40.420



SUPERFAMILY
Cupredoxins
Cupredoxins
25-134



SSF49503


Unintegrated
PRODOM
Q6NLD7_ARATH_Q6NLD7;

60-130



PD003122



SignalP
signal-peptide
signal-peptide
1-27



TMHMM
Tmhmm
transmembrane_regions
170-190 









4.6. Glomalin Polypeptides

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. Pfam is a large collection of multiple sequence alignments and hidden Markov models covering many common protein domains and families. Pfam is hosted at the Sanger Institute server in the United Kingdom. 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: 546 are presented in Table C6.







TABLE C6







InterPro scan results (major accession numbers) of the


polypeptide sequence as represented by SEQ ID NO: 546.













Amino acid



Accession
Accession
coordinates on


Database
number
name
SEQ ID NO: 546














InterPro
IPR001844
Chaperonin Cpn60




PRINTS
PR00298
CHAPERONIN60
1.7E−71
[118-144]T





1.7E−71
[174-201]T





1.7E−71
[359-382]T





1.7E−71
[441-466]T





1.7E−71
[489-510]T


TIGRFAMs
TIGR02348
GroEL
0.0
[94-619]T


InterPro
IPR002423
Chaperonin Cpn60/TCP-1


PANTHER
PTHR11353
Cpn60/TCP-1
0.0
[77-633]T


PFAM
PF00118
Cpn60_TCP1
0.0
[114-617]T


SUPERFAMILY
SSF48592
GroEL-ATPase
1.4E−74
[101-614]T


InterPro
IPR018370
Chaperonin Cpn60,




conserved site


PROSITE
PS00296
CHAPERONINS_CPN60
0.0
[496-507]T


noIPR
unintegrated


GENE3D
G3DSA:1.10.560.10
G3DSA:1.10.560.10
6.0E−33
[483-617]T


GENE3D
G3DSA:3.50.7.10
G3DSA:3.50.7.10
3.4E−83
[265-467]T


PANTHER
PTHR11353:SF9
PTHR11353:SF9
0.0
[77-633]T





0.0
[77-633]T


SUPERFAMILY
SSF52029
SSF52029
4.3E−62
[275-467]T


SUPERFAMILY
SSF54849
SSF54849
3.3E−19
[228-289]T









In addition, SEQ ID NO: 546 has 3 N-glycosylation sites (Prosite PS00001): 110-NATN-113 113-NDTA-116, and 432-NATK-435.
Example 5
Topology Prediction of the Polypeptide Sequences Useful in Performing the Methods of the Invention
5.1. eRF1 Polypeptides
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 are 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).
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
        PSORT (URL: psort.org)
        PLOC (Park and Kanehisa, Bioinformatics, 19, 1656-1663, 2003).

5.2. SCAMP-Like Polypeptides

    
    


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.
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
        PSORT (URL: psort.org)
        PLOC (Park and Kanehisa, Bioinformatics, 19, 1656-1663, 2003).

5.3. Fibrillin Polypeptides

    
    


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 pre-sequence a potential cleavage site can also be predicted. A number of parameters are 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).
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
        PSORT (URL: psort.org)
        PLOC (Park and Kanehisa, Bioinformatics, 19, 1656-1663, 2003).

5.4. PLATZ Polypeptides

    
    


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: 261 are presented Table D1. 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: 261 may be the cytoplasm or nucleus, no transit peptide is predicted.







TABLE D1







TargetP 1.1 analysis of the polypeptide sequence


as represented by SEQ ID NO: 261.















Name
Len
cTP
mTP
SP
other
Loc
RC
TPlen





Pt583639
243
0.052
0.241
0.110
0.740
—
3
—


cutoff

0.000
0.000
0.000
0.000





Abbreviations: Len, Length; cTP, Chloroplastic transit peptide; mTP, Mitochondrial transit peptide, SP, Secretory pathway signal peptide, other, Other subcellular targeting, Loc, Predicted Location; RC, Reliability class; TPlen, 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
        PSORT (URL: psort.org)
        PLOC (Park and Kanehisa, Bioinformatics, 19, 1656-1663, 2003).

5.5. PLST-Like Polypeptides

    
    


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.










Targetp v1.1 prediction results















Name
Len
cTP
mTP
SP
other
Loc
RC
TPlen





Sequence
191
0.001
0.483
0.951
0.006
S
3
27


cutoff

0.620
0.760
0.000
0.530





Number of query sequences: 1


Cleavage site predictions included.


Using PLANT networks.






A number of parameters are 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).
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
        PSORT (URL: psort.org)
        PLOC (Park and Kanehisa, Bioinformatics, 19, 1656-1663, 2003).

5.6. Glomalin Polypeptides

    
    


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: 546 are presented Table D2. 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: 546 is most likely the mitochondrion (which is in agreement with results from PSORT), but possibly also the chloroplast.







TABLE D2







TargetP 1.1 analysis of the polypeptide sequence


as represented by SEQ ID NO: 546.















Name
Len
cTP
mTP
SP
other
Loc
RC
TPlen





SEQ ID NO: 546
574
0.379
0.856
0.011
0.013
M
3
32


cutoff

0.000
0.000
0.000
0.000





Abbreviations: Len, Length; cTP, Chloroplastic transit peptide; mTP, Mitochondrial transit peptide, SP, Secretory pathway signal peptide, other, Other subcellular targeting, Loc, Predicted Location; RC, Reliability class; TPlen, 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
        PSORT (URL: psort.org)
        PLOC (Park and Kanehisa, Bioinformatics, 19, 1656-1663, 2003).
    
    


Example 6
Assay Related to the Polypeptide Sequences Useful in Performing the Methods of the Invention
6.1. PLATZ Polypeptides
An assay for DNA binding by PLATZ proteins is described in Nagano et al. (2001). Briefly, a standard electrophoretic mobility shift assay (EMSA) is performed using a [γ-32P]ATP labelled probe derived from the −734 to −667 region downstream of the pea pra2 transcriptional start site in binding buffer (20 μl) containing 2 μg of poly(dl-dC)-poly(dl-dC), bovine serum albumin (500 μg/μl), and competitor DNA. Using recombinantly produced and subsequently purified PLATZ protein, the protein-DNA complex is formed by incubating this mixture at 25° C. for 20 min with the 32P-labeled probe. Electrophoresis is conducted at 4° C. in a 5% polyacrylamide Tris-borate/EDTA gel. After drying, the gel is subjected to autoradiography. The requirement of zinc ions for the binding of the PLATZ protein to DNA can be tested by addition of 1,10-o-phenanthroline at concentrations from 0 to 5 mM.
Example 7
Cloning of the Nucleic Acid Sequence Used in the Methods of the Invention
7.1. eRF1 Polypeptides
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: prm 09556 (fw) (SEQ ID NO: 86; sense, start codon in bold): 5′-gg ggacaagtttgtacaaaaaagcaggcttaaacaatgggagacaaaaacgatgac-3′ and prm 09557 (rev) (SEQ ID NO: 87; reverse, complementary): 5′-ggggaccactttgtacaagaaagctgggttttgattgattgtca ttccga-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 recombined in vivo with the pDONR201 plasmid to produce, according to the Gateway terminology, an “entry clone”, peRF1. Plasmid pDONR201 was purchased from Invitrogen, as part of the Gateway® technology.
The entry clone comprising SEQ ID NO: 1 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: 85) for constitutive specific expression was located upstream of this Gateway cassette.
After the LR recombination step, the resulting expression vector pGOS2::eRF1 (FIG. 2) was transformed into Agrobacterium strain LBA4044 according to methods well known in the art.
7.2. SCAMP-Like Polypeptides
The nucleic acid sequence used in the methods of the invention was amplified by PCR using as template a custom-made Arabidopsis thaliana 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 prm 13820 (SEQ ID NO: 657; sense): 5′-ggggacaagtttgtacaaaaaagcaggcttaaacaatggcacg acacgatccta-3′ and prm 13821 (SEQ ID NO: 658: reverse, complementary) 5′-ggggaccactttgtacaagaaagctgggttgatttcttcatagtgcacgc-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 recombined in vivo with the pDONR201 plasmid to produce, according to the Gateway terminology, an “entry clone”, pSCAMP. Plasmid pDONR201 was purchased from Invitrogen, as part of the Gateway® technology.
The entry clone comprising SEQ ID NO: 88 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: 203) for constitutive specific expression was located upstream of this Gateway cassette.
After the LR recombination step, the resulting expression vector pGOS2::SCAMP-LIKE (FIG. 4) was transformed into Agrobacterium strain LBA4044 according to methods well known in the art.
A further nucleic acid sequence used in the methods of the invention was amplified by PCR using as template a custom-made Arabidopsis thaliana seedlings cDNA library. 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 prm 13818 (SEQ ID NO: 659; sense): 5′-ggggacaagtttgtacaaaaaagcaggcttaaacaatgaatcgccaccacgat-3′ and prm 13819 (SEQ ID NO: 660: reverse, complementary) 5′-ggggaccactttgtacaagaaagctgggttctccttttcacttgtttccc-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 recombined in vivo with the pDONR201 plasmid to produce, according to the Gateway terminology, an “entry clone”, pSCAMP. Plasmid pDONR201 was purchased from Invitrogen, as part of the Gateway® technology.
The entry clone comprising SEQ ID NO: 663 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: 203) for constitutive specific expression was located upstream of this Gateway cassette.
After the LR recombination step, the resulting expression vector pGOS2::SCAMP-LIKEx was transformed into Agrobacterium strain LBA4044 according to methods well known in the art.
Another further nucleic acid sequence used in the methods of the invention was amplified by PCR using as template a custom-made Arabidopsis thaliana seedlings cDNA library. 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 prm 13816 (SEQ ID NO: 661; sense): 5′-ggggacaagtttgtacaaaaaagcaggcttaaacaatggctaatcgttatgatcca-3′ and prm 13817 (SEQ ID NO: 662: reverse, complementary) 5′-ggggaccactttgtacaagaaagctgggtcaaggagaactctca aacagc-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 recombined in vivo with the pDONR201 plasmid to produce, according to the Gateway terminology, an “entry clone”, pSCAMP. Plasmid pDONR201 was purchased from Invitrogen, as part of the Gateway® technology.
The entry clone comprising SEQ ID NO: 665 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: 203) for constitutive specific expression was located upstream of this Gateway cassette.
After the LR recombination step, the resulting expression vector pGOS2::SCAMP-LIKEy was transformed into Agrobacterium strain LBA4044 according to methods well known in the art.
7.3. Fibrillin Polypeptides
The nucleic acid sequence used in the methods of the invention was amplified by PCR using a Lycopersicon esculentum (Solanum lycopersicum) cDNA library. 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 prm10420 (SEQ ID NO: 258; sense, start codon in bold): 5′-ggggacaagtttgtacaaaaaagcaggcttaaacaatgatttcagcaggtttcg-3′ and prm10421 (SEQ ID NO: 259; reverse, complementary): 5′-ggggaccactttgtacaagaaagctgggtggctggttag caaataagagt-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 recombined in vivo with the pDONR201 plasmid to produce, according to the Gateway terminology, an “entry clone”, pfibrillin. Plasmid pDONR201 was purchased from Invitrogen, as part of the Gateway® technology.
The entry clone comprising SEQ ID NO: 204 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: 257) for constitutive specific expression was located upstream of this Gateway cassette.
After the LR recombination step, the resulting expression vector pGOS2::fibrillin (FIG. 9) was transformed into Agrobacterium strain LBA4044 according to methods well known in the art.
7.4. PLATZ Polypeptides
Example A
In this example, a nucleic acid sequence for use in the methods of the invention was amplified by PCR using as template a custom-made Populus sp. 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 prm13426 (SEQ ID NO: 274; sense, start codon in bold): 5′-ggggacaagtttgtacaaaaaagcaggcttaaacaatgggaactcaaaagcctgcat-3′ and prm13427 (SEQ ID NO: 275; reverse, complementary): 5′-ggggaccactttgtacaagaaagctgggtttttcctaacataaagg agatcgatga-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 recombined in vivo with the pDONR201 plasmid to produce, according to the Gateway terminology, an “entry clone”, pPLATZ. Plasmid pDONR201 was purchased from Invitrogen, as part of the Gateway® technology.
The entry clone comprising SEQ ID NO: 260 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: 273) for constitutive expression was located upstream of this Gateway cassette.
After the LR recombination step, the resulting expression vector pGOS2::PLATZ (FIG. 13) was transformed into Agrobacterium strain LBA4044 according to methods well known in the art.
Example B
In another example another nucleic acid sequence was amplified by PCR using as template a custom-made Arabidospis seedlings cDNA library. 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 prm17901: (SEQ ID NO: 613; sense): 5′-ggggacaagtttgtacaaaaaagca ggcttaaacaatggttagagaaggtgaagaa-3′, and prm17902 (SEQ ID NO: 614; reverse, complementary): 5′-ggggaccactttgtacaagaaagctgggttatgatttttgcctatggtta-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 recombined in vivo with the pDONR201 plasmid to produce another “entry clone”, pPLATZAt, according to the Gateway terminology. Plasmid pDONR201 was purchased from Invitrogen, as part of the Gateway® technology.
The entry clone comprising SEQ ID NO: 356 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: 273) for constitutive expression was located upstream of this Gateway cassette.
After the LR recombination step, the resulting expression vector pGOS2::PLATZAt was transformed into Agrobacterium strain LBA4044 according to methods well known in the art.
7.5. PLST-Like Polypeptides
The nucleic acid sequence used in the methods of the invention was amplified by PCR using as template a custom-made Populus sp. 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: prm13192 (fw) (SEQ ID NO: 543; sense, start codon in bold): 5′-ggggacaagtttgtacaaaaa agcaggcttaaacaatggtcaatcttagaagtcct-3′ and prm13193 (rev) (SEQ ID NO: 544; reverse, complementary): 5′-ggggaccactttgtacaagaaagctgggtctcatctgggttcttg taata-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 recombined in vivo with the pDONR201 plasmid to produce, according to the Gateway terminology, an “entry clone”, pPLST-like. Plasmid pDONR201 was purchased from Invitrogen, as part of the Gateway® technology.
The entry clone comprising SEQ ID NO: 410 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: 542) for constitutive specific expression was located upstream of this Gateway cassette.
After the LR recombination step, the resulting expression vector pGOS2::PLST-like (FIG. 2) was transformed into Agrobacterium strain LBA4044 according to methods well known in the art.
7.6. Glomalin Polypeptides
The nucleic acid sequence 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 prm13455 (SEQ ID NO: 609; sense, start codon in bold): 5′-ggggacaagtttgtacaaaaaagcaggcttaaacaatgtaccgcgcggc-3′ and prm13456 (SEQ ID NO: 610; reverse, complementary): 5′-ggggaccactttgtacaagaaagctgggtttagtaatccattccac ccat-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 recombined in vivo with the pDONR201 plasmid to produce, according to the Gateway terminology, an “entry clone”, pGlomalin. Plasmid pDONR201 was purchased from Invitrogen, as part of the Gateway® technology.
The entry clone comprising SEQ ID NO: 545 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 RCc3 promoter (SEQ ID NO: 611) for root specific expression was located upstream of this Gateway cassette.
After the LR recombination step, the resulting expression vector pRCc3::Glomalin (FIG. 19) was transformed into Agrobacterium strain LBA4044 according to methods well known in the art.
Example 8
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% HgC12, 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 the expression vector was used 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 one construct. The primary transformants were transferred from a tissue culture chamber to a greenhouse. After a quantitative PCR analysis to verify copy number of the T-DNA insert, only single copy transgenic plants that exhibit tolerance to the selection agent were kept for harvest of T1 seed. Seeds were then harvested three to five months after transplanting. The method yielded single locus transformants at a rate of over 50% (Aldemita and Hodges 1996, Chan et al. 1993, Hiei et al. 1994).
Example 9
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 is transformed using Agrobacterium tumefaciens according to the method described in U.S. Pat. No. 5,159,135. Cotton seeds are surface sterilised in 3% sodium hypochlorite solution during 20 minutes and washed in distilled water with 500 μg/ml cefotaxime. The seeds are then transferred to SH-medium with 50 μg/ml benomyl for germination. Hypocotyls of 4 to 6 days old seedlings are removed, cut into 0.5 cm pieces and are placed on 0.8% agar. An Agrobacterium suspension (approx. 108 cells per ml, diluted from an overnight culture transformed with the gene of interest and suitable selection markers) is used for inoculation of the hypocotyl explants. After 3 days at room temperature and lighting, the tissues are transferred to a solid medium (1.6 g/l Gelrite) with Murashige and Skoog salts with B5 vitamins (Gamborg et al., Exp. Cell Res. 50:151-158 (1968)), 0.1 mg/l 2,4-D, 0.1 mg/l 6-furfurylaminopurine and 750 μg/ml MgCL2, and with 50 to 100 μg/ml cefotaxime and 400-500 μg/ml carbenicillin to kill residual bacteria. Individual cell lines are isolated after two to three months (with subcultures every four to six weeks) and are further cultivated on selective medium for tissue amplification (30° C., 16 hr photoperiod). Transformed tissues are subsequently further cultivated on non-selective medium during 2 to 3 months to give rise to somatic embryos. Healthy looking embryos of at least 4 mm length are transferred to tubes with SH medium in fine vermiculite, supplemented with 0.1 mg/l indole acetic acid, 6 furfurylaminopurine and gibberellic acid. The embryos are cultivated at 30° C. with a photoperiod of 16 hrs, and plantlets at the 2 to 3 leaf stage are transferred to pots with vermiculite and nutrients. The plants are hardened and subsequently moved to the greenhouse for further cultivation.
Example 10
Phenotypic Evaluation Procedure
10.1 Evaluation Setup
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%. Plants grown under non-stress conditions were watered at regular intervals to ensure that water and nutrients were not limiting and to satisfy plant needs to complete growth and development.
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.
Drought Screen
Plants from T2 seeds 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 goes below certain thresholds, the plants are automatically re-watered continuously until a normal level is reached again. The plants are then re-transferred again 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.
Nitrogen Use Efficiency Screen
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 were 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 are harvested. Seed-related parameters are then measured.
10.2 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.
Where 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.
10.3 Parameters Measured
Biomass-Related Parameter Measurement
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).
Root biomass can be determined using a method as described in WO 2006/029987.
Early vigour is a parameter related to development time of a plant. 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.
The “flowering time” of the plant can be determined using the method as described in WO 2007/093444.
Seed-Related Parameter Measurements
The mature primary panicles were harvested, counted, bagged, barcode-labelled and then dried for three days in an oven at 37° C. The panicles were then threshed and all the seeds were collected and counted. The filled husks were separated from the empty ones using an air-blowing device. The empty husks were discarded and the remaining fraction was counted again. The filled husks were weighed on an analytical balance. The number of filled seeds was determined by counting the number of filled husks that remained after the separation step. The total seed yield was measured by weighing all filled husks harvested from a plant. Total seed number per plant was measured by counting the number of husks harvested from a plant. Thousand Kernel Weight (TKW) is extrapolated from the number of filled seeds counted and their total weight. The Harvest Index (HI) in the present invention is defined as the ratio between the total seed yield and the above ground area (mm2), multiplied by a factor 106. The total number of flowers per panicle as defined in the present invention is the ratio between the total number of seeds and the number of mature primary panicles. The seed fill rate as defined in the present invention is the proportion (expressed as a %) of the number of filled seeds over the total number of seeds (or florets).
Examples 11
Results of the Phenotypic Evaluation of the Transgenic Plants
11.1. eRF1 Polypeptides
The results of the evaluation of transgenic rice plants in the T2 generation and expressing a nucleic acid comprising the longest Open Reading Frame in SEQ ID NO: 1 under non-stress conditions are presented below.
The results of the evaluation of transgenic rice plants under non-stress conditions are presented below (Table E1). An increase of (at least-more than) 5% was observed for aboveground biomass (AreaMax), seedling vigour (EmerVigor), yield per plant (totalwgseeds), total number of seeds per plant (nrtotalseed), number of panicles in the first flush (firstpan), number of filled seeds of a plant (nrfilledseed), harvest index, which is totalwgseeds/AreaMax (harvestindex).







TABLE E1







Non-Stress conditions










Parameter
Overall







AreaMax
11.3



EmerVigor
20.0



totalwgseeds
14.6



nrtotalseed
12.2



firstpan
11.4



nrfilledseed
14.6










For each parameter, the percentage overall is shown if it reaches p<0:05 and above the 5% threshold.
11.2. SCAMP-Like Polypeptides
The results of the evaluation of transgenic rice plants in the T1 generation and expressing a nucleic acid comprising the longest Open Reading Frame in SEQ ID NO: 88 under the control of the rice GOS2 promoter and cultivated under the Nitrogen use efficiency screen conditions are presented below(Table E2A). See previous Examples for details on the generations of the transgenic plants.
An increase of (at least-more than) 5% was observed, as compared to control plants, for aboveground biomass (AreaMax), Rootbiomass (RootMax), emergence vigour (Emervigor, early vigour), total seed yield (totalwgseeds), number of filled seeds (nrfilledseed), fill rate, number of seeds per plant (nrtotalseeds), harvest index (harvestindex), and number of primary panicles (firstpan).










TABLE E2A








% increased in transgenic plant



yield-related trait
compared to control nullyzygous plant



















Areamax
21.5



Emervigor
10.9



RootMax
65.6



totalwgseeds
28.25



nrtotalseeds
26.4



harvestindex
30.7



firstpan
62.7



nrfilledseed
68.8










The results of the evaluation of transgenic rice plants in the T1 generation and expressing a nucleic acid comprising the longest Open Reading Frame in SEQ ID NO: 663 under the control of the rice GOS2 promoter and cultivated under the Nitrogen use efficiency screen conditions are presented below. See previous Examples for details on the generations of the transgenic plants.
An increase of at least 5% was observed, as compared to control plants, for Greeness before Flowering (GNbfFlow) for 3 lines, for Gravity Yield Max (GravityYMax) for 2 lines. Furthermore we observed an increase of at least 5% for fillrate, thousand kernel weight (TKW), number of filled seeds (nrfilledseed), flowers per panicle (flowerperpan) and maximum height of the plant, i.e. the highest tip of the plant, in at least one line.
The results of the evaluation of transgenic rice plants in the T1 generation and expressing a nucleic acid comprising the longest Open Reading Frame in SEQ ID NO: 665 under the control of the rice GOS2 promoter and cultivated under the nitrogen Nitrogen use efficiency screen conditions are presented below (Table E2B). See previous Examples for details on the generations of the transgenic plants.
An increase of (at least-more than) 5% was, observed as compared to control plants, for aboveground biomass (Rootbiomass (RootMax), total seed yield (totalwgseeds), harvest index (harvestindex), thousand kernel weight (TKW) and number of filled seeds (nrfilledseed).










TABLE E2B








% increased in transgenic plant



yield-related trait
compared to control nullyzygous plant



















RootMax
5.0



totalwgseeds
25.5



harvestindex
23.9



TKW
5.0



nrfilledseed
22.5










11.3. Fibrillin Polypeptides

Each parameter shown in the tables below gives a percentage overall difference with a p value of p<0.05. The results of the evaluation show an increase in the total seed weight, seed fill rate, harvest index and in the number of filled seeds compared to control plants (corresponding nullizygotes).







TABLE E3







T1 Generation










Parameter
Overall














Total weight seeds
17.9%



Fill rate
9.6%



Harvest index
10.5%



No. filled seeds
15.1%

















TABLE E4







T2 Generation










Parameter
Overall














Total weight seeds
12.3%



Fill rate
8.5%



Harvest index
9.8%



No. filled seed
9.8%










11.4. PLATZ Polypeptides

Example A
The results of an evaluation of transgenic rice plants in the T1 generation expressing an PLATZ nucleic acid of SEQ ID NO: 260 (see point 7.4 above-example A) under non-stress conditions are presented hereunder. An increase was observed for above ground biomass (AreaMax) and root biomass (RootThickMax), total seed weight (totalwgseeds), harvest index, thousand-kernel weight (TKW) (Table E5A).







TABLE E5A







Data summary for transgenic rice plants; for each parameter,


the overall percent increase as compared to control plants


is shown, for each parameter the p-value is ≦0.05.










Parameter
Overall increase














AreaMax
12.6



totalwgseeds
19.8



harvestindex
7.6



TKW
17.9



RootThickMax
5.9










Example B
The results of an evaluation of transgenic rice plants in the T1 generation expressing a PLATZ nucleic acid of SEQ ID NO: 356 (see point 7.4 above-example B) under non-stress conditions are presented hereunder. An increase was observed for yield-related parameters such as an increased rate of filled seeds over the number of total seeds (fillrate) and an increased increased thousand-kernel weight (TKW). The transgenic plants also showed a quicker early development as compared to control plants (Table E5B). The latter parameter is indicated by AreaEmer in Table E5B and represents the Ratio in % between the time a plant needs to make 30% of the final biomass and the time the plant needs to make 90 of its final biomass.







TABLE E5B







Data summary for transgenic rice plants; for each parameter,


the overall percent increase as compared to control plants


is shown, for each parameter the p-value is ≦0.05.










Parameter
Overall increase







Fillrate
5.7%



TKW
3.5%



AreaEmer
5.3%










In addition, plants expressing a PLATZ protein grown under conditions of abiotic stress such as drought stress, nutrient stress, salt stress, show improved yield-related traits, comprising one or more of increased biomass, such as above-ground or/and below-ground; increased seed yield as defined in the definitions section; early vigour.
11.5. PLST-Like Polypeptides
The results of the evaluation of transgenic rice plants in the T2 generation and expressing a nucleic acid comprising the longest Open Reading Frame in SEQ ID NO: 410 under non-stress conditions are presented below.
The results of the evaluation of transgenic rice plants expressing the PLST-like polypeptide represented by SEQ ID NO 411 under drought conditions are presented below (Table E6). An increase of more than 5% was observed for total seed weight (totalwgseeds), rate of filled seeds over the number of total seeds (fillrate), harvest index, which is totalwgseeds/AreaMax (harvestindex) and number of filled seeds (nrfilledseed).







TABLE E6







Data summary for transgenic rice plants grown under


drought stress conditions; the overall increase is


shown and for each parameter the p-value is ≦0.05.










Parameter
Overall







totalwgseeds
19.2



fillrate
27.2



harvestindex
21.6



nrfilledseed
17.1










11.6. Glomalin Polypeptides
Transgenic rice plants expressing the Glomalin gene under control of the root-specific RCc3 promoter had increased seed yield, in particular increased 1000 kernel weight (overall increase 2.8% with a p-value of 0.035). Three out of six lines also had increased harvest index.

## Claims

1. A method for increasing seed yield and/or biomass in a plant relative to a corresponding control plant, comprising introducing and expressing in a plant a nucleic acid encoding a polypeptide having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 261, and selecting for a plant having increased seed yield and/or biomass relative to a corresponding control plant.

2. The method of claim 1, wherein said polypeptide comprises one or more of the motifs of SEQ ID NO: 264, 265, 266, 267, 268, 269, 270, 271 and 272.

3. The method of claim 1, wherein said nucleic acid is:
(i) a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 260; or
(ii) a nucleic acid encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 261.

4. The method of claim 1, wherein said increased seed yield comprises increased total seed weight, increased number of flowers per plant, increased number of seeds, increased number of filled seeds, increased seed filling rate, increased harvest index, and/or increased thousand kernel weight (TKW).

5. The method of claim 1, wherein said increased seed yield and/or biomass is obtained under non-stress conditions.

6. The method of claim 1, wherein said increased seed yield and/or biomass is obtained under conditions of drought stress, salt stress, or nitrogen deficiency.

7. A plant obtained by the method of claim 1, or a plant part, seed or progeny of said plant, wherein said plant, or said plant part, seed or progeny, comprises said nucleic acid.

8. A plant, plant part, or plant cell comprising a construct comprising:
(i) a nucleic acid sequence encoding a polypeptide having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 261; and
(ii) one or more heterologous control sequences capable of driving expression of the nucleic acid sequence of (i); and optionally
(iii) a transcription termination sequence,
wherein the plant is a crop plant selected from the group consisting of soybean, sunflower, canola, alfalfa, rapeseed, linseed, cotton, tomato, potato, tobacco and beet, a monocot, or a cereal.

9. A method for the production of a transgenic plant having increased yield, increased biomass, and/or increased seed yield relative to a corresponding control plant, comprising:
(i) introducing and expressing in a plant a nucleic acid encoding a polypeptide having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 261; and
(iii) selecting for a transgenic plant having increased yield, increased biomass, and/or increased seed yield relative to a corresponding control plant.

10. A transgenic plant having increased yield, increased biomass, and/or increased seed yield relative to a corresponding control plant, resulting from introducing and expressing a nucleic acid encoding a polypeptide having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 261 in said plant, wherein the plant is a crop plant selected from the group consisting of soybean, sunflower, canola, alfalfa, rapeseed, linseed, cotton, tomato, potato, tobacco and beet, a monocot, or a cereal.

11. The transgenic plant of claim 10, wherein said nucleic acid is:
(i) a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 260; or
(ii) a nucleic acid encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 261.

12. The transgenic plant of claim 10, wherein said plant is rice, maize, wheat, barley, millet, rye, triticale, sorghum, emmer, spelt, secale, einkorn, teff, milo, or oats.

13. Harvestable parts of the transgenic plant of claim 10, wherein said harvestable parts comprises said nucleic acid.

14. Products obtained from the transgenic plant of claim 10 and/or from harvestable parts of said plant, wherein said products comprises said nucleic acid.

15. The method of claim 1, wherein said nucleic acid encodes a polypeptide having at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 261.

16. The method of claim 1, wherein said nucleic acid encodes a polypeptide having at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 261.

17. The transgenic plant of claim 10, wherein said nucleic acid encodes a polypeptide having at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 261.

18. The transgenic plant of claim 10, wherein said nucleic acid encodes a polypeptide having at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 261.

19. The method of claim 1, wherein said polypeptide comprises a first zinc binding region at the N-terminal and a second zinc binding region in the central region.

20. The method of claim 1, wherein said polypeptide comprises:
(i) at least one or more of the motifs of SEQ ID NO: 264, 265 and 266;
(ii) at least one or more of the motifs of SEQ ID NO: 267, 268 and 269; or
(iii) at least one or more of the motifs of SEQ ID NO: 270, 271 and 272.

21. The transgenic plant of claim 10, wherein said polypeptide comprises a first zinc binding region at the N-terminal and a second zinc binding region in the central region.

22. The transgenic plant of claim 10, wherein said polypeptide comprises:
(i) at least one or more of the motifs of SEQ ID NO: 264, 265 and 266;
(ii) at least one or more of the motifs of SEQ ID NO: 267, 268 and 269; or
(iii) at least one or more of the motifs of SEQ ID NO: 270, 271 and 272.