# 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 plant yield-related traits relative to control plants. More specifically, the present invention concerns a method for enhancing yield related traits in plants relative to control plants, by modulating, preferably increasing, expression in the roots of a plant, of a nucleic acid sequence encoding a 2-cysteine peroxiredoxin (2-Cys PRX); or by modulating expression of a nucleic acid encoding an ANN polypeptide in a plant. The present invention also concerns plants having modulated, preferably increased, expression in the roots, of a nucleic acid sequence encoding a 2-Cys PRX, or having modulated expression of a nucleic acid encoding an ANN polypeptide, which plants have enhanced yield-related traits relative to control plants. The invention also provides constructs useful in the methods of the invention.

## Background
RELATED APPLICATIONS
This application is a national stage application (under 35 U.S.C. §371) of PCT/EP2008/056381, filed May 23, 2008, which claims benefit of European Application 07108768.8, filed May 23, 2007; U.S. Provisional Application 60/932,226, filed May 29, 2007, European Application 07110086.1, filed Jun. 12, 2007, and U.S. Provisional Application 60/937,994, filed Jun. 29, 2007.


SUBMISSION OF SEQUENCE LISTING
The Sequence Listing associated with this application is filed in electronic format via EFS-Web and hereby incorporated by reference into the specification in its entirety. The name of the text file containing the Sequence Listing is Revised_Sequence_Listing—14546—00054_US. The size of the text file is 355 KB, and the text file was created on Nov. 4, 2011.
The present invention relates generally to the field of molecular biology and concerns a method for enhancing plant yield-related traits relative to control plants. More specifically, the present invention concerns a method for enhancing yield related traits in plants relative to control plants, by modulating, preferably increasing, expression in the roots of a plant, of a nucleic acid sequence encoding a 2-cysteine peroxiredoxin (2-Cys PRX); or by modulating expression of a nucleic acid encoding an ANN polypeptide in a plant. The present invention also concerns plants having modulated, preferably increased, expression in the roots, of a nucleic acid sequence encoding a 2-Cys PRX, or having modulated expression of a nucleic acid encoding an ANN polypeptide, which plants have enhanced yield-related traits relative to control plants. The invention also provides constructs useful in the methods of the invention.
The ever-increasing world population and the dwindling supply of arable land available for agriculture fuels research towards increasing the efficiency of agriculture. Conventional means for crop and horticultural improvements utilise selective breeding techniques to identify plants having desirable characteristics. However, such selective breeding techniques have several drawbacks, namely that these techniques are typically labour intensive and result in plants that often contain heterogeneous genetic components that may not always result in the desirable trait being passed on from parent plants. Advances in molecular biology have allowed mankind to modify the germplasm of animals and plants. Genetic engineering of plants entails the isolation and manipulation of genetic material (typically in the form of DNA or RNA) and the subsequent introduction of that genetic material into a plant. Such technology has the capacity to deliver crops or plants having various improved economic, agronomic or horticultural traits.
A trait of particular economic interest is increased yield. Yield is normally defined as the measurable produce of economic value from a crop. This may be defined in terms of quantity and/or quality. Yield is directly dependent on several factors, for example, the number and size of the organs, plant architecture (for example, the number of branches), seed production, leaf senescence and more. Root development, nutrient uptake, stress tolerance and early vigour may also be important factors in determining yield. Optimizing the above-mentioned factors may therefore contribute to increasing crop yield.
Seed yield is a particularly important trait, since the seeds of many plants are important for human and animal nutrition. Crops such as corn, rice, wheat, canola and soybean account for over half the total human caloric intake, whether through direct consumption of the seeds themselves or through consumption of meat products raised on processed seeds. They are also a source of sugars, oils and many kinds of metabolites used in industrial processes. Seeds contain an embryo (the source of new shoots and roots) and an endosperm (the source of nutrients for embryo growth during germination and during early growth of seedlings). The development of a seed involves many genes, and requires the transfer of metabolites from the roots leaves and stems into the growing seed. The endosperm, in particular, assimilates the metabolic precursors of carbohydrates, oils and proteins and synthesizes them into storage macromolecules to fill out the grain.
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.
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.
Harvest index, the ratio of seed yield to aboveground dry weight, is relatively stable under many environmental conditions and so a robust correlation between plant size and grain yield can often be obtained (e.g. Rebetzke et al 2002 Crop Science 42:739). These processes are intrinsically linked because the majority of grain biomass is dependent on current or stored photosynthetic productivity by the leaves and stem of the plant (Gardener et al 1985 Physiology of Crop Plants. Iowa State University Press, pp 68-73). Therefore, selecting for plant size, even at early stages of development, has been used as an indicator for future potential yield (e.g. Tittonell et al 2005 Agric Ecosys & Environ 105: 213). When testing for the impact of genetic differences on stress tolerance, the ability to standardize soil properties, temperature, water and nutrient availability and light intensity is an intrinsic advantage of greenhouse or plant growth chamber environments compared to the field. However, artificial limitations on yield due to poor pollination due to the absence of wind or insects, or insufficient space for mature root or canopy growth, can restrict the use of these controlled environments for testing yield differences. Therefore, measurements of plant size in early development, under standardized conditions in a growth chamber or greenhouse, are standard practices to provide indication of potential genetic yield advantages.
Another trait of importance is that of improved abiotic stress tolerance. Abiotic stress is a primary cause of crop loss worldwide, reducing average yields for most major crop plants by more than 50% (Wang et al. (2003) Planta 218: 1-14). Abiotic stresses may be caused by drought, salinity, extremes of temperature, chemical toxicity, excess or lack of nutrients (macroelements and/or microelements), radiation and oxidative stress. The ability to increase plant tolerance to abiotic stress would be of great economic advantage to farmers worldwide and would allow for the cultivation of crops during adverse conditions and in territories where cultivation of crops may not otherwise be possible.
Crop yield may therefore be increased by optimising one of the above-mentioned factors.
Depending on the end use, the modification of certain yield traits may be favoured over others. For example for applications such as forage or wood production, or bio-fuel resource, an increase in the vegetative parts of a plant may be desirable, and for applications such as flour, starch or oil production, an increase in seed parameters may be particularly desirable. Even amongst the seed parameters, some may be favoured over others, depending on the application. Various mechanisms may contribute to increasing seed yield, whether that is in the form of increased seed size or increased seed number.
One approach to enhance yield-related traits (for example increasing yield, in particular seed yield and/or biomass) in plants may be through modification of the inherent growth mechanisms of a plant, such as the cell cycle or various signalling pathways involved in plant growth or in defense mechanisms.
Surprisingly, it has now been found that various plant yield-related traits may be enhanced relative to control plants, by modulating, preferably increasing, expression in the roots of a plant, of a nucleic acid sequence encoding a 2-cysteine peroxiredoxin (2-Cys PRX), or by modulating expression of a nucleic acid encoding an ANN polypeptide in a plant.
According to one embodiment, there is provided a method for enhancing various yield-related traits relative to control plants, comprising modulating, preferably increasing, expression in the roots of a plant, of a nucleic acid sequence encoding a 2-cysteine peroxiredoxin (2-Cys PRX), or by modulating expression of a nucleic acid encoding an ANN polypeptide, in a plant.
BACKGROUND
1. 2-Cysteine Peroxiredoxin (2-Cys PRX)
Thiol peroxidases (PRX) are ubiquitous heme-free peroxidases, which catalyze the reduction of peroxynitrites and of various peroxides by catalytic cysteine residues and thiol-containing proteins as reductants. In plants, five different classes can be distinguished, according to the number and the position of conserved catalytic cysteines. Four classes are defined as peroxiredoxins and were already identified by phylogenetic sequence analysis, 1-Cys, 2-Cys, type II, and type Q peroxiredoxins, and the fifth is represented by glutathione peroxidases, which were recently shown to possess a thioredoxin-dependent activity in plants (Rouhier & Jacquot (2005) Free Radic Biol Med 38(11): 1413-21). The analysis of the Arabidopsis thaliana genome indicates that at least 17 isoforms of thioredoxin-dependent peroxidases are expressed in various plant compartments.
2-Cysteine peroxiredoxin (2-Cys PRX) are a group of proteins that participate in cell proliferation, differentiation, apoptosis, and photosynthesis. These enzymes reduce H2O2, peroxinitrite and alkyl hydroperoxide to water or alcohol, respectively (Netto et al., (1996) J Biol Chem 271(26): 15315-15321) with thioredoxin (Trx) as electron donor. By doing so, 2-Cys PRXs regulate signal transduction pathways or protect macromolecules against oxidative damage. These proteins are homodimers and each subunit has the two conserved cysteines (Choi et al., (1998) Nature Struct Biol 5:400-406). The peroxide oxidizes the N-terminal cysteine of one subunit to sulphenic acid, which reacts with the C-terminal cysteine of the other subunit to form an intermolecular disulphide. To complete the catalytic cycle the enzyme is reduced via a thiol/disulphide redox interchange (Chae et al., (1994) Proc Natl Acad Sci USA 91: 7017-7021).
Transgenic Arabidopsis thaliana plants (Baier et al. (2000) Plant Physiol 124(2): 823-32) with reduced levels of 2-Cys PRX were generated by antisense suppression. The suppression of 2-Cys PRX expression lead to increased expression of other anti-oxidative genes, demonstrating that the enzyme forms an integral part of the anti-oxidant network of chloroplasts and is functionally interconnected with other defense systems.
International patent application WO05/116082 describes the obtention of transgenic Arabidopsis plants overexpressing an Arabidopsis 2-Cys PRX (named BAS1) using the constitutive cauliflower mosaic virus 35S promoter. The transgenic plants are described as having more potential resistance to heat shock and pathogens than the wild type plants.
Surprisingly, it has now been found that modulating, preferably increasing, expression in the roots of a plant, of a nucleic acid sequence encoding a 2-Cys PRX 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, preferably increasing, expression in the roots of a plant, of a nucleic acid sequence encoding a 2-Cys PRX polypeptide. The enhanced yield related traits comprise one or more of: (i) improved early vigour; (ii) increased aboveground biomass; (iii) increased root (thick and thin) biomass; (iv) increase number of flowers per panicle; (v) increased seed fill rate; (vi) increased total seed yield per plant; (vii) increased number of (filled) seeds; (viii) increased harvest index; or (ix) increased thousand kernel weight (TKW).
2. Annexin-Like (ANN)
Annexins form a family of calcium dependent phospholipid binding proteins and are found in plants and animals. In all plant species tested, the presence of at least two different annexins has been demonstrated. Structurally, plant annexins are less divergent than animal annexins. Comparative studies revealed that plant annexins share significant homology in a core domain which comprises at least one, usually four or more conserved repeats which are approximately 70 amino acids in length. As calcium-binding proteins, annexins are postulated to play a role in calcium signalling pathways. Although the structure of annexins is well known nowadays, functionally they are not well characterised. In plants, annexins are reported to be involved in Golgi-mediated secretion, cell expansion, vacuole biogenesis, chloroplast membrane binding, cell cycle, nodulation signalling, stress signalling.
US20050089872 describes T-DNA insertion mutants (anx1 and anx4-1) for respectively the Annexin 1 and Annexin 4 encoding genes from Arabidopsis thaliana. The mutants were sensitive to salt stress and osmotic stress. Also abscisic acid had a negative effect on germination and growth of the anx1 and anx4-1 mutants. Expression analysis revealed that the ANX1 protein was predominantly expressed in the root, but not in flower, stem or leaf tissues. It is postulated that the ANX1 and ANX4 proteins play a role in the transduction of osmotic stress and ABA signals.
Surprisingly, it has now been found that modulating expression of a nucleic acid encoding an ANN polypeptide gives plants having enhanced yield-related traits, in particular increased yield relative to control plants.
According one embodiment, there is provided a method for enhancing yield-related traits of a plant relative to control plants, comprising modulating expression of a nucleic acid encoding an ANN polypeptide in a plant. The improved yield related traits comprise increased seed yield.
DEFINITIONS
Polypeptide(s)/Protein(s)
The terms “polypeptide” and “protein” are used interchangeably herein and refer to amino acids in a polymeric form of any length, linked together by peptide bonds.
Polynucleotide(s)/Nucleic Acid(s)/Nucleic Acid Sequence(s)/Nucleotide Sequence(s)
The terms “polynucleotide(s)”, “nucleic acid sequence(s)”, “nucleotide sequence(s)”, “nucleic acid(s)”, “nucleic acid molecule(s)” are used interchangeably herein and refer to nucleotides, either ribonucleotides or deoxyribonucleotides or a combination of both, in a polymeric unbranched form of any length.
Control Plant(s)
The choice of suitable control plants is a routine part of an experimental setup and may include corresponding wild type plants or corresponding plants without the gene of interest. The control plant is typically of the same plant species or even of the same variety as the plant to be assessed. The control plant may also be a nullizygote of the plant to be assessed. A “control plant” as used herein refers not only to whole plants, but also to plant parts, including seeds and seed parts.
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; 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.
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
The term “domain” refers to a set of amino acids conserved at specific positions along an alignment of sequences of evolutionarily related proteins. While amino acids at other positions can vary between homologues, amino acids that are highly conserved at specific positions indicate amino acids that are likely essential in the structure, stability or function of a protein. Identified by their high degree of conservation in aligned sequences of a family of protein homologues, they can be used as identifiers to determine if any polypeptide in question belongs to a previously identified polypeptide family.
Motif/Consensus Sequence/Signature
The term “motif” or “consensus sequence” or “signature” refers to a short conserved region in the sequence of evolutionarily related proteins. Motifs are frequently highly conserved parts of domains, but may also include only part of the domain, or be located outside of conserved domain (if all of the amino acids of the motif fall outside of a defined domain).
Hybridisation
The term “hybridisation” as defined herein is a process wherein substantially homologous complementary nucleotide sequences anneal to each other. The hybridisation process can occur entirely in solution, i.e. both complementary nucleic acid molecules are in solution. The hybridisation process can also occur with one of the complementary nucleic acid molecules immobilised to a matrix such as magnetic beads, Sepharose beads or any other resin. The hybridisation process can furthermore occur with one of the complementary nucleic acid molecules immobilised to a solid support such as a nitro-cellulose or nylon membrane or immobilised by e.g. photolithography to, for example, a siliceous glass support (the latter known as nucleic acid 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 acid molecules.
The term “stringency” refers to the conditions under which a hybridisation takes place. The stringency of hybridisation is influenced by conditions such as temperature, salt concentration, ionic strength and hybridisation buffer composition. Generally, low stringency conditions are selected to be about 30° C. lower than the thermal melting 2-Cys PRXnt (Tm) for the specific sequence at a defined ionic strength and pH. Medium stringency conditions are when the temperature is 20° C. below Tm, and high stringency conditions are when the temperature is 10° C. below Tm. High stringency hybridisation conditions are typically used for isolating hybridising sequences that have high sequence similarity to the target nucleic acid sequence. However, nucleic acid sequences may deviate in sequence composition 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

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.

2) DNA-RNA or RNA-RNA hybrids:

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

3) oligo-DNA or oligo-RNAd hybrids: d oligo, oligonucleotide; In, =effective length of primer=2×(no. of G/C)+(no. of A/T).

    
    
        For <20 nucleotides: Tm=2 (In)
        For 20-35 nucleotides: Tm=22+1.46 (In)
    
    


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 molecule. When nucleic acid molecules of known sequence are hybridised, the hybrid length may be determined by aligning the sequences and identifying the conserved regions described herein. 1×SSC is 0.15M NaCl and 15 mM sodium citrate; the hybridisation solution and wash solutions may additionally include 5×Denhardt's reagent, 0.5-1.0% SDS, 100 μg/ml denatured, fragmented salmon sperm DNA, 0.5% sodium pyrophosphate.
For the purposes of defining the level of stringency, reference can be made to Sambrook et al. (2001) Molecular Cloning: a laboratory manual, 3rd Edition, Cold Spring Harbor Laboratory Press, CSH, N.Y. or to Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989 and yearly updates).
Splice Variant
The term “splice variant” as used herein encompasses variants of a nucleic acid sequence in which selected introns and/or exons have been excised, replaced, displaced or added, or in which introns have been shortened or lengthened. Such variants will be ones in which the biological activity of the protein is substantially retained; this may be achieved by selectively retaining functional segments of the protein. Such splice variants may be found in nature or may be manmade. Methods for predicting and isolating such splice variants are well known in the art (see for example Foissac and Schiex (2005) BMC Bioinformatics 6: 25).
Allelic Variant
Alleles or allelic variants are alternative forms of a given gene, located at the same chromosomal position. Allelic variants encompass Single Nucleotide Polymorphisms (SNPs), as well as Small Insertion/Deletion Polymorphisms (INDELs). The size of INDELs is usually less than 100 bp. SNPs and INDELs form the largest set of sequence variants in naturally occurring polymorphic strains of most organisms.
Gene Shuffling/Directed Evolution
Gene shuffling or directed evolution consists of iterations of DNA shuffling followed by appropriate screening and/or selection to generate variants of nucleic acid sequences or portions thereof encoding proteins having a modified biological activity (Castle et al., (2004) Science 304(5674): 1151-4; U.S. Pat. Nos. 5,811,238 and 6,395,547).
Regulatory Element/Control Sequence/Promoter
The terms “regulatory element”, “control sequence” and “promoter” are all used interchangeably herein and are to be taken in a broad context to refer to regulatory nucleic acid sequences capable of effecting expression of the sequences to which they are ligated. The term “promoter” typically refers to a nucleic acid 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 sequence. 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 sequence 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 sequence must, as described above, be linked operably to or comprise a suitable promoter which expresses the gene at the right moment in time and with the required spatial expression pattern.
For the identification of functionally equivalent promoters, the promoter strength and/or expression pattern of a candidate promoter may be analysed for example by operably linking the promoter to a reporter gene and assaying the expression level and pattern of the reporter gene in various tissues of the plant. Suitable well-known reporter genes include for example beta-glucuronidase or beta-galactosidase. The promoter activity is assayed by measuring the enzymatic activity of the beta-glucuronidase or beta-galactosidase. The promoter strength and/or expression pattern may then be compared to that of a reference promoter (such as the one used in the methods of the present invention). Alternatively, promoter strength may be assayed by quantifying mRNA levels or by comparing mRNA levels of the nucleic acid sequence used in the methods of the present invention, with mRNA levels of housekeeping genes such as 18S rRNA, using methods known in the art, such as Northern blotting with densitometric analysis of autoradiograms, quantitative real-time PCR or RT-PCR (Heid et al., 1996 Genome Methods 6: 986-994). Generally by “weak promoter” is intended a promoter that drives expression of a coding sequence at a low level. By “low level” is intended at levels of about 1/10,000 transcripts to about 1/100,000 transcripts, to about 1/500,0000 transcripts per cell. Conversely, a “strong promoter” drives expression of a coding sequence at high level, or at about 1/10 transcripts to about 1/100 transcripts to about 1/1000 transcripts per cell.
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


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


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


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


Rubisco small
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





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


Arabidopsis phosphate
Kovama et al., 2005


transporter PHT1


Medicago phosphate
Xiao et al., 2006


transporter


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


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


RB7, RD2, RD5, RH12
1203-1211


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



124-129


Root-specific hydroxy-
Keller & Lamb (1989) Genes & Dev 3:


proline rich protein
1639-1646


Arabidopsis CDC27B/
Blilou et al. (2002) Genes & Dev 16:


hobbit
2566-2575









A seed-specific promoter is transcriptionally active predominantly in seed tissue, but not necessarily exclusively in seed tissue (in cases of leaky expression). The seed-specific promoter may be active during seed development and/or during germination. The seed specific promoter may be specific to one or more of: endosperm, aleurone, or embryo specific. Examples of seed-specific promoters are shown in Tables 2c, 2d, 2e, 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 glutenin-1
Mol Gen Genet 216: 81-90, 1989; NAR 17: 461-2, 1989


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


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


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


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



Plant J 4: 343-55, 1993; Mol Gen Genet 250: 750-60, 1996


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


blz2
EP99106056.7


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


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


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


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



1996


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


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


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


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


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


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


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


PRO0117, putative rice 40S
WO 2004/070039


ribosomal protein


PRO0136, rice alanine
unpublished


aminotransferase


PRO0147, trypsin inhibitor ITR1
Unpublished


(barley)


PRO0151, rice WSI18
WO 2004/070039


PRO0175, rice RAB21
WO 2004/070039


PRO005
WO 2004/070039


PRO0095
WO 2004/070039


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



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


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


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


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


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
















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 and
Colot et al. (1989) Mol Gen Genet 216: 81-90;


HMW glutenin-1
Anderson et al. (1989) NAR 17: 461-2


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


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


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


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 Glb-1
Wu et al. (1998) Plant Cell Physiol 39(8) 885-889


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





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



Skriver et al, Proc Natl Acad Sci USA 88:



7266-7270, 1991


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


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


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


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









A green tissue-specific promoter as defined herein is a promoter that is transcriptionally active predominantly in green tissue, substantially to the exclusion of any other parts of a plant, whilst still allowing for any leaky expression in these other plant parts.
Examples of green tissue-specific promoters which may be used to perform the methods of the invention are shown in Table 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, from
Sato et al. (1996)



embryo globular stage to
Proc. Natl. Acad. Sci.



seedling stage
USA, 93: 8117-8122


Rice
Meristem specific
BAD87835.1


metallothionein


WAK1 & WAK 2
Shoot and root apical
Wagner & Kohorn



meristems, and in expanding
(2001) Plant Cell



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.
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, preferably the expression level is increased. 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 enhanced yield-related traits (for example, increased yield and/or increased growth) of the plants.
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 acid sequences 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 sequence 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).
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 gene/nucleic acid sequence) in an isolated form subsequently (re)introduced into a plant (a transgene). For example, a transgenic plant containing such a transgene may encounter a substantial reduction of the transgene expression and/or substantial reduction of expression of the endogenous gene. The isolated gene may be isolated from an organism or may be manmade, for example by chemical synthesis.
Decreased Expression
Reference herein to “decreased 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 sequence encoding the protein of interest (target gene), or from any nucleic acid sequence capable of encoding an orthologue, paralogue or homologue of the protein of interest. Preferably, the stretch of substantially contiguous nucleotides is capable of forming hydrogen bonds with the target gene (either sense or antisense strand), more preferably, the stretch of substantially contiguous nucleotides has, in increasing order of preference, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity to the target gene (either sense or antisense strand). A nucleic acid sequence encoding a (functional) polypeptide is not a requirement for the various methods discussed herein for the reduction or substantial elimination of expression of an endogenous gene.
This reduction or substantial elimination of expression may be achieved using routine tools and techniques. A method for the reduction or substantial elimination of endogenous gene expression is by RNA-mediated silencing using an inverted repeat of a nucleic acid sequence or a part thereof (in this case a stretch of substantially contiguous nucleotides derived from the gene of interest, or from any nucleic acid sequence capable of encoding an orthologue, paralogue or homologue of the protein of interest), preferably capable of forming a hairpin structure. Another example of an RNA silencing method involves the introduction of nucleic acid sequences or parts thereof (in this case a stretch of substantially contiguous nucleotides derived from the gene of interest, or from any nucleic acid sequence capable of encoding an orthologue, paralogue or homologue of the protein of interest) in a sense orientation into a plant. Another example of an RNA silencing method involves the use of antisense nucleic acid sequences. Gene silencing may also be achieved by insertion mutagenesis (for example, T-DNA insertion or transposon insertion) or by strategies as described by, among others, Angell and Baulcombe ((1999) Plant J 20(3): 357-62), (Amplicon VIGS WO 98/36083), or Baulcombe (WO 99/15682). Other methods, such as the use of antibodies directed to an endogenous polypeptide for inhibiting its function in planta, or interference in the signalling pathway in which a polypeptide is involved, will be well known to the skilled man. Artificial and/or natural microRNAs (miRNAs) may be used to knock out gene expression and/or mRNA translation. Endogenous miRNAs are single stranded small RNAs of typically 19-24 nucleotides long. Artificial microRNAs (amiRNAs), which are typically 21 nucleotides in length, can be genetically engineered specifically to negatively regulate gene expression of single or multiple genes of interest. Determinants of plant microRNA target selection are well known in the art. Empirical parameters for target recognition have been defined and can be used to aid in the design of specific amiRNAs (Schwab et al., (2005) Dev Cell 8(4):517-27). Convenient tools for design and generation of amiRNAs and their precursors are also available to the public (Schwab et al., (2006) Plant Cell 18(5):1121-33).
For optimal performance, the gene silencing techniques used for reducing expression in a plant of an endogenous gene requires the use of nucleic acid sequences from monocotyledonous plants for transformation of monocotyledonous plants, and from dicotyledonous plants for transformation of dicotyledonous plants. Preferably, a nucleic acid sequence from any given plant species is introduced into that same species. For example, a nucleic acid sequence from rice is transformed into a rice plant. However, it is not an absolute requirement that the nucleic acid sequence to be introduced originates from the same plant species as the plant in which it will be introduced. It is sufficient that there is substantial homology between the endogenous target gene and the nucleic acid sequence to be introduced.
Described above are examples of various methods for the reduction or substantial elimination of expression in a plant of an endogenous gene. A person skilled in the art would readily be able to adapt the aforementioned methods for silencing so as to achieve reduction of expression of an endogenous gene in a whole plant or in parts thereof through the use of an appropriate promoter, for example.
Selectable Marker (Gene)/Reporter Gene
“Selectable marker”, “selectable marker gene” or “reporter gene” includes any gene that confers a phenotype on a cell in which it is expressed to facilitate the identification and/or selection of cells that are transfected or transformed with a nucleic acid 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 acid sequences into plant cells, only a minority of the cells takes up the foreign DNA and, if desired, integrates it into its genome, depending on the expression vector used and the transfection technique used. To identify and select these integrants, a gene coding for a selectable marker (such as the ones described above) is usually introduced into the host cells together with the gene of interest. These markers can for example be used in mutants in which these genes are not functional by, for example, deletion by conventional methods. Furthermore, nucleic acid molecules encoding a selectable marker can be introduced into a host cell on the same vector that comprises the sequence encoding the polypeptides of the invention or used in the methods of the invention, or else in a separate vector. Cells which have been stably transfected with the introduced nucleic acid sequence can be identified for example by selection (for example, cells which have integrated the selectable marker survive whereas the other cells die).
Since the marker genes, particularly genes for resistance to antibiotics and herbicides, are no longer required or are undesired in the transgenic host cell once the nucleic acid sequences have been introduced successfully, the process according to the invention for introducing the nucleic acid sequences advantageously employs techniques which enable the removal or excision of these marker genes. One such a method is what is known as co-transformation. The co-transformation method employs two vectors simultaneously for the transformation, one vector bearing the nucleic acid sequence according to the invention and a second bearing the marker gene(s). A large proportion of transformants receives or, in the case of plants, comprises (up to 40% or more of the transformants), both vectors. In case of transformation with Agrobacteria, the transformants usually receive only a part of the vector, i.e. the sequence flanked by the T-DNA, which usually represents the expression cassette. The marker genes can subsequently be removed from the transformed plant by performing crosses. In another method, marker genes integrated into a transposon are used for the transformation together with desired nucleic acid sequence (known as the Ac/Ds technology). The transformants can be crossed with a transposase source or the transformants are transformed with a nucleic acid construct conferring expression of a transposase, transiently or stable. In some cases (approx. 10%), the transposon jumps out of the genome of the host cell once transformation has taken place successfully and is lost. In a further number of cases, the transposon jumps to a different location. In these cases the marker gene must be eliminated by performing crosses. In microbiology, techniques were developed which make possible, or facilitate, the detection of such events. A further advantageous method relies on what is known as recombination systems; whose advantage is that elimination by crossing can be dispensed with. The best-known system of this type is what is known as the Cre/lox system. Cre1 is a recombinase that removes the sequences located between the loxP sequences. If the marker gene is integrated between the loxP sequences, it is removed once transformation has taken place successfully, by expression of the recombinase. Further recombination systems are the HIN/HIX, FLP/FRT and REP/STB system (Tribble et al., J. Biol. Chem., 275, 2000: 22255-22267; Velmurugan et al., J. Cell Biol., 149, 2000: 553-566). A site-specific integration into the plant genome of the nucleic acid sequences according to the invention is possible. Naturally, these methods can also be applied to microorganisms such as yeast, fungi or bacteria.
Transgenic/Transgene/Recombinant
For the purposes of the invention, “transgenic”, “transgene” or “recombinant” means with regard to, for example, a nucleic acid sequence, an expression cassette, gene construct or a vector comprising the nucleic acid sequence or an organism transformed with the nucleic acid sequences, expression cassettes or vectors according to the invention, all those constructions brought about by recombinant methods in which either

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

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

    
    


A transgenic plant for the purposes of the invention is thus understood as meaning, as above, that the nucleic acid sequences used in the method of the invention are not at their natural locus in the genome of said plant, it being possible for the nucleic acid sequences to be expressed homologously or heterologously. However, as mentioned, transgenic also means that, while the nucleic acid sequences according to the invention or used in the inventive method are at their natural position in the genome of a plant, the sequence has been modified with regard to the natural sequence, and/or that the regulatory sequences of the natural sequences have been modified. Transgenic is preferably understood as meaning the expression of the nucleic acid sequences according to the invention at an unnatural locus in the genome, i.e. homologous or, preferably, heterologous expression of the nucleic acid sequences takes place. Preferred transgenic plants are mentioned herein.
Transformation
The term “introduction” or “transformation” as referred to herein encompasses the transfer of an exogenous polynucleotide into a host cell, irrespective of the method used for transfer. Plant tissue capable of subsequent clonal propagation, whether by organogenesis or embryogenesis, may be transformed with a genetic construct of the present invention and a whole plant regenerated there from. The particular tissue chosen will vary depending on the clonal propagation systems available for, and best suited to, the particular species being transformed. Exemplary tissue targets include leaf disks, pollen, embryos, cotyledons, hypocotyls, megagametophytes, callus tissue, existing meristematic tissue (e.g., apical meristem, axillary buds, and root meristems), and induced meristem tissue (e.g., cotyledon meristem and hypocotyl meristem). The polynucleotide may be transiently or stably introduced into a host cell and may be maintained non-integrated, for example, as a plasmid. Alternatively, it may be integrated into the host genome. The resulting transformed plant cell may then be used to regenerate a transformed plant in a manner known to persons skilled in the art.
The transfer of foreign genes into the genome of a plant is called transformation. Transformation of plant species is now a fairly routine technique. Advantageously, any of several transformation methods may be used to introduce the gene of interest into a suitable ancestor cell. The methods described for the transformation and regeneration of plants from plant tissues or plant cells may be utilized for transient or for stable transformation. Transformation methods include the use of liposomes, electroporation, chemicals that increase free DNA uptake, injection of the DNA directly into the plant, particle gun bombardment, transformation using viruses or pollen and microprojection. Methods may be selected from the calcium/polyethylene glycol method for protoplasts (Krens, F. A. et al., (1982) Nature 296, 72-74; Negrutiu I et al. (1987) Plant Mol Biol 8: 363-373); electroporation of protoplasts (Shillito R. D. et al. (1985) Bio/Technol 3, 1099-1102); microinjection into plant material (Crossway A et al., (1986) Mol. Gen. Genet. 202: 179-185); DNA or RNA-coated particle bombardment (Klein T M et al., (1987) Nature 327: 70) infection with (non-integrative) viruses and the like. Transgenic plants, including transgenic crop plants, are preferably produced via Agrobacterium-mediated transformation. An advantageous transformation method is the transformation in planta. To this end, it is possible, for example, to allow the agrobacteria to act on plant seeds or to inoculate the plant meristem with agrobacteria. It has proved particularly expedient in accordance with the invention to allow a suspension of transformed agrobacteria to act on the intact plant or at least on the flower primordia. The plant is subsequently grown on until the seeds of the treated plant are obtained (Clough and Bent, Plant J. (1998) 16, 735-743). Methods for Agrobacterium-mediated transformation of rice include well known methods for rice transformation, such as those described in any of the following: European patent application EP 1198985 A1, Aldemita and Hodges (Planta 199: 612-617, 1996); Chan et al. (Plant Mol Biol 22 (3): 491-506, 1993), Hiei et al. (Plant J 6 (2): 271-282, 1994), which disclosures are incorporated by reference herein as if fully set forth. In the case of corn transformation, the preferred method is as described in either Ishida et al. (Nat. Biotechnol 14(6): 745-50, 1996) or Frame et al. (Plant Physiol 129(1): 13-22, 2002), which disclosures are incorporated by reference herein as if fully set forth. Said methods are further described by way of example in B. Jenes et al., Techniques for Gene Transfer, in: Transgenic Plants, Vol. 1, Engineering and Utilization, eds. S. D. Kung and R. Wu, Academic Press (1993) 128-143 and in Potrykus Annu. Rev. Plant Physiol. Plant Molec. Biol. 42 (1991) 205-225). The nucleic acid sequences or the construct to be expressed is preferably cloned into a vector, which is suitable for transforming Agrobacterium tumefaciens, for example pBin19 (Bevan et al., Nucl. Acids Res. 12 (1984) 8711). Agrobacteria transformed by such a vector can then be used in known manner for the transformation of plants, such as plants used as a model, like Arabidopsis (Arabidopsis thaliana is within the scope of the present invention not considered as a crop plant), or crop plants such as, by way of example, tobacco plants, for example by immersing bruised leaves or chopped leaves in an agrobacterial solution and then culturing them in suitable media. The transformation of plants by means of Agrobacterium tumefaciens is described, for example, by Höfgen and Willmitzer in Nucl. Acid Res. (1988) 16, 9877 or is known inter alia from F. F. White, Vectors for Gene Transfer in Higher Plants; in Transgenic Plants, Vol. 1, Engineering and Utilization, eds. S. D. Kung and R. Wu, Academic Press, 1993, pp. 15-38.
In addition to the transformation of somatic cells, which then have to be regenerated into intact plants, it is also possible to transform the cells of plant meristems and in particular those cells which develop into gametes. In this case, the transformed gametes follow the natural plant development, giving rise to transgenic plants. Thus, for example, seeds of Arabidopsis are treated with agrobacteria and seeds are obtained from the developing plants of which a certain proportion is transformed and thus transgenic [Feldman, K A and Marks M D (1987). Mol Gen Genet 208:274-289; Feldmann K (1992). In: C Koncz, N-H Chua and J Shell, eds, Methods in Arabidopsis Research. Word Scientific, Singapore, pp. 274-289]. Alternative methods are based on the repeated removal of the inflorescences and incubation of the excision site in the center of the rosette with transformed agrobacteria, whereby transformed seeds can likewise be obtained at a later moment in time (Chang (1994). Plant J. 5: 551-558; Katavic (1994). Mol Gen Genet, 245: 363-370). However, an especially effective method is the vacuum infiltration method with its modifications such as the “floral dip” method. In the case of vacuum infiltration of Arabidopsis, intact plants under reduced pressure are treated with an agrobacterial suspension [Bechthold, N (1993). C R Acad Sci Paris Life Sci, 316: 1194-1199], while in the case of the “floral dip” method the developing floral tissue is incubated briefly with a surfactant-treated agrobacterial suspension [Clough, S J and Bent A F (1998) The Plant J. 16, 735-743]. A certain proportion of transgenic seeds are harvested in both cases, and these seeds can be distinguished from non-transgenic seeds by growing under the above-described selective conditions. In addition the stable transformation of plastids is of advantages because plastids are inherited maternally is most crops reducing or eliminating the risk of transgene flow through pollen. The transformation of the chloroplast genome is generally achieved by a process which has been schematically displayed in Klaus et al., 2004 [Nature Biotechnology 22 (2), 225-229]. Briefly the sequences to be transformed are cloned together with a selectable marker gene between flanking sequences homologous to the chloroplast genome. These homologous flanking sequences direct site specific integration into the plastome. Plastidal transformation has been described for many different plant species and an overview is given in Bock (2001) Transgenic plastids in basic research and plant biotechnology. J Mol Biol. 2001 Sep. 21; 312 (3):425-38 or Maliga, P (2003) Progress towards commercialization of plastid transformation technology. Trends Biotechnol. 21, 20-28. Further biotechnological progress has recently been reported in form of marker free plastid transformants, which can be produced by a transient co-integrated maker gene (Klaus et al., 2004, Nature Biotechnology 22(2), 225-229).
T-DNA Activation Tagging
T-DNA activation tagging (Hayashi et al. Science (1992) 1350-1353), involves insertion of T-DNA, usually containing a promoter (may also be a translation 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 acid sequences encoding proteins with modified expression and/or activity. TILLING also allows selection of plants carrying such mutant variants. These mutant variants may exhibit modified expression, either in strength or in location or in timing (if the mutations affect the promoter for example). These mutant variants may exhibit higher activity than that exhibited by the gene in its natural form. TILLING combines high-density mutagenesis with high-throughput screening methods. The steps typically followed in TILLING are: (a) EMS mutagenesis (Redei G P and Koncz C (1992) In Methods in Arabidopsis Research, Koncz C, Chua N H, Schell J, eds. Singapore, World Scientific Publishing Co, pp. 16-82; Feldmann et al., (1994) In Meyerowitz E M, Somerville C R, eds, Arabidopsis. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., pp 137-172; Lightner J and Caspar T (1998) In J Martinez-Zapater, J Salinas, eds, Methods on Molecular Biology, Vol. 82. Humana Press, Totowa, N.J., pp 91-104); (b) DNA preparation and pooling of individuals; (c) PCR amplification of a region of interest; (d) denaturation and annealing to allow formation of heteroduplexes; (e) DHPLC, where the presence of a heteroduplex in a pool is detected as an extra peak in the chromatogram; (f) identification of the mutant individual; and (g) sequencing of the mutant PCR product. Methods for TILLING are well known in the art (McCallum et al., (2000) Nat Biotechnol 18: 455-457; reviewed by Stemple (2004) Nat Rev Genet 5(2): 145-50).
Homologous Recombination
Homologous recombination allows introduction in a genome of a selected nucleic acid sequence at a defined selected position. Homologous recombination is a standard technology used routinely in biological sciences for lower organisms such as yeast or the moss Physcomitrella. Methods for performing homologous recombination in plants have been described not only for model plants (Offringa et al. (1990) EMBO J 9(10): 3077-84) but also for crop plants, for example rice (Terada et al. (2002) Nat Biotech 20(10): 1030-4; lida and Terada (2004) Curr Opin Biotech 15(2): 132-8); Terada et al., (2007) Plant Physiol).
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 acre for a crop and year, which is determined by dividing total production (includes both harvested and appraised production) by planted acres. The term “yield” of a plant may relate to vegetative biomass (root and/or shoot biomass), to reproductive organs, and/or to propagules (such as seeds) of that plant.
Early Vigour
“Early vigour” refers to active healthy well-balanced growth especially during early stages of plant growth, and may result from increased plant fitness due to, for example, the plants being better adapted to their environment (i.e. optimizing the use of energy resources and partitioning between shoot and root). Plants having early vigour also show increased seedling survival and a better establishment of the crop, which often results in highly uniform fields (with the crop growing in uniform manner, i.e. with the majority of plants reaching the various stages of development at substantially the same time), and often better and higher yield. Therefore, early vigour may be determined by measuring various factors, such as thousand kernel weight, percentage germination, percentage emergence, seedling growth, seedling height, root length, root and shoot biomass and many more.
Increase/Improve/Enhance
The terms “increase”, “improve” or “enhance” are interchangeable and shall mean in the sense of the application at least a 5%, 6%, 7%, 8%, 9% or 10%, preferably at least 15% or 20%, more preferably 25%, 30%, 35% or 40% more yield and/or growth in comparison to control plants as defined herein.
Seed Yield
Increased seed yield may manifest itself as one or more of the following: a) an increase in seed biomass (total seed weight) which may be on an individual seed basis and/or per plant and/or per hectare or acre; b) increased number of flowers per plant; c) increased number of (filled) seeds; d) increased seed filling rate (which is expressed as the ratio between the number of filled seeds divided by the total number of seeds); e) increased harvest index, which is expressed as a ratio of the yield of harvestable parts, such as seeds, divided by the total biomass; 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. Increased yield may also result in modified architecture, or may occur because of modified architecture.
Greenness Index
The “greenness index” as used herein is calculated from digital images of plants. For each pixel belonging to the plant object on the image, the ratio of the green value versus the red value (in the RGB model for encoding color) is calculated. The greenness index is expressed as the percentage of pixels for which the green-to-red ratio exceeds a given threshold. Under normal growth conditions, under salt stress growth conditions, and under reduced nutrient availability growth conditions, the greenness index of plants is measured in the last imaging before flowering. In contrast, under drought stress growth conditions, the greenness index of plants is measured in the first imaging after drought.
Plant
The term “plant” as used herein encompasses whole plants, ancestors and progeny of the plants and plant parts, including seeds, shoots, stems, leaves, roots (including tubers), flowers, and tissues and organs, wherein each of the aforementioned comprise the gene/nucleic acid sequence of interest. The term “plant” also encompasses plant cells, suspension cultures, callus tissue, embryos, meristematic regions, gametophytes, sporophytes, pollen and microspores, again wherein each of the aforementioned comprises the gene/nucleic acid sequence of interest.
Plants that are particularly useful in the methods of the invention include all plants which belong to the superfamily Viridiplantae, in particular monocotyledonous and dicotyledonous plants including fodder or forage legumes, ornamental plants, food crops, trees or shrubs selected from the list comprising Acer spp., Actinidia spp., Abelmoschus spp., Agave sisalana, Agropyron spp., Agrostis stolonifera, Allium spp., Amaranthus spp., Ammophila arenaria, Ananas comosus, Annona spp., Apium graveolens, Arachis spp, Artocarpus spp., Asparagus officinalis, Avena spp. (e.g. Avena sativa, Avena fatua, Avena byzantina, Avena fatua var. sativa, Avena hybrida), Averrhoa carambola, Bambusa sp., Benincasa hispida, Bertholletia excelsea, Beta vulgaris, Brassica spp. (e.g. Brassica napus, Brassica rapa ssp. [canola, oilseed rape, turnip rape]), Cadaba farinosa, Camellia sinensis, Canna indica, Cannabis sativa, Capsicum spp., Carex 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, 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., Triticosecale rimpaui, Triticale (Triticum secale), Triticum spp. (e.g. Triticum aestivum, Triticum durum, Triticum turgidum, Triticum hybernum, Triticum macha, Triticum sativum or Triticum vulgare), Tropaeolum minus, Tropaeolum majus, Vaccinium spp., Vicia spp., Vigna spp., Viola odorata, Vitis spp., Zea mays, Zizania palustris, Ziziphus spp., amongst others.
DETAILED DESCRIPTION OF THE INVENTION
1. 2-Cysteine Peroxiredoxin (2-Cys PRX)
Surprisingly, it has now been found that modulating, preferably increasing, expression in the roots of a plant, of a nucleic acid sequence encoding a 2-Cys PRX 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, preferably increasing, expression in the roots of a plant, of a nucleic acid sequence encoding a 2-Cys PRX polypeptide.
A preferred method for modulating, preferably increasing, expression of a nucleic acid sequence encoding a 2-Cys PRX polypeptide is by introducing and expressing in the roots of a plant, a nucleic acid sequence encoding a 2-Cys PRX polypeptide.
Any reference hereinafter to a “protein useful in the methods of the invention” is taken to mean a 2-Cys PRX polypeptide as defined herein. Any reference hereinafter to a “nucleic acid sequence useful in the methods of the invention” is taken to mean a nucleic acid sequence capable of encoding such a 2-Cys PRX polypeptide. The nucleic acid sequence to be introduced into a plant (and therefore useful in performing the methods of the invention) is any nucleic acid sequence encoding the type of polypeptide, which will now be described, hereafter also named “2-Cys PRX nucleic acid sequence” or “2-Cys PRX gene”.
A “2-Cys PRX polypeptide” as defined herein refers to any polypeptide comprising from N-terminus to C-terminus: (1) a plastidic transit peptide; and (2) a 2-Cys PRX conserved domain.
Additionally, a “2-Cys PRX polypeptide” comprises one or both of: (i) Motif1 as represented by SEQ ID NO: 77, or a motif having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more amino acid sequence identity to SEQ ID NO: 77; or (ii) Motif 2 as represented by SEQ ID NO: 78, or a motif having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more amino acid sequence identity to SEQ ID NO: 78.
Alternatively or additionally, a “2-Cys PRX polypeptide” as defined herein refers to any polypeptide sequence which when used in the construction of a phylogenetic tree, such as the one depicted in FIG. 3, tends to cluster with the 2-Cys PRX clade of polypeptides comprising the polypeptide sequence as represented by SEQ ID NO: 2, rather than with any other PRX clade.
Alternatively or additionally, a “2-Cys PRX polypeptide” as defined herein refers to any polypeptide having in increasing order of preference at least 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more sequence identity to the 2-Cys PRX polypeptide as represented by SEQ ID NO: 2 or to any of the polypeptide sequences given in Table A1 herein.
The term “domain” and “motif” is defined in the “definitions” section herein. Specialist databases exist for the identification of domains, for example, SMART (Schultz et al. (1998) Proc. Natl. Acad. Sci. USA 95, 5857-5864; Letunic et al. (2002) Nucleic Acids Res 30, 242-244, InterPro (Mulder et al., (2003) Nucl. Acids. Res. 31, 315-318, Prosite (Bucher and Bairoch (1994), A generalized profile syntax for biomolecular sequences motifs and its function in automatic sequence interpretation. (In) ISMB-94; Proceedings 2nd International Conference on Intelligent Systems for Molecular Biology. Altman R., Brutlag D., Karp P., Lathrop R., Searls D., Eds., pp 53-61, AAAI Press, Menlo Park; Hulo et al., Nucl. Acids. Res. 32:D134-D137, (2004), or Pfam (Bateman et al., Nucleic Acids Research 30(1): 276-280 (2002). A set of tools for in silico analysis of protein sequences is available on the ExPASy proteomics server (Swiss Institute of Bioinformatics (Gasteiger et al., ExPASy: the proteomics server for in-depth protein knowledge and analysis, Nucleic Acids Res. 31:3784-3788 (2003)). Domains may also be identified using routine techniques, such as by sequence alignment. Analysis of the polypeptide sequence of SEQ ID NO: 2 is presented below in Examples 2 and 4.
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, which are indicated below in Example 3 as a percentage were determined over the entire nucleic acid sequence or polypeptide sequence (Table A2 herein), but may also be determined over selected domains or conserved motif(s) (such as Motif 1 as represented by SEQ ID NO: 77, and such as Motif 2 as represented by SEQ ID NO: 78, both Motif1 and Motif2 comprised in SEQ ID NO: 2), using the programs mentioned above using the default parameters.
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). 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, Predotar, LipoP, MITOPROT, PATS, PTS1, SignalP and others. The identification of subcellular localisation of the polypeptide of the invention is shown in Example 5. In particular SEQ ID NO: 2 of the present invention is assigned to the plastidic (chloroplastic) compartment of photosynthetic (autotrophic) cells.
Methods for targeting proteins to plastids are well known in the art and include the use of transit peptides. Table 3 below shows examples of transit peptides which can be used to target any 2-Cys PRX polypeptide to a plastid, which 2-Cys PRX polypeptide is not, in its natural form, normally targeted to a plastid, or which 2-Cys PRX polypeptide in its natural form is targeted to a plastid by virtue of a different transit peptide (for example, its natural transit peptide). For example, a nucleic acid sequence encoding a cyanobacterial 2-Cys PRX polypeptide may also be suitable for use in the methods of the invention as long as the 2-Cys PRX polypeptide is targeted to a plastid, preferably to a chloroplast.







TABLE 3







Examples of transit peptide sequences


useful in targeting polypeptides to plastids











NCBI



SEQ


Accession



ID


Number
Source Organism
Protein Function
Transit Peptide Sequence
NO:





P07839
Chlamydomonas
Ferredoxin
MAMAMRSTFAARVGAKPAVRGARPASRMSCMA
156


 


AAR23425
Chlamydomonas
Rubisco activase
MQVTMKSSAVSGQRVGGARVATRSVRRAQLQV
157


 


CAA56932
Arabidopsis thaliana
Aspartate amino
MASLMLSLGSTSLLPREINKDKLKLGTSASNPFLKAK
158




transferase
SFSRVTMTVAVKPSR



 


CAA31991
Arabidopsis thaliana
Acyl carrier protein1
MATQFSASVSLQTSCLATTRISFQKPALISNHGKTNL
159





SFNLRRSIPSRRLSVSC



 


CAB63798
Arabidopsis thaliana
Acyl carrier protein2
MASIAASASISLQARPRQLAIAASQVKSFSNGRRSSL
160





SFNLRQLPTRLTVSCAAKPETVDKVCAVVRKQL



 


CAB63799
Arabidopsis thaliana
Acyl carrier protein3
MASIATSASTSLQARPRQLVIGAKQVKSFSYGSRSNL
161





SFNLRQLPTRLTVYCAAKPETVDKVCAVVRKQLSLKE









A 2-Cys PRX polypeptide is targeted and active in the plastid, i.e., the 2-Cys PRX polypeptide (at least in its native form) is capable of catalyzing the removal of H2O2, in the chloroplast. Assays for testing this activity are well known in the art. Further details are provided in Example 6.
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 2-Cys PRX-encoding nucleic acid sequence or 2-Cys PRX polypeptide sequence as defined herein.
Examples of nucleic acid sequences encoding 2-Cys PRX polypeptides are given in Table A1 of Example 1 herein. Such nucleic acid sequences are useful in performing the methods of the invention. The polypeptide sequences given in Table A1 of Example 1 are example sequences of orthologues and paralogues of the 2-Cys PRX 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 Example 1) against any sequence database, such as the publicly available NCBI database. BLASTN or TBLASTX (using standard default values) are generally used when starting from a nucleotide sequence, and BLASTP or TBLASTN (using standard default values) when starting from a protein sequence.
The BLAST results may optionally be filtered. The full-length sequences of either the filtered results or non-filtered results are then BLASTed back (second BLAST) against sequences from the organism from which the query sequence is derived (where the query sequence is SEQ ID NO: 1 or SEQ ID NO: 2, the second BLAST would therefore be against Brassica 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.
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 (see FIG. 3).
Nucleic acid variants may also be useful in practising the methods of the invention. Examples of such variants include nucleic acid sequences encoding homologues and derivatives of any one of the polypeptide sequences given in Table A1 of Example 1, the terms “homologue” and “derivative” being as defined herein. Also useful in the methods of the invention are nucleic acid sequences encoding homologues and derivatives of orthologues or paralogues of any one of the polypeptide sequences given in Table A1 of Example 1. Homologues and derivatives useful in the methods of the present invention have substantially the same biological and functional activity as the unmodified protein from which they are derived.
Further nucleic acid variants useful in practising the methods of the invention include portions of nucleic acid sequences encoding 2-Cys PRX polypeptides, nucleic acid sequences hybridising to nucleic acid sequences encoding 2-Cys PRX polypeptides, splice variants of nucleic acid sequences encoding 2-Cys PRX polypeptides, allelic variants of nucleic acid sequences encoding 2-Cys PRX polypeptides and variants of nucleic acid sequences encoding 2-Cys PRX polypeptides obtained by gene shuffling. The terms hybridising sequence, splice variant, allelic variant and gene shuffling are as described herein.
Nucleic acid sequences encoding 2-Cys PRX polypeptides need not be full-length nucleic acid sequences, since performance of the methods of the invention does not rely on the use of full-length nucleic acid sequences. According to the present invention, there is provided a method for enhancing yield-related traits in plants, comprising introducing and expressing in the roots of a plant, a portion of any one of the nucleic acid sequences given in Table A1 of Example 1, or a portion of a nucleic acid sequence encoding an orthologue, paralogue or homologue of any of the polypeptide sequences given in Table A1 of Example 1.
A portion of a nucleic acid sequence may be prepared, for example, by making one or more deletions to the nucleic acid sequence. The portions may be used in isolated form or they may be fused to other coding (or non-coding) sequences in order to, for example, produce a protein that combines several activities. When fused to other coding sequences, the resultant polypeptide produced upon translation may be bigger than that predicted for the protein portion.
Portions useful in the methods of the invention, encode a 2-Cys PRX polypeptide as defined herein, and have substantially the same biological activity as the polypeptide sequences given in Table A1 of Example 1. Preferably, the portion is a portion of any one of the nucleic acid sequences given in Table A1 of Example 1, or is a portion of a nucleic acid sequence encoding an orthologue or paralogue of any one of the polypeptide sequences given in Table A1 of Example 1. Preferably the portion is at least 500, 550, 600, 650, 700, 750, 800, consecutive nucleotides in length, the consecutive nucleotides being of any one of the nucleic acid sequences given in Table A1 of Example 1, or of a nucleic acid sequence encoding an orthologue or paralogue of any one of the polypeptide sequences given in Table A1 of Example 1. Most preferably the portion is a portion of the nucleic acid sequence of SEQ ID NO: 1. Preferably, the portion encodes a polypeptide sequence which when used in the construction of a phylogenetic tree, such as the one depicted in FIG. 3, tends to cluster with the group of 2-Cys PRX polypeptides comprising the polypeptide sequence represented by SEQ ID NO: 2 rather than with any other group.
Another nucleic acid variant useful in the methods of the invention is a nucleic acid sequence capable of hybridising, under reduced stringency conditions, preferably under stringent conditions, with a nucleic acid sequence encoding a 2-Cys PRX 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 the roots of a plant, a nucleic acid sequence capable of hybridizing to any one of the nucleic acid sequences given in Table A1 of Example 1, or comprising introducing and expressing in the roots of a plant, a nucleic acid sequence capable of hybridising to a nucleic acid sequence encoding an orthologue, paralogue or homologue of any of the nucleic acid sequences given in Table A1 of Example 1.
Hybridising sequences useful in the methods of the invention encode a 2-Cys PRX polypeptide as defined herein, having substantially the same biological activity as the polypeptide sequences given in Table A1 of Example 1. Preferably, the hybridising sequence is capable of hybridising to any one of the nucleic acid sequences given in Table A1 of Example 1, or to a portion of any of these sequences, a portion being as defined above, or the hybridising sequence is capable of hybridising to a nucleic acid sequence encoding an orthologue or paralogue of any one of the polypeptide sequences given in Table A1 of Example 1. Most preferably, the hybridising sequence is capable of hybridising to a nucleic acid sequence as represented by SEQ ID NO: 1 or to a portion thereof.
Preferably, the hybridising sequence encodes a polypeptide sequence which when used in the construction of a phylogenetic tree, such as the one depicted in FIG. 3, tends to cluster with the group of 2-Cys PRX polypeptides comprising the polypeptide sequence represented by SEQ ID NO: 2 rather than with any other group.
Another nucleic acid variant useful in the methods of the invention is a splice variant encoding a 2-Cys PRX 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 the roots of a plant, a splice variant of any one of the nucleic acid sequences given in Table A1 of Example 1, or a splice variant of a nucleic acid sequence encoding an orthologue, paralogue or homologue of any of the polypeptide sequences given in Table A1 of Example 1.
Preferred splice variants are splice variants of a nucleic acid sequence represented by SEQ ID NO: 1, or a splice variant of a nucleic acid sequence encoding an orthologue or paralogue of SEQ ID NO: 2. Preferably, the polypeptide sequence encoded by the splice variant, when used in the construction of a phylogenetic tree, such as the one depicted in FIG. 3, tends to cluster with the group of 2-Cys PRX polypeptides comprising the polypeptide sequence represented by SEQ ID NO: 2 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 sequence encoding a 2-Cys PRX 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 the roots of a plant, an allelic variant of any one of the nucleic acid sequences given in Table A1 of Example 1, or comprising introducing and expressing in the roots of a plant, an allelic variant of a nucleic acid sequence encoding an orthologue, paralogue or homologue of any of the polypeptide sequences given in Table A1 of Example 1.
The allelic variants useful in the methods of the present invention have substantially the same biological activity as the 2-Cys PRX polypeptide of SEQ ID NO: 2 and any of the polypeptide sequences depicted in Table A1 of Example 1. Allelic variants exist in nature, and encompassed within the methods of the present invention is the use of these natural alleles. Preferably, the allelic variant is an allelic variant of SEQ ID NO: 1 or an allelic variant of a nucleic acid sequence encoding an orthologue or paralogue of SEQ ID NO: 2. Preferably, the polypeptide sequence encoded by the allelic variant, when used in the construction of a phylogenetic tree, such as the one depicted in FIG. 3, tends to cluster with the 2-Cys PRX polypeptides comprising the polypeptide sequence represented by SEQ ID NO: 2 rather than with any other group.
Gene shuffling or directed evolution may also be used to generate variants of nucleic acid sequences encoding 2-Cys PRX polypeptides 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 the roots of a plant, a variant of any one of the nucleic acid sequences given in Table A1 of Example 1, or comprising introducing and expressing in the roots of a plant, a variant of a nucleic acid sequence encoding an orthologue, paralogue or homologue of any of the polypeptide sequences given in Table A1 of Example 1, which variant nucleic acid sequence is obtained by gene shuffling.
Preferably, the polypeptide sequence encoded by the variant nucleic acid sequence obtained by gene shuffling, when used in the construction of a phylogenetic tree such as the one depicted in FIG. 3, tends to cluster with the group of 2-Cys PRX polypeptides comprising the polypeptide sequence represented by SEQ ID NO: 2 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 acid sequences encoding 2-Cys PRX polypeptides may be from a natural source, such as from eubacteria and eukaryotes (fungi, plants, or animals). The nucleic acid sequence derived from any artificial source, or may be modified from its native form in composition and/or genomic environment through deliberate human manipulation. Preferably the 2-Cys PRX polypeptide-encoding nucleic acid sequence is from a plant, further preferably from a dicotyledonous plant, more preferably from the family Brassicaceae, most preferably the nucleic acid sequence is from Brassica rapa. 
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 increased yield, especially increased seed yield relative to control plants. The terms “yield” and “seed yield” are described in more detail in the “definitions” section herein.
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 seeds, and performance of the methods of the invention results in plants having 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 hectare or acre, an increase in the number of ears per plant, an increase in the number of rows, number of kernels per row, kernel weight, thousand kernel weight, ear length/diameter, increase in the seed filling rate (which is the number of filled seeds divided by the total number of seeds and multiplied by 100), among others. Taking rice as an example, a yield increase may manifest itself as an increase in one or more of the following: number of plants per hectare or acre, number of panicles per plant, number of spikelets per panicle, number of flowers (florets) per panicle (which is expressed as a ratio of the number of filled seeds over the number of primary panicles), increase in the seed filling rate (which is the number of filled seeds divided by the total number of seeds and multiplied by 100), increase in thousand kernel weight, among others.
The present invention provides a method for enhancing yield-related traits in plants, especially seed yield of plants, relative to control plants, which method comprises modulating, preferably increasing, expression in the roots of a plant, of a nucleic acid sequence encoding a 2-Cys PRX polypeptide as defined herein.
Since the transgenic plants according to the present invention have enhanced yield-related traits, it is likely that these plants exhibit an increased growth rate (during at least part of their life cycle), relative to the growth rate of control plants at a corresponding stage in their life cycle.
The increased growth rate may be specific to one or more parts of a plant (including seeds), or may be throughout substantially the whole plant. Plants having an increased growth rate may have a shorter life cycle. The life cycle of a plant may be taken to mean the time needed to grow from a dry mature seed up to the stage where the plant has produced dry mature seeds, similar to the starting material. This life cycle may be influenced by factors such as early vigour, growth rate, greenness index, flowering time and speed of seed maturation. The increase in growth rate may take place at one or more stages in the life cycle of a plant or during substantially the whole plant life cycle. Increased growth rate during the early stages in the life cycle of a plant may reflect 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 acre (due to an increase in the number of times (say in a year) that any particular plant may be grown and harvested). An increase in growth rate may also allow for the cultivation of transgenic plants in a wider geographical area than their wild-type counterparts, since the territorial limitations for growing a crop are often determined by adverse environmental conditions either at the time of planting (early season) or at the time of harvesting (late season). Such adverse conditions may be avoided if the harvest cycle is shortened. The growth rate may be determined by deriving various parameters from growth curves, such parameters may be: T-Mid (the time taken for plants to reach 50% of their maximal size) and T-90 (time taken for plants to reach 90% of their maximal size), amongst others.
According to a preferred feature of the present invention, performance of the methods of the invention gives plants having an increased growth rate relative to control plants. Therefore, according to the present invention, there is provided a method for increasing the growth rate of plants, which method comprises modulating, preferably increasing, expression in the roots of a plant, of a nucleic acid sequence encoding a 2-Cys PRX 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% or 30%, preferably less than 25%, 20% or 15%, more preferably less than 14%, 13%, 12%, 11% or 10% or less in comparison to the control plant under non-stress conditions. Due to advances in agricultural practices (irrigation, fertilization, pesticide treatments) severe stresses are not often encountered in cultivated crop plants. As a consequence, the compromised growth induced by mild stress is often an undesirable feature for agriculture. Mild stresses are the everyday biotic and/or abiotic (environmental) stresses to which a plant is exposed. Abiotic stresses may be due to drought or excess water, anaerobic stress, salt stress, chemical toxicity, oxidative stress and hot, cold or freezing temperatures. The abiotic stress may be an osmotic stress caused by a water stress (particularly due to drought), salt stress, oxidative stress or an ionic stress. Biotic stresses are typically those stresses caused by pathogens, such as bacteria, viruses, fungi 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 enhanced yield-related tarits relative to control plants grown under comparable conditions. As reported in Wang et al. (Planta (2003) 218: 1-14), abiotic stress leads to a series of morphological, physiological, biochemical and molecular changes that adversely affect plant growth and productivity. Drought, salinity, extreme temperatures and oxidative stress are known to be interconnected and may induce growth and cellular damage through similar mechanisms. Rabbani et al. (Plant Physiol (2003) 133: 1755-1767) describes a particularly high degree of “cross talk” between drought stress and high-salinity stress. For example, drought and/or salinisation are manifested primarily as osmotic stress, resulting in the disruption of homeostasis and ion distribution in the cell. Oxidative stress, which frequently accompanies high or low temperature, salinity or drought stress, may cause denaturing of functional and structural proteins. As a consequence, these diverse environmental stresses often activate similar cell signalling pathways and cellular responses, such as the production of stress proteins, up-regulation of anti-oxidants, accumulation of compatible solutes and growth arrest. 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.
Performance of the methods of the invention gives plants grown under non-stress conditions or under mild drought conditions enhanced yield-related traits relative to control plants grown under comparable conditions. Therefore, according to the present invention, there is provided a method for enhanced yield-related traits in plants grown under non-stress conditions or under mild drought conditions, which method comprises modulating, preferably increasing, expression in the roots a plant of a nucleic acid sequence encoding a 2-Cys PRX polypeptide.
Performance of the methods according to the present invention results in plants grown under abiotic stress conditions having enhanced yield-related traits relative to control plants grown under comparable stress conditions. As reported in Wang et al. (Planta (2003) 218: 1-14), abiotic stress leads to a series of morphological, physiological, biochemical and molecular changes that adversely affect plant growth and productivity. Drought, salinity, extreme temperatures and oxidative stress are known to be interconnected and may induce growth and cellular damage through similar mechanisms. For example, drought and/or salinisation are manifested primarily as osmotic stress, resulting in the disruption of homeostasis and ion distribution in the cell. Oxidative stress, which frequently accompanies high or low temperature, salinity or drought stress may cause denaturation of functional and structural proteins. As a consequence, these diverse environmental stresses often activate similar cell signaling pathways and cellular responses, such as the production of stress proteins, up-regulation of anti-oxidants, accumulation of compatible solutes and growth arrest. Since diverse environmental stresses activate similar pathways, the exemplification of the present invention with drought stress should not be seen as a limitation to drought stress, but more as a screen to indicate the involvement of 2-Cys PRX polypeptides as defined above, in enhancing yield-related traits relative to control plants grown in comparable stress conditions, in abiotic stresses in general.
A particularly high degree of “cross talk” is reported between drought stress and high-salinity stress (Rabbani et al. (2003) Plant Physiol 133: 1755-1767). Therefore, it would be apparent that a 2-Cys PRX polypeptides would, along with its usefulness in enhancing yield-related traits in plants, relative to control plants grown under drought stress conditions, also find use in enhancing yield-related traits in plants, relative to control plants grown under various other abiotic stress conditions.
The term “abiotic stress” as defined herein is taken to mean any one or more of: water stress (due to drought or excess water), anaerobic stress, salt stress, temperature stress (due to hot, cold or freezing temperatures), chemical toxicity stress and oxidative stress. According to one aspect of the invention, the abiotic stress is an osmotic stress, selected from water stress, salt stress, oxidative stress and ionic stress. Preferably, the water stress is drought stress. The term salt stress is not restricted to common salt (NaCl), but may be any stress caused by one or more of: NaCl, KCl, LiCl, MgCl2, CaCl2, amongst others.
In particular, the enhanced yield-related traits in plants grown under abiotic stress conditions (preferably under drought stress conditions) relative to control plants grown in comparable stress conditions, may include one or more of the following: (i) improved early vigour; (ii) increased aboveground biomass; (iii) increased root (thick and thin) biomass; (iv) increase number of flowers per panicle; (v) increased seed fill rate; (vi) increased total seed yield per plant; (vii) increased number of (filled) seeds; (viii) increased harvest index; or (ix) increased thousand kernel weight (TKW).
Performance of the methods of the invention gives plants having enhanced yield-related traits under abiotic stress conditions relative to control plants grown in comparable stress conditions. Therefore, according to the present invention, there is provided a method for enhancing yield-related traits in plants grown under abiotic stress conditions, which method comprises modulating, preferably increasing, expression in the roots of a plant, of a nucleic acid sequence encoding a 2-Cys PRX polypeptide. According to one aspect of the invention, the abiotic stress is an osmotic stress, selected from one or more of the following: water stress, salt stress, oxidative stress and ionic stress. Preferably, the water stress is drought stress.
Another example of abiotic environmental stress is the reduced availability of one or more nutrients that need to be assimilated by the plants for growth and development. Because of the strong influence of nutrition utilization efficiency on plant yield and product quality, a huge amount of fertilizer is poured onto fields to optimize plant growth and quality. Productivity of plants ordinarily is limited by three primary nutrients, phosphorous, potassium and nitrogen, which is usually the rate-limiting element in plant growth of these three. Therefore the major nutritional element required for plant growth is nitrogen (N). It is a constituent of numerous important compounds found in living cells, including amino acids, proteins (enzymes), nucleic acids, and chlorophyll. 1.5% to 2% of plant dry matter is nitrogen and approximately 16% of total plant protein. Thus, nitrogen availability is a major limiting factor for crop plant growth and production (Frink et al. (1999) Proc Natl Acad Sci USA 96(4): 1175-1180), and has as well a major impact on protein accumulation and amino acid composition. Therefore, of great interest are crop plants with an increased yield when grown under nitrogen-limiting conditions.
Performance of the methods of the invention gives plants grown under conditions of nutrient deficiency, particularly under conditions of nitrogen deficiency, having enhanced yield-related traits relative to control plants grown under comparable conditions. Therefore, according to the present invention, there is provided a method for enhancing yield-related traits in plants grown under conditions of nutrient deficiency, which method comprises modulating, preferably increasing, expression in the roots of a plant, of a nucleic acid sequence encoding a 2-Cys PRX polypeptide. Nutrient deficiency may result from a lack or excess of nutrients such as nitrogen, phosphates and other phosphorous-containing compounds, potassium, calcium, cadmium, magnesium, manganese, iron and boron, amongst others.
The present invention encompasses plants or parts thereof (including seeds) obtainable by the methods according to the present invention. The plants or parts thereof comprise a nucleic acid transgene encoding a 2-Cys PRX polypeptide as defined above, operably linked to a root-specific promoter.
The invention also provides genetic constructs and vectors to facilitate introduction and/or expression in plants of nucleic acid sequences encoding 2-Cys PRX polypeptides. 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 sequence encoding a 2-Cys PRX 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 sequence encoding a 2-Cys PRX polypeptide is as defined above. The term “control sequence” and “termination sequence” are as defined herein.
In one embodiment, one of the control sequences of a construct is a organ-specific promoter, preferably a promoter for expression in the roots of a plant. An example of a root-specific promoter is a Rcc3 promoter, for example a rice Rcc3 promoter as represented by SEQ ID NO: 80.
Plants are transformed with a vector comprising any of the nucleic acid sequences described above. The skilled artisan is well aware of the genetic elements that must be present on the vector in order to successfully transform, select and propagate host cells containing the sequence of interest. The sequence of interest is operably linked to one or more control sequences (at least to a promoter).
Advantageously, any type of promoter, whether natural or synthetic, may be used to drive expression of the nucleic acid sequence. According to a preferred feature of the invention, the nucleic acid sequence encoding a 2-Cys PRX polypeptide is operably linked to a root-specific promoter. 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: 80, most preferably the promoter is as represented by SEQ ID NO: 80. 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.
It should be clear that the applicability of the present invention is not restricted to the 2-Cys PRX polypeptide-encoding nucleic acid sequence represented by SEQ ID NO: 1, nor is the applicability of the invention restricted to expression of a 2-Cys PRX polypeptide-encoding nucleic acid sequence when driven by a root-specific promoter.
Other organ-specific promoters, for example for preferred expression in leaves, stems, tubers, meristems, seeds (embryo and/or endosperm), are useful in performing the methods of the invention. See the “Definitions” section herein for definitions of the various promoter types.
Optionally, one or more terminator sequences may be used in the construct introduced into a plant. Additional regulatory elements may include transcriptional as well as translational 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 acid sequences, it is advantageous to use marker genes (or reporter genes). Therefore, the genetic construct may optionally comprise a selectable marker gene. Selectable markers are described in more detail in the “definitions” section herein.
It is known that upon stable or transient integration of nucleic acid sequences into plant cells, only a minority of the cells takes up the foreign DNA and, if desired, integrates it into its genome, depending on the expression vector used and the transfection technique used. To identify and select these integrants, a gene coding for a selectable marker (such as the ones described above) is usually introduced into the host cells together with the gene of interest. These markers can for example be used in mutants in which these genes are not functional by, for example, deletion by conventional methods. Furthermore, nucleic acid molecules encoding a selectable marker can be introduced into a host cell on the same vector that comprises the sequence encoding the polypeptides of the invention or used in the methods of the invention, or else in a separate vector. Cells which have been stably transfected with the introduced nucleic acid sequence can be identified for example by selection (for example, cells which have integrated the selectable marker survive whereas the other cells die). The marker genes may be removed or excised from the transgenic cell once they are no longer needed. Techniques for marker gene removal are known in the art, useful techniques are described above in the definitions section.
The invention also provides a method for the production of transgenic plants having enhanced yield-related traits relative to control plants, comprising introduction and expression in the roots of a plant, of any nucleic acid sequence encoding a 2-Cys PRX polypeptide as defined hereinabove.
More specifically, the present invention provides a method for the production of transgenic plants having enhanced yield-related traits, which method comprises:

    
    
        (i) introducing and expressing in a plant, plant part, or plant cell a nucleic acid sequence encoding 2-Cys PRX polypeptide, under the control of a root-specific promoter; and
        (ii) cultivating the plant cell under conditions promoting plant growth and development.
    
    


The nucleic acid sequence of (i) may be any of the nucleic acid sequences capable of encoding a 2-Cys PRX polypeptide as defined herein.
The nucleic acid sequence may be introduced directly into a plant cell or into the plant itself (including introduction into a tissue, organ or any other part of a plant). According to a preferred feature of the present invention, the nucleic acid sequence is preferably introduced into a plant by transformation. The term “transformation” is described in more detail in the “definitions” section herein.
The genetically modified plant cells can be regenerated via all methods with which the skilled worker is familiar. Suitable methods can be found in the abovementioned publications by S. D. Kung and R. Wu, Potrykus or Höfgen and Willmitzer.
Generally after transformation, plant cells or cell groupings are selected for the presence of one or more markers which are encoded by plant-expressible genes co-transferred with the gene of interest, following which the transformed material is regenerated into a whole plant. To select transformed plants, the plant material obtained in the transformation is, as a rule, subjected to selective conditions so that transformed plants can be distinguished from untransformed plants. For example, the seeds obtained in the above-described manner can be planted and, after an initial growing period, subjected to a suitable selection by spraying. A further possibility consists in growing the seeds, if appropriate after sterilization, on agar plates using a suitable selection agent so that only the transformed seeds can grow into plants. Alternatively, the transformed plants are screened for the presence of a selectable marker such as the ones described above.
Following DNA transfer and regeneration, putatively transformed plants may also be evaluated, for instance using Southern analysis, for the presence of the gene of interest, copy number and/or genomic organisation. Alternatively or additionally, expression levels of the newly introduced DNA may be monitored using Northern and/or Western analysis, both techniques being well known to persons having ordinary skill in the art.
The generated transformed plants may be propagated by a variety of means, such as by clonal propagation or classical breeding techniques. For example, a first generation (or T1) transformed plant may be selfed and homozygous second-generation (or T2) transformants selected, and the T2 plants may then further be propagated through classical breeding techniques. The generated transformed organisms may take a variety of forms. For example, they may be chimeras of transformed cells and non-transformed cells; clonal transformants (e.g., all cells transformed to contain the expression cassette); grafts of transformed and untransformed tissues (e.g., in plants, a transformed rootstock grafted to an untransformed scion).
The present invention clearly extends to any plant cell or plant produced by any of the methods described herein, and to all plant parts and propagules thereof. The present invention extends further to encompass the progeny of a primary transformed or transfected cell, tissue, organ or whole plant that has been produced by any of the aforementioned methods, the only requirement being that progeny exhibit the same genotypic and/or phenotypic characteristic(s) as those produced by the parent in the methods according to the invention.
The invention also includes host cells containing an isolated nucleic acid sequence encoding a 2-Cys PRX polypeptide as defined hereinabove. Preferred host cells according to the invention are plant cells. Host plants for the nucleic acid sequences or the vector used in the method according to the invention, the expression cassette or construct or vector are, in principle, advantageously all plants, which are capable of synthesizing the polypeptides used in the inventive method.
The methods of the invention are advantageously applicable to any plant. Plants that are particularly useful in the methods of the invention include all plants which belong to the superfamily Viridiplantae, in particular monocotyledonous and dicotyledonous plants including fodder or forage legumes, ornamental plants, food crops, trees or shrubs. According to a preferred embodiment of the present invention, the plant is a crop plant. Examples of crop plants include soybean, sunflower, canola, alfalfa, rapeseed, cotton, tomato, potato and tobacco. Further preferably, the plant is a monocotyledonous plant. Examples of monocotyledonous plants include sugarcane. More preferably the plant is a cereal. Examples of cereals include rice, maize, wheat, barley, millet, rye, triticale, sorghum and oats.
The invention also extends to harvestable parts of a plant such as, but not limited to seeds, leaves, fruits, flowers, stems, rhizomes, tubers and bulbs. The invention furthermore relates to products derived, preferably directly derived, from a harvestable part of such a plant, such as dry pellets or powders, oil, fat and fatty acids, starch or proteins.
According to a preferred feature of the invention, the modulated expression is increased expression. Methods for increasing expression of nucleic acid sequences 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, preferably increasing, expression of a nucleic acid sequence encoding a 2-Cys PRX polypeptide is by introducing and expressing in the roots of a plant, a nucleic acid sequence encoding a 2-Cys PRX 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 acid sequences encoding 2-Cys PRX polypeptides as described herein and use of these 2-Cys PRX polypeptides in enhancing any of the aforementioned yield-related traits in plants.
Nucleic acid sequences encoding 2-Cys PRX polypeptide described herein, or the 2-Cys PRX polypeptides themselves, may find use in breeding programmes in which a DNA marker is identified which may be genetically linked to a 2-Cys PRX polypeptide-encoding gene. The genes/nucleic acid sequences, or the 2-Cys PRX 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 2-Cys PRX polypeptide-encoding gene/nucleic acid sequence 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 enhance yield-related traits. Selection is typically carried out by monitoring growth performance of plants containing different allelic variants of the sequence in question. Growth performance may be monitored in a greenhouse or in the field. Further optional steps include crossing plants in which the superior allelic variant was identified with another plant. This could be used, for example, to make a combination of interesting phenotypic features.
Nucleic acid sequences encoding 2-Cys PRX polypeptides 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 2-Cys PRX polypeptide-encoding nucleic acid sequences requires only a nucleic acid sequence of at least 15 nucleotides in length. The 2-Cys PRX polypeptide-encoding nucleic acid sequences 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 2-Cys PRX-encoding nucleic acid sequences. The resulting banding patterns may then be subjected to genetic analyses using computer programs such as MapMaker (Lander et al. (1987) Genomics 1: 174-181) in order to construct a genetic map. In addition, the nucleic acid sequences may be used to probe Southern blots containing restriction endonuclease-treated genomic DNAs of a set of individuals representing parent and progeny of a defined genetic cross. Segregation of the DNA polymorphisms is noted and used to calculate the position of the 2-Cys PRX polypeptide-encoding nucleic acid sequence 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 acid sequences. Examples include allele-specific amplification (Kazazian (1989) J. Lab. Clin. Med 11:95-96), polymorphism of PCR-amplified fragments (CAPS; Sheffield et al. (1993) Genomics 16:325-332), allele-specific ligation (Landegren et al. (1988) Science 241:1077-1080), nucleotide extension reactions (Sokolov (1990) Nucleic Acid 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.
2. Annexin-Like (ANN)
Surprisingly, it has now been found that modulating expression in a plant of a nucleic acid encoding an annexin-like (hereafter named ANN) 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 ANN polypeptide.
A preferred method for modulating (preferably, increasing) expression of a nucleic acid encoding an ANN polypeptide is by introducing and expressing in a plant a nucleic acid encoding an ANN polypeptide.
Any reference hereinafter to a “protein useful in the methods of the invention” is taken to mean an ANN polypeptide as defined herein. Any reference hereinafter to a “nucleic acid useful in the methods of the invention” is taken to mean a nucleic acid capable of encoding such an ANN polypeptide. The nucleic acid to be introduced into a plant (and therefore useful in performing the methods of the invention) is any nucleic acid encoding the type of protein which will now be described, hereafter also named “ANN nucleic acid” or “ANN gene”.
An “ANN polypeptide” as defined herein refers to any polypeptide comprising in its native form (i.e. the protein as it is encoded in the genome) at least one, preferably two or more of the following conserved signature sequences:







Signature sequence 1


(SEQ ID NO: 87)


(A/L/V) (M/V/L/I) (L/V/M/I/C) X (W/F) (I/V/M/T/A)


 


(L/P/Y/M/F) (D/E/S/H) (P/A) X (G/S/E/A) RDA






wherein X on position 4 may be any amino acid, preferably one of L, S, I, V, Q, or M; and X on position 10 may be any amino acid, preferably one of A, V, P, G, S, T or W.

Preferably, signature sequence 1 is







(A/L/V) (V/L/I) (L/V/M/I) X (W/F) (V/T/A)


 


(L/Y/M/F) (D/E/S/H) PX (E/A) RDA


 


Signature sequence 2 (SEQ ID NO: 88):


A (F/I/V/C/G) XG (F/R/W/M) G (C/T/V) (D/N)


 


(S/A/T/E) X (T/A/V/L/M) (V/I/L) (I/T) X (I/V/T) L


 


(T/A/G) (H/Q/K) (R/S)






wherein X in position 3 may be any amino acid, preferably one of K, R, Q, S, E, A or M; X on position 10 may be any amino acid, preferably one of T, S, K, N, G, D, A, E, Q, or R; X on position 14 may be any amino acid, preferably one of N, A, R, E, D, S, Q,

Preferably, signature sequence 2 is







A (F/I/V/C/G) XG (W/M) G (T/V) (D/N) EX (A/L/M)


 


(I/L) (I/T) X (I/V/T) L (A/G) (H/Q/K) (R/S)


 


Signature sequence 3 (SEQ ID NO: 89):


(T/S) (D/N/E/T) (D/E/K) XXL (I/T/S/N) R (V/I/A/G)


 


(V/I/F) (V/T/C/S/A) (T/S) R (T/A) (E/D)


 


(I/V/F/L/K/H) (D/S)






wherein X on position 4 may be any amino acid, preferably one of S, T, D, E, G, W, N, K; X on position 5 may be any amino acid, preferably one of T, A, S, M, H, D, G, W.

Preferably, signature sequence 3 is







(T/S) (D/E/T) (D/E/K) XXL (T/S/N) R (V/I/A/G)


 


(V/I/F) (V/T/C/S/A) (T/S) R (T/A) (E/D)


 


(I/V/F/L/K/H) (D/S)


 


Signature sequence 4 (SEQ ID NO: 90):


(Y/H) (F/Y) (A/E/V/S) (K/E/D) (V/A/L/I) (L/V/I)


 


(R/H/D) X (S/A) (M/I/L)






wherein X on position 8 may be any amino acid, preferably one of K, E, D, T, L, S, Q, R, N, or A.

Preferably, signature sequence 4 is







(Y/H) (F/Y) (A/E/V/S) (K/E/D) (V/L/I) (L/V/I)


 


(R/D) X (S/A) (I/L)


 


Signature sequence 5 (SEQ ID NO: 91):


(Y/G/K/S) (L/I/M) E (H/E) (D/H) (I/V/L) (G/A/E)






Preferably, signature sequence 5 is









S (L/I/M) EE (D/H) (I/V/L) A


 



Signature sequence 6 (SEQ ID NO: 92):



(F/L/V/I/T) (I/L/V) (R/Q/Y) (I/V) (F/L/V/I)


 



(T/S/G/A) (E/D/T) RS






Preferably, signature sequence 6 is







(F/L/V/I/T) (I/L/V) (R/Y) (I/V) (L/V/I) (T/S/G/A)


 


TRS


 


Signature sequence 7 (SEQ ID NO: 93):


Y (R/K/M/E/Q) X (F/T/L/M/I) (L/I) (L/I/V)


 


(S/T/V/A) L (V/I/L/A/M) (G/S)






wherein X on position 3 may be any amino acid, preferably one of T, D, N, K, S, R, A

Since the ANN polypeptide is related to annexins, the ANN polypeptide useful in the methods of the invention preferably also has one or more annexin domains (Pfam entry PF00191, SMART entry SM00335, InterPro IPR001464, see also FIGS. 7 A and 7 B).
Preferably, the polypeptide sequence which when used in the construction of a phylogenetic tree, such as the one depicted in FIG. 8 (taken from Cantero et al., Plant Physiol. Biochem. 44, 13-24, 2006), tends to cluster with the group of ANN polypeptides comprising the amino acid sequence represented by SEQ ID NO: 84 and SEQ ID NO: 135, rather than with any other group.
The term “domain”, “motif” and “signature” is defined in the “definitions” section herein. Specialist databases exist for the identification of domains, for example, SMART (Schultz et al. (1998) Proc. Natl. Acad. Sci. USA 95, 5857-5864; Letunic et al. (2002) Nucleic Acids Res 30, 242-244, InterPro (Mulder et al., (2003) Nucl. Acids. Res. 31, 315-318, Prosite (Bucher and Bairoch (1994), A generalized profile syntax for biomolecular sequences motifs and its function in automatic sequence interpretation. (In) ISMB-94; Proceedings 2nd International Conference on Intelligent Systems for Molecular Biology. Altman R., Brutlag D., Karp P., Lathrop R., Searls D., Eds., pp 53-61, AAAI Press, Menlo Park; Hulo et al., Nucl. Acids. Res. 32:D134-D137, (2004), or Pfam (Bateman et al., Nucleic Acids Research 30(1): 276-280 (2002). A set of tools for in silico analysis of protein sequences is available on the ExPASy proteomics server (Swiss Institute of Bioinformatics (Gasteiger et al., ExPASy: the proteomics server for in-depth protein knowledge and analysis, Nucleic Acids Res. 31:3784-3788 (2003)). Domains 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.
Furthermore, ANN polypeptides (at least in their native form) typically have calcium binding activity and capability of binding to membranes. Tools and techniques for measuring membrane binding activity are well known in the art, and include measurement of effects on membrane surface hydrophobicity, vesicle leakage or vesicle aggregation. In addition, ANN polypeptides may exhibit enzymatic activity; for example, Annexin 1 from Arabidopsis thaliana is reported to display peroxidase activity (Gorecka et al., Biochem. Biophys. Res. Comm. 336, 868-875, 2005). Further details are provided in Example 19.
The present invention is illustrated by transforming plants with the nucleic acid sequence represented by SEQ ID NO: 83, encoding respectively the polypeptide sequences of SEQ ID NO: 84. However, performance of the invention is not restricted to these sequences; the methods of the invention may advantageously be performed using any ANN-encoding nucleic acid or ANN polypeptide as defined herein.
Examples of nucleic acids encoding ANN polypeptides are given in Table B1 of Example 14 herein. Such nucleic acids are useful in performing the methods of the invention. The amino acid sequences given in Table B1 of Example 14 are example sequences of orthologues and paralogues of the ANN polypeptide represented by SEQ ID NO: 84, 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 B1 of Example 14) 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: 83 or SEQ ID NO: 84, 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.
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 B1 of Example 14, 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 B1 of Example 14. 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 nucleic acid variants useful in practising the methods of the invention include portions of nucleic acids encoding ANN polypeptides, nucleic acids hybridising to nucleic acids encoding ANN polypeptides, splice variants of nucleic acids encoding ANN polypeptides, allelic variants of nucleic acids encoding ANN polypeptides and variants of nucleic acids encoding ANN polypeptides obtained by gene shuffling. The terms hybridising sequence, splice variant, allelic variant and gene shuffling are as described herein.
Nucleic acids encoding ANN polypeptides 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 B1 of Example 14, or a portion of a nucleic acid encoding an orthologue, paralogue or homologue of any of the amino acid sequences given in Table B1 of Example 14.
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.
Portions useful in the methods of the invention, encode an ANN polypeptide as defined herein, and have substantially the same biological activity as the amino acid sequences given in Table B1 of Example 14. Preferably, the portion is a portion of any one of the nucleic acids given in Table B1 of Example 14, or is a portion of a nucleic acid encoding an orthologue or paralogue of any one of the amino acid sequences given in Table B1 of Example 14. Preferably the portion is at least 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 B1 of Example 14, or of a nucleic acid encoding an orthologue or paralogue of any one of the amino acid sequences given in Table B1 of Example 14. Most preferably the portion is a portion of the nucleic acid of SEQ ID NO: 83. Preferably, the portion encodes an amino acid sequence which when used in the construction of a phylogenetic tree, such as the one depicted in FIG. 8, tends to cluster with the group of ANN comprising the amino acid sequence represented by SEQ ID NO: 84 and SEQ ID NO: 135 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 ANN 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 B1 of Example 14, 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 B1 of Example 14.
Hybridising sequences useful in the methods of the invention encode an ANN polypeptide as defined herein, and have substantially the same biological activity as the amino acid sequences given in Table B1 of Example 14. Preferably, the hybridising sequence is capable of hybridising to any one of the nucleic acids given in Table B1 of Example 14, or to a portion of any of these sequences, a portion being as defined above, or wherein the hybridising sequence is capable of hybridising to a nucleic acid encoding an orthologue or paralogue of any one of the amino acid sequences given in Table B1 of Example 14. Most preferably, the hybridising sequence is capable of hybridising to a nucleic acid as represented by SEQ ID NO: 83 or to a portion thereof.
Preferably, the hybridising sequence encodes an amino acid sequence which when used in the construction of a phylogenetic tree, such as the one depicted in FIG. 8, tends to cluster with the group of ANN polypeptides comprising the amino acid sequence represented by SEQ ID NO: 84 and SEQ ID NO: 135, rather than with any other group.
Another nucleic acid variant useful in the methods of the invention is a splice variant encoding an ANN 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 B1 of Example 14, or a splice variant of a nucleic acid encoding an orthologue, paralogue or homologue of any of the amino acid sequences given in Table B1 of Example 14.
Preferred splice variants are splice variants of a nucleic acid represented by SEQ ID NO: 83, or a splice variant of a nucleic acid encoding an orthologue or paralogue of SEQ ID NO: 84. Preferably, the amino acid sequence encoded by the splice variant, when used in the construction of a phylogenetic tree, such as the one depicted in FIG. 8, tends to cluster with the group of ANN polypeptides comprising the amino acid sequence represented by SEQ ID NO: 84 and SEQ ID NO: 135 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 ANN 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 B1 of Example 14, 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 B1 of Example 14.
The allelic variants useful in the methods of the present invention have substantially the same biological activity as the ANN polypeptide of SEQ ID NO: 84 and any of the amino acids depicted in Table B1 of Example 14. 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: 83 or an allelic variant of a nucleic acid encoding an orthologue or paralogue of SEQ ID NO: 84. Preferably, the amino acid sequence encoded by the allelic variant, when used in the construction of a phylogenetic tree, such as the one depicted in FIG. 8, tends to cluster with the ANN polypeptides comprising the amino acid sequence represented by SEQ ID NO: 84 and SEQ ID NO: 135, rather than with any other group.
Gene shuffling or directed evolution may also be used to generate variants of nucleic acids encoding ANN polypeptides 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 B1 of Example 14, 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 B1 of Example 14, which variant nucleic acid is obtained by gene shuffling.
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. 8, tends to cluster with the group of ANN polypeptides comprising the amino acid sequence represented by SEQ ID NO: 84 and SEQ ID NO: 135, 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 ANN 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 ANN polypeptide-encoding nucleic acid is from a plant, further preferably from a dicotyledonous plant, more preferably from the family Brassicaceae, most preferably the nucleic acid is from Arabidopsis thaliana. 
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 increased yield, especially increased seed yield relative to control plants. The terms “yield” and “seed yield” are described in more detail in the “definitions” section herein.
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 seeds, and performance of the methods of the invention results in plants having 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 hectare or acre, an increase in the number of ears per plant, an increase in the number of rows, number of kernels per row, kernel weight, thousand kernel weight, ear length/diameter, increase in the seed filling rate (which is the number of filled seeds divided by the total number of seeds and multiplied by 100), among others. Taking rice as an example, a yield increase may manifest itself as an increase in one or more of the following: number of plants per hectare or acre, number of panicles per plant, number of spikelets per panicle, number of flowers (florets) per panicle (which is expressed as a ratio of the number of filled seeds over the number of primary panicles), increase in the seed filling rate (which is the number of filled seeds divided by the total number of seeds and multiplied by 100), increase in thousand kernel weight, among others.
The present invention provides a method for increasing yield, especially seed yield of plants, relative to control plants, which method comprises modulating expression, preferably increasing expression, in a plant of a nucleic acid encoding an ANN polypeptide as defined herein.
Since the transgenic plants according to the present invention have increased yield, it is likely that these plants exhibit an increased growth rate (during at least part of their life cycle), relative to the growth rate of 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 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 acre (due to an increase in the number of times (say in a year) that any particular plant may be grown and harvested). An increase in growth rate may also allow for the cultivation of transgenic plants in a wider geographical area than their wild-type counterparts, since the territorial limitations for growing a crop are often determined by adverse environmental conditions either at the time of planting (early season) or at the time of harvesting (late season). Such adverse conditions may be avoided if the harvest cycle is shortened. The growth rate may be determined by deriving various parameters from growth curves, such parameters may be: T-Mid (the time taken for plants to reach 50% of their maximal size) and T-90 (time taken for plants to reach 90% of their maximal size), amongst others.
According to a preferred feature of the present invention, performance of the methods of the invention gives plants having an increased growth rate relative to control plants. Therefore, according to the present invention, there is provided a method for increasing the growth rate of plants, which method comprises modulating expression, preferably increasing expression, in a plant of a nucleic acid encoding an ANN 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% or 30%, preferably less than 25%, 20% or 15%, more preferably less than 14%, 13%, 12%, 11% or 10% or less in comparison to the control plant under non-stress conditions. Due to advances in agricultural practices (irrigation, fertilization, pesticide treatments) severe stresses are not often encountered in cultivated crop plants. As a consequence, the compromised growth induced by mild stress is often an undesirable feature for agriculture. Mild stresses are the everyday biotic and/or abiotic (environmental) stresses to which a plant is exposed. Abiotic stresses may be due to drought or excess water, anaerobic stress, salt stress, chemical toxicity, oxidative stress and hot, cold or freezing temperatures. The abiotic stress may be an osmotic stress caused by a water stress (particularly due to drought), salt stress, oxidative stress or an ionic stress. Biotic stresses are typically those stresses caused by pathogens, such as bacteria, viruses, fungi 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.
Performance of the methods of the invention gives plants grown under non-stress conditions or under mild drought conditions increased yield relative to control plants grown under comparable conditions. Therefore, according to the present invention, there is provided a method for increasing yield in plants grown under non-stress conditions or under mild drought conditions, which method comprises increasing expression in a plant of a nucleic acid encoding an ANN polypeptide.
Performance of the methods of the invention gives plants grown under conditions of nutrient deficiency, particularly under conditions of nitrogen deficiency, increased yield relative to control plants grown under comparable conditions. Therefore, according to the present invention, there is provided a method for increasing yield in plants grown under conditions of nutrient deficiency, which method comprises increasing expression in a plant of a nucleic acid encoding an ANN polypeptide. Nutrient deficiency may result from a lack of nutrients such as nitrogen, phosphates and other phosphorous-containing compounds, potassium, calcium, cadmium, magnesium, manganese, iron and boron, amongst others.
The present invention encompasses plants or parts thereof (including seeds) obtainable by the methods according to the present invention. The plants or parts thereof comprise a nucleic acid transgene encoding an ANN polypeptide as defined above.
The invention also provides genetic constructs and vectors to facilitate introduction and/or expression in plants of nucleic acids encoding ANN polypeptides. 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 ANN 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 ANN 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. A constitutive promoter is particularly useful in the methods. See the “Definitions” section herein for definitions of the various promoter types. Also useful in the methods of the invention is a green tissue-specific promoter.
It should be clear that the applicability of the present invention is not restricted to the ANN polypeptide-encoding nucleic acid represented by SEQ ID NO: 83, nor is the applicability of the invention restricted to expression of an ANN polypeptide-encoding nucleic acid when driven by a constitutive promoter, or when driven by a green tissue-specific promoter.
The constitutive promoter is preferably a GOS2 promoter, preferably a GOS2 promoter from rice. Further preferably the constitutive promoter is represented by a nucleic acid sequence substantially similar to SEQ ID NO: 94, most preferably the constitutive promoter is as represented by SEQ ID NO: 94. See Table 2a in the “Definitions” section herein for further examples of constitutive promoters.
According to another preferred feature of the invention, the nucleic acid encoding an ANN polypeptide is operably linked to a green tissue-specific promoter. The green tissue specific promoter is preferably an expansin promoter, further preferably an expansin promoter from rice. Further preferably the green tissue-specific promoter is represented by a nucleic acid sequence substantially similar to SEQ ID NO: 95, most preferably the green tissue-specific promoter is as represented by SEQ ID NO: 95. See Table 2g in the “Definitions” section herein for further examples of green tissue-specific promoters.
Optionally, one or more terminator sequences may be used in the construct introduced into a plant. Additional regulatory elements may include transcriptional as well as translational 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.
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). The marker genes may be removed or excised from the transgenic cell once they are no longer needed. Techniques for marker gene removal are known in the art, useful techniques are described above in the definitions section.
The invention also provides a method for the production of transgenic plants having enhanced yield-related traits relative to control plants, comprising introduction and expression in a plant of any nucleic acid encoding an ANN polypeptide as defined hereinabove.
More specifically, the present invention provides a method for the production of transgenic plants having increased enhanced yield-related traits, particularly increased seed yield, which method comprises:

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


The nucleic acid of (i) may be any of the nucleic acids capable of encoding an ANN 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 abovementioned publications by S. D. Kung and R. Wu, Potrykus or Höfgen and Willmitzer.
Generally after transformation, plant cells or cell groupings are selected for the presence of one or more markers which are encoded by plant-expressible genes co-transferred with the gene of interest, following which the transformed material is regenerated into a whole plant. To select transformed plants, the plant material obtained in the transformation is, as a rule, subjected to selective conditions so that transformed plants can be distinguished from untransformed plants. For example, the seeds obtained in the above-described manner can be planted and, after an initial growing period, subjected to a suitable selection by spraying. A further possibility consists in growing the seeds, if appropriate after sterilization, on agar plates using a suitable selection agent so that only the transformed seeds can grow into plants. Alternatively, the transformed plants are screened for the presence of a selectable marker such as the ones described above.
Following DNA transfer and regeneration, putatively transformed plants may also be evaluated, for instance using Southern analysis, for the presence of the gene of interest, copy number and/or genomic organisation. Alternatively or additionally, expression levels of the newly introduced DNA may be monitored using Northern and/or Western analysis, both techniques being well known to persons having ordinary skill in the art.
The generated transformed plants may be propagated by a variety of means, such as by clonal propagation or classical breeding techniques. For example, a first generation (or T1) transformed plant may be selfed and homozygous second-generation (or T2) transformants selected, and the T2 plants may then further be propagated through classical breeding techniques. The generated transformed organisms may take a variety of forms. For example, they may be chimeras of transformed cells and non-transformed cells; clonal transformants (e.g., all cells transformed to contain the expression cassette); grafts of transformed and untransformed tissues (e.g., in plants, a transformed rootstock grafted to an untransformed scion).
The present invention clearly extends to any plant cell or plant produced by any of the methods described herein, and to all plant parts and propagules thereof. The present invention extends further to encompass the progeny of a primary transformed or transfected cell, tissue, organ or whole plant that has been produced by any of the aforementioned methods, the only requirement being that progeny exhibit the same genotypic and/or phenotypic characteristic(s) as those produced by the parent in the methods according to the invention.
The invention also includes host cells containing an isolated nucleic acid encoding an ANN 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, 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 and oats.
The invention also extends to harvestable parts of a plant such as, but not limited to seeds, leaves, fruits, flowers, stems, rhizomes, tubers and bulbs. The invention furthermore relates to products derived, preferably directly derived, from a harvestable part of such a plant, such as dry pellets or powders, oil, fat and fatty acids, starch or proteins.
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 (preferably, increasing) expression of a nucleic acid encoding an ANN polypeptide is by introducing and expressing in a plant a nucleic acid encoding an ANN 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 ANN polypeptides as described herein and use of these ANN polypeptides in enhancing any of the aforementioned yield-related traits in plants.
Nucleic acids encoding ANN polypeptide described herein, or the ANN polypeptides themselves, may find use in breeding programmes in which a DNA marker is identified which may be genetically linked to an ANN polypeptide-encoding gene. The nucleic acids/genes, or the ANN 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 an ANN polypeptide-encoding nucleic acid/gene may also find use in marker-assisted breeding programmes. Such breeding programmes sometimes require introduction of allelic variation by mutagenic treatment of the plants, using for example EMS mutagenesis; alternatively, the programme may start with a collection of allelic variants of so called “natural” origin caused unintentionally. Identification of allelic variants then takes place, for example, by PCR. This is followed by a step for selection of superior allelic variants of the sequence in question and which give increased yield. Selection is typically carried out by monitoring growth performance of plants containing different allelic variants of the sequence in question. Growth performance may be monitored in a greenhouse or in the field. Further optional steps include crossing plants in which the superior allelic variant was identified with another plant. This could be used, for example, to make a combination of interesting phenotypic features.
Nucleic acids encoding ANN polypeptides 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 ANN polypeptide-encoding nucleic acids requires only a nucleic acid sequence of at least 15 nucleotides in length. The ANN polypeptide-encoding 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 ANN-encoding nucleic acids. 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 ANN polypeptide-encoding nucleic acid 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. A method for enhancing yield-related traits in plants relative to control plants, comprising modulating, preferably increasing, expression in the roots a plant, of a nucleic acid sequence encoding 2-cysteine peroxiredoxin (2-Cys PRX), which 2-Cys PRX polypeptide comprises from N-terminus to C-terminus: (1) a plastidic transit peptide; and (2) a 2-Cys PRX domain, and optionally selecting for plants having increased yield.
    2. Method according to item 1, wherein said 2-Cys PRX polypeptide additionally comprise one or both of: (i) Motif 1 as represented by SEQ ID NO: 77, or a motif having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more amino acid sequence identity to SEQ ID NO: 77; or (ii) Motif 2 as represented by SEQ ID NO: 78, or a motif having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more amino acid sequence identity to SEQ ID NO: 78.
    3. Method according to item 1 or 2, wherein said 2-Cys PRX polypeptide, when used in the construction of a phylogenetic tree, such as the one depicted in FIG. 3, tends to cluster with the 2-Cys PRX Glade of polypeptides comprising the polypeptide sequence as represented by SEQ ID NO: 2, rather than with any other PRX Glade.
    4. Method according to any preceding item wherein said 2-Cys PRX polypeptide is a polypeptide having in increasing order of preference at least 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more sequence identity to the 2-Cys PRX polypeptide as represented by SEQ ID NO: 2 or to any of the polypeptide sequences given in Table A1 herein.
    5. Method according to any preceding item, wherein said nucleic acid sequence encoding a 2-Cys PRX polypeptide is represented by any one of the nucleic acid sequences listed in Table A1, or is a portion thereof, or is a sequence capable of hybridising with any one of the nucleic acid sequences listed in Table A1.
    6. Method according to any preceding item, wherein said nucleic acid sequence encodes an orthologue or paralogue of any of the polypeptides listed in Table A1.
    7. Method according to any preceding item, wherein said modulated, preferably increased, expression is effected by any one or more of: T-DNA activation tagging, TILLING, or homologous recombination.
    8. Method according to any preceding item, wherein said modulated, preferably increased, expression is effected by introducing and expressing in the roots of a plant a nucleic acid sequence encoding a 2-Cys PRX polypeptide.
    9. Method according to any preceding item, wherein said enhanced yield-related traits is one or more of: (i) improved early vigour; (ii) increased aboveground biomass; (iii) increased root biomass; (iv) increase number of flowers per panicle; (v) increased seed fill rate; (vi) increased total seed yield per plant; (vii) increased number of (filled) seeds; (viii) increased harvest index; or (ix) increased thousand kernel weight (TKW).
    10. Method according to any preceding item, wherein said enhanced yield-related traits are obtained under abiotic stress.
    11. Method according to item 10, wherein said abiotic stress is osmotic stress, selected from one or more of: water stress, salt stress, oxidative stress and ionic stress; preferably wherein said water stress is drought stress and/or reduced nutrient availability, preferably reduced nitrogen availability.
    12. Method according to item 10 or 11, wherein said abiotic stress tolerance is manifested as enhanced yield-related trait selected from one or more of: (i) improved early vigour; (ii) increased aboveground biomass; (iii) increased root (thick and thin) biomass; (iv) increase number of flowers per panicle; (v) increased seed fill rate; (vi) increased total seed yield per plant; (vii) increased number of (filled) seeds; (viii) increased harvest index; or (ix) increased thousand kernel weight (TKW), each relative to control plants.
    13. Method according to any of items 8 to 12, wherein said nucleic acid sequence is operably linked to a root-specific promoter, preferably to an RCc3 promoter, further preferably to an RCc3 promoter substantially similar to SEQ ID NO: 80, most preferably to a promoter as represented by SEQ ID NO: 80.
    14. Method according to any preceding item, wherein said nucleic acid sequence encoding a 2-Cys PRX polypeptide is of plant origin, preferably from a dicotyledonous plant, more preferably from the family Brassicaceae, most preferably from Brassica rapa. 
    15. Plant or part thereof, including seeds, obtainable by a method according to any preceding item, wherein said plant or part thereof comprises a nucleic acid transgene encoding a 2-Cys PRX polypeptide, operably linked to a root-specific promoter.
    16. Construct comprising:
    
        (a) a nucleic acid sequence encoding a 2-Cys PRX polypeptide as defined in any one of items 1 to 6;
        (b) one or more control sequences capable of driving expression of the nucleic acid sequence of (a); and optionally
        (c) a transcription termination sequence;
        wherein at least one of the control sequences is a root-specific promoter, preferably an Rcc3 promoter.
    
    
    17. Use of a construct according to item 16 in a method for making plants having enhanced yield-related traits, which enhanced yield-related traits is preferably one or more of: (i) improved early vigour; (ii) increased aboveground biomass; (iii) increased root (thick and thin) biomass; (iv) increase number of flowers per panicle; (v) increased seed fill rate; (vi) increased total seed yield per plant; (vii) increased number of (filled) seeds; (viii) increased harvest index; or (ix) increased thousand kernel weight (TKW), relative to control plants.
    18. Plant, plant part or plant cell transformed with a construct according to item 16.
    19. Method for the production of a transgenic plant having enhanced yield-related traits relative to control plants, comprising:
    
        (i) introducing and expressing in a plant, plant part or plant cell a nucleic acid sequence encoding a 2-Cys PRX polypeptide as defined in any one of items 1 to 5, under the control of a root-specific promoter; and
        (ii) cultivating the plant cell under conditions promoting plant growth and development.
    
    
    20. Method according to item 19 wherein said enhanced yield-related traits occur under increased abiotic stress.
    21. Transgenic plant having enhanced yield-related traits relative to control plants, resulting from modulated, preferably increased, expression in the roots, of a nucleic acid sequence encoding a 2-Cys PRX polypeptide as defined in any one of items 1 to 6, or a transgenic plant cell or plant part derived from said transgenic plant.
    22. Transgenic plant according to item 15, 18 or 21, wherein said plant is a crop plant or a monocot or a cereal, such as rice, maize, wheat, barley, millet, rye, triticale, sorghum and oats, or a transgenic plant cell or plant part derived from said transgenic plant.
    23. Harvestable parts of a plant comprising a nucleic acid sequence encoding a 2-Cys PRX polypeptide according to item 22, wherein said harvestable parts are preferably seeds.
    24. Products derived from a plant according to item 22 and/or from harvestable parts of a plant according to item 23.
    25. Use of a nucleic acid sequence encoding a 2-Cys PRX polypeptide as defined in any one of items 1 to 6 in enhancing yield-related traits in plants, preferably in increasing one or more of: (i) increased seed fill rate; (ii) increased total seed yield per plant; (iii) increased number of filled seeds; (iv) increased total number of seeds; (v) increased thousand kernel weight (TKW) or (vi) increased harvest index, relative to control plants.
    26. Use according to item 25, wherein said enhanced yield-related traits occur under abiotic stress.
    27. 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 ANN polypeptide, wherein said ANN polypeptide comprises one or more of the following motifs:
    
        (i) Signature sequence 1 (SEQ ID NO: 87),
        (ii) Signature sequence 2 (SEQ ID NO: 88),
        (iii) Signature sequence 3 (SEQ ID NO: 89),
        (iv) Signature sequence 4 (SEQ ID NO: 90),
        (v) Signature sequence 5 (SEQ ID NO: 91),
        (vi) Signature sequence 6 (SEQ ID NO: 92),
        (vii) Signature sequence 7 (SEQ ID NO: 93).
    
    
    28. Method according to item 27, wherein said ANN polypeptide comprises at least an annexin domain.
    29. Method according to item 27 or 28, wherein said modulated expression is effected by introducing and expressing in a plant a nucleic acid encoding an ANN polypeptide.
    30. Method according to any one of items 27 to 29, wherein said nucleic acid encoding an ANN polypeptide encodes any one of the proteins listed in Table B1 or is a portion of such a nucleic acid, or a nucleic acid capable of hybridising with such a nucleic acid.
    31. Method according to any one of items 27 to 30, wherein said nucleic acid sequence encodes an orthologue or paralogue of any of the proteins given in Table B1.
    32. Method according to any one of items 27 to 31, wherein said enhanced yield-related traits comprise increased yield, preferably increased seed yield relative to control plants.
    33. Method according to any one of items 27 to 32, wherein said enhanced yield-related traits are obtained under non-stress conditions.
    34. Method according to any one of items 27 to 33, wherein said enhanced yield-related traits are obtained under conditions of drought.
    35. Method according to any one of items 29 to 34, wherein said nucleic acid is operably linked to a constitutive promoter, preferably to a GOS2 promoter, most preferably to a GOS2 promoter from rice.
    36. Method according to any one of items 29 to 34, wherein said nucleic acid is operably linked to a green-tissue specific promoter, preferably to an expansin promoter, most preferably to an expansin promoter from rice.
    37. Method according to any preceding item, wherein said nucleic acid encoding an ANN 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. 
    38. Plant or part thereof, including seeds, obtainable by a method according to any preceding item, wherein said plant or part thereof comprises a recombinant nucleic acid encoding an ANN polypeptide.
    39. Construct comprising:
    
        (i) nucleic acid encoding an ANN polypeptide as defined in items 27 or 28;
        (ii) one or more control sequences capable of driving expression of the nucleic acid sequence of (i); and optionally
        (iii) a transcription termination sequence.
    
    
    40. Construct according to item 39, wherein one of said control sequences is a constitutive promoter, preferably a GOS2 promoter, most preferably a GOS2 promoter from rice.
    41. Construct according to item 39, wherein one of said control sequences is a green-tissue specific promoter, preferably an expansin promoter, most preferably an expansin promoter from rice.
    42. Use of a construct according to any one of items 39 to 41 in a method for making plants having increased yield, particularly increased biomass and/or increased seed yield relative to control plants.
    43. Plant, plant part or plant cell transformed with a construct according to any one of items 39 to 41.
    44. Method for the production of a transgenic plant having increased yield, particularly increased seed yield relative to control plants, comprising:
    
        (i) introducing and expressing in a plant a nucleic acid encoding an ANN polypeptide as defined in item 27 or 28; and
        (ii) cultivating the plant cell under conditions promoting plant growth and development.
    
    
    45. Transgenic plant having increased yield, particularly increased seed yield, relative to control plants, resulting from increased expression of a nucleic acid encoding an ANN polypeptide as defined in item 27 or 28, or a transgenic plant cell derived from said transgenic plant.
    46. Transgenic plant according to item 38, 43, or 45, or a transgenic plant cell derived thereof, wherein said plant is a crop plant or a monocot or a cereal, such as rice, maize, wheat, barley, millet, rye, triticale, sorghum and oats.
    47. Harvestable parts of a plant according to item 46, wherein said harvestable parts are preferably seeds.
    48. Products derived from a plant according to item 46 and/or from harvestable parts of a plant according to item 47.
    49. Use of a nucleic acid encoding an ANN 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 catalytic and inactivation/reactivation cycles of 2-Cys Prx enzymes, according to Rhee et al., (2005) Free radical Biology and Medicine 38: 1543-1552.
FIG. 2 represents the output of a search (using default values) of the Conserved Domains Database (CDD) at NCBI, using a 2-Cys PRX as represented by SEQ ID NO: 2. The top hit is entry CD3015, PRX_Typ2cys.
FIG. 3 shows a phylogenetic tree built using the neighbour joining clustering method, after a ClustalW (1.83) multiple sequence alignment of 2-Cys PRX from eubacteria, plant algae, animals, and 1-Cys PRX from plants. The 2-Cys PRX class is marked with an accolade. The 2-Cys PRX as represented by SEQ ID NO: 2 is boxed.
FIG. 4 shows a CLUSTAL W (1.83) multiple sequence alignment of 2-Cys PRX from eubacteria, plant algae, animals, and 1-Cys PRX from plants, using default values. Motif 1 as represented by SEQ ID NO: 77 and Motif 2 as represented by SEQ ID NO: 78 are boxed. Sequences shown are: gi|113596153|dbj|BAF20027.1| (SEQ ID NO: 162); gi|113535696|dbj|BAF08079.1| (SEQ ID NO: 163); gi|113533376|dbj|BAF05759.1| (SEQ ID NO: 164); gi|115439131|ref|NP—001043845. (SEQ ID NO: 165); gi|113532211|dbi|BAF04594.1| (SEQ ID NO: 166); gi|115435844|ref|NP—001042680. (SEQ ID NO: 167); gi|113611943|dbj|BAF22321.1| (SEQ ID NO: 168); gi|113611944|dbj|BAF22322.1| (SEQ ID NO: 169); gi|12499469|sp|Q61171|PRDX2_MOU (SEQ ID NO: 170); gi|8394432|ref|NP—058865.1| (SEQ ID NO: 52); gi|21553667|gb|AAM62760.1| (SEQ ID NO: 10); gi|13265490|gb|AAG40040.2|AF32 (SEQ ID NO: 171); gi|11119229|gb|AAG30570.1|AF31 (SEQ ID NO: 8); gi|113564335|dbj|BAF14678.1| (SEQ ID NO: 172) gi|15229806|ref|NP—187769.1| (SEQ ID NO: 173); gi|21592588|gb|AAM64537.1| (SEQ ID NO: 6); gi|3121825|sp|O24364|BAS1_SPIO (SEQ ID NO: 174); gi|1498198|emb|CAA63909.1| (SEQ ID NO: 175); gi|6002472|gb|AAF00001.1|AF052 (SEQ ID NO: 4); gi|21912927|emb|CAC84143.2| (SEQ ID NO: 14); gi|47027073|gb|AAT08751.1| (SEQ ID NO: 176); gi|11558242|emb|CAC17803.1| (SEQ ID NO: 16); gi|15131688|emb|CAC48323.1| (SEQ ID NO: 18); gi|3328221|gb|AAC78473.1| (SEQ ID NO: 177); gi|2499477|sp|Q96468|BAS1_HORV (SEQ ID NO: 178); gi|2829687|sp|P80602|BAS1_WHEA (SEQ ID NO: 179); gi|1076722|pir∥S49173 (SEQ ID NO: 180); gi|115446541|ref|NP—001047050. (SEQ ID NO: 20); gi|113536581|dbj|BAF08964.1| (SEQ ID NO: 181); gi|125539780|gb|EAY86175.1| (SEQ ID NO: 182); gi|7339568|emb|CAB82860.1| (SEQ ID NO: 24); gi|17232133|ref|NP—488681.1| (SEQ ID NO: 28); gi|119509654|ref|ZP—01628800.1 (SEQ ID NO: 32); gi|86609696|ref|YP—478458.1| (SEQ ID NO: 30); gi|86605254|ref|YP—474017.1| (SEQ ID NO: 38); gi|22298997|ref|NP—682244.1| (SEQ ID NO: 34); gi|11465738|ref|NP—053882.1| (SEQ ID NO: 46); gi|51209959|ref|YP—063623.1| (SEQ ID NO: 48); gi|81301118|ref|YP—401326.1| (SEQ ID NO: 42); gi|33865747|ref|NP—897306.1| (SEQ ID NO: 40); gi|84518029|ref|ZP—01005378.1| (SEQ ID NO: 44); gi|116059461|emb|CAL55168.1| (SEQ ID NO: 36); gi|74272711|gb|ABA01151.1| (SEQ ID NO: 26); gi|11995220|emb|CAC19677.1| (SEQ ID NO: 183); gi|115455107|ref|NP—001051154. (SEQ ID NO: 184); gi|115455105|ref|NP—001051153. (SEQ ID NO: 185); gi|113595092|dbj|BAF18966.1| (SEQ ID NO: 186); gi|115452325|ref|NP—001049763. (SEQ ID NO: 187); and gi|113610859|dbj|BAF21237.1| (SEQ ID NO: 188).
FIG. 5 represents the binary vector for increased expression in Oryza sativa of a 2-Cys PRX-encoding nucleic acid sequence under the control of either a rice GOS2 promoter (pGOS2; SEQ ID NO: 79), or a rice Rcc3 (pRcc3; SEQ ID NO: 80) promoter.
FIG. 6 details examples of sequences useful in performing the methods according to the present invention.
FIG. 7 A represents SEQ ID NO: 84 with the annexin domains as predicted by SMART indicated in bold underlined; FIG. 7 B shows the annexin domains (predicted by SMART) in ANNEXIN 4 of Arabidopsis thaliana (SEQ ID NO: 135).
FIG. 8 shows a phylogenetic tree (Cantero et al., Plant Physiol. Biochem. 44, 13-24, 2006) of plant annexin proteins useful in the methods of the present invention. The arrows indicate annexin 1 (SEQ ID NO: 84) and annexin 4 (SEQ ID NO: 135) both from Arabidopsis thaliana. 
FIG. 9 represents a multiple alignment of various plant annexin proteins. The identifiers refer to the database accessions; NP—1174810 corresponds to SEQ ID NO: 84. Conserved residues are indicated by colons or dots. Sequences shown are: NP—001063096 (SEQ ID NO: 141); NP—001061839 (SEQ ID NO: 145); ABE65753 (SEQ ID NO: 133); NP—181410 (SEQ ID NO: 131); NP—001055408 (SEQ ID NO: 151); AAC33305 (SEQ ID NO: 97); AAB71830 (SEQ ID NO: 99); CAB92956 (SEQ ID NO: 113); ABB55363 (SEQ ID NO: 123); AAC97494 (SEQ ID NO: 115); 1DK5 (SEQ ID NO: 105); CAA75213 (SEQ ID NO: 111); CAA75214 (SEQ ID NO: 121); NP—174810 (SEQ ID NO: 84); AAD24540 (SEQ ID NO: 107); AAC97493 (SEQ ID NO: 117); AAB67994 (SEQ ID NO: 103); AAR13288 (SEQ ID NO: 109); AAZ41833 (SEQ ID NO: 101); NP—201307 (SEQ ID NO: 119); NP—196585 (SEQ ID NO: 125); AAZ67605 (SEQ ID NO: 129); NP—196584 (SEQ ID NO: 137); CAA52903 (SEQ ID NO: 127); NP—001048149 (SEQ ID NO: 143); NP—001057176 (SEQ ID NO: 147); NP—568271 (SEQ ID NO: 139); NP—181409 (SEQ ID NO: 135); NP—001063343 (SEQ ID NO: 149); NP—001061661 (SEQ ID NO: 153); and NP—001051711 (SEQ ID NO: 155).
FIG. 10 represents the binary vector for increased expression in Oryza sativa of an ANN-encoding nucleic acid under the control of a rice GOS2 promoter (pGOS2).
FIG. 11 details examples of sequences useful in performing the methods according to the present invention.



EXAMPLES
The present invention will now be described with reference to the following examples, which are by way of illustration alone. The following examples are not intended to completely define or otherwise limit the scope of the invention.
DNA manipulation: unless otherwise stated, recombinant DNA techniques are performed according to standard protocols described in (Sambrook (2001) Molecular Cloning: a laboratory manual, 3rd Edition Cold Spring Harbor Laboratory Press, CSH, N.Y.) or in Volumes 1 and 2 of Ausubel et al. (1994), Current Protocols in Molecular Biology, Current Protocols. Standard materials and methods for plant molecular work are described in Plant Molecular Biology Labfax (1993) by R. D. D. Croy, published by BIOS Scientific Publications Ltd (UK) and Blackwell Scientific Publications (UK).
Example 1
Identification of Sequences Related to the Nucleic Acid Sequence Used in the Methods of the Invention
Sequences (full length cDNA, ESTs or genomic) related to the nucleic acid sequence used in the methods of the present invention were identified amongst those maintained in the Entrez Nucleotides database at the National Center for Biotechnology Information (NCBI) using database sequence search tools, such as the Basic Local Alignment Tool (BLAST) (Altschul et al. (1990) J. Mol. Biol. 215:403-410; and Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402). The program is used to find regions of local similarity between sequences by comparing nucleic acid or polypeptide sequences to sequence databases and by calculating the statistical significance of matches. For example, the polypeptide encoded by the nucleic acid sequence 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.
Table A1 provides a list of nucleic acid sequences related to the nucleic acid sequence used in the methods of the present invention.







TABLE A1







Examples of 2-Cys PRX nucleic acid and polypeptide sequences:












Nucleic acid sequence NCBI
SEQ
Polypeptide sequence
SEQ


Species
accession
ID NO
NCBI accession
ID NO














Brassica sp.
SEQ ID NO: 1
1
SEQ ID NO: 2
2


Brassica rapa
AF052202.1
3
AAF00001.1
4


Arabidopsis thaliana
AY086974.1
5
AAM64537.1
6


Brassica napus
AF311863.1
7
AAG30570.1
8


Arabidopsis thaliana
NM_120712.2
9
AAM62760.1
10


Spinacia oleracea
X94219.1
11
O24364
12


Nicotiana tabacum
AJ309009.2
13
CAC84143.2
14


Phaseolus vulgaris
AJ288895.1
15
CAC17803.1
16


Pisum sativum
AJ315851.1|
17
CAC48323.1
18


Oryza sativa
NM_001053585.1
19
NP_001047050.1
20





Os02g0537700


Secale cereale
AF076920.1
21
AAC78473.1
22


Riccia fluitans
AJ005006.1
23
CAB82860.1
24


Chlamydomonas incerta
DQ122920.1
25
ABA01151.1
26


Nostoc
gi|17227497: 5544705-5545316
27
NP_488681.1
28


Synechococcus sp
gi|86607503: 2357237-2357845
29
YP_478458.1
30


Nodularia spumigena
gi|119509627: 35267-35878
31
ZP_01628800.1
32


Thermosynechococcus elongatus
gi|22297544: 1516844-1517437
33
NP_682244.1
34


Ostreococcus tauri
gi|118721427: 14610-14671,
35
CAL55168.1
36



14874-14913, 15068-15658


Synechococcus
gi|86604733: 552727-553335
37
YP_474017.1
38


Synechococcus
gi|33864539: 1204065-1204667
39
NP_897306.1
40


Synechococcus elongatus
gi|81298811: 2377107-2377703
41
YP_401326.1
42


Prochlorococcus marinus
gi|84517401: 555567-556163
43
ZP_01005378.1
44


Porphyra purpurea
gi|11465652: 76976-77575
45
NP_053882.1
46


Gracilaria tenuistipitata
gi|51209843: 100291-100971
47
YP_063623.1
48


Mus musculus
X82067.1
49
Q61171
50


Rattus norvegicus
NM_017169.1
51
NP_058865.1
52









In some instances, related sequences have tentatively been assembled and publicly disclosed by research institutions, such as The Institute for Genomic Research (TIGR). The Eukaryotic Gene Orthologs (EGO) database may be used to identify such related sequences, either by keyword search or by using the BLAST algorithm with the nucleic acid sequence or polypeptide sequence of interest.
Example 2
Alignment of 2-Cys PRX Polypeptide Sequences
Alignment of 2-Cys PRX polypeptide sequences from eubacteria, plant algae, animals, and 1-Cys PRX polypeptide sequences from plants (as outliers) was performed the Clustal algorithm (1.83) of progressive alignment, using default values (Thompson et al. (1997) Nucleic Acids Res 25:4876-4882; Chenna et al. (2003). Nucleic Acids Res 31:3497-3500). Minor manual editing may be done to further optimise the alignment. The 2-Cys PRX polypeptides are aligned in FIG. 4. Motif 1 as represented by SEQ ID NO: 77 and Motif 2 as represented by SEQ ID NO: 78 are boxed.
A phylogenetic tree of 2-Cys PRX polypeptide sequences from eubacteria, plant algae, animals, and 1-Cys PRX polypeptide sequences from plants (as outliers) was constructed using a neighbour-joining clustering algorithm, well known in the art (FIG. 3). The 2-Cys PRX class is marked with an accolade. The 2-Cys PRX as represented by SEQ ID NO: 2 is boxed.
Example 3
Calculation of Global Percentage Identity Between Polypeptide Sequences Useful in Performing the Methods of the Invention
Global percentages of similarity and identity between full length polypeptide sequences useful in performing the methods of the invention were determined using one of the methods available in the art, the MatGAT (Matrix Global Alignment Tool) software (BMC Bioinformatics. 2003 4:29. MatGAT: an application that generates similarity/identity matrices using protein or DNA sequences. Campanella J J, Bitincka L, Smalley J; software hosted by Ledion Bitincka). MatGAT software generates similarity/identity matrices for DNA or protein sequences without needing pre-alignment of the data. The program performs a series of pair-wise alignments using the Myers and Miller global alignment algorithm (with a gap opening penalty of 12, and a gap extension penalty of 2), calculates similarity and identity using for example Blosum 62 (for polypeptides), and then places the results in a distance matrix. Sequence similarity is shown in the bottom half of the dividing line and sequence identity is shown in the top half of the diagonal dividing line.
Parameters used in the comparison were:
















Scoring matrix:
Blosum 62



First Gap:
12



Extending gap:
 2










Results of the software analysis are shown in Table A2 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 2-Cys PRX polypeptide sequences useful in performing the methods of the invention can be as low as 35% amino acid identity compared to SEQ ID NO: 2.







TABLE A2





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




































1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20





01. seqidno02

91
89
86
86
88
76
78
76
76
74
66
61
55
53
55
54
57
52
53


02. seqidno04
92

85
80
80
85
71
74
71
72
69
61
56
52
50
52
51
54
49
49


03. seqidno06
93
88

85
86
97
75
77
74
74
74
66
61
55
52
55
53
58
51
52


04. seqidno08
92
86
92

89
85
74
77
74
73
73
64
60
56
52
56
52
59
51
52


05. seqidno10
91
86
91
93

85
77
76
74
75
74
66
61
57
53
56
53
59
52
53


06. seqidno12
92
87
98
91
90

76
77
75
75
75
66
61
55
53
55
54
58
52
52


07. seqidno14
83
80
85
86
86
85

79
79
75
75
66
63
57
54
55
54
57
53
53


08. seqidno16
84
79
84
83
82
83
83

86
77
76
66
63
58
57
57
56
61
55
55


09. seqidno18
85
81
85
83
83
85
85
91

75
74
67
63
57
54
56
55
59
53
53


10. seqidno20
82
77
81
81
83
83
83
85
86

79
66
64
57
55
57
57
60
54
53


11. seqidno22
84
78
83
82
82
83
83
84
82
85

67
64
58
56
57
56
61
55
54


12. seqidno24
79
75
79
78
80
79
81
77
78
79
77

60
56
51
53
52
58
51
50


13. seqidno26
75
70
74
74
75
74
75
75
75
75
74
72

66
62
63
65
69
61
64


14. seqidno28
65
61
65
64
65
65
65
66
65
67
67
63
75

83
91
83
63
82
76


15. seqidno30
63
59
63
64
64
63
64
66
65
66
68
63
72
92

80
84
61
96
74


16. seqidno32
65
61
65
64
65
65
65
67
64
67
67
62
75
97
92

79
63
80
76


17. seqidno34
62
58
62
62
61
62
62
64
63
66
65
60
73
88
90
88

64
83
75


18. seqidno36
67
63
67
68
67
67
67
68
67
71
70
65
79
74
72
74
73

59
63


19. seqidno38
63
59
63
63
63
63
63
66
64
66
68
62
72
92
100 
93
89
71

72


20. seqidno40
62
58
61
60
61
61
61
64
62
62
63
58
73
84
85
85
84
71
85


21. seqidno42
61
58
61
61
61
62
61
65
63
63
64
60
73
89
90
89
89
73
91
88


22. seqidno44
61
58
61
60
60
61
61
64
62
62
63
57
73
85
85
85
84
73
85
96


23. seqidno46
62
58
61
61
61
62
61
63
62
63
65
60
72
87
90
88
88
71
90
84


24. seqidno48
64
61
65
64
65
65
64
67
66
64
67
62
73
74
73
74
71
71
72
71


25. seqidno50
58
54
58
57
58
58
56
58
57
58
59
55
64
73
73
73
73
64
72
75


26. seqidno52
57
54
57
56
56
57
55
57
56
58
58
55
63
72
72
72
72
64
72
74


27. seqidno54
41
39
40
40
40
40
40
42
40
39
41
40
39
45
45
46
46
40
45
46


28. seqidno56
50
48
50
50
48
49
49
50
50
52
53
49
53
55
52
55
52
49
52
53


29. seqidno58
36
36
35
34
35
36
36
36
37
36
37
36
40
43
43
44
41
38
42
43


30. seqidno60
41
41
41
40
40
40
40
40
37
42
41
39
43
28
28
28
27
39
25
28


31. seqidno62
41
41
43
40
41
43
41
40
40
42
41
40
46
42
39
42
40
43
39
40


32. seqidno64
27
30
27
27
27
31
27
29
30
33
28
30
35
38
38
37
40
35
40
38


33. seqidno66
36
37
33
35
35
34
38
33
33
37
34
36
33
30
29
30
27
30
29
29


34. seqidno68
39
40
39
39
41
39
39
41
38
41
40
35
42
39
34
39
34
40
34
34


35. seqidno70
34
38
34
34
34
33
32
36
32
35
34
31
33
26
31
26
25
33
26
25


36. seqidno72
34
33
35
34
34
35
32
36
33
34
36
34
33
27
25
26
25
28
25
26


37. seqidno74
33
33
32
36
32
33
33
34
32
35
33
33
34
27
29
29
28
31
31
26


38. seqidno76
30
28
29
30
31
29
30
34
29
32
30
30
35
36
35
38
36
36
35
36































21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38







01. seqidno02
53
51
50
47
47
46
20
46
19
24
25
16
18
23
18
19
17
17



02. seqidno04
51
48
47
46
45
44
21
45
19
22
26
16
19
21
20
18
18
18



03. seqidno06
52
50
50
48
46
46
19
46
18
24
25
16
16
22
18
21
18
16



04. seqidno08
53
52
50
50
47
45
20
46
19
23
24
15
17
21
17
21
16
16



05. seqidno10
53
51
51
49
47
46
20
47
18
24
24
15
18
22
18
20
16
17



06. seqidno12
52
51
50
48
48
47
21
46
19
25
25
16
17
22
18
21
19
16



07. seqidno14
55
52
51
50
46
45
22
45
20
23
24
15
18
22
18
20
17
16



08. seqidno16
57
53
53
51
48
48
21
48
19
21
23
17
17
22
20
20
18
17



09. seqidno18
56
51
52
51
46
45
20
46
21
22
24
18
15
22
17
20
16
14



10. seqidno20
55
51
53
49
47
46
19
48
18
24
26
16
19
22
20
20
21
19



11. seqidno22
56
53
53
49
48
47
20
50
19
26
25
18
18
24
22
22
20
16



12. seqidno24
54
50
49
46
44
45
20
43
20
24
25
17
18
21
17
20
20
17



13. seqidno26
67
64
59
56
53
53
21
45
22
23
25
20
18
24
19
18
18
18



14. seqidno28
83
75
74
61
60
60
27
44
27
13
21
22
15
19
16
17
16
16



15. seqidno30
83
72
76
60
58
58
25
43
25
15
19
23
17
15
18
16
17
16



16. seqidno32
80
73
73
63
61
61
27
44
27
12
21
22
16
18
16
17
18
17



17. seqidno34
83
74
77
60
60
60
27
44
26
16
19
23
15
17
18
17
19
15



18. seqidno36
63
63
60
57
50
50
22
44
21
24
22
20
15
20
20
19
17
20



19. seqidno38
82
72
76
59
57
57
27
42
27
11
19
23
17
17
17
16
19
17



20. seqidno40
81
90
71
57
64
63
29
44
28
16
19
23
16
17
15
17
17
16



21. seqidno42

78
76
58
62
62
26
44
25
15
18
21
15
15
18
17
18
15



22. seqidno44
88

70
56
61
61
28
43
27
17
19
20
16
19
17
13
17
18



23. seqidno46
90
86

63
58
57
27
39
27
12
18
21
13
18
14
15
16
17



24. seqidno48
71
72
73

46
45
23
34
22
18
20
17
12
18
13
12
11
17



25. seqidno50
74
75
73
61

99
27
36
26
18
19
22
17
18
18
17
19
16



26. seqidno52
73
74
72
60
99

27
36
26
18
19
22
16
18
18
17
19
17



27. seqidno54
43
47
47
41
46
46

17
81
12
16
20
15
17
15
16
16
13



28. seqidno56
53
53
53
47
47
46
30

19
16
19
15
15
15
13
14
16
17



29. seqidno58
42
43
46
40
41
41
90
31

16
17
20
16
17
14
14
17
17



30. seqidno60
31
30
26
38
34
34
28
28
28

23
42
20
73
24
20
22
34



31. seqidno62
38
40
39
43
38
38
26
29
28
38

17
22
22
20
19
19
18



32. seqidno64
36
37
35
35
39
38
34
35
34
53
30

18
44
17
16
19
35



33. seqidno66
29
28
24
31
25
25
25
26
27
34
35
27

21
21
19
20
21



34. seqidno68
32
33
34
42
34
34
30
29
31
81
42
54
34

20
18
22
31



35. seqidno70
26
27
28
30
27
27
27
27
27
38
32
26
33
33

57
33
20



36. seqidno72
25
23
27
29
26
26
28
24
26
36
32
26
33
33
73

30
19



37. seqidno74
27
27
29
27
31
30
29
24
28
36
30
30
35
35
46
48

17



38. seqidno76
32
38
35
35
36
39
28
34
29
50
37
49
35
48
32
32
33










Example 4
Identification of Domains Comprised in Polypeptide Sequences Useful in Performing the Methods of the Invention
The Conserved Domain Search service (CD-Search) is a web-based tool for the detection of structural and functional domains in protein sequences, hosted at NCBI. CD-Search uses BLAST® to search a comprehensive collection of domain models. Search results are displayed as domain architecture cartoons and pairwise alignments between the query and domain-model consensus sequences (Marchler-Bauer A, Bryant S H (2004), “CD-Search: protein domain annotations on the fly”, Nucleic Acids Res. 32 (W) 327-331). A search (using default values) performed using a 2-Cys PRX as represented by SEQ ID NO: 2, gives as top hit is entry CD3015, PRX_Typ2cys (FIG. 2, Table A3).







TABLE A3







CDD of the polypeptide sequence as represented by SEQ ID NO: 2.











Accession
Accession



Database
number
name
Description





CDD
CD3015
PRX_Typ2cys
Peroxiredoxin (PRX) family,





Typical 2-Cys PRX subfamily;





PRXs are thiol-specific





antioxidant (TSA) proteins,





which confer a protective role in





cells through its peroxidase





activity by reducing hydrogen





peroxide, peroxynitrite, and





organic hydroperoxides









Example 5
Subcellular Localisation Prediction of the Polypeptide Sequences Useful in Performing the Methods of the Invention
TargetP 1.1 predicts the subcellular location of eukaryotic proteins. The location assignment is based on the predicted presence of any of the N-terminal pre-sequences: chloroplast transit peptide (cTP), mitochondrial targeting peptide (mTP) or secretory pathway signal peptide (SP). Scores on which the final prediction is based are not really probabilities, and they do not necessarily add to one. However, the location with the highest score is the most likely according to TargetP, and the relationship between the scores (the reliability class) may be an indication of how certain the prediction is. The reliability class (RC) ranges from 1 to 5, where 1 indicates the strongest prediction. TargetP is maintained at the server of the Technical University of Denmark.
For the sequences predicted to contain an N-terminal presequence a potential cleavage site can also be predicted.
A number of parameters were selected, such as organism group (non-plant or plant), cutoff sets (none, predefined set of cutoffs, or user-specified set of cutoffs), and the calculation of prediction of cleavage sites (yes or no).
The results of TargetP 1.1 analysis of the polypeptide sequence as represented by SEQ ID NO: 2 are presented Table A4. The “plant” organism group has been selected, no cutoffs defined, and the predicted length of the transit peptide requested. The predicted subcellular localization of the polypeptide sequence as represented by SEQ ID NO: 2 is the plastidic compartment.







TABLE A4





TargetP 1.1 analysis of the polypeptide sequence as represented by


SEQ ID NO: 2


















Length (AA)
268



Chloroplastic transit peptide
0.987



Mitochondrial transit peptide
0.014



Secretory pathway signal peptide
0.023



Other subcellular targeting
0.018



Predicted Location
Chloro



Reliability class
1



Predicted transit peptide length
58










The predicted length according to TargetP1.1 is of 58 amino acids (starting from the N-terminus), but this can only be verified experimentally by sequencing the mature protein. Cheong et al (1999) predict a 65 amino acid transit peptide for the polypeptide as represented by SEQ ID NO: 2 (Plant Molec Biol 40: 825-834).
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
    
    


Example 6
Functional Assay for a 2-Cys PRX Polypeptide
2-Cys PRX polypeptides present peroxidase activity on hydrogen peroxide, for example. Enzyme assays for 2-Cys PRX proteins have been extensively described in the literature, and are well-know to a person skilled in the art. Huang et al. (2007; Appl Microbiol Biotechnol 74(1):84-92), Bernier-Villamor et al. (2004; J Exp Bot 55(406):2191-9) and Caporaletti et al. (2007; Biochem Biophys Res Commun 355(3):722-7) are recent publications describing the enzymatic assay of 2-Cys PRX proteins.
Example 7
Cloning of the Nucleic Acid Sequence Used in the Methods of the Invention
The nucleic acid sequence used in the methods of the invention was amplified by PCR using as template a Brassica rapa 2-Cys PRX clone as described in Cheong et al., (1999; Plant Molec Biol). 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 prm08756 (SEQ ID NO: 81; sense, start codon in bold): 5′-ggggacaagtttgtacaaaaaagcaggcttaaacaatggcgt ctgttgcttctt-3′ and prm08757 (SEQ ID NO: 82; reverse, complementary): 5′-gg ggaccactttgtacaagaaagctgggttcgagctaaatagctgagaagag-3′, which include the AttB sites for Gateway recombination. The amplified PCR fragment was purified also using standard methods. The first step of the Gateway procedure, the BP reaction, was then performed, during which the PCR fragment recombines in vivo with the pDONR201 plasmid to produce, according to the Gateway terminology, an “entry clone”, p2-Cys PRX. 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 two destination vectors used for Oryza sativa transformation. The vectors 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. The first destination vector comprised upstream of this Gateway cassette the rice GOS2 promoter (SEQ ID NO: 79) for strong constitutive expression, and the second destination vector comprised the rice Rcc3 promoter for root-specific expression (SEQ ID NO: 80).
After the LR recombination step, the resulting expression vectors pGOS2::2-Cys PRX and pRcc3::2-Cys PRX (FIG. 5) were separately transformed into Agrobacterium strain LBA4044 according to methods well known in the art.
Example 8
Plant Transformation
Rice Transformation
The two Agrobacterium strains each containing one of the expression vectors as described in Example 7, used to transform Oryza sativa plants. Mature dry seeds of the rice japonica cultivar Nipponbare were dehusked. Sterilization was carried out by incubating for one minute in 70% ethanol, followed by 30 minutes in 0.2% HgCl2, followed by a 6 times 15 minutes wash with sterile distilled water. The sterile seeds were then germinated on a medium containing 2,4-D (callus induction medium). After incubation in the dark for four weeks, embryogenic, scutellum-derived calli were excised and propagated on the same medium. After two weeks, the calli were multiplied or propagated by subculture on the same medium for another 2 weeks. Embryogenic callus pieces were sub-cultured on fresh medium 3 days before co-cultivation (to boost cell division activity).
Agrobacterium strain LBA4404 containing 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
Phenotypic Evaluation Procedure
9.1 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%.
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 from 4 events were grown in potting soil under normal conditions until they approached the heading stage. They were then transferred to a “dry” section where irrigation was withheld. Humidity probes were inserted in randomly chosen pots to monitor the soil water content (SWC). When SWC went below certain thresholds, the plants were automatically re-watered continuously until a normal level was reached again. The plants were then re-transferred again to normal conditions. The rest of the cultivation (plant maturation, seed harvest) was the same as for plants not grown under abiotic stress conditions. Growth and yield parameters were recorded as detailed for growth under normal conditions.
9.2 Statistical Analysis: F Test
A two factor ANOVA (analysis of variants) was used as a statistical model for the overall evaluation of plant phenotypic characteristics. An F test was carried out on all the parameters measured of all the plants of all the events transformed with the gene of the present invention. The F test was carried out to check for an effect of the gene over all the transformation events and to verify for an overall effect of the gene, also known as a global gene effect. The threshold for significance for a true global gene effect was set at a 5% probability level for the F test. A significant F test value points to a gene effect, meaning that it is not only the mere presence or position of the gene that is causing the differences in phenotype.
Because two experiments with overlapping events were carried out, a combined analysis was performed. This is useful to check consistency of the effects over the two experiments, and if this is the case, to accumulate evidence from both experiments in order to increase confidence in the conclusion. The method used was a mixed-model approach that takes into account the multilevel structure of the data (i.e. experiment-event-segregants). P values were obtained by comparing likelihood ratio test to chi square distributions.
9.3 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 2-Cys PRXnt digital images (2048×1536 pixels, 16 million colours) were taken of each plant from at least 6 different angles.
The early vigour is the plant (seedling) aboveground area three weeks post-germination. 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 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.
To measure root-related parameters, plants were grown in specially designed pots with transparent bottoms to allow visualization of the roots. A digital camera recorded images through the bottom of the pot during plant growth. Root features such as total projected area (which can be correlated to total root volume), average diameter and length of roots above a certain thickness threshold (length of thick roots, or thick root length) were deduced from the picture using of appropriate software. 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).
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).
Example 10
Results of the Phenotypic Evaluation of the Transgenic Plants, Grown Under Normal Growth Conditions
The results of the evaluation of transgenic rice plants expressing a nucleic acid sequence encoding a 2-Cys PRX polypeptide under the control of a root-specific promoter, and grown under normal growth conditions, are presented below. Improved early vigour was observed, as well as increased seed fill rate, increased total seed yield per plant, increased number of filled seeds, increased harvest index, and increased thousand kernel weight.







TABLE A5







Results of the evaluation of transgenic rice plants expressing the nucleic


acid sequence useful in performing the methods of the invention, under


the control of a root-specific-promoter, grown under normal growth


conditions.










% Increase for
% Increase for



the three
the three



best events
best events


Trait
in T1 generation
in T2 generation












Early vigour (seedlings)
7%
5%


Seed fill rate
1%
10%


Total seed yield per plant
8%
10%


Total numbers of filled seeds
6%
8%


Harvest index
7%
9%


Thousand kernel weight
3%
3%









Transgenic rice plants expressing a nucleic acid sequence encoding a 2-Cys PRX polypeptide under the control of a constitutive promoter (a rice GOS2 promoter), and grown under normal growth conditions, showed no difference in any of the traits phenotypically examined as compared to control plants grown under comparable normal growth conditions (data not shown).
Example 11
Results of the Phenotypic Evaluation of the Transgenic Plants, Grown Under Stress Growth Conditions
The results of the evaluation of transgenic rice plants expressing a nucleic acid sequence encoding a 2-Cys PRX polypeptide under the control of a root-specific promoter, and grown under drought-stress growth conditions, are presented below. Improved early vigour was observed, as well as increased aboveground biomass, increased root biomass, increased number of flowers per panicle, increased seed fill rate, increased total seed yield per plant, increased number of seeds, increased number of filled seeds, and increased harvest index.







TABLE A6







Results of the evaluation of transgenic rice plants expressing the nucleic


acid sequence useful in performing the methods of the invention, under


the control of a root-specific-promoter, grown under drought-stress


growth conditions.











% Increase in



Trait
T2 generation (all events)














Early vigour (seedlings)
31%



boveground biomass
11%



Root biomass
6%



Flowers per panicle
8%



Seed fill rate
8%



Total seed yield per plant
18%



Total number of seeds
10%



Total numbers of filled seeds
17%



Harvest index
8%










Example 12
Examples of Transformation of Other Crops
Corn Transformation
Transformation of maize (Zea mays) is performed with a modification of the method described by Ishida et al. (1996) Nature Biotech 14(6): 745-50. Transformation is genotype-dependent in corn and only specific genotypes are amenable to transformation and regeneration. The inbred line A188 (University of Minnesota) or hybrids with A188 as a parent are good sources of donor material for transformation, but other genotypes can be used successfully as well. Ears are harvested from corn plant approximately 11 days after pollination (DAP) when the length of the immature embryo is about 1 to 1.2 mm. Immature embryos are cocultivated with Agrobacterium tumefaciens containing the expression vector, and transgenic plants are recovered through organogenesis. Excised embryos are grown on callus induction medium, then maize regeneration medium, containing the selection agent (for example imidazolinone but various selection markers can be used). The Petri plates are incubated in the light at 25° C. for 2-3 weeks, or until shoots develop. The green shoots are transferred from each embryo to maize rooting medium and incubated at 25° C. for 2-3 weeks, until roots develop. The rooted shoots are transplanted to soil in the greenhouse. T1 seeds are produced from plants that exhibit tolerance to the selection agent and that contain a single copy of the T-DNA insert.
Wheat Transformation
Transformation of wheat is performed with the method described by Ishida et al. (1996) Nature Biotech 14(6): 745-50. The cultivar Bobwhite (available from CIMMYT, Mexico) is commonly used in transformation. Immature embryos are co-cultivated with Agrobacterium tumefaciens containing the expression vector, and transgenic plants are recovered through organogenesis. After incubation with Agrobacterium, the embryos are grown in vitro on callus induction medium, then regeneration medium, containing the selection agent (for example imidazolinone but various selection markers can be used). The Petri plates are incubated in the light at 25° C. for 2-3 weeks, or until shoots develop. The green shoots are transferred from each embryo to rooting medium and incubated at 25° C. for 2-3 weeks, until roots develop. The rooted shoots are transplanted to soil in the greenhouse. T1 seeds are produced from plants that exhibit tolerance to the selection agent and that contain a single copy of the T-DNA insert.
Soybean Transformation
Soybean is transformed according to a modification of the method described in the Texas A&M 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 D C W and A Atanassov (1985. Plant Cell Tissue Organ Culture 4: 111-112). Alternatively, the RA3 variety (University of Wisconsin) has been selected for use in tissue culture (Walker et al., 1978 μm J Bot 65:654-659). Petiole explants are cocultivated with an overnight culture of Agrobacterium tumefaciens C58C1 pMP90 (McKersie et al., 1999 Plant Physiol 119: 839-847) or LBA4404 containing the expression vector. The explants are cocultivated for 3 d in the dark on SH induction medium containing 288 mg/L Pro, 53 mg/L thioproline, 4.35 g/L K2SO4, and 100 μm acetosyringinone. The explants are washed in half-strength Murashige-Skoog medium (Murashige and Skoog, 1962) and plated on the same SH induction medium without acetosyringinone but with a suitable selection agent and suitable antibiotic to inhibit Agrobacterium growth. After several weeks, somatic embryos are transferred to BOi2Y development medium containing no growth regulators, no antibiotics, and 50 g/L sucrose. Somatic embryos are subsequently germinated on half-strength Murashige-Skoog medium. Rooted seedlings were transplanted into pots and grown in a greenhouse. T1 seeds are produced from plants that exhibit tolerance to the selection agent and that contain a single copy of the T-DNA insert.
Cotton Transformation
Cotton (Gossypium hirsutum L.) transformation is performed using Agrobacterium tumefaciens, on hypocotyls explants. The commercial cultivars such as Coker 130 or Coker 312 (SeedCo, Lubbock, Tex.) are standard varieties used for transformation, but other varieties can also be used. The seeds are surface sterilized and germinated in the dark. Hypocotyl explants are cut from the germinated seedlings to lengths of about 1-1.5 centimeter. The hypotocyl explant is submersed in the Agrobacterium tumefaciens inoculum containing the expression vector, for 5 minutes then co-cultivated for about 48 hours on MS+1.8 mg/l KNO3+2% glucose at 24° C., in the dark. The explants are transferred the same medium containing appropriate bacterial and plant selectable markers (renewed several times), until embryogenic calli is seen. The calli are separated and subcultured until somatic embryos appear. Plantlets derived from the somatic embryos are matured on rooting medium until roots develop. The rooted shoots are transplanted to potting soil in the greenhouse. T1 seeds are produced from plants that exhibit tolerance to the selection agent and that contain a single copy of the T-DNA insert.
Example 13
Examples of Other Stress Screens
Salt Stress Screen
Plants are grown on a substrate made of coco fibers and argex (3 to 1 ratio). A normal nutrient solution is used during the first two weeks after transplanting the plantlets in the greenhouse. After the first two weeks, 25 mM of salt (NaCl) is added to the nutrient solution, until the plants were harvested.
Reduced Nutrient (Nitrogen) Availability Screen
Rice plants from T2 seeds are grown in potting soil under normal conditions except for the nutrient solution. The pots are watered from transplantation to maturation with a specific nutrient solution containing reduced N nitrogen (N) content, usually between 7 to 8 times less. The rest of the cultivation (plant maturation, seed harvest) is the same as for plants not grown under abiotic stress. Growth and yield parameters are recorded as detailed for growth under normal conditions.
Example 14
Identification of Sequences Related to the Nucleic Acid Sequence Used in the Methods of the Invention
Sequences (full length cDNA, ESTs or genomic) related to the nucleic acid sequence used in the methods of the present invention were identified amongst those maintained in the Entrez Nucleotides database at the National Center for Biotechnology Information (NCBI) using database sequence search tools, such as the Basic Local Alignment Tool (BLAST) (Altschul et al. (1990) J. Mol. Biol. 215:403-410; and Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402). The program is used to find regions of local similarity between sequences by comparing nucleic acid 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.
Table B1 provides a list of nucleic acid sequences related to the nucleic acid sequence useful in the methods of the present invention.







TABLE B1







Examples of ANN polypeptides:










Nucleic




acid
Protein



SEQ
SEQ


Plant Source
ID NO:
ID NO:












Arabidopsis thaliana ANNAT1
83
84


Gossypium hirsutum fiber annexin
96
97


Lavatera thuringiaca annexin
98
99


Brassica rapa subsp. Pekinensis 80C09_22
100
101


Gossypium hirsutum annexin
102
103


Capsicum annuum Annexin 24
104
105


Nicotiana tabacum VCaB42
106
107


Gossypium hirsutum Anx1
108
109


Nicotiana tabacum annexin
110
111


Solanum tuberosum annexin p34
112
113


Lycopersicon esculentum annexin p34
114
115


Lycopersicon esculentum annexin p35
116
117


Arabidopsis thaliana ANNAT2
118
119


Nicotiana tabacum annexin
120
121


Solanum tuberosum annexin p34-like
122
123


Arabidopsis thaliana ANN7
124
125


Medicago sativa annexin
126
127


Brassica rapa subsp. pekinensis 80A08_20
128
129


Arabidopsis thaliana ANNAT3
130
131


Arabidopsis thaliana annexin 5
132
133


Arabidopsis thaliana ANNAT4
134
135


Arabidopsis thaliana ANN6
136
137


Arabidopsis thaliana ANN8 At5g12380
138
139


Oryza sativa (japonica cultivar-group) Os09g0394900
140
141


Oryza sativa (japonica cultivar-group) Os02g0753800
142
143


Oryza sativa (japonica cultivar-group) Os08g0425700
144
145


Oryza sativa (japonica cultivar-group) Os06g0221200
146
147


Oryza sativa (japonica cultivar-group) Os09g0453300
148
149


Oryza sativa (japonica cultivar-group) Os05g0382900
150
151


Oryza sativa (japonica cultivar-group) Os08g0372900
152
153


Oryza sativa (japonica cultivar-group) Os03g0819300
154
155









In some instances, related sequences have tentatively been assembled and publicly disclosed by research institutions, such as The Institute for Genomic Research (TIGR). The Eukaryotic Gene Orthologs (EGO) database may be used to identify such related sequences, either by keyword search or by using the BLAST algorithm with the nucleic acid or polypeptide sequence of interest.
Example 15
Alignment of ANN Polypeptide Sequences
Alignment of polypeptide sequences was performed using the AlignX programme from the Vector NTI (Invitrogen) which is based on the popular Clustal W algorithm of progressive alignment (Thompson et al. (1997) Nucleic Acids Res 25:4876-4882; Chenna et al. (2003). Nucleic Acids Res 31:3497-3500). Default values are for the gap open penalty of 10, for the gap extension penalty of 0, 1 and the selected weight matrix is Blosum 62 (if polypeptides are aligned). Minor manual editing was done to further optimise the alignment. Sequence conservation among ANN polypeptides is found throughout the whole sequence. The ANN polypeptides are aligned in FIG. 9.
A phylogenetic tree of ANN polypeptides (such as the one from FIG. 8) may be constructed using a neighbour-joining clustering algorithm as provided in the AlignX programme from the Vector NTI (Invitrogen).
Example 16
Calculation of Global Percentage Identity Between Polypeptide Sequences Useful in Performing the Methods of the Invention
Global percentages of similarity and identity between full length polypeptide sequences useful in performing the methods of the invention were determined using one of the methods available in the art, the MatGAT (Matrix Global Alignment Tool) software (BMC Bioinformatics. 2003 4:29. MatGAT: an application that generates similarity/identity matrices using protein or DNA sequences. Campanella J J, Bitincka L, Smalley J; software hosted by Ledion Bitincka). MatGAT software generates similarity/identity matrices for DNA or protein sequences without needing pre-alignment of the data. The program performs a series of pair-wise alignments using the Myers and Miller global alignment algorithm (with a gap opening penalty of 12, and a gap extension penalty of 2), calculates similarity and identity using for example Blosum 62 (for polypeptides), and then places the results in a distance matrix. Sequence similarity is shown in the bottom half of the dividing line and sequence identity is shown in the top half of the diagonal dividing line.
Parameters used in the comparison were:
















Scoring matrix:
Blosum 62



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 in bold and percentage similarity is given below the diagonal (normal face).
The percentage identity between the ANN polypeptide sequences useful in performing the methods of the invention can be as low as about 30% amino acid identity compared to SEQ ID NO: 84 (NP—174810).







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





 1. NP_174810

72.6
71.0
29.9
68.6
65.2
67.5
67.8
65.6
66.2
65.9


 2. AAC33305
84.5

89.9
32.1
71.6
71.6
68.0
72.8
72.8
72.8
73.4


 3. AAB71830
84.5
94.3

30.6
68.8
67.0
64.2
68.7
69.0
68.7
69.0


 4. AAZ41833
36.4
37.4
36.8

32.6
29.4
33.4
33.9
29.6
30.0
30.2


 5. AAB67994
82.3
84.5
83.2
38.7

63.1
72.2
85.5
63.4
63.1
63.1


 6. 1DK5
83.9
83.9
81.7
37.7
79.2

63.6
64.5
91.0
91.9
91.6


 7. AAD24540
81.1
82.6
81.0
38.7
83.9
77.6

74.4
63.9
64.2
64.9


 8. AAR13288
81.7
84.5
82.6
39.3
92.7
78.6
84.8

65.2
65.2
66.5


 9. CAA75213
84.2
85.1
83.2
37.4
81.0
95.7
79.1
80.4

92.4
92.7


10. CAB92956
83.9
85.4
83.5
37.6
80.6
95.3
79.1
80.4
97.8

98.4


11. AAC97494
83.9
85.4
83.5
37.6
80.6
95.3
79.1
80.4
97.8
100.0


12. AAC97493
80.8
80.4
79.4
37.8
82.9
77.6
92.1
85.8
79.4
79.4
79.4


13. NP_201307
79.8
81.4
80.1
44.5
85.5
79.2
83.9
84.5
80.1
80.4
80.4


14. CAA75214
83.6
84.2
82.3
37.1
80.0
94.7
78.2
79.7
98.4
97.5
97.5


15. ABB55363
83.3
84.8
82.9
37.3
79.7
94.7
78.5
79.7
96.5
98.7
98.7


16. NP_196585
80.4
80.4
79.1
41.2
84.2
78.9
82.0
82.6
80.4
81.0
81.0


17. CAA52903
80.8
82.0
81.6
38.6
83.8
79.2
82.3
83.2
81.5
81.5
81.5


18. AAZ67605
79.2
79.1
79.1
41.1
82.6
78.0
81.6
82.0
79.4
79.7
79.7


19. NP_181410
59.2
58.6
58.6
27.8
61.4
58.4
61.7
63.2
58.6
58.9
58.9


20. ABE65753
58.4
56.3
54.7
27.0
58.5
54.0
56.6
59.5
54.7
55.1
55.1


21. NP_181409
56.4
55.2
53.6
25.3
53.9
54.7
54.5
54.2
55.8
55.8
55.8


22. NP_196584
77.4
78.9
78.6
41.2
81.8
78.0
81.1
81.8
78.3
79.2
78.9


23. NP_568271
69.4
67.1
67.7
30.9
68.4
66.5
66.8
69.0
66.8
68.0
67.7


24. NP_001063096
55.8
55.7
53.8
27.4
59.0
54.7
57.0
58.5
54.9
54.6
54.6


25. NP_001048149
76.0
78.5
77.5
35.2
79.4
74.5
75.3
79.1
77.1
77.1
77.1


26. NP_001061839
55.1
54.5
52.6
27.1
57.6
53.7
57.0
56.1
55.1
53.9
53.9


27. NP_001057176
78.2
79.8
80.1
35.2
79.5
75.8
77.0
79.5
76.7
77.0
77.0


28. NP_001063343
53.0
53.6
52.4
26.1
53.6
50.3
52.0
52.0
50.8
51.1
51.1


29. NP_001055408
54.3
52.7
53.0
30.2
53.8
55.1
53.8
54.3
53.0
53.2
53.2


30. NP_001061661
27.8
27.6
27.3
24.9
26.8
24.2
28.4
26.8
28.1
27.3
27.3


31. NP_001051711
17.7
16.8
17.1
10.2
16.5
17.4
14.2
16.8
17.2
17.5
17.8






12
13
14
15
16
17
18
19
20
21
22





 1. NP_174810
65.9
64.0
65.6
64.9
63.1
65.0
62.8
38.5
34.3
32.3
60.8


 2. AAC33305
65.5
67.5
71.8
71.4
66.5
69.3
64.9
41.0
35.6
34.2
63.8


 3. AAB71830
63.3
64.4
68.0
67.3
63.3
68.4
62.3
39.8
34.1
33.0
61.3


 4. AAZ41833
32.4
41.8
29.3
29.5
35.9
33.1
35.3
19.9
17.5
16.8
34.8


 5. AAB67994
70.3
70.1
63.1
62.1
68.1
70.5
67.5
39.6
34.9
35.1
64.9


 6. 1DK5
62.0
61.8
90.7
90.4
62.0
63.6
61.1
40.0
33.5
34.2
59.8


 7. AAD24540
87.0
70.3
63.6
62.9
69.3
69.6
69.3
42.5
36.9
33.7
67.6


 8. AAR13288
73.7
71.0
65.2
63.8
70.3
69.3
68.7
43.5
36.6
35.3
67.3


 9. CAA75213
62.0
64.4
97.1
90.8
64.6
64.2
62.7
41.9
35.6
35.0
61.9


10. CAB92956
63.6
64.4
92.4
98.4
63.9
65.2
62.7
41.3
35.3
34.1
61.6


11. AAC97494
64.2
64.4
92.7
96.8
64.9
65.8
63.6
42.2
35.0
34.7
62.3


12. AAC97493

68.1
62.7
62.3
68.0
66.5
67.4
40.4
36.0
33.4
65.4


13. NP_201307
81.4

63.1
63.0
79.2
69.7
78.9
42.2
37.4
34.9
74.9


14. CAA75214
79.0
79.5

90.8
63.9
63.0
62.3
41.0
35.0
34.7
61.6


15. ABB55363
78.8
79.8
96.2

62.6
63.8
61.3
40.4
34.5
34.1
60.3


16. NP_196585
80.7
89.9
79.7
80.4

69.0
89.9
41.9
38.2
34.7
82.4


17. CAA52903
80.3
83.3
80.9
80.4
81.0

68.4
40.8
35.1
33.8
67.0


18. AAZ67605
80.1
89.9
78.8
79.1
95.6
81.3

40.4
36.0
33.8
82.4


19. NP_181410
60.1
57.6
58.3
58.9
59.5
58.3
58.3

31.9
33.6
41.4


20. ABE65753
55.4
58.0
55.7
55.1
58.9
54.7
58.5
51.7

27.4
36.1


21. NP_181409
54.9
53.9
55.8
55.8
53.0
51.4
52.0
54.2
47.6

32.6


22. NP_196584
80.2
88.4
77.4
78.6
91.2
80.8
91.5
58.6
58.5
51.7


23. NP_568271
66.5
66.9
66.8
68.0
67.7
68.0
68.4
63.2
56.6
55.8
66.0


24. NP_001063096
57.5
59.0
55.2
54.4
59.2
57.1
58.9
55.1
72.2
46.1
59.1


25. NP_001048149
74.9
76.0
76.1
75.9
76.6
77.1
75.9
60.1
57.9
51.1
74.2


26. NP_001061839
56.1
58.3
54.8
53.6
57.9
55.5
57.0
54.5
69.2
46.4
58.6


27. NP_001057176
74.8
76.3
75.7
76.7
74.8
76.3
74.4
60.4
56.8
48.6
74.2


28. NP_001063343
51.4
52.4
50.8
50.8
53.0
48.3
52.0
48.6
48.9
50.2
52.4


29. NP_001055408
53.5
53.8
53.2
53.2
53.8
52.4
53.5
60.2
44.9
47.3
53.0


30. NP_001061661
29.1
29.4
28.1
29.4
28.1
25.8
27.3
26.3
26.0
28.9
27.3


31. NP_001051711
16.8
16.4
16.2
17.1
15.2
19.2
16.8
16.8
17.1
17.2
16.7




















23
24
25
26
27
28
29
30
31





 1. NP_174810
49.4
35.5
59.6
36.8
60.2
28.4
37.2
12.4
11.3


 2. AAC33305
51.1
37.4
63.3
38.8
64.5
30.2
38.9
14.1
10.1


 3. AAB71830
49.2
35.3
59.8
36.0
61.9
29.1
37.8
15.1
9.2


 4. AAZ41833
22.7
19.2
29.0
19.0
28.9
14.6
21.5
14.4
8.0


 5. AAB67994
51.3
36.8
63.1
39.0
61.8
30.6
39.0
12.5
10.1


 6. 1DK5
48.6
35.2
60.8
33.9
59.5
29.0
41.3
12.2
10.5


 7. AAD24540
51.4
37.2
61.1
40.7
62.6
28.3
39.4
14.9
9.5


 8. AAR13288
51.7
37.5
63.6
38.8
63.5
30.2
40.8
14.3
7.9


 9. CAA75213
48.6
36.5
62.0
35.6
60.7
30.5
39.1
13.0
10.5


10. CAB92956
49.2
35.5
62.0
34.8
61.0
29.5
39.1
12.5
9.9


11. AAC97494
49.5
35.5
63.0
34.8
61.9
29.5
39.7
12.5
8.6


12. AAC97493
49.8
36.9
59.5
39.8
59.4
28.0
38.3
13.7
11.6


13. NP_201307
48.1
40.4
59.6
39.6
60.8
27.9
40.1
12.9
10.7


14. CAA75214
48.9
35.3
60.8
35.3
59.4
29.8
38.9
13.0
9.9


15. ABB55363
48.3
35.3
61.0
34.0
60.6
29.2
38.4
13.2
10.2


16. NP_196585
50.5
38.7
61.4
38.8
58.2
28.3
39.4
12.9
11.4


17. CAA52903
49.1
36.3
60.3
36.0
59.6
27.5
37.6
13.0
9.4


18. AAZ67605
50.2
37.5
60.1
37.9
56.6
28.3
37.5
12.8
11.1


19. NP_181410
45.5
33.4
41.1
30.3
42.4
25.5
41.4
15.0
11.1


20. ABE65753
34.3
53.2
37.0
50.2
35.5
27.0
28.8
14.7
12.3


21. NP_181409
34.7
30.1
33.0
28.2
31.0
25.4
30.2
14.1
10.3


22. NP_196584
49.5
37.9
59.1
37.5
58.1
29.0
37.6
12.8
11.3


23. NP_568271

36.5
51.1
35.9
52.4
29.2
38.5
13.3
12.3


24. NP_001063096
58.9

37.8
68.5
38.6
30.4
29.3
14.0
12.1


25. NP_001048149
67.1
59.7

37.7
83.9
29.8
37.4
14.6
10.8


26. NP_001061839
57.3
84.4
57.3

38.4
31.8
30.9
16.8
15.5


27. NP_001057176
69.1
57.4
90.2
58.3

28.1
39.8
15.4
11.4


28. NP_001063343
50.2
50.2
49.8
49.2
49.2

19.8
14.6
10.9


29. NP_001055408
55.6
46.5
51.6
46.5
54.8
38.4

15.5
12.1


30. NP_001061661
27.6
26.5
29.4
27.6
27.3
28.6
29.1

13.4


31. NP_001051711
19.0
19.0
17.8
20.6
17.7
19.4
16.4
19.6









Example 17
Identification of Domains Comprised in Polypeptide Sequences Useful in Performing the Methods of the Invention
The Integrated Resource of Protein Families, Domains and Sites (InterPro) database is an integrated interface for the commonly used signature databases for text- and sequence-based searches. The InterPro database combines these databases, which use different methodologies and varying degrees of biological information about well-characterized proteins to derive protein signatures. Collaborating databases include SWISS-PROT, PROSITE, TrEMBL, PRINTS, ProDom and Pfam, Smart and TIGRFAMs. 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: 84 are presented in Table B3.







TABLE B3







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


represented by SEQ ID NO: 84.













Amino acid coordinates on


Database
Accession number
Accession name
SEQ ID NO 84





PRODOM
PD000143
Annexin
[16-78]T-[88-153]T-[172-239]T-





[246-313]T


PRINTS
PR00196
ANNEXIN
[25-47]T-[65-81]T-[92-113]T-





[255-275]T-[299-312]T


GENE3D
G3DSA:1.10.220.10
Annexin
[13-82]T-[83-161]T-[166-238]T-





[243-317]T


PANTHER
PTHR10502
Annexin
[9-312]T


PFAM
PF00191
Annexin
[15-80]T-[87-152]T-[170-236]T-





[246-311]T


SMART
SM00335
ANX
[28-80]T-[100-152]T-[183-232]T-





[259-311]T


PROFILE
PS00223
ANNEXIN
[259-311]T


SUPERFAMILY
SSF47874
Annexin
[1-316]T


PRINTS
PR01814
ANNEXINPLANT
[119-133]T-[161-181]T-[227-245]T


PANTHER
PTHR10502:SF10
Annexin_like
[9-312]T









Example 18
Topology Prediction of the Polypeptide Sequences Useful in Performing the Methods of the Invention
TargetP 1.1 predicts the subcellular location of eukaryotic proteins. The location assignment is based on the predicted presence of any of the N-terminal pre-sequences: chloroplast transit peptide (cTP), mitochondrial targeting peptide (mTP) or secretory pathway signal peptide (SP). Scores on which the final prediction is based are not really probabilities, and they do not necessarily add to one. However, the location with the highest score is the most likely according to TargetP, and the relationship between the scores (the reliability class) may be an indication of how certain the prediction is. The reliability class (RC) ranges from 1 to 5, where 1 indicates the strongest prediction. TargetP is maintained at the server of the Technical University of Denmark.
For the sequences predicted to contain an N-terminal presequence a potential cleavage site can also be predicted.
A number of parameters were selected, such as organism group (non-plant or plant), cutoff sets (none, predefined set of cutoffs, or user-specified set of cutoffs), and the calculation of prediction of cleavage sites (yes or no).
The results of TargetP 1.1 analysis of the polypeptide sequence as represented by SEQ ID NO: 84 are presented Table B4. 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: 84 may be the cytoplasm or nucleus, no transit peptide is predicted. This prediction is in agreement with earlier reports that indicate that annexin proteins are associated to the plasma membrane, vacuole and nuclear periphery (Clark & Roux, Plant Physiol. 109, 1133-1139, 1995).







TABLE B4





TargetP 1.1 analysis of the polypeptide sequence as represented by


SEQ ID NO: 84


















Length (AA)
317



Chloroplastic transit peptide
0.102



Mitochondrial transit peptide
0.126



Secretory pathway signal peptide
0.045



Other subcellular targeting
0.905



Predicted Location
/



Reliability class
2



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
    
    


Example 19
Functional Assay for the ANN Polypeptide
Assay for annexin-membrane interactions (Dabitz et al. Biochemistry 44, 16292-16300, 2005):
Membrane Binding Assay (Copelleting Assay).
Phospholipid vesicles are prepared following the protocol of Reeves and Dowben (J. Cell. Physiol. 73, 49-60, 1969). To assess the plant annexin-membrane binding behavior, a copelleting assay is conducted (Hofmann and Huber, Methods Enzymol. 372, 186-216, 2003).
A total amount of 0.2 μmol of phospholipids is used for each individual sample (500 μL), composed of 0.5 nmol of protein in liposome buffer and varying amounts of calcium. As a control, a sample of 0.1 nmol of protein in 100 μL of 10% SDS is prepared at this stage. All samples are centrifuged (23 000 rpm at 4° C. for 45 min), and the pellets are resuspended with 50 μL of 10% SDS and then subjected to SDS-PAGE. Gels are stained with Coomassie and analyzed densitometrically using ImageJ (Rasband, W. ImageJ, version 1.30, National Institutes of Health, Bethesda, Md., 2005). Each calcium concentration is assessed three times independently. Curve fitting is performed with SigmaPlot using a standard binding equation.
Phospholipid Vesicle Preparation.
For experiments assessing membrane surface hydrophobicity and liposome leakage, the following protocols are used. Brain phosphatidylserine (PS), egg phosphatidylcholine (PC), egg phosphatidylethanolamine (PE), nitrobenzoxadiazole phosphatidylethanolamine (NBD-PE), and lissamine rhodamine B sulfonyl phosphatidylethanolamine (Rh-PE) are from a commercial supplier. Multilamellar phospholipid vesicles (MLVs) are prepared using the method of Bangham et al. (Preparation and use of liposomes as models of biological membranes, in Methods in Membrane Biology (Korn, E. D., Ed.) pp 1-68, Plenum Press, New York, 1974). The MLVs are converted into large unilamellar vesicles (LUVs) by five freeze-thaw cycles and subsequent extrusion (five times) through 0.1 μm Nucleopore filter membranes using an extruder (Lipex Biomembranes, Vancouver, BC) at 30° C. Phosphate determination is performed according to the method of Chen et al. (Anal. Chem. 28, 1756-1758, 1956).
Membrane Surface Hydrophobicity.
An increase in membrane surface hydrophobicity is the result of dehydration of the phospholipid headgroups by either the binding of protein to the membrane surface or the creation of water-free interfaces between two vesicles, which occurs during aggregation. Changing membrane surface hydrophobicity can be observed by labeling vesicles with N-[5-(dimethylamino)-naphthalene-2-sulfonyl)-1,2-dioleoylyl-sn-glycero-3-PE (dansyl-PE), whose emission wavelength is proportional to the dielectric constant of the probe environment. In this context, pure PS, PS/PE (3:1), and PS/PC (1:1) LUVs containing 1 mol % dansyl-PE are prepared and added to a 900 μL buffer solution (final phospholipid concentration of 45 μM). The effect of annexin on these vesicles is observed at different pH values by injecting 200 nM (0.18 nmol) protein into the calcium-free sample. The samples are excited at 340 nm, and the fluorescence emission is recorded from 400 to 600 nm (23). The calcium-dependent behaviour of surface hydrophobicity is observed after monitoring the effect of protein alone.
Liposome Leakage Assay.
Annexin-phospholipid interactions may cause the destabilization of phospholipid vesicles which results in leakage of the vesicle's interior. Vesicle leakage is monitored by the fluorescence quenching of 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS) in the presence of p-xylen-bis-pyridiniumbromid (DPX). The watersoluble fluorophore ANTS and its quencher DPX are added to the buffer solution while the vesicles are prepared. Excess ANTS/DPX buffer solution is removed by gel filtration using a Sephadex G-50 column. In the undisturbed vesicles, the fluorophore and quencher are spatially close so that DPX quenches the fluorescence of ANTS. With an increasing level of vesicle leakage, ANTS and DPX are diluted into the outer buffer solution, resulting in an increase in the fluorescence of ANTS (Ellens et al., Biochemistry 24, 3099-3106, 1985).
Assay for Peroxidase Activity of Annexin 1 (Gorecka et al. 2005).
Peroxidase activity of recombinant annexin1 proteins expressed in eukaryotic or prokaryotic systems may be tested with two methods. The first method is based on the chemiluminescence of oxidized luminal. Samples containing the proteins to be analyzed, separated by non-denaturating electrophoresis, are transferred onto a nitrocellulose membrane, covered with the developing solution (ECL kit, Amersham) containing luminol, and exposed to X-ray medical film for 1 hr according to the manufacturer's protocol. Alternatively, peroxidase activity of recombinant annexin1 proteins is determined using a fluorometric method with Amplex Red reagent (Molecular Probes) on a Fluorolog 3 spectrofluorimeter (Jobin Yvon Spex, Edison, N.J.) with 1-nm slits for both excitation and emission. The assay medium (total volume of 100 μl) contains 50 mM potassium phosphate buffer, pH 7.4, 2 mM H2O2, Amplex Red reagent at a final concentration of 100 μM. Measurements are made in quartz cuvettes of optical path length of 10 mm (0.1 ml volume). Fluorescence emission of the product of Amplex Red reagent oxidation, resorufin, is recorded at λem 590 nm (λexc 560 nm). For the determination of the effect of protein phosphorylation on peroxidase activityAnnAt1 is prior to measurements incubated with alkaline phosphatase (Sigma, 15 U/ml) at 36° C. for 10 min in a potassium phosphate buffer, pH 7.4. A sample without AnnAt1 is used as a control.
Example 20
Cloning of the Nucleic Acid Sequence Used in the Methods of the Invention
The nucleic acid sequence used in the methods of the invention was amplified by PCR using as template a custom-made 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 prm08727 (SEQ ID NO: 85; sense, start codon in bold): 5′-ggggacaagtttgtacaaaaaagcaggctta aacaatggcgactcttaaggtttct-3′ and prm09025 (SEQ ID NO: 86; reverse, complementary): 5′-ggggaccactttgtacaagaaagctgggtttaagcatcatcttcaccg ag-3′, which include the AttB sites for Gateway recombination. The amplified PCR fragment was purified also using standard methods. The first step of the Gateway procedure, the BP reaction, was then performed, during which the PCR fragment recombines in vivo with the pDONR201 plasmid to produce, according to the Gateway terminology, an “entry clone”, pANN. Plasmid pDONR201 was purchased from Invitrogen, as part of the Gateway® technology.
The entry clone comprising SEQ ID NO: 83 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 expansin promoter (SEQ ID NO: 95) for green-tissue specific expression was located upstream of this Gateway cassette.
In alternative embodiment, a destination vector comprising the GOS2 promoter (SEQ ID NO: 94) was used resulting in the expression vector pGOS2::ANN.
After the LR recombination step, the resulting expression vector pEXP::ANN (FIG. 10) or pGOS2::ANN was transformed into Agrobacterium strain LBA4044 according to methods well known in the art.
Example 21
Plant Transformation
Transformation of rice plants was carried out according to the procedure outlined in Example 8 herein.
Example 22
Phenotypic Evaluation Procedure
22.1 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%.
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 were grown in potting soil under normal conditions until they approached the heading stage. They were then transferred to a “dry” section where irrigation was withheld. Humidity probes were inserted in randomly chosen pots to monitor the soil water content (SWC). When SWC went below certain thresholds, the plants were automatically re-watered continuously until a normal level was reached again. The plants were then re-transferred again to normal conditions. The rest of the cultivation (plant maturation, seed harvest) was the same as for plants not grown under abiotic stress conditions. Growth and yield parameters are recorded as detailed for growth under normal conditions.
Nitrogen Use Efficiency Screen
Rice plants from T2 seeds are grown in potting soil under normal conditions except for the nutrient solution. The pots are watered from transplantation to maturation with a specific nutrient solution containing reduced N nitrogen (N) content, usually between 7 to 8 times less. The rest of the cultivation (plant maturation, seed harvest) is the same as for plants not grown under abiotic stress. Growth and yield parameters are recorded as detailed for growth under normal conditions.
22.2 Statistical Analysis: F Test
A two factor ANOVA (analysis of variants) was used as a statistical model for the overall evaluation of plant phenotypic characteristics. An F test was carried out on all the parameters measured of all the plants of all the events transformed with the gene of the present invention. The F test was carried out to check for an effect of the gene over all the transformation events and to verify for an overall effect of the gene, also known as a global gene effect. The threshold for significance for a true global gene effect was set at a 5% probability level for the F test. A significant F test value points to a gene effect, meaning that it is not only the mere presence or position of the gene that is causing the differences in phenotype.
Because two experiments with overlapping events were carried out, a combined analysis was performed. This is useful to check consistency of the effects over the two experiments, and if this is the case, to accumulate evidence from both experiments in order to increase confidence in the conclusion. The method used was a mixed-model approach that takes into account the multilevel structure of the data (i.e. experiment-event-segregants). P values were obtained by comparing likelihood ratio test to chi square distributions.
22.3 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.
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).
Example 23
Results of the Phenotypic Evaluation of the Transgenic Plants
Evaluation of transgenic rice plants expressing an ANN nucleic acid operably linked to a constitutive promoter, grown under non-stress conditions revealed an increase of more than 5% was observed for total seed yield, number of filled seeds, fill rate, harvest index and more than 3% for TKW. Under drought stress conditions, an increase was observed for total seed yield, number of filled seeds and fill rate.
Plants expressing an ANN nucleic acid operably linked to a green-tissue specific promoter also exhibited an increased yield, in particular increased TKW.
Example 24
Examples of Transformation of Other Crops
Transformation of other crops is described in Example 12 hereinabove.

## Claims

1. A method for enhancing seed yield in a plant relative to a control plant, comprising:
(a) introducing and expressing in a plant, plant part, or plant cell a nucleic acid encoding a 2-cysteine peroxiredoxin (2-Cys PRX) polypeptide, wherein said nucleic acid is operably linked to a root-specific promoter; and
(b) selecting a plant having increased seed yield relative to a control plant on the basis of said plant showing increased seed yield relative to said control plant, wherein said 2-Cys PRX polypeptide comprises from N-terminus to C-terminus:
(1) a plastidic transit peptide; and
(2) a 2-Cys PRX domain,

and has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 2, and wherein said 2-Cys PRX polypeptide further comprises a motif comprising the amino acid sequence of SEQ ID NO: 77 and a motif comprising the amino acid sequence of SEQ ID NO: 78.

2. The method according to claim 1, wherein said nucleic acid encoding a 2-Cys PRX polypeptide comprises the nucleic acid sequence of SEQ ID NO: 1 or a sequence capable of hybridising with the nucleic acid sequence of SEQ ID NO: 1 under conditions comprising 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.

3. The method according to claim 1, wherein said increased seed yield is one or more of: (i) increase number of flowers per panicle; (ii) increased seed fill rate; (iii) increased total seed yield per plant; (iv) increased number of (filled) seeds; (v) increased harvest index; or (vi) increased thousand kernel weight (TKW).

4. The method according to claim 1, wherein said increased seed yield is obtained under an abiotic stress.

5. The method according to claim 4, wherein said abiotic stress is osmotic stress, selected from one or more of: water stress, salt stress, oxidative stress, ionic stress, drought stress, and/or reduced nutrient availability.

6. The method according to claim 4, wherein tolerance to said abiotic stress is manifested as an enhanced yield-related trait selected from one or more of: (i) increased number of flowers per panicle; (ii) increased seed fill rate; (iii) increased total seed yield per plant; (iv) increased number of filled seeds; (v) increased harvest index; or (vi) increased thousand kernel weight (TKW), each relative to a control plant.

7. The method according to claim 1, wherein said nucleic acid encoding a 2-Cys PRX polypeptide is of plant origin.

8. A plant having increased seed yield obtained by the method according to claim 1, or a part or seed thereof, wherein said plant, or said part or seed thereof, comprises the nucleic acid encoding the 2-Cys PRX polypeptide operably linked to a root-specific promoter.

9. A method for making a plant having enhanced seed yield relative to a control plant, said method comprising the steps of:
(i) transforming a plant, plant part, or plant cell with a DNA construct, wherein said DNA construct comprises:
(a) a nucleic acid sequence encoding a 2-Cys PRX polypeptide comprising from N-terminus to C-terminus: (1) a plastidic transit peptide, and (2) a 2-Cys PRX domain, and having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 2;
(b) one or more control sequences capable of driving expression of the nucleic acid sequence of (a); and optionally
(c) a transcription termination sequence;
wherein said 2-Cys PRX polypeptide further comprises a motif comprising the amino acid sequence of SEQ ID NO: 77 and a motif comprising the amino acid sequence of SEQ ID NO: 78;
wherein at least one of the control sequences is a root-specific promoter; and
(ii) selecting a plant having increased seed yield relative to a control plant on the basis of said plant showing increased seed yield relative to said control plant.

10. A plant, plant part or plant cell obtained by the method according to claim 9.

11. A transgenic plant having enhanced seed yield relative to a control plant, produced by the method of claim 9, or a transgenic plant cell or plant part derived from said transgenic plant, wherein said transgenic plant or said transgenic plant cell or plant part thereof comprises the nucleic acid encoding a 2-Cys PRX polypeptide operably linked to a root-specific promoter.

12. The transgenic plant according to claim 11, wherein said plant is a crop plant or a monocot or a cereal, or a transgenic plant cell, plant part, or harvestable part derived from said transgenic plant.

13. Products derived from the plant and/or from harvestable parts of the plant according to claim 12, wherein said harvestable parts are seeds.

14. The method according to claim 1, wherein the nucleic acid comprises the nucleic acid sequence of SEQ ID NO: 1 or a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 2.

15. The method according to claim 9, wherein the nucleic acid sequence comprises the nucleic acid sequence of SEQ ID NO: 1 or a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 2.

16. The plant or part thereof according to claim 8, wherein the plant is a crop plant, a monocot or a cereal.

17. A progeny of the plant according to claim 8, wherein the progeny comprises the nucleic acid transgene and has increased seed yield relative to a control plant on the basis of said progeny showing increased seed yield relative to said control plant.

18. The plant, plant part or plant cell according to claim 10, wherein the plant is a crop plant, a monocot or a cereal.

19. A progeny of the plant according to claim 10, wherein the progeny comprises the construct and has increased seed yield relative to a control plant on the basis of said progeny showing increased seed yield relative to said control plant.

20. A progeny of the transgenic plant according to claim 11, wherein the progeny comprises the nucleic acid encoding a 2-Cys PRX polypeptide operably linked to a root-specific promoter, and has increased seed yield relative to a control plant on the basis of said progeny showing increased seed yield relative to said control plant.

21. The method according to claim 9, wherein said nucleic acid sequence encoding a 2-Cys PRX polypeptide comprises the nucleic acid sequence of SEQ ID NO: 1 or a sequence capable of hybridising with the nucleic acid sequence of SEQ ID NO: 1 under conditions comprising 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.

22. The method of claim 9, wherein the increased seed yield is one or more of: (i) increased number of flowers per panicle; (ii) increased seed fill rate; (iii) increased total seed yield per plant; (iv) increased number of filled seeds; (v) increased harvest index; or (vi) increased thousand kernel weight (TKW).