(en)A method for enhancing yield-related traits in plants by modulating expression in a plant of a nucleic acid encoding a GRP (Growth Related Protein) polypeptide is provided. Plants having modulated expression of a nucleic acid encoding a GRP polypeptide, which plants have enhanced yield-related traits relative to control plants, and constructs comprising the GRP-encoding nucleic acid, useful in performing the methods for enhancing yield-related traits in plants are also provided.

1.ApplicationNumber: US-201214357678-A
1.PublishNumber: US-2014352004-A1
2.Date Publish: 20141127
3.Inventor: VAN CAMP WIM
VANDENABEELE STEVEN
LOUWERS MARIEKE
MATIN JEROME

4.Inventor Harmonized: VAN CAMP WIM(BE)
VANDENABEELE STEVEN(BE)
LOUWERS MARIEKE(BE)
MATIN JEROME(BE)

5.Country: US
6.Claims:
(en)A method for enhancing yield-related traits in plants by modulating expression in a plant of a nucleic acid encoding a GRP (Growth Related Protein) polypeptide is provided. Plants having modulated expression of a nucleic acid encoding a GRP polypeptide, which plants have enhanced yield-related traits relative to control plants, and constructs comprising the GRP-encoding nucleic acid, useful in performing the methods for enhancing yield-related traits in plants are also provided.

7.Description:
(en)BACKGROUND
The present invention relates generally to the field of molecular biology and concerns a method for enhancing yield-related traits in plants by modulating expression in a plant of a nucleic acid encoding a GRP (Growth Related Protein) polypeptide. The present invention also concerns plants having modulated expression of a nucleic acid encoding a GRP polypeptide, which plants have enhanced yield-related traits relative to corresponding wild type plants or other control plants. The invention also provides constructs useful in the methods of the invention.
The ever-increasing world population and the dwindling supply of arable land available for agriculture fuels research towards increasing the efficiency of agriculture. Conventional means for crop and horticultural improvements utilise selective breeding techniques to identify plants having desirable characteristics. However, such selective breeding techniques have several drawbacks, namely that these techniques are typically labour intensive and result in plants that often contain heterogeneous genetic components that may not always result in the desirable trait being passed on from parent plants. Advances in molecular biology have allowed mankind to modify the germplasm of animals and plants. Genetic engineering of plants entails the isolation and manipulation of genetic material (typically in the form of DNA or RNA) and the subsequent introduction of that genetic material into a plant. Such technology has the capacity to deliver crops or plants having various improved economic, agronomic or horticultural traits.
A trait of particular economic interest is 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 abovementioned factors may therefore contribute to increasing crop yield.
Seed yield is a particularly important trait, since the seeds of many plants are important for human and animal nutrition. Crops such as corn, rice, wheat, canola and soybean account for over half the total human caloric intake, whether through direct consumption of the seeds themselves or through consumption of meat products raised on processed seeds. They are also a source of sugars, oils and many kinds of metabolites used in industrial processes. Seeds contain an embryo (the source of new shoots and roots) and an endosperm (the source of nutrients for embryo growth during germination and during early growth of seedlings). The development of a seed involves many genes, and requires the transfer of metabolites from 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.
A further important trait is that of improved abiotic stress tolerance. Abiotic stress is a primary cause of crop loss worldwide, reducing average yields for most major crop plants by more than 50% (Wang et al., Planta 218, 1-14, 2003). Abiotic stresses may be caused by drought, salinity, extremes of temperature, chemical toxicity and oxidative stress. The ability to improve plant tolerance to abiotic stress would be of great economic advantage to farmers worldwide and would allow for the cultivation of crops during adverse conditions and in territories where cultivation of crops may not otherwise be possible.
Crop yield may therefore be increased by optimising one of the above-mentioned factors.
Depending on the end use, the modification of certain yield traits may be favoured over others. For example for applications such as forage or wood production, or bio-fuel resource, an increase in the vegetative parts of a plant may be desirable, and for applications such as flour, starch or oil production, an increase in seed parameters may be particularly desirable. Even amongst the seed parameters, some may be favoured over others, depending on the application. Various mechanisms may contribute to increasing seed yield, whether that is in the form of increased seed size or increased seed number.
It has now been found that various yield-related traits may be improved in plants by modulating expression in a plant of a nucleic acid encoding a GRP (Growth Related Protein) polypeptide in a plant.


DETAILED DESCRIPTION OF THE INVENTION
The present invention shows that modulating expression in a plant of a nucleic acid encoding a GRP polypeptide gives plants having enhanced yield-related traits relative to control plants.
According to a first embodiment, the present invention provides a method for enhancing yield-related traits in plants relative to control plants, comprising modulating expression in a plant of a nucleic acid encoding a GRP polypeptide and optionally selecting for plants having enhanced yield-related traits. According to another embodiment, the present invention provides a method for producing plants having enhanced yield-related traits relative to control plants, wherein said method comprises the steps of modulating expression in said plant of a nucleic acid encoding a GRP polypeptide as described herein and optionally selecting for plants having enhanced yield-related traits.
In one embodiment, the preferred method for modulating expression of a nucleic acid encoding a GRP polypeptide is increasing expression by introducing and expressing in a plant a nucleic acid encoding a GRP polypeptide, preferably a recombinant nucleic acid encoding a GRP polypeptide. In another embodiment, the preferred method of modulating expression is by introducing a genetic construct designed for decreasing expression of a nucleic acid encoding a GRP.
Any reference hereinafter to a “protein useful in the methods of the invention” is taken to mean a GRP polypeptide as defined herein. Any reference hereinafter to a “nucleic acid useful in the methods of the invention” is taken to mean a nucleic acid capable of encoding such a GRP 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 “GRP nucleic acid” or “GRP gene”.
A “GRP polypeptide” as defined herein refers to a polypeptide selected from Table A in the Examples section, and to homologues and derivatives thereof.
In a particular embodiment, preferred GRP proteins for use in the methods of the invention include Zeama_GRMZM2G180172, Zeama_GRMZM2G012224_T01, Zeama_GRMZM2G055585, Zeama_GRMZM2G049398_T01, Zeama_GRMZM2G127850_T01, Zm protein kinase-related (ZM1s60983186), Orysa_LOC_Os03g46490, Zeama_GRMZM2G126566, Zeama_GRMZM2G031724, Zeama_GRMZM2G047684, Zm indole-3-acetic acid amido synthetase-like 1, Zm indole-3-acetic acid amido synthetase-like 2, Zeama_GRMZM2G031983_T01, Zeama_GRMZM2G347043, Orysa_LOC_Os06g01440, Zeama_GRMZM2G108716, Zm embryo-abundant protein-related, Zm ATP-binding protein, Zeama_unknown protein b.
Homologues of polypeptides from Table A preferably have in increasing order of preference at least 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 81%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% overall sequence identity to one of the sequences of the proteins listed in Table A. The overall sequence identity is determined using a global alignment algorithm, such as the Needleman Wunsch algorithm in the program GAP (GCG Wisconsin Package, Accelrys), preferably with default parameters and preferably with sequences of mature proteins (i.e. without taking into account secretion signals or transit peptides). In one embodiment the sequence identity level is determined by comparison of the polypeptide sequences over the entire length of the sequence of the proteins listed in Table A.
Alternatively or additionally, homologues of polypeptides from Table A can be identified by screening for the presence of domains or motifs in common with one of the polypeptides from Table A or by screening for the same biological activity as that of one of the polypeptides from Table A. Tools for identifying domains are known in the art and comprise querying databases like InterPro (Hunter et al., Nucleic Acids Res. 37 (Database Issue):D224-228, 2009) with a protein sequence from Table A or of homologous sequences, as exemplified in Example 4. Also the identification of motifs is known in the art, for example by using the MEME algorithm (Bailey and Elkan, Proceedings of the Second International Conference on Intelligent Systems for Molecular Biology, pp. 28-36, AAAI Press, Menlo Park, Calif., 1994). At each position within a MEME motif, the residues are shown that are present in the query set of sequences with a frequency higher than 0.2. Residues within square brackets represent alternatives.
Alternatively or additionally, the sequence identity level is determined by comparison of one or more conserved domains or motifs in a given protein sequence provided in Table A with corresponding conserved domains or motifs in other GRP polypeptides. Compared to overall sequence identity, the sequence identity will generally be higher when only conserved domains or motifs are considered. Preferably the motifs in a GRP polypeptide have, in increasing order of preference, at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one or more of the motifs present in a protein sequence of Table A.
The terms “domain”, “signature” and “motif” are defined in the “definitions” section herein. Preferably the homologue of a given protein from Table A has the same domain and motif organisation as in that particular protein from Table A.
According one embodiment, there is provided a method for improving yield-related traits as provided herein in plants relative to control plants, comprising modulating expression in a plant of a nucleic acid encoding a GRP polypeptide as defined herein, preferably a nucleic acid encoding a GRP polypeptide listed in Table A.
Preferably, GRP polypeptides (at least in their native form) typically have a biological function similar or identical to that of one of the polypeptides of Table A. Tools and techniques for measuring biological functions are known in the art. In addition, nucleic acids encoding GRP polypeptides, when expressed in rice according to the methods of the present invention as outlined in Examples 7 and 9, give plants having increased yield related traits, such a those provided in Example 10. Another function of the nucleic acid sequences encoding GRP polypeptides is to confer information for synthesis of the GRP protein that increases yield or yield related traits as described herein, when such a nucleic acid sequence of the invention is transcribed and translated in a living plant cell.
The present invention is illustrated by transforming plants with a nucleic acid sequence selected from Table A, encoding the GRP polypeptides. However, performance of the invention is not restricted to these sequences; the methods of the invention may advantageously be performed using any GRP-encoding nucleic acid or GRP polypeptide as defined herein. The term “GRP” or “GRP polypeptide” as used herein also intends to include homologues as defined hereunder of the proteins given in Table A. Preferably the homologue is an orthologue or a paralogue of a protein given in Table A, the terms “orthologues” and “paralogues” being as defined herein. Orthologues and paralogues may readily be identified by performing a so-called reciprocal blast search as described in the definitions section; where the query sequence is one of the protein or DNA sequences listed in Table A, the second BLAST (back-BLAST) would be against maize, rice or Arabidopsis sequences.
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 A of the Examples section, the terms “homologue” and “derivative” being as defined herein. Also useful in the methods of the invention are nucleic acids encoding homologues and derivatives of orthologues or paralogues of any one of the amino acid sequences given in Table A of the Examples section. Homologues and derivatives useful in the methods of the present invention have substantially the same biological and functional activity as the unmodified protein from which they are derived. Further variants useful in practising the methods of the invention are variants in which codon usage is optimised or in which miRNA target sites are removed.
Further nucleic acid variants useful in practising the methods of the invention include portions of nucleic acids encoding GRP polypeptides, nucleic acids hybridising to nucleic acids encoding GRP polypeptides, splice variants of nucleic acids encoding GRP polypeptides, allelic variants of nucleic acids encoding GRP polypeptides and variants of nucleic acids encoding GRP polypeptides obtained by gene shuffling. The terms hybridising sequence, splice variant, allelic variant and gene shuffling are as described herein.
Nucleic acids encoding GRP 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 A of the Examples section, or a portion of a nucleic acid encoding an orthologue, paralogue or homologue of any of the amino acid sequences given in Table A of the Examples section.
A portion of a nucleic acid may be prepared, for example, by making one or more deletions to the nucleic acid. The portions may be used in isolated form or they may be fused to other coding (or non-coding) sequences in order to, for example, produce a protein that combines several activities. When fused to other coding sequences, the resultant polypeptide produced upon translation may be bigger than that predicted for the protein portion.
Portions useful in the methods of the invention, encode a GRP polypeptide as defined herein or at least part thereof, and have substantially the same biological activity as the amino acid sequences given in Table A of the Examples section. Preferably, the portion is a portion of any one of the nucleic acids given in Table A of the Examples section, or is a portion of a nucleic acid encoding an orthologue or paralogue of any one of the amino acid sequences given in Table A of the Examples section. Preferably the portion is at least 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200 consecutive nucleotides in length, the consecutive nucleotides being of any one of the nucleic acid sequences given in Table A of the Examples section, or of a nucleic acid encoding an orthologue or paralogue of any one of the amino acid sequences given in Table A of the Examples section. Most preferably the portion is a portion of a nucleic acid listed in Table A. Preferably, the portion encodes a fragment of an amino acid sequence which has at least 25% sequence identity to one of the proteins listed in Table A.
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, more preferably under conditions of high stringency, with a nucleic acid encoding a GRP 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 the complement of a nucleic acid encoding any one of the proteins given in Table A of the Examples section, or to a nucleic acid encoding an orthologue, paralogue or homologue of any one of the proteins given in Table A.
Hybridising sequences useful in the methods of the invention encode a GRP polypeptide as defined herein, having substantially the same biological activity as any one of the amino acid sequences given in Table A of the Examples section. Preferably, the hybridising sequence is capable of hybridising to the complement of a nucleic acid encoding any one of the proteins given in Table A of the Examples section, or to a portion of any of these sequences, a portion being as defined herein, or the hybridising sequence is capable of hybridising to the complement of a nucleic acid encoding an orthologue or paralogue of any one of the amino acid sequences given in Table A of the Examples section. Most preferably, the hybridising sequence is capable of hybridising to the complement of a nucleic acid encoding the polypeptide as represented by one of the sequences given in Table A or to a portion thereof. In one embodiment, the hybridization conditions are of medium stringency, preferably of high stringency, as defined herein.
Preferably, the hybridising sequence encodes a polypeptide with an amino acid sequence which has at least 25% sequence identity to one of the proteins listed in Table A.
In another embodiment, there is provided a method for enhancing yield-related traits in plants, comprising introducing and expressing in a plant a splice variant of a nucleic acid encoding any one of the proteins given in Table A of the Examples section, or a splice variant of a nucleic acid encoding an orthologue, paralogue or homologue of any of the amino acid sequences given in Table A of the Examples section.
Preferred splice variants are splice variants of a nucleic acid provided in Table A, or a splice variant of a nucleic acid encoding an orthologue or paralogue of one of the proteins listed in Table A. Preferably, the amino acid sequence encoded by the splice variant has at least 25% sequence identity to one of the proteins listed in Table A.
In yet another embodiment, there is provided a method for enhancing yield-related traits in plants, comprising introducing and expressing in a plant an allelic variant of a nucleic acid encoding any one of the proteins given in Table A of the Examples section, or comprising introducing and expressing in a plant an allelic variant of a nucleic acid encoding an orthologue, paralogue or homologue of any of the amino acid sequences given in Table A of the Examples section.
The polypeptides encoded by allelic variants useful in the methods of the present invention have substantially the same biological activity as one of the GRP polypeptides listed in Table A of the Examples section. Allelic variants exist in nature, and encompassed within the methods of the present invention is the use of these natural alleles. Preferably, the allelic variant is an allelic variant of one of the nucleic acids provided in Table A or an allelic variant of a nucleic acid encoding an orthologue or paralogue of a protein listed in Table A. Preferably, the amino acid sequence encoded by the allelic variant has at least 25% sequence identity to one of the proteins listed in Table A.
In yet another embodiment, 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 A of the Examples section, or comprising introducing and expressing in a plant a variant of a nucleic acid encoding an orthologue, paralogue or homologue of any of the amino acid sequences given in Table A of the Examples section, which variant nucleic acid is obtained by gene shuffling.
Preferably, the amino acid sequence encoded by the variant nucleic acid obtained by gene shuffling has at least 25% sequence identity to one of the proteins listed in Table A.
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.). GRP polypeptides differing from the sequence of a protein listed in Table A by one or several amino acids (substitution(s), insertion(s) and/or deletion(s) as defined herein) may equally be useful to increase the yield of plants in the methods and constructs and plants of the invention.
Nucleic acids encoding GRP 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 GRP polypeptide-encoding nucleic acid is from a plant, further preferably from a monocotyledonous plant, more preferably from the family Poaceae, most preferably the nucleic acid is from Oryza sativa or Zea mays . In another embodiment, the GRP encoding nucleic acid is from a dicotyledonous plant, more preferably from the family Brassicaceae, most preferably the nucleic acid is from Arabidopsis thaliana . In a particular embodiment, the expression of the endogenous GRP encoding nucleic acid is downregulated. For optimal performance, the gene silencing techniques used for the reduction or substantial elimination of endogenous GRP gene expression require the use of GRP sequences that are substantially homologous to the endogenous GRP gene to be downregulated. For example, a GRP nucleic acid from rice (be it a full length GRP sequence or a fragment) is transformed into a rice plant. The GRP nucleic acid need not be introduced into the same plant variety. Most preferably, the GRP nucleic acid from rice is a sufficient length of substantially contiguous nucleotides of any one of the SEQ ID NOs listed in Table A or a sufficient length of substantially contiguous nucleotides of a nucleic acid sequence encoding an orthologue or paralogue of a GRP listed in Table A. A person skilled in the art would be well aware of what would constitute a sufficient length of substantially contiguous nucleotides to perform any of the gene silencing methods defined hereinabove, this may be as little as 20 or fewer substantially contiguous nucleotides in some cases.
In another embodiment the present invention extends to recombinant chromosomal DNA comprising a nucleic acid sequence useful in the methods of the invention, wherein said nucleic acid is present in the chromosomal DNA as a result of recombinant methods, but is not in its natural genetic environment. In a further embodiment the recombinant chromosomal DNA of the invention is comprised in a plant cell.
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 early vigour and/or increased yield, especially increased biomass and/or increased seed yield relative to control plants. The terms “early vigour” “yield” and “seed yield” are described in more detail in the “definitions” section herein.
The present invention thus provides a method for increasing yield-related traits, especially one or more of early vigour, root biomass, shoot biomass, and seed yield of plants, relative to control plants, which method comprises modulating expression in a plant of a nucleic acid encoding a GRP polypeptide as defined herein.
According to a preferred feature of the present invention, performance of the methods of the invention gives plants having an increased growth rate relative to control plants. Therefore, according to the present invention, there is provided a method for increasing the growth rate of plants, which method comprises modulating expression in a plant of a nucleic acid encoding a GRP polypeptide as defined herein.
Performance of the methods of the invention gives plants grown under non-stress conditions or under mild drought conditions increased yield-related traits relative to control plants grown under comparable conditions. Therefore, according to the present invention, there is provided a method for increasing yield-related traits in plants grown under non-stress conditions or under mild drought conditions, which method comprises modulating expression in a plant of a nucleic acid encoding a GRP polypeptide.
Performance of the methods of the invention gives plants grown under conditions of drought, increased yield-related traits relative to control plants grown under comparable conditions. Therefore, according to the present invention, there is provided a method for increasing yield-related traits in plants grown under conditions of drought which method comprises modulating expression in a plant of a nucleic acid encoding a GRP polypeptide.
Performance of the methods of the invention gives plants grown under conditions of nutrient deficiency, particularly under conditions of nitrogen deficiency, increased yield-related traits relative to control plants grown under comparable conditions. Therefore, according to the present invention, there is provided a method for increasing yield-related traits in plants grown under conditions of nutrient deficiency, which method comprises modulating expression in a plant of a nucleic acid encoding a GRP polypeptide.
Performance of the methods of the invention gives plants grown under conditions of salt stress, increased yield-related traits relative to control plants grown under comparable conditions. Therefore, according to the present invention, there is provided a method for increasing yield-related traits in plants grown under conditions of salt stress, which method comprises modulating expression in a plant of a nucleic acid encoding a GRP polypeptide.
The invention also provides genetic constructs and vectors to facilitate introduction and/or expression in plants of nucleic acids encoding GRP polypeptides. The gene constructs may be inserted into vectors, which may be commercially available, suitable for transforming into plants or host cells 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 a GRP 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 a GRP polypeptide is as defined above. The term “control sequence” and “termination sequence” are as defined herein.
The genetic construct of the invention may be comprised in a host cell, plant cell, seed, agricultural product or plant. Plants or host cells are transformed with a genetic construct such as a vector or an expression cassette comprising any of the nucleic acids described above. Thus the invention furthermore provides plants or host cells transformed with a construct as described above. In particular, the invention provides plants transformed with a construct as described above, which plants have increased yield-related traits as described herein.
In one embodiment the genetic construct of the invention confers increased yield or yield related traits(s) to a plant when it has been introduced into said plant, which plant expresses the nucleic acid encoding the GRP comprised in the genetic construct. In another embodiment the genetic construct of the invention confers increased yield or yield related traits(s) to a plant comprising plant cells in which the construct has been introduced, which plant cells express the nucleic acid encoding the GRP comprised in the genetic construct.
The skilled artisan is well aware of the genetic elements that must be present on the genetic construct in order to successfully transform, select and propagate host cells containing the sequence of interest. The sequence of interest is operably linked to one or more control sequences (at least to a promoter).
Advantageously, any type of promoter, whether natural or synthetic, may be used to drive expression of the nucleic acid sequence, but preferably the promoter is of plant origin. A constitutive promoter is particularly useful in the methods. See the “Definitions” section herein for definitions of the various promoter types.
In one embodiment, the constitutive promoter is a ubiquitous constitutive promoter of medium strength. Preferably it is a plant derived promoter, e.g. a promoter of plant chromosomal origin, such as a GOS2 promoter or a promoter of substantially the same strength and having substantially the same expression pattern (a functionally equivalent promoter), more preferably the promoter is the promoter GOS2 promoter from rice. Further preferably the constitutive promoter is represented by a nucleic acid sequence substantially similar to SEQ ID NO: 643, most preferably the constitutive promoter is as represented by SEQ ID NO: 643. In another embodiment, the constitutive promoter is a CaMV-35S promoter, preferably the 35S promoter is substantially similar to SEQ ID NO: 644. See the “Definitions” section herein for further examples of constitutive promoters.
According to another preferred embodiment of the invention, the nucleic acid encoding a GRP polypeptide is operably linked to a root-specific promoter. The root-specific promoter is preferably a pericycle specific UAS promoter (Brand and Perrimon Development 118(2): 401-415, 1993; Laplaze et al., The Plant cell, 19, 3889-38900, 2007) or a promoter of substantially the same strength and having substantially the same expression pattern (a functionally equivalent promoter). 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.
It should be clear that the applicability of the present invention is not restricted to the GRP polypeptide-encoding nucleic acid represented by those listed in Table A, nor is the applicability of the invention restricted to the rice GOS2 promoter or to the 35S promoter when expression of a GRP polypeptide-encoding nucleic acid is driven by a constitutive promoter.
Optionally, one or more terminator sequences may be used in the construct introduced into a plant. Those skilled in the art will be aware of terminator sequences that may be suitable for use in performing the invention. In one embodiment, the construct comprises an expression cassette comprising a GOS2 promoter, substantially similar to SEQ ID NO: 643, operably linked to the nucleic acid encoding the GRP polypeptide. More preferably, the construct furthermore comprises a zein terminator (t-zein) linked to the 3′ end of the GRP coding sequence. In another embodiment, the construct comprises an expression cassette comprising a CaMV-35S promoter substantially similar to SEQ ID NO: 644, operably linked to the nucleic acid encoding the GRP polypeptide.
Furthermore, one or more sequences encoding selectable markers may be present on the construct introduced into a plant.
According to one embodiment 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. In another embodiment, the modulated expression is decreased expression. Methods for decreasing expression of nucleic acids or genes, or gene products, are well documented in the art and examples are provided in the definitions section.
As mentioned above, a preferred method for modulating expression of a nucleic acid encoding a GRP polypeptide is by introducing and expressing in a plant a nucleic acid encoding a GRP polypeptide comprised in a genetic construct designed for either increased expression or for decreased expression; 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 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 a GRP polypeptide as defined herein.
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 or plant cell a GRP polypeptide-encoding nucleic acid or a genetic construct comprising a GRP polypeptide-encoding nucleic acid; and (ii) cultivating the plant cell under conditions promoting plant growth and development.

The nucleic acid of (i) may be any of the nucleic acids capable of encoding a GRP polypeptide as defined herein.
Cultivating the plant cell under conditions promoting plant growth and development, may or may not include regeneration and/or growth to maturity. Accordingly, in a particular embodiment of the invention, the plant cell transformed by the method according to the invention is regenerable into a transformed plant. In another particular embodiment, the plant cell transformed by the method according to the invention is not regenerable into a transformed plant, i.e. cells that are not capable to regenerate into a plant using cell culture techniques known in the art. While plants cells generally have the characteristic of totipotency, some plant cells can not be used to regenerate or propagate intact plants from said cells. In one embodiment of the invention the plant cells of the invention are such cells. In another embodiment the plant cells of the invention are plant cells that do not sustain themselves in an autotrophic way.
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 or plant cell by transformation. The term “transformation” is described in more detail in the “definitions” section herein.
In one embodiment the present invention 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 encompasses plants or parts thereof (including plant cells and seeds) obtainable by the methods according to the present invention. The plants or plant parts or plant cells comprise a nucleic acid transgene encoding a GRP polypeptide as defined above, preferably in a genetic construct such as an expression cassette. 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.
In a further embodiment the invention extends to seeds comprising the expression cassettes of the invention, the genetic constructs of the invention, or the nucleic acids encoding the GRP and/or the GRP polypeptides as described above.
The invention also includes host cells containing an isolated nucleic acid encoding a GRP polypeptide as defined above. In one embodiment host cells according to the invention are plant cells, yeasts, bacteria or fungi. Host plants for the nucleic acids, construct, expression cassette or the vector used in the method according to the invention are, in principle, advantageously all plants which are capable of synthesizing the polypeptides used in the inventive method. In a particular embodiment the plant cells of the invention overexpress the nucleic acid molecule of the invention.
The methods of the invention are advantageously applicable to any plant, in particular to any plant as defined herein. 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 an embodiment of the present invention, the plant is a crop plant. Examples of crop plants include but are not limited to chicory, carrot, cassava, trefoil, soybean, beet, sugar beet, sunflower, canola, alfalfa, rapeseed, linseed, cotton, tomato, potato and tobacco. According to another embodiment of the present invention, the plant is a monocotyledonous plant. Examples of monocotyledonous plants include sugarcane. According to another embodiment of the present invention, the plant is a cereal. Examples of cereals include rice, maize, wheat, barley, millet, rye, triticale, sorghum, emmer, spelt, einkorn, teff, milo and oats. In a particular embodiment the plants used in the methods of the invention are selected from the group consisting of maize, wheat, rice, soybean, cotton, oilseed rape including canola, sugarcane, sugar beet and alfalfa. Advantageously the methods of the invention are more efficient than the known methods, because the plants of the invention have increased yield and/or tolerance to an environmental stress compared to control plants used in comparable methods.
The invention also extends to plants, plant parts or harvestable parts of a plant such as, but not limited to seeds, leaves, fruits, flowers, stems, roots, rhizomes, tubers and bulbs, which plants or plant parts or harvestable parts comprise a recombinant nucleic acid encoding a GRP polypeptide. The invention furthermore relates to products derived or produced, preferably directly derived or produced, from a harvestable part of such a plant, such as dry pellets, meal or powders, oil, fat and fatty acids, starch or proteins.
The invention also includes methods for manufacturing a product comprising a) growing the plants of the invention and b) producing said product from or by the plants of the invention or parts thereof, including seeds. In a further embodiment the methods comprise the steps of a) growing the plants of the invention, b) removing the harvestable parts as described herein from the plants and c) producing said product from, or with the harvestable parts of plants according to the invention.
In one embodiment the products produced by the methods of the invention are plant products such as, but not limited to, a foodstuff, feedstuff, a food supplement, feed supplement, fiber, cosmetic or pharmaceutical. In another embodiment the methods for production are used to make agricultural products such as, but not limited to, plant extracts, proteins, amino acids, carbohydrates, fats, oils, polymers, vitamins, and the like.
In yet another embodiment the polynucleotides or the polypeptides of the invention are comprised in an agricultural product. In a particular embodiment the nucleic acid sequences and protein sequences of the invention may be used as product markers, for example where an agricultural product was produced by the methods of the invention. Such a marker can be used to identify a product to have been produced by an advantageous process resulting not only in a greater efficiency of the process but also improved quality of the product due to increased quality of the plant material and harvestable parts used in the process. Such markers can be detected by a variety of methods known in the art, for example but not limited to PCR based methods for nucleic acid detection or antibody based methods for protein detection.
The present invention also encompasses use of nucleic acids encoding GRP polypeptides as described herein and use of these GRP polypeptides in enhancing any of the aforementioned yield-related traits in plants. For example, nucleic acids encoding GRP polypeptide described herein, or the GRP polypeptides themselves, may find use in breeding programmes in which a DNA marker is identified which may be genetically linked to a GRP polypeptide-encoding gene. The nucleic acids/genes, or the GRP 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 herein in the methods of the invention. Furthermore, allelic variants of a GRP polypeptide-encoding nucleic acid/gene may find use in marker-assisted breeding programmes. Nucleic acids encoding GRP 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.
Moreover, the present invention relates to the following specific embodiments:
1. A method for enhancing yield-related traits in plants relative to control plants, comprising modulating expression in a plant of a nucleic acid encoding a GRP polypeptide listed in Table A. 2. Method according to embodiment 1, wherein said modulated expression is effected by introducing and expressing in a plant said nucleic acid encoding said GRP polypeptide. 3. Method according to embodiment 1 or 2, wherein said enhanced yield-related traits comprise increased yield and/or increased early vigour relative to control plants, wherein said increased yield preferably comprises increased biomass and/or increased seed yield relative to control plants. 4. Method according to any one of embodiments 1 to 3, wherein said enhanced yield-related traits are obtained under non-stress conditions. 5. Method according to any one of embodiments 1 to 3, wherein said enhanced yield-related traits are obtained under conditions of drought stress, salt stress or nitrogen deficiency. 6. Method according to any one of embodiments 1 to 5, wherein said nucleic acid encoding a GRP is of plant origin, preferably from a monocotyledonous or a dicotyledonous plant. 7. Method according to any one of embodiments 1 to 6, wherein said nucleic acid encoding a GRP listed in Table A is a portion of such a nucleic acid, or a nucleic acid capable of hybridising with such a nucleic acid. 8. Method according to any one of embodiments 1 to 7, wherein said nucleic acid sequence encodes an orthologue or paralogue of any of the polypeptides given in Table A. 9. Method according to any one of embodiments 1 to 8, wherein said nucleic acid is operably linked to a constitutive promoter of plant origin, preferably to a medium strength constitutive promoter of plant origin, more preferably to a GOS2 promoter, most preferably to a GOS2 promoter from rice. 10. Method according to any one of embodiments 1 to 8, wherein said nucleic acid is operably linked to a strong constitutive promoter, preferably the CaMV-35S promoter. 11. Plant, or part thereof, or plant cell, obtainable by a method according to any one of embodiments 1 to 10, wherein said plant, plant part or plant cell comprises a recombinant nucleic acid encoding a GRP polypeptide as defined in any of embodiments 1 and 6 to 8. 12. Construct comprising:
(i) nucleic acid encoding an GRP as defined in any of embodiments 1 and 6 to 8; (ii) one or more control sequences capable of driving expression of the nucleic acid sequence of (i); and optionally (iii) a transcription termination sequence.
13. Construct according to embodiment 12, wherein one of said control sequences is a constitutive promoter of plant origin, preferably to a medium strength constitutive promoter of plant origin, more preferably to a GOS2 promoter, most preferably to a GOS2 promoter from rice. 14. Construct according to embodiment 12, wherein one of said control sequences is a strong constitutive promoter, preferably the CaMV-35S promoter. 15. Use of a construct according to any one of embodiments 12 to 14 in a method for making plants having enhanced yield-related traits, preferably increased yield and/or early vigour relative to control plants. 16. Plant, plant part or plant cell transformed with a construct according to any one of embodiments 12 to 14. 17. Method for the production of a transgenic plant having enhanced yield-related traits relative to control plants, preferably increased yield and/or increased early vigour relative to control plants, and more preferably increased seed yield and/or increased biomass relative to control plants, comprising:
(i) introducing and expressing in a plant cell or plant a nucleic acid encoding an GRP polypeptide as defined in any of embodiments 1 and 6 to 10; and (ii) cultivating said plant cell or plant under conditions promoting plant growth and development.
18. Transgenic plant having enhanced yield-related traits relative to control plants, preferably increased yield and/or early vigour relative to control plants, resulting from modulated expression of a nucleic acid encoding an GRP polypeptide as defined in any of embodiments 1 and 6 to 8 or a transgenic plant cell derived from said transgenic plant. 19. Transgenic plant according to embodiment 11, 16 or 18, or a transgenic plant cell derived therefrom, wherein said plant is a crop plant, such as beet, sugarbeet or alfalfa; or a monocotyledonous plant such as sugarcane; or a cereal, such as rice, maize, wheat, barley, millet, rye, triticale, sorghum, emmer, spelt, einkorn, teff, milo or oats. 20. Harvestable parts of a plant according to embodiment 19, wherein said harvestable parts are preferably shoot biomass and/or root biomass and/or seeds. 21. Products derived from a plant according to embodiment 19 and/or from harvestable parts of a plant according to embodiment 20. 22. Use of a nucleic acid encoding an GRP polypeptide as defined in any of embodiments 1 and 6 to 8 for enhancing yield-related traits in plants relative to control plants, preferably for increasing yield and/or early vigour, relative to control plants. 23. A method for manufacturing a product comprising the steps of growing the plants according to embodiment 12, 16, 19 or 20 and producing said product from or by said plants; or parts thereof, including seeds. 24. Products produced from a plant according to embodiment 19 and/or from harvestable parts of a plant according to embodiment 20. 25. Construct according to any one of embodiments 12 to 14 comprised in a plant cell. 26. Recombinant chromosomal DNA comprising the construct according to any one of embodiments 12 to 14.

DEFINITIONS
The following definitions will be used throughout the present application. The section captions and headings in this application are for convenience and reference purpose only and should not affect in any way the meaning or interpretation of this application. The technical terms and expressions used within the scope of this application are generally to be given the meaning commonly applied to them in the pertinent art of plant biology, molecular biology, bioinformatics and plant breeding. All of the following term definitions apply to the complete content of this application. The term “essentially”, “about”, “approximately” and the like in connection with an attribute or a value, particularly also define exactly the attribute or exactly the value, respectively. The term “about” in the context of a given numeric value or range relates in particular to a value or range that is within 20%, within 10%, or within 5% of the value or range given.
Peptide(s)/Protein(s)
The terms “peptides”, “oligopeptides”, “polypeptide” and “protein” are used interchangeably herein and refer to amino acids in a polymeric form of any length, linked together by peptide bonds, unless mentioned herein otherwise.
Polynucleotide(s)/Nucleic Acid(s)/Nucleic Acid Sequence(s)/Nucleotide Sequence(s)
The terms “polynucleotide(s)”, “nucleic acid sequence(s)”, “nucleotide sequence(s)”, “nucleic acid(s)”, “nucleic acid molecule” are used interchangeably herein and refer to nucleotides, either ribonucleotides or deoxyribonucleotides or a combination of both, in a polymeric unbranched form of any length.
Homologue(s)
“Homologues” of a protein encompass peptides, oligopeptides, polypeptides, proteins and enzymes having amino acid substitutions, deletions and/or insertions relative to the unmodified protein in question and having similar biological and functional activity as the unmodified protein from which they are derived.
Orthologues and paralogues are two different forms of homologues and 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.
A “deletion” refers to removal of one or more amino acids from a protein.
An “insertion” refers to one or more amino acid residues being introduced into a predetermined site in a protein. Insertions may comprise N-terminal and/or C-terminal fusions as well as intra-sequence insertions of single or multiple amino acids. Generally, insertions within the amino acid sequence will be smaller than N- or C-terminal fusions, of the order of about 1 to 10 residues. Examples of N- or C-terminal fusion proteins or peptides include the binding domain or activation domain of a transcriptional activator as used in the yeast two-hybrid system, phage coat proteins, (histidine)-6-tag, glutathione S-transferase-tag, protein A, maltose-binding protein, dihydrofolate reductase, Tag•100 epitope, c-myc epitope, FLAG®-epitope, lacZ, CMP (calmodulin-binding peptide), HA epitope, protein C epitope and VSV epitope.
A “substitution” refers to replacement of amino acids of the protein with other amino acids having similar properties (such as similar hydrophobicity, hydrophilicity, antigenicity, propensity to form or break α-helical structures or β-sheet structures). Amino acid substitutions are typically of single residues, but may be clustered depending upon functional constraints placed upon the polypeptide and may range from 1 to 10 amino acids. The amino acid substitutions are preferably conservative amino acid substitutions. Conservative substitution tables are well known in the art (see for example Creighton (1984) Proteins. W.H. Freeman and Company (Eds) and Table 1 below).





TABLE 1



Examples of conserved amino acid substitutions







Conservative


Residue
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; Gin


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 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 (see Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989 and yearly updates)).
Derivatives
“Derivatives” include peptides, oligopeptides, polypeptides which may, compared to the amino acid sequence of the naturally-occurring form of the protein, such as the protein of interest, comprise substitutions of amino acids with non-naturally occurring amino acid residues, or additions of non-naturally occurring amino acid residues. “Derivatives” of a protein also encompass peptides, oligopeptides, polypeptides which comprise naturally occurring altered (glycosylated, acylated, prenylated, phosphorylated, myristoylated, sulphated etc.) or non-naturally altered amino acid residues compared to the amino acid sequence of a naturally-occurring form of the polypeptide. A derivative may also comprise one or more non-amino acid substituents or additions compared to the amino acid sequence from which it is derived, for example a reporter molecule or other ligand, covalently or non-covalently bound to the amino acid sequence, such as a reporter molecule which is bound to facilitate its detection, and non-naturally occurring amino acid residues relative to the amino acid sequence of a naturally-occurring protein. Furthermore, “derivatives” also include fusions of the naturally-occurring form of the protein with tagging peptides such as FLAG, HIS6 or thioredoxin (for a review of tagging peptides, see Terpe, Appl. Microbiol. Biotechnol. 60, 523-533, 2003).
Domain, Motif/Consensus Sequence/Signature
The term “domain” refers to a set of amino acids conserved at specific positions along an alignment of sequences of evolutionarily related proteins. While amino acids at other positions can vary between homologues, amino acids that are highly conserved at specific positions indicate amino acids that are likely essential in the structure, stability or function of a protein. Identified by their high degree of conservation in aligned sequences of a family of protein homologues, they can be used as identifiers to determine if any polypeptide in question belongs to a previously identified polypeptide family.
The term “motif” or “consensus sequence” or “signature” refers to a short conserved region in the sequence of evolutionarily related proteins. Motifs are frequently highly conserved parts of domains, but may also include only part of the domain, or be located outside of conserved domain (if all of the amino acids of the motif fall outside of a defined domain).
Specialist databases exist for the identification of domains, for example, SMART (Schultz et al. (1998) Proc. Natl. Acad. Sci. USA 95, 5857-5864; Letunic et al. (2002) Nucleic Acids Res 30, 242-244), InterPro (Mulder et al., (2003) Nucl. Acids. Res. 31, 315-318), Prosite (Bucher and Bairoch (1994), A generalized profile syntax for biomolecular sequences motifs and its function in automatic sequence interpretation. (In) ISMB-94; Proceedings 2nd International Conference on Intelligent Systems for Molecular Biology. Altman R., Brutlag D., Karp P., Lathrop R., Searls D., Eds., pp 53-61, AAAI Press, Menlo Park; Hulo et al., Nucl. Acids. Res. 32:D134-D137, (2004)), or Pfam (Bateman et al., Nucleic Acids Research 30(1): 276-280 (2002)). A set of tools for in silico analysis of protein sequences is available on the ExPASy proteomics server (Swiss Institute of Bioinformatics (Gasteiger et al., ExPASy: the proteomics server for in-depth protein knowledge and analysis, Nucleic Acids Res. 31:3784-3788(2003)). Domains or motifs may also be identified using routine techniques, such as by sequence alignment.
Methods for the alignment of sequences for comparison are well known in the art, such methods include GAP, BESTFIT, BLAST, FASTA and TFASTA. GAP uses the algorithm of Needleman and Wunsch ((1970) J Mol Biol 48: 443-453) to find the global (i.e. spanning the complete sequences) alignment of two sequences that maximizes the number of matches and minimizes the number of gaps. The BLAST algorithm (Altschul et al. (1990) J Mol Biol 215: 403-10) calculates percent sequence identity and performs a statistical analysis of the similarity between the two sequences. The software for performing BLAST analysis is publicly available through the National Centre for Biotechnology Information (NCBI). Homologues may readily be identified using, for example, the ClustalW multiple sequence alignment algorithm (version 1.83), with the default pairwise alignment parameters, and a scoring method in percentage. Global percentages of similarity and identity may also be determined using one of the methods available in the MatGAT software package (Campanella et al., BMC Bioinformatics. 2003 Jul. 10; 4:29. MatGAT: an application that generates similarity/identity matrices using protein or DNA sequences.). Minor manual editing may be performed to optimise alignment between conserved motifs, as would be apparent to a person skilled in the art. Furthermore, instead of using full-length sequences for the identification of homologues, specific domains may also be used. The sequence identity values may be determined over the entire nucleic acid or amino acid sequence or over selected domains or conserved motif(s), using the programs mentioned above using the default parameters. For local alignments, the Smith-Waterman algorithm is particularly useful (Smith T F, Waterman M S (1981) J. Mol. Biol 147(1); 195-7).
Reciprocal BLAST
Typically, this involves a first BLAST involving BLASTing a query sequence (for example using any of the sequences listed in Table A of the Examples section) against any sequence database, such as the publicly available NCBI database. BLASTN or TBLASTX (using standard default values) are generally used when starting from a nucleotide sequence, and BLASTP or TBLASTN (using standard default values) when starting from a protein sequence. The BLAST results may optionally be filtered. The full-length sequences of either the filtered results or non-filtered results are then BLASTed back (second BLAST) against sequences from the organism from which the query sequence is derived. The results of the first and second BLASTs are then compared. A paralogue is identified if a high-ranking hit from the first blast is from the same species as from which the query sequence is derived, a BLAST back then ideally results in the query sequence amongst the highest hits; an orthologue is identified if a high-ranking hit in the first BLAST is not from the same species as from which the query sequence is derived, and preferably results upon BLAST back in the query sequence being among the highest hits.
High-ranking hits are those having a low E-value. The lower the E-value, the more significant the score (or in other words the lower the chance that the hit was found by chance). Computation of the E-value is well known in the art. In addition to E-values, comparisons are also scored by percentage identity. Percentage identity refers to the number of identical nucleotides (or amino acids) between the two compared nucleic acid (or polypeptide) sequences over a particular length. In the case of large families, ClustalW may be used, followed by a neighbour joining tree, to help visualize clustering of related genes and to identify orthologues and paralogues.
Hybridisation
The term “hybridisation” as defined herein is a process wherein substantially homologous complementary nucleotide sequences anneal to each other. The hybridisation process can occur entirely in solution, i.e. both complementary nucleic acids are in solution. The hybridisation process can also occur with one of the complementary nucleic acids immobilised to a matrix such as magnetic beads, Sepharose beads or any other resin. The hybridisation process can furthermore occur with one of the complementary nucleic acids immobilised to a solid support such as a nitro-cellulose or nylon membrane or immobilised by e.g. photolithography to, for example, a siliceous glass support (the latter known as nucleic acid arrays or microarrays or as nucleic acid chips). In order to allow hybridisation to occur, the nucleic acid molecules are generally thermally or chemically denatured to melt a double strand into two single strands and/or to remove hairpins or other secondary structures from single stranded nucleic acids.
The term “stringency” refers to the conditions under which a hybridisation takes place. The stringency of hybridisation is influenced by conditions such as temperature, salt concentration, ionic strength and hybridisation buffer composition. Generally, low stringency conditions are selected to be about 30° C. lower than the thermal melting point (T m ) for the specific sequence at a defined ionic strength and pH. Medium stringency conditions are when the temperature is 20° C. below T m , and high stringency conditions are when the temperature is 10° C. below T m . High stringency hybridisation conditions are typically used for isolating hybridising sequences that have high sequence similarity to the target nucleic acid sequence. However, nucleic acids may deviate in sequence and still encode a substantially identical polypeptide, due to the degeneracy of the genetic code. Therefore medium stringency hybridisation conditions may sometimes be needed to identify such nucleic acid molecules.
The T m is the temperature under defined ionic strength and pH, at which 50% of the target sequence hybridises to a perfectly matched probe. The T m is dependent upon the solution conditions and the base composition and length of the probe. For example, longer sequences hybridise specifically at higher temperatures. The maximum rate of hybridisation is obtained from about 16° C. up to 32° C. below T m . The presence of monovalent cations in the hybridisation solution reduce the electrostatic repulsion between the two nucleic acid strands thereby promoting hybrid formation; this effect is visible for sodium concentrations of up to 0.4M (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 T m 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):
T m =81.5° C.+16.6 x log 10 [Na + ] a +0.41 x %[ G/C b ]−500 x[L c ] −1 −0.61 x % formamide

2) DNA-RNA or RNA-RNA hybrids:
T m =79.8° C.+18.5(log 10 [Na + ] a )+0.58(% G/C b )+11.8(% G/C b ) 2 −820/ L c

3) oligo-DNA or oligo-RNAs hybrids:
For <20 nucleotides: T m =2 (I n ) For 20-35 nucleotides: T m =22+1.46 (I n )

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 =length of duplex in base pairs.

d oligo, oligonucleotide; I n ,=effective length of primer=2×(no. of G/C)+(no. of A/T).


Non-specific binding may be controlled using any one of a number of known techniques such as, for example, blocking the membrane with protein containing solutions, additions of heterologous RNA, DNA, and SDS to the hybridisation buffer, and treatment with Rnase. For non-homologous probes, a series of hybridizations may be performed by varying one of (i) progressively lowering the annealing temperature (for example from 68° C. to 42° C.) or (ii) progressively lowering the formamide concentration (for example from 50% to 0%). The skilled artisan is aware of various parameters which may be altered during hybridisation and which will either maintain or change the stringency conditions.
Besides the hybridisation conditions, specificity of hybridisation typically also depends on the function of post-hybridisation washes. To remove background resulting from non-specific hybridisation, samples are washed with dilute salt solutions. Critical factors of such washes include the ionic strength and temperature of the final wash solution: the lower the salt concentration and the higher the wash temperature, the higher the stringency of the wash. Wash conditions are typically performed at or below hybridisation stringency. A positive hybridisation gives a signal that is at least twice of that of the background. Generally, suitable stringent conditions for nucleic acid hybridisation assays or gene amplification detection procedures are as set forth above. More or less stringent conditions may also be selected. The skilled artisan is aware of various parameters which may be altered during washing and which will either maintain or change the stringency conditions.
For example, typical high stringency hybridisation conditions for DNA hybrids longer than 50 nucleotides encompass hybridisation at 65° C. in 1×SSC or at 42° C. in 1×SSC and 50% formamide, followed by washing at 65° C. in 0.3×SSC. Examples of medium stringency hybridisation conditions for DNA hybrids longer than 50 nucleotides encompass hybridisation at 50° C. in 4×SSC or at 40° C. in 6×SSC and 50% formamide, followed by washing at 50° C. in 2×SSC. The length of the hybrid is the anticipated length for the hybridising nucleic acid. When nucleic acids of known sequence are hybridised, the hybrid length may be determined by aligning the sequences and identifying the conserved regions described herein. 1×SSC is 0.15M NaCl and 15 mM sodium citrate; the hybridisation solution and wash solutions may additionally include 5×Denhardt's reagent, 0.5-1.0% SDS, 100 μg/ml denatured, fragmented salmon sperm DNA, 0.5% sodium pyrophosphate.
For the purposes of defining the level of stringency, reference can be made to Sambrook et al. (2001) Molecular Cloning: a laboratory manual, 3 rd Edition, Cold Spring Harbor Laboratory Press, CSH, New York or to Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989 and yearly updates).
Splice Variant
The term “splice variant” as used herein encompasses variants of a nucleic acid sequence in which selected introns and/or exons have been excised, replaced, displaced or added, or in which introns have been shortened or lengthened. Such variants will be ones in which the biological activity of the protein is substantially retained; this may be achieved by selectively retaining functional segments of the protein. Such splice variants may be found in nature or may be manmade. Methods for predicting and isolating such splice variants are well known in the art (see for example Foissac and Schiex (2005) BMC Bioinformatics 6: 25).
Allelic Variant
“Alleles” or “allelic variants” are alternative forms of a given gene, located at the same chromosomal position. Allelic variants encompass Single Nucleotide Polymorphisms (SNPs), as well as Small Insertion/Deletion Polymorphisms (INDELs). The size of INDELs is usually less than 100 bp. SNPs and INDELs form the largest set of sequence variants in naturally occurring polymorphic strains of most organisms.
Endogenous Gene
Reference herein to an “endogenous” gene not only refers to the gene in question as found in a plant in its natural form (i.e., without there being any human intervention), but also refers to that same gene (or a substantially homologous nucleic acid/gene) in an isolated form subsequently (re)introduced into a plant (a transgene). For example, a transgenic plant containing such a transgene may encounter a substantial reduction of the transgene expression and/or substantial reduction of expression of the endogenous gene. The isolated gene may be isolated from an organism or may be manmade, for example by chemical synthesis.
Gene Shuffling/Directed Evolution
“Gene shuffling” or “directed evolution” consists of iterations of DNA shuffling followed by appropriate screening and/or selection to generate variants of nucleic acids or portions thereof encoding proteins having a modified biological activity (Castle et al., (2004) Science 304(5674): 1151-4; U.S. Pat. Nos. 5,811,238 and 6,395,547).
Construct
Artificial DNA (such as but, not limited to plasmids or viral DNA) capable of replication in a host cell and used for introduction of a DNA sequence of interest into a host cell or host organism. Host cells of the invention may be any cell selected from bacterial cells, such as Escherichia coli or Agrobacterium species cells, yeast cells, fungal, algal or cyanobacterial cells or plant cells. The skilled artisan is well aware of the genetic elements that must be present on the genetic construct 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) as described herein. Additional regulatory elements may include transcriptional as well as translational enhancers. Those skilled in the art will be aware of terminator and enhancer sequences that may be suitable for use in performing the invention. An intron sequence may also be added to the 5′ untranslated region (UTR) or in the coding sequence to increase the amount of the mature message that accumulates in the cytosol, as described in the definitions section. Other control sequences (besides promoter, enhancer, silencer, intron sequences, 3′UTR and/or 5′UTR regions) may be protein and/or RNA stabilizing elements. Such sequences would be known or may readily be obtained by a person skilled in the art.
The genetic constructs of the invention may further include an origin of replication sequence that is required for maintenance and/or replication in a specific cell type. One example is when a genetic construct is required to be maintained in a bacterial cell as an episomal genetic element (e.g. plasmid or cosmid molecule). Preferred origins of replication include, but are not limited to, the f1-ori and colE1.
For the detection of the successful transfer of the nucleic acid sequences as used in the methods of the invention and/or selection of transgenic plants comprising these nucleic acids, it is advantageous to use marker genes (or reporter genes). Therefore, the genetic construct may optionally comprise a selectable marker gene. Selectable markers are described in more detail in the “definitions” section herein. The marker genes may be removed or excised from the transgenic cell once they are no longer needed. Techniques for marker removal are known in the art, useful techniques are described above in the definitions section.
Regulatory Element/Control Sequence/Promoter
The terms “regulatory element”, “control sequence” and “promoter” are all used interchangeably herein and are to be taken in a broad context to refer to regulatory nucleic acid sequences capable of effecting expression of the sequences to which they are ligated. The term “promoter” typically refers to a nucleic acid control sequence located upstream from the transcriptional start of a gene and which is involved in recognising and binding of RNA polymerase and other proteins, thereby directing transcription of an operably linked nucleic acid. Encompassed by the aforementioned terms are transcriptional regulatory sequences derived from a classical eukaryotic genomic gene (including the TATA box which is required for accurate transcription initiation, with or without a CCAAT box sequence) and additional regulatory elements (i.e. upstream activating sequences, enhancers and silencers) which alter gene expression in response to developmental and/or external stimuli, or in a tissue-specific manner. Also included within the term is a transcriptional regulatory sequence of a classical prokaryotic gene, in which case it may include a −35 box sequence and/or −10 box transcriptional regulatory sequences. The term “regulatory element” also encompasses a synthetic fusion molecule or derivative that confers, activates or enhances expression of a nucleic acid molecule in a cell, tissue or organ.
A “plant promoter” comprises regulatory elements, which mediate the expression of a coding sequence segment in plant cells. Accordingly, a plant promoter need not be of plant origin, but may originate from viruses or micro-organisms, for example from viruses which attack plant cells. The “plant promoter” can also originate from a plant cell, e.g. from the plant which is transformed with the nucleic acid sequence to be expressed in the inventive process and described herein. This also applies to other “plant” regulatory signals, such as “plant” terminators. The promoters upstream of the nucleotide sequences useful in the methods of the present invention can be modified by one or more nucleotide substitution(s), insertion(s) and/or deletion(s) without interfering with the functionality or activity of either the promoters, the open reading frame (ORF) or the 3′-regulatory region such as terminators or other 3′ regulatory regions which are located away from the ORF. It is furthermore possible that the activity of the promoters is increased by modification of their sequence, or that they are replaced completely by more active promoters, even promoters from heterologous organisms. For expression in plants, the nucleic acid molecule must, as described above, be linked operably to or comprise a suitable promoter which expresses the gene at the right point in time and with the required spatial expression pattern.
For the identification of functionally equivalent promoters, the promoter strength and/or expression pattern of a candidate promoter may be analysed for example by operably linking the promoter to a reporter gene and assaying the expression level and pattern of the reporter gene in various tissues of the plant. Suitable well-known reporter genes include for example beta-glucuronidase or beta-galactosidase. The promoter activity is assayed by measuring the enzymatic activity of the beta-glucuronidase or beta-galactosidase. The promoter strength and/or expression pattern may then be compared to that of a reference promoter (such as the one used in the methods of the present invention). Alternatively, promoter strength may be assayed by quantifying mRNA levels or by comparing mRNA levels of the nucleic acid used in the methods of the present invention, with mRNA levels of housekeeping genes such as 18S rRNA, using methods known in the art, such as Northern blotting with densitometric analysis of autoradiograms, quantitative real-time PCR or RT-PCR (Heid et al., 1996 Genome Methods 6: 986-994). Generally by “weak promoter” is intended a promoter that drives expression of a coding sequence at a low level. By “low level” is intended at levels of about 1/10,000 transcripts to about 1/100,000 transcripts, to about 1/500,0000 transcripts per cell. Conversely, a “strong promoter” drives expression of a coding sequence at high level, or at about 1/10 transcripts to about 1/100 transcripts to about 1/1000 transcripts per cell. Generally, by “medium strength promoter” is intended a promoter that drives expression of a coding sequence at a lower level than a strong promoter, in particular at a level that is in all instances below that obtained when under the control of a 35S CaMV promoter.
Operably Linked
The term “operably linked” as used herein refers to a functional linkage between the promoter sequence and the gene of interest, such that the promoter sequence is able to initiate transcription of the gene of interest.
Constitutive Promoter
A “constitutive promoter” refers to a promoter that is transcriptionally active during most, but not necessarily all, phases of growth and development and under most environmental conditions, in at least one cell, tissue or organ. Table 2a below gives examples of constitutive promoters.





TABLE 2a



Examples of constitutive promoters




Gene Source
Reference



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

HMGP
WO 2004/070039

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

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

GOS2
de Pater et al, Plant J Nov;


2 (6): 837-44, 1992, WO 2004/065596

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

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

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

Alfalfa H3 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
U.S. Pat. No. 4,962,028

small subunit


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

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

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

nos
Shaw et al. (1984) Nucleic


Acids Res. 12 (20): 7831-7846

V-ATPase
WO 01/14572

Super promoter
WO 95/14098

G-box proteins
WO 94/12015










































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





TABLE 2b



Examples of root-specific promoters




Gene Source
Reference



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

Arabidopsis PHT1
Koyama et al. J Biosci Bioeng.


2005 January; 99 (1): 38-42. ;


Mudge et al. (2002, Plant J. 31:341)

Medicago phosphate
Xiao et al., 2006, Plant Biol (Stuttg).

transporter
2006 July; 8 (4): 439-49

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

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

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

gene
16, 983, 1991.

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

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

genes


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

SbPRP1
Suzuki et al., Plant Mol. Biol. 21: 109-119, 1993.

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

BTG-26 Brassica napus
US 20050044585

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

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

class I patatin gene
Liu et al., Plant Mol. Biol. 17 (6): 1139-1154

(potato)


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

TobRB7 gene
W Song (1997) PhD Thesis,


North Carolina State University,


Raleigh, NC USA

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

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

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

plumbaginifolia )

















































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





TABLE 2c



Examples of seed-specific promoters




Gene source
Reference



seed-specific genes
Simon et al.,


Plant Mol. Biol. 5: 191, 1985;


Scofield et al.,


J. Biol. Chem. 262: 12202, 1987.;


Baszczynski et al.,


Plant Mol. Biol. 14: 633, 1990.

Brazil Nut albumin
Pearson et al.,


Plant Mol. Biol. 18: 235-245, 1992.

legumin
Ellis et al.,


Plant Mol. Biol. 10: 203-214, 1988.

glutelin (rice)
Takaiwa et al.,


Mol. Gen. Genet. 208: 15-22, 1986;


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

zein
Matzke et al


Plant Mol Biol, 14(3): 323-32 1990

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

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

glutenin-1
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,
Theor Appl Gen 98: 1253-62, 1999;

D, hordein
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 α-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
Nakase et al.

REB/OHP-1
Plant Mol. Biol. 33: 513-522, 1997

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

pyrophos-


phorylase


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

sorghum α-kafirin
DeRose et al., Plant


Mol. Biol 32: 1029-35, 1996

KNOX
Postma-Haarsma et al,


Plant Mol. Biol. 39: 257-71, 1999

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

sunflower oleosin
Cummins et al.,


Plant Mol. Biol. 19: 873-876, 1992

PRO0117,
WO 2004/070039

putative rice 40S


ribosomal protein


PRO0136, rice alanine
unpublished

aminotransferase


PRO0147,
unpublished

trypsin inhibitor


ITR1 (barley)


PRO0151, rice WSI18
WO 2004/070039

PRO0175, rice RAB21
WO 2004/070039

PRO005
WO 2004/070039

PRO0095
WO 2004/070039

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

(Amy32b)
Skriver et al,


Proc Natl Acad Sci


USA 88: 7266-7270, 1991

cathepsin β-like gene
Cejudo et al,


Plant Mol Biol 20: 849-856, 1992

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

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

Maize B-Peru
Selinger et al.,


Genetics 149; 1125-38, 1998

































































































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

C, D, 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 NRP33
Wu et al, (1998) Plant Cell Physiol 39(8) 885-889

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

family


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
















































TABLE 2e



Examples of embryo specific promoters:




Gene source
Reference



rice OSH1
Sato et al, Proc. Natl. Acad. Sci. USA, 93: 8117-8122, 1996

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

PRO0151
WO 2004/070039

PRO0175
WO 2004/070039

PRO005
WO 2004/070039

PRO0095
WO 2004/070039































TABLE 2f



Examples of aleurone-specific promoters:






Gene source
Reference





α-amylase
Lanahan et al, Plant


(Amy32b)
Cell 4: 203-211, 1992;



Skriver et al, Proc Natl



Acad Sci USA 88: 7266-7270, 1991


cathepsin β-like
Cejudo et al, Plant


gene
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
Leaf specific
Fukavama et al., Plant Physiol.

Orthophosphate

2001 November; 127 (3):

dikinase

1136-46

Maize
Leaf specific
Kausch et al., Plant Mol Biol.

Phosphoenolpyruvate

2001 January; 45 (1): 1-15

carboxylase



Rice
Leaf specific
Lin et al., 2004 DNA Seq. 2004

Phosphoenolpyruvate

August; 15 (4): 269-76

carboxylase



Rice small subunit
Leaf specific
Nomura et al., Plant Mol Biol.

Rubisco

2000 September; 44 (1): 99-106

rice beta expansin
Shoot specific
WO 2004/070039

EXBP9



Pigeonpea
Leaf specific
Panguluri et al., Indian J Exp

small subunit Rubisco

Biol. 2005 April; 43 (4): 369-72

Pea RBCS3A
Leaf specific




































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





TABLE 2h



Examples of meristem-specific promoters





Gene source
Expression pattern
Reference



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


from embryo globular
Natl. Acad. Sci. USA,


stage to seedling stage
93: 8117-8122

Rice
Meristem specific
BAD87835.1

metallothionein



WAK1 &
Shoot and root apical
Wagner & Kohorn (2001)

WAK 2
meristems, and in
Plant Cell 13(2): 303-318


expanding leaves and



sepals





























Terminator
The term “terminator” encompasses a control sequence which is a DNA sequence at the end of a transcriptional unit which signals 3′ processing and polyadenylation of a primary transcript and termination of transcription. The terminator can be derived from the natural gene, from a variety of other plant genes, or from T-DNA. The terminator to be added may be derived from, for example, the nopaline synthase or octopine synthase genes, or alternatively from another plant gene, or less preferably from any other eukaryotic gene.
Selectable Marker (Gene)/Reporter Gene
“Selectable marker”, “selectable marker gene” or “reporter gene” includes any gene that confers a phenotype on a cell in which it is expressed to facilitate the identification and/or selection of cells that are transfected or transformed with a nucleic acid construct of the invention. These marker genes enable the identification of a successful transfer of the nucleic acid molecules via a series of different principles. Suitable markers may be selected from markers that confer antibiotic or herbicide resistance, that introduce a new metabolic trait or that allow visual selection. Examples of selectable marker genes include genes conferring resistance to antibiotics (such as nptII that phosphorylates neomycin and kanamycin, or hpt, phosphorylating hygromycin, or genes conferring resistance to, for example, bleomycin, streptomycin, tetracyclin, chloramphenicol, ampicillin, gentamycin, geneticin (G418), spectinomycin or blasticidin), to herbicides (for example bar which provides resistance to Basta®; aroA or gox providing resistance against glyphosate, or the genes conferring resistance to, for example, imidazolinone, phosphinothricin or sulfonylurea), or genes that provide a metabolic trait (such as manA that allows plants to use mannose as sole carbon source or xylose isomerase for the utilisation of xylose, or antinutritive markers such as the resistance to 2-deoxyglucose). Expression of visual marker genes results in the formation of colour (for example β-glucuronidase, GUS or β-galactosidase with its coloured substrates, for example X-Gal), luminescence (such as the luciferin/luceferase system) or fluorescence (Green Fluorescent Protein, GFP, and derivatives thereof). This list represents only a small number of possible markers. The skilled worker is familiar with such markers. Different markers are preferred, depending on the organism and the selection method.
It is known that upon stable or transient integration of nucleic acids into plant cells, only a minority of the cells takes up the foreign DNA and, if desired, integrates it into its genome, depending on the expression vector used and the transfection technique used. To identify and select these integrants, a gene coding for a selectable marker (such as the ones described above) is usually introduced into the host cells together with the gene of interest. These markers can for example be used in mutants in which these genes are not functional by, for example, deletion by conventional methods. Furthermore, nucleic acid molecules encoding a selectable marker can be introduced into a host cell on the same vector that comprises the sequence encoding the polypeptides of the invention or used in the methods of the invention, or else in a separate vector. Cells which have been stably transfected with the introduced nucleic acid can be identified for example by selection (for example, cells which have integrated the selectable marker survive whereas the other cells die).
Since the marker genes, particularly genes for resistance to antibiotics and herbicides, are no longer required or are undesired in the transgenic host cell once the nucleic acids have been introduced successfully, the process according to the invention for introducing the nucleic acids advantageously employs techniques which enable the removal or excision of these marker genes. One such a method is what is known as co-transformation. The co-transformation method employs two vectors simultaneously for the transformation, one vector bearing the nucleic acid according to the invention and a second bearing the marker gene(s). A large proportion of transformants receives or, in the case of plants, comprises (up to 40% or more of the transformants), both vectors. In case of transformation with Agrobacteria, the transformants usually receive only a part of the vector, i.e. the sequence flanked by the T-DNA, which usually represents the expression cassette. The marker genes can subsequently be removed from the transformed plant by performing crosses. In another method, marker genes integrated into a transposon are used for the transformation together with desired nucleic acid (known as the Ac/Ds technology). The transformants can be crossed with a transposase source or the transformants are transformed with a nucleic acid construct conferring expression of a transposase, transiently or stable. In some cases (approx. 10%), the transposon jumps out of the genome of the host cell once transformation has taken place successfully and is lost. In a further number of cases, the transposon jumps to a different location. In these cases the marker gene must be eliminated by performing crosses. In microbiology, techniques were developed which make possible, or facilitate, the detection of such events. A further advantageous method relies on what is known as recombination systems; whose advantage is that elimination by crossing can be dispensed with. The best-known system of this type is what is known as the Cre/Iox system. Cre1 is a recombinase that removes the sequences located between the IoxP sequences. If the marker gene is integrated between the IoxP sequences, it is removed once transformation has taken place successfully, by expression of the recombinase. Further recombination systems are the HIN/HIX, FLP/FRT and REP/STB system (Tribble et al., J. Biol. Chem., 275, 2000: 22255-22267; Velmurugan et al., J. Cell Biol., 149, 2000: 553-566). A site-specific integration into the plant genome of the nucleic acid sequences according to the invention is possible. Naturally, these methods can also be applied to microorganisms such as yeast, fungi or bacteria.
Transgenic/Transgene/Recombinant
For the purposes of the invention, “transgenic”, “transgene” or “recombinant” means with regard to, for example, a nucleic acid sequence, an expression cassette, gene construct or a vector comprising the nucleic acid sequence or an organism transformed with the nucleic acid sequences, expression cassettes or vectors according to the invention, all those constructions brought about by recombinant methods in which either
(a) the nucleic acid sequences encoding proteins useful in the methods of the invention, or (b) genetic control sequence(s) which is operably linked with the nucleic acid sequence according to the invention, for example a promoter, or (c) a) and b)

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


A transgenic plant for the purposes of the invention is thus understood as meaning, as above, that the nucleic acids used in the method of the invention are not present in, or originating from, the genome of said plant, or are present in the genome of said plant but not at their natural locus in the genome of said plant, it being possible for the nucleic acids to be expressed homologously or heterologously. However, as mentioned, transgenic also means that, while the nucleic acids according to the invention or used in the inventive method are at their natural position in the genome of a plant, the sequence has been modified with regard to the natural sequence, and/or that the regulatory sequences of the natural sequences have been modified. Transgenic is preferably understood as meaning the expression of the nucleic acids according to the invention at an unnatural locus in the genome, i.e. homologous or, preferably, heterologous expression of the nucleic acids takes place. Preferred transgenic plants are mentioned herein.
It shall further be noted that in the context of the present invention, the term “isolated nucleic acid” or “isolated polypeptide” may in some instances be considered as a synonym for a “recombinant nucleic acid” or a “recombinant polypeptide”, respectively and refers to a nucleic acid or polypeptide that is not located in its natural genetic environment and/or that has been modified by recombinant methods.
Modulation
The term “modulation” means in relation to expression or gene expression, a process in which the expression level is changed by said gene expression in comparison to the control plant, the expression level may be increased or decreased. The original, unmodulated expression may be of any kind of expression of a structural RNA (rRNA, tRNA) or mRNA with subsequent translation. For the purposes of this invention, the original unmodulated expression may also be absence of any expression. The term “modulating the activity” shall mean any change of the expression of the inventive nucleic acid sequences or encoded proteins, which leads to increased yield and/or increased growth of the plants. The expression can increase from zero (absence of, or immeasurable expression) to a certain amount, or can decrease from a certain amount to immeasurable small amounts or zero.
Expression
The term “expression” or “gene expression” means the transcription of a specific gene or specific genes or specific genetic construct. The term “expression” or “gene expression” in particular means the transcription of a gene or genes or genetic construct into structural RNA (rRNA, tRNA) or mRNA with or without subsequent translation of the latter into a protein. The process includes transcription of DNA and processing of the resulting mRNA product.
Increased Expression/Overexpression
The term “increased expression” or “overexpression” as used herein means any form of expression that is additional to the original wild-type expression level. For the purposes of this invention, the original wild-type expression level might also be zero, i.e. absence of expression or immeasurable expression.
Methods for increasing expression of genes or gene products are well documented in the art and include, for example, overexpression driven by appropriate promoters, the use of transcription enhancers or translation enhancers. Isolated nucleic acids which serve as promoter or enhancer elements may be introduced in an appropriate position (typically upstream) of a non-heterologous form of a polynucleotide so as to upregulate expression of a nucleic acid encoding the polypeptide of interest. For example, endogenous promoters may be altered in vivo by mutation, deletion, and/or substitution (see, Kmiec, U.S. Pat. No. 5,565,350; Zarling et al., WO9322443), or isolated promoters may be introduced into a plant cell in the proper orientation and distance from a gene of the present invention so as to control the expression of the gene.
If polypeptide expression is desired, it is generally desirable to include a polyadenylation region at the 3′-end of a polynucleotide coding region. The polyadenylation region can be derived from the natural gene, from a variety of other plant genes, or from T-DNA. The 3′ end sequence to be added may be derived from, for example, the nopaline synthase or octopine synthase genes, or alternatively from another plant gene, or less preferably from any other eukaryotic gene.
An intron sequence may also be added to the 5′ untranslated region (UTR) or the coding sequence of the partial coding sequence to increase the amount of the mature message that accumulates in the cytosol. Inclusion of a spliceable intron in the transcription unit in both plant and animal expression constructs has been shown to increase gene expression at both the mRNA and protein levels up to 1000-fold (Buchman and Berg (1988) Mol. Cell biol. 8: 4395-4405; Callis et al. (1987) Genes Dev 1:1183-1200). Such intron enhancement of gene expression is typically greatest when placed near the 5′ end of the transcription unit. Use of the maize introns Adh1-S intron 1, 2, and 6, the Bronze-1 intron are known in the art. For general information see: The Maize Handbook, Chapter 116, Freeling and Walbot, Eds., Springer, N.Y. (1994).
Decreased Expression
Reference herein to “decreased expression” or “reduction or substantial elimination” of expression is taken to mean a decrease in endogenous gene expression and/or polypeptide levels and/or polypeptide activity relative to control plants. The reduction or substantial elimination is in increasing order of preference at least 10%, 20%, 30%, 40% or 50%, 60%, 70%, 80%, 85%, 90%, or 95%, 96%, 97%, 98%, 99% or more reduced compared to that of control plants.
For the reduction or substantial elimination of expression an endogenous gene in a plant, a sufficient length of substantially contiguous nucleotides of a nucleic acid sequence is required. In order to perform gene silencing, this may be as little as 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10 or fewer nucleotides, alternatively this may be as much as the entire gene (including the 5′ and/or 3′ UTR, either in part or in whole). The stretch of substantially contiguous nucleotides may be derived from the nucleic acid encoding the protein of interest (target gene), or from any nucleic acid capable of encoding an orthologue, paralogue or homologue of the protein of interest. Preferably, the stretch of substantially contiguous nucleotides is capable of forming hydrogen bonds with the target gene (either sense or antisense strand), more preferably, the stretch of substantially contiguous nucleotides has, in increasing order of preference, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity to the target gene (either sense or antisense strand). A nucleic acid sequence encoding a (functional) polypeptide is not a requirement for the various methods discussed herein for the reduction or substantial elimination of expression of an endogenous gene.
This reduction or substantial elimination of expression may be achieved using routine tools and techniques. A preferred method for the reduction or substantial elimination of endogenous gene expression is by introducing and expressing in a plant a genetic construct into which the nucleic acid (in this case a stretch of substantially contiguous nucleotides derived from the gene of interest, or from any nucleic acid capable of encoding an orthologue, paralogue or homologue of any one of the protein of interest) is cloned as an inverted repeat (in part or completely), separated by a spacer (non-coding DNA).
In such a preferred method, expression of the endogenous gene is reduced or substantially eliminated through RNA-mediated silencing using an inverted repeat of a nucleic acid or a part thereof (in this case a stretch of substantially contiguous nucleotides derived from the gene of interest, or from any nucleic acid capable of encoding an orthologue, paralogue or homologue of the protein of interest), preferably capable of forming a hairpin structure. The inverted repeat is cloned in an expression vector comprising control sequences. A non-coding DNA nucleic acid sequence (a spacer, for example a matrix attachment region fragment (MAR), an intron, a polylinker, etc.) is located between the two inverted nucleic acids forming the inverted repeat. After transcription of the inverted repeat, a chimeric RNA with a self-complementary structure is formed (partial or complete). This double-stranded RNA structure is referred to as the hairpin RNA (hpRNA). The hpRNA is processed by the plant into siRNAs that are incorporated into an RNA-induced silencing complex (RISC). The RISC further cleaves the mRNA transcripts, thereby substantially reducing the number of mRNA transcripts to be translated into polypeptides. For further general details see for example, Grierson et al. (1998) WO 98/53083; Waterhouse et al. (1999) WO 99/53050).
Performance of the methods of the invention does not rely on introducing and expressing in a plant a genetic construct into which the nucleic acid is cloned as an inverted repeat, but any one or more of several well-known “gene silencing” methods may be used to achieve the same effects.
One such method for the reduction of endogenous gene expression is RNA-mediated silencing of gene expression (downregulation). Silencing in this case is triggered in a plant by a double stranded RNA sequence (dsRNA) that is substantially similar to the target endogenous gene. This dsRNA is further processed by the plant into about 20 to about 26 nucleotides called short interfering RNAs (siRNAs). The siRNAs are incorporated into an RNA-induced silencing complex (RISC) that cleaves the mRNA transcript of the endogenous target gene, thereby substantially reducing the number of mRNA transcripts to be translated into a polypeptide. Preferably, the double stranded RNA sequence corresponds to a target gene.
Another example of an RNA silencing method involves the introduction of nucleic acid sequences or parts thereof (in this case a stretch of substantially contiguous nucleotides derived from the gene of interest, or from any nucleic acid capable of encoding an orthologue, paralogue or homologue of the protein of interest) in a sense orientation into a plant. “Sense orientation” refers to a DNA sequence that is homologous to an mRNA transcript thereof. Introduced into a plant would therefore be at least one copy of the nucleic acid sequence. The additional nucleic acid sequence will reduce expression of the endogenous gene, giving rise to a phenomenon known as co-suppression. The reduction of gene expression will be more pronounced if several additional copies of a nucleic acid sequence are introduced into the plant, as there is a positive correlation between high transcript levels and the triggering of co-suppression.
Another example of an RNA silencing method involves the use of antisense nucleic acid sequences. An “antisense” nucleic acid sequence comprises a nucleotide sequence that is complementary to a “sense” nucleic acid sequence encoding a protein, i.e. complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA transcript sequence. The antisense nucleic acid sequence is preferably complementary to the endogenous gene to be silenced. The complementarity may be located in the “coding region” and/or in the “non-coding region” of a gene. The term “coding region” refers to a region of the nucleotide sequence comprising codons that are translated into amino acid residues. The term “non-coding region” refers to 5′ and 3′ sequences that flank the coding region that are transcribed but not translated into amino acids (also referred to as 5′ and 3′ untranslated regions).
Antisense nucleic acid sequences can be designed according to the rules of Watson and Crick base pairing. The antisense nucleic acid sequence may be complementary to the entire nucleic acid sequence (in this case a stretch of substantially contiguous nucleotides derived from the gene of interest, or from any nucleic acid capable of encoding an orthologue, paralogue or homologue of the protein of interest), but may also be an oligonucleotide that is antisense to only a part of the nucleic acid sequence (including the mRNA 5′ and 3′ UTR). For example, the antisense oligonucleotide sequence may be complementary to the region surrounding the translation start site of an mRNA transcript encoding a polypeptide. The length of a suitable antisense oligonucleotide sequence is known in the art and may start from about 50, 45, 40, 35, 30, 25, 20, 15 or 10 nucleotides in length or less. An antisense nucleic acid sequence according to the invention may be constructed using chemical synthesis and enzymatic ligation reactions using methods known in the art. For example, an antisense nucleic acid sequence (e.g., an antisense oligonucleotide sequence) may be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acid sequences, e.g., phosphorothioate derivatives and acridine substituted nucleotides may be used. Examples of modified nucleotides that may be used to generate the antisense nucleic acid sequences are well known in the art. Known nucleotide modifications include methylation, cyclization and ‘caps’ and substitution of one or more of the naturally occurring nucleotides with an analogue such as inosine. Other modifications of nucleotides are well known in the art.
The antisense nucleic acid sequence can be produced biologically using an expression vector into which a nucleic acid sequence has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest). Preferably, production of antisense nucleic acid sequences in plants occurs by means of a stably integrated nucleic acid construct comprising a promoter, an operably linked antisense oligonucleotide, and a terminator.
The nucleic acid molecules used for silencing in the methods of the invention (whether introduced into a plant or generated in situ) hybridize with or bind to mRNA transcripts and/or genomic DNA encoding a polypeptide to thereby inhibit expression of the protein, e.g., by inhibiting transcription and/or translation. The hybridization can be by conventional nucleotide complementarity to form a stable duplex, or, for example, in the case of an antisense nucleic acid sequence which binds to DNA duplexes, through specific interactions in the major groove of the double helix. Antisense nucleic acid sequences may be introduced into a plant by transformation or direct injection at a specific tissue site. Alternatively, antisense nucleic acid sequences can be modified to target selected cells and then administered systemically. For example, for systemic administration, antisense nucleic acid sequences can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, e.g., by linking the antisense nucleic acid sequence to peptides or antibodies which bind to cell surface receptors or antigens. The antisense nucleic acid sequences can also be delivered to cells using the vectors described herein.
According to a further aspect, the antisense nucleic acid sequence is an a-anomeric nucleic acid sequence. An a-anomeric nucleic acid sequence forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual b-units, the strands run parallel to each other (Gaultier et al. (1987) Nucl Ac Res 15: 6625-6641). The antisense nucleic acid sequence may also comprise a 2′-o-methylribonucleotide (Inoue et al. (1987) Nucl Ac Res 15, 6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987) FEBS Lett. 215, 327-330).
The reduction or substantial elimination of endogenous gene expression may also be performed using ribozymes. Ribozymes are catalytic RNA molecules with ribonuclease activity that are capable of cleaving a single-stranded nucleic acid sequence, such as an mRNA, to which they have a complementary region. Thus, ribozymes (e.g., hammerhead ribozymes (described in Haselhoff and Gerlach (1988) Nature 334, 585-591) can be used to catalytically cleave mRNA transcripts encoding a polypeptide, thereby substantially reducing the number of mRNA transcripts to be translated into a polypeptide. A ribozyme having specificity for a nucleic acid sequence can be designed (see for example: Cech et al. U.S. Pat. No. 4,987,071; and Cech et al. U.S. Pat. No. 5,116,742). Alternatively, mRNA transcripts corresponding to a nucleic acid sequence can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules (Bartel and Szostak (1993) Science 261, 1411-1418). The use of ribozymes for gene silencing in plants is known in the art (e.g., Atkins et al. (1994) WO 94/00012; Lenne et al. (1995) WO 95/03404; Lutziger et al. (2000) WO 00/00619; Prinsen et al. (1997) WO 97/13865 and Scott et al. (1997) WO 97/38116).
Gene silencing may also be achieved by insertion mutagenesis (for example, T-DNA insertion or transposon insertion) or by strategies as described by, among others, Angell and Baulcombe ((1999) Plant J 20(3): 357-62), (Amplicon VIGS WO 98/36083), or Baulcombe (WO 99/15682).
Gene silencing may also occur if there is a mutation on an endogenous gene and/or a mutation on an isolated gene/nucleic acid subsequently introduced into a plant. The reduction or substantial elimination may be caused by a non-functional polypeptide. For example, the polypeptide may bind to various interacting proteins; one or more mutation(s) and/or truncation(s) may therefore provide for a polypeptide that is still able to bind interacting proteins (such as receptor proteins) but that cannot exhibit its normal function (such as signalling ligand).
A further approach to gene silencing is by targeting nucleic acid sequences complementary to the regulatory region of the gene (e.g., the promoter and/or enhancers) to form triple helical structures that prevent transcription of the gene in target cells. See Helene, C., Anticancer Drug Res. 6, 569-84, 1991; Helene et al., Ann. N.Y. Acad. Sci. 660, 27-36 1992; and Maher, L. J. Bioassays 14, 807-15, 1992.
Other methods, such as the use of antibodies directed to an endogenous polypeptide for inhibiting its function in planta, or interference in the signalling pathway in which a polypeptide is involved, will be well known to the skilled man. In particular, it can be envisaged that manmade molecules may be useful for inhibiting the biological function of a target polypeptide, or for interfering with the signalling pathway in which the target polypeptide is involved.
Alternatively, a screening program may be set up to identify in a plant population natural variants of a gene, which variants encode polypeptides with reduced activity. Such natural variants may also be used for example, to perform homologous recombination.
Artificial and/or natural microRNAs (miRNAs) may be used to knock out gene expression and/or mRNA translation. Endogenous miRNAs are single stranded small RNAs of typically 19-24 nucleotides long. They function primarily to regulate gene expression and/or mRNA translation. Most plant microRNAs (miRNAs) have perfect or near-perfect complementarity with their target sequences. However, there are natural targets with up to five mismatches. They are processed from longer non-coding RNAs with characteristic fold-back structures by double-strand specific RNases of the Dicer family. Upon processing, they are incorporated in the RNA-induced silencing complex (RISC) by binding to its main component, an Argonaute protein. MiRNAs serve as the specificity components of RISC, since they base-pair to target nucleic acids, mostly mRNAs, in the cytoplasm. Subsequent regulatory events include target mRNA cleavage and destruction and/or translational inhibition. Effects of miRNA overexpression are thus often reflected in decreased mRNA levels of target genes.
Artificial microRNAs (amiRNAs), which are typically 21 nucleotides in length, can be genetically engineered specifically to negatively regulate gene expression of single or multiple genes of interest. Determinants of plant microRNA target selection are well known in the art. Empirical parameters for target recognition have been defined and can be used to aid in the design of specific amiRNAs, (Schwab et al., Dev. Cell 8, 517-527, 2005). Convenient tools for design and generation of amiRNAs and their precursors are also available to the public (Schwab et al., Plant Cell 18, 1121-1133, 2006).
For optimal performance, the gene silencing techniques used for reducing expression in a plant of an endogenous gene requires the use of nucleic acid sequences from monocotyledonous plants for transformation of monocotyledonous plants, and from dicotyledonous plants for transformation of dicotyledonous plants. Preferably, a nucleic acid sequence from any given plant species is introduced into that same species. For example, a nucleic acid sequence from rice is transformed into a rice plant. However, it is not an absolute requirement that the nucleic acid sequence to be introduced originates from the same plant species as the plant in which it will be introduced. It is sufficient that there is substantial homology between the endogenous target gene and the nucleic acid to be introduced.
Described above are examples of various methods for the reduction or substantial elimination of expression in a plant of an endogenous gene. A person skilled in the art would readily be able to adapt the aforementioned methods for silencing so as to achieve reduction of expression of an endogenous gene in a whole plant or in parts thereof through the use of an appropriate promoter, for example.
Transformation
The term “introduction” or “transformation” as referred to herein encompasses the transfer of an exogenous polynucleotide into a host cell, irrespective of the method used for transfer. Plant tissue capable of subsequent clonal propagation, whether by organogenesis or embryogenesis, may be transformed with a genetic construct of the present invention and a whole plant regenerated there from. The particular tissue chosen will vary depending on the clonal propagation systems available for, and best suited to, the particular species being transformed. Exemplary tissue targets include leaf disks, pollen, embryos, cotyledons, hypocotyls, megagametophytes, callus tissue, existing meristematic tissue (e.g., apical meristem, axillary buds, and root meristems), and induced meristem tissue (e.g., cotyledon meristem and hypocotyl meristem). The polynucleotide may be transiently or stably introduced into a host cell and may be maintained non-integrated, for example, as a plasmid. Alternatively, it may be integrated into the host genome. The resulting transformed plant cell may then be used to regenerate a transformed plant in a manner known to persons skilled in the art. Alternatively, a plant cell that cannot be regenerated into a plant may be chosen as host cell, i.e. the resulting transformed plant cell does not have the capacity to regenerate into a (whole) plant.
The transfer of foreign genes into the genome of a plant is called transformation. Transformation of plant species is now a fairly routine technique. Advantageously, any of several transformation methods may be used to introduce the gene of interest into a suitable ancestor cell. The methods described for the transformation and regeneration of plants from plant tissues or plant cells may be utilized for transient or for stable transformation. Transformation methods include the use of liposomes, electroporation, chemicals that increase free DNA uptake, injection of the DNA directly into the plant, particle gun bombardment, transformation using viruses or pollen and microprojection. Methods may be selected from the calcium/polyethylene glycol method for protoplasts (Krens, F. A. et al., (1982) Nature 296, 72-74; Negrutiu I et al. (1987) Plant Mol Biol 8: 363-373); electroporation of protoplasts (Shillito R. D. et al. (1985) Bio/Technol 3, 1099-1102); microinjection into plant material (Crossway A et al., (1986) Mol. Gen Genet 202: 179-185); DNA or RNA-coated particle bombardment (Klein T M et al., (1987) Nature 327: 70) infection with (non-integrative) viruses and the like. Transgenic plants, including transgenic crop plants, are preferably produced via Agrobacterium -mediated transformation. An advantageous transformation method is the transformation in planta. To this end, it is possible, for example, to allow the agrobacteria to act on plant seeds or to inoculate the plant meristem with agrobacteria. It has proved particularly expedient in accordance with the invention to allow a suspension of transformed agrobacteria to act on the intact plant or at least on the flower primordia. The plant is subsequently grown on until the seeds of the treated plant are obtained (Clough and Bent, Plant J. (1998) 16, 735-743). Methods for Agrobacterium -mediated transformation of rice include well known methods for rice transformation, such as those described in any of the following: European patent application EP 1198985 A1, Aldemita and Hodges (Planta 199: 612-617, 1996); Chan et al. (Plant Mol Biol 22 (3): 491-506, 1993), Hiei et al. (Plant J 6 (2): 271-282, 1994), which disclosures are incorporated by reference herein as if fully set forth. In the case of corn transformation, the preferred method is as described in either Ishida et al. (Nat. Biotechnol 14(6): 745-50, 1996) or Frame et al. (Plant Physiol 129(1): 13-22, 2002), which disclosures are incorporated by reference herein as if fully set forth. Said methods are further described by way of example in B. Jenes et al., Techniques for Gene Transfer, in: Transgenic Plants, Vol. 1, Engineering and Utilization, eds. S. D. Kung and R. Wu, Academic Press (1993) 128-143 and in Potrykus Annu. Rev. Plant Physiol. Plant Molec. Biol. 42 (1991) 205-225). The nucleic acids or the construct to be expressed is preferably cloned into a vector, which is suitable for transforming Agrobacterium tumefaciens , for example pBin19 (Bevan et al., Nucl. Acids Res. 12 (1984) 8711). Agrobacteria transformed by such a vector can then be used in known manner for the transformation of plants, such as plants used as a model, like Arabidopsis ( Arabidopsis thaliana is within the scope of the present invention not considered as a crop plant), or crop plants such as, by way of example, tobacco plants, for example by immersing bruised leaves or chopped leaves in an agrobacterial solution and then culturing them in suitable media. The transformation of plants by means of Agrobacterium tumefaciens is described, for example, by Hofgen 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 208:1-9; Feldmann K (1992). In: C Koncz, N-H Chua and J Shell, eds, Methods in Arabidopsis Research. Word Scientific, Singapore, pp. 274-289]. Alternative methods are based on the repeated removal of the inflorescences and incubation of the excision site in the center of the rosette with transformed agrobacteria, whereby transformed seeds can likewise be obtained at a later point in time (Chang (1994). Plant J. 5: 551-558; Katavic (1994). Mol Gen Genet, 245: 363-370). However, an especially effective method is the vacuum infiltration method with its modifications such as the “floral dip” method. In the case of vacuum infiltration of Arabidopsis , intact plants under reduced pressure are treated with an agrobacterial suspension [Bechthold, N (1993). C R Acad Sci Paris Life Sci, 316: 1194-1199], while in the case of the “floral dip” method the developing floral tissue is incubated briefly with a surfactant-treated agrobacterial suspension [Clough, S J and Bent A F (1998) The Plant J. 16, 735-743]. A certain proportion of transgenic seeds are harvested in both cases, and these seeds can be distinguished from non-transgenic seeds by growing under the above-described selective conditions. In addition the stable transformation of plastids is of advantages because plastids are inherited maternally is most crops reducing or eliminating the risk of transgene flow through pollen. The transformation of the chloroplast genome is generally achieved by a process which has been schematically displayed in Klaus et al., 2004 [Nature Biotechnology 22 (2), 225-229]. Briefly the sequences to be transformed are cloned together with a selectable marker gene between flanking sequences homologous to the chloroplast genome. These homologous flanking sequences direct site specific integration into the plastome. Plastidal transformation has been described for many different plant species and an overview is given in Bock (2001) Transgenic plastids in basic research and plant biotechnology. J Mol Biol. 2001 Sep. 21; 312 (3):425-38 or Maliga, P (2003) Progress towards commercialization of plastid transformation technology. Trends Biotechnol. 21, 20-28. Further biotechnological progress has recently been reported in form of marker free plastid transformants, which can be produced by a transient co-integrated maker gene (Klaus et al., 2004, Nature Biotechnology 22(2), 225-229).
The genetically modified plant cells can be regenerated via all methods with which the skilled worker is familiar. Suitable methods can be found in the abovementioned publications by S. D. Kung and R. Wu, Potrykus or Hofgen and Willmitzer. Alternatively, the genetically modified plant cells are non-regenerable into a whole plant.
Generally after transformation, plant cells or cell groupings are selected for the presence of one or more markers which are encoded by plant-expressible genes co-transferred with the gene of interest, following which the transformed material is regenerated into a whole plant. To select transformed plants, the plant material obtained in the transformation is, as a rule, subjected to selective conditions so that transformed plants can be distinguished from untransformed plants. For example, the seeds obtained in the above-described manner can be planted and, after an initial growing period, subjected to a suitable selection by spraying. A further possibility consists in growing the seeds, if appropriate after sterilization, on agar plates using a suitable selection agent so that only the transformed seeds can grow into plants. Alternatively, the transformed plants are screened for the presence of a selectable marker such as the ones described above.
Following DNA transfer and regeneration, putatively transformed plants may also be evaluated, for instance using Southern analysis, for the presence of the gene of interest, copy number and/or genomic organisation. Alternatively or additionally, expression levels of the newly introduced DNA may be monitored using Northern and/or Western analysis, both techniques being well known to persons having ordinary skill in the art.
The generated transformed plants may be propagated by a variety of means, such as by clonal propagation or classical breeding techniques. For example, a first generation (or T1) transformed plant may be selfed and homozygous second-generation (or T2) transformants selected, and the T2 plants may then further be propagated through classical breeding techniques. The generated transformed organisms may take a variety of forms. For example, they may be chimeras of transformed cells and non-transformed cells; clonal transformants (e.g., all cells transformed to contain the expression cassette); grafts of transformed and untransformed tissues (e.g., in plants, a transformed rootstock grafted to an untransformed scion).
T-DNA Activation Tagging
“T-DNA activation” tagging (Hayashi et al. Science (1992) 1350-1353), involves insertion of T-DNA, usually containing a promoter (may also be a translation enhancer or an intron), in the genomic region of the gene of interest or 10 kb up- or downstream of the coding region of a gene in a configuration such that the promoter directs expression of the targeted gene. Typically, regulation of expression of the targeted gene by its natural promoter is disrupted and the gene falls under the control of the newly introduced promoter. The promoter is typically embedded in a T-DNA. This T-DNA is randomly inserted into the plant genome, for example, through Agrobacterium infection and leads to modified expression of genes near the inserted T-DNA. The resulting transgenic plants show dominant phenotypes due to modified expression of genes close to the introduced promoter.
TILLING
The term “TILLING” is an abbreviation of “Targeted Induced Local Lesions In Genomes” and refers to a mutagenesis technology useful to generate and/or identify nucleic acids encoding proteins with modified expression and/or activity. TILLING also allows selection of plants carrying such mutant variants. These mutant variants may exhibit modified expression, either in strength or in location or in timing (if the mutations affect the promoter for example). These mutant variants may exhibit higher activity than that exhibited by the gene in its natural form. TILLING combines high-density mutagenesis with high-throughput screening methods. The steps typically followed in TILLING are: (a) EMS mutagenesis (Redei G P and Koncz C (1992) In Methods in Arabidopsis Research, Koncz C, Chua N H, Schell J, eds. Singapore, World Scientific Publishing Co, pp. 16-82; Feldmann et al., (1994) In Meyerowitz E M, Somerville 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 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), and approaches exist that are generally applicable regardless of the target organism (Miller et al, Nature Biotechnol. 25, 778-785, 2007).
Yield Related Trait(s)
A “Yield related trait” is a trait or feature which is related to plant yield. Yield-related traits may comprise one or more of the following non-limitative list of features: early flowering time, yield, biomass, seed yield, early vigour, greenness index, growth rate, agronomic traits, such as e.g. tolerance to submergence (which leads to yield in rice), Water Use Efficiency (WUE), Nitrogen Use Efficiency (NUE), etc.
Reference herein to enhanced yield-related traits, relative to of control plants is taken to mean one or more of an increase in early vigour and/or in biomass (weight) of one or more parts of a plant, which may include (i) aboveground parts and preferably aboveground harvestable parts and/or (ii) parts below ground and preferably harvestable below ground. In particular, such harvestable parts are seeds.
Yield
The term “yield” in general means a measurable produce of economic value, typically related to a specified crop, to an area, and to a period of time. Individual plant parts directly contribute to yield based on their number, size and/or weight, or the actual yield is the yield per square meter for a crop and year, which is determined by dividing total production (includes both harvested and appraised production) by planted square meters.
The terms “yield” of a plant and “plant yield” are used interchangeably herein and are meant to refer to vegetative biomass such as root and/or shoot biomass, to reproductive organs, and/or to propagules such as seeds of that plant.
Flowers in maize are unisexual; male inflorescences (tassels) originate from the apical stem and female inflorescences (ears) arise from axillary bud apices. The female inflorescence produces pairs of spikelets on the surface of a central axis (cob). Each of the female spikelets encloses two fertile florets, one of them will usually mature into a maize kernel once fertilized. Hence a yield increase in maize may be manifested as one or more of the following: increase in the number of plants established per square meter, an increase in the number of ears per plant, an increase in the number of rows, number of kernels per row, kernel weight, thousand kernel weight, ear length/diameter, increase in the seed filling rate, which is the number of filled florets (i.e. florets containing seed) divided by the total number of florets and multiplied by 100), among others.
Inflorescences in rice plants are named panicles. The panicle bears spikelets, which are the basic units of the panicles, and which consist of a pedicel and a floret. The floret is borne on the pedicel and includes a flower that is covered by two protective glumes: a larger glume (the lemma) and a shorter glume (the palea). Hence, taking rice as an example, a yield increase may manifest itself as an increase in one or more of the following: number of plants per square meter, number of panicles per plant, panicle length, number of spikelets per panicle, number of flowers (or florets) per panicle; an increase in the seed filling rate which is the number of filled florets (i.e. florets containing seeds) divided by the total number of florets and multiplied by 100; an increase in thousand kernel weight, among others.
Early Flowering Time
Plants having an “early flowering time” as used herein are plants which start to flower earlier than control plants. Hence this term refers to plants that show an earlier start of flowering. Flowering time of plants can be assessed by counting the number of days (“time to flower”) between sowing and the emergence of a first inflorescence. The “flowering time” of a plant can for instance be determined using the method as described in WO 2007/093444.
Early Vigour
“Early vigour” refers to active healthy well-balanced growth especially during early stages of plant growth, and may result from increased plant fitness due to, for example, the plants being better adapted to their environment (i.e. optimizing the use of energy resources and partitioning between shoot and root). Plants having early vigour also show increased seedling survival and a better establishment of the crop, which often results in highly uniform fields (with the crop growing in uniform manner, i.e. with the majority of plants reaching the various stages of development at substantially the same time), and often better and higher yield. Therefore, early vigour may be determined by measuring various factors, such as thousand kernel weight, percentage germination, percentage emergence, seedling growth, seedling height, root length, root and shoot biomass and many more.
Increased Growth Rate
The increased growth rate may be specific to one or more parts of a plant (including seeds), or may be throughout substantially the whole plant. Plants having an increased growth rate may have a shorter life cycle. The life cycle of a plant may be taken to mean the time needed to grow from a mature seed up to the stage where the plant has produced mature seeds, similar to the starting material. This life cycle may be influenced by factors such as speed of germination, early vigour, growth rate, greenness index, flowering time and speed of seed maturation. The increase in growth rate may take place at one or more stages in the life cycle of a plant or during substantially the whole plant life cycle. Increased growth rate during the early stages in the life cycle of a plant may reflect enhanced vigour. The increase in growth rate may alter the harvest cycle of a plant allowing plants to be sown later and/or harvested sooner than would otherwise be possible (a similar effect may be obtained with earlier flowering time). If the growth rate is sufficiently increased, it may allow for the further sowing of seeds of the same plant species (for example sowing and harvesting of rice plants followed by sowing and harvesting of further rice plants all within one conventional growing period). Similarly, if the growth rate is sufficiently increased, it may allow for the further sowing of seeds of different plants species (for example the sowing and harvesting of corn plants followed by, for example, the sowing and optional harvesting of soybean, potato or any other suitable plant). Harvesting additional times from the same rootstock in the case of some crop plants may also be possible. Altering the harvest cycle of a plant may lead to an increase in annual biomass production per square meter (due to an increase in the number of times (say in a year) that any particular plant may be grown and harvested). An increase in growth rate may also allow for the cultivation of transgenic plants in a wider geographical area than their wild-type counterparts, since the territorial limitations for growing a crop are often determined by adverse environmental conditions either at the time of planting (early season) or at the time of harvesting (late season). Such adverse conditions may be avoided if the harvest cycle is shortened. The growth rate may be determined by deriving various parameters from growth curves, such parameters may be: T-Mid (the time taken for plants to reach 50% of their maximal size) and T-90 (time taken for plants to reach 90% of their maximal size), amongst others.
Stress Resistance
An increase in yield and/or growth rate occurs whether the plant is under non-stress conditions or whether the plant is exposed to various stresses compared to control plants. Plants typically respond to exposure to stress by growing more slowly. In conditions of severe stress, the plant may even stop growing altogether. Mild stress on the other hand is defined herein as being any stress to which a plant is exposed which does not result in the plant ceasing to grow altogether without the capacity to resume growth. Mild stress in the sense of the invention leads to a reduction in the growth of the stressed plants of less than 40%, 35%, 30% or 25%, more preferably less than 20% or 15% in comparison to the control plant under non-stress conditions. Due to advances in agricultural practices (irrigation, fertilization, pesticide treatments) severe stresses are not often encountered in cultivated crop plants. As a consequence, the compromised growth induced by mild stress is often an undesirable feature for agriculture. Abiotic stresses may be due to drought or excess water, anaerobic stress, salt stress, chemical toxicity, oxidative stress and hot, cold or freezing temperatures.
“Biotic stresses” are typically those stresses caused by pathogens, such as bacteria, viruses, fungi, nematodes and insects.
The “abiotic stress” may be an osmotic stress caused by a water stress, e.g. due to drought, salt stress, or freezing stress. Abiotic stress may also be an oxidative stress or a cold stress. “Freezing stress” is intended to refer to stress due to freezing temperatures, i.e. temperatures at which available water molecules freeze and turn into ice. “Cold stress”, also called “chilling stress”, is intended to refer to cold temperatures, e.g. temperatures below 10°, or preferably below 5° C., but at which water molecules do not freeze. As reported in Wang et al. (Planta (2003) 218: 1-14), abiotic stress leads to a series of morphological, physiological, biochemical and molecular changes that adversely affect plant growth and productivity. Drought, salinity, extreme temperatures and oxidative stress are known to be interconnected and may induce growth and cellular damage through similar mechanisms. Rabbani et al. (Plant Physiol (2003) 133: 1755-1767) describes a particularly high degree of “cross talk” between drought stress and high-salinity stress. For example, drought and/or salinisation are manifested primarily as osmotic stress, resulting in the disruption of homeostasis and ion distribution in the cell. Oxidative stress, which frequently accompanies high or low temperature, salinity or drought stress, may cause denaturing of functional and structural proteins. As a consequence, these diverse environmental stresses often activate similar cell signalling pathways and cellular responses, such as the production of stress proteins, up-regulation of anti-oxidants, accumulation of compatible solutes and growth arrest. The term “non-stress” conditions as used herein are those environmental conditions that allow optimal growth of plants. Persons skilled in the art are aware of normal soil conditions and climatic conditions for a given location. Plants with optimal growth conditions, (grown under non-stress conditions) typically yield in increasing order of preference at least 97%, 95%, 92%, 90%, 87%, 85%, 83%, 80%, 77% or 75% of the average production of such plant in a given environment. Average production may be calculated on harvest and/or season basis. Persons skilled in the art are aware of average yield productions of a crop.
In particular, the methods of the present invention may be performed under non-stress conditions. In an example, the methods of the present invention may be performed under non-stress conditions such as mild drought to give plants having increased yield relative to control plants.
In another embodiment, the methods of the present invention may be performed under stress conditions.
In an example, the methods of the present invention may be performed under stress conditions such as drought to give plants having increased yield relative to control plants.
In another example, the methods of the present invention may be performed under stress conditions such as nutrient deficiency to give plants having increased yield relative to control plants.
Nutrient deficiency may result from a lack of nutrients such as nitrogen, phosphates and other phosphorous-containing compounds, potassium, calcium, magnesium, manganese, iron and boron, amongst others.
In yet another example, the methods of the present invention may be performed under stress conditions such as salt stress to give plants having increased yield relative to control plants. The term salt stress is not restricted to common salt (NaCl), but may be any one or more of: NaCl, KCl, LiCl, MgCl 2 , CaCl 2 , amongst others.
In yet another example, the methods of the present invention may be performed under stress conditions such as cold stress or freezing stress to give plants having increased yield relative to control plants.
Increase/Improve/Enhance
The terms “increase”, “improve” or “enhance” are interchangeable and shall mean in the sense of the application at least a 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10%, preferably at least 15% or 20%, more preferably 25%, 30%, 35% or 40% more yield and/or growth in comparison to control plants as defined herein.
Seed Yield
Increased seed yield may manifest itself as one or more of the following:
(a) an increase in seed biomass (total seed weight) which may be on an individual seed basis and/or per plant and/or per square meter; (b) increased number of flowers per plant; (c) increased number of seeds; (d) increased seed filling rate (which is expressed as the ratio between the number of filled florets divided by the total number of florets); (e) increased harvest index, which is expressed as a ratio of the yield of harvestable parts, such as seeds, divided by the biomass of aboveground plant parts; and (f) increased thousand kernel weight (TKW), which is extrapolated from the number of 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.

The terms “filled florets” and “filled seeds” may be considered synonyms.
An increase in seed yield may also be manifested as an increase in seed size and/or seed volume. Furthermore, an increase in seed yield may also manifest itself as an increase in seed area and/or seed length and/or seed width and/or seed perimeter.
Greenness Index
The “greenness index” as used herein is calculated from digital images of plants. For each pixel belonging to the plant object on the image, the ratio of the green value versus the red value (in the RGB model for encoding color) is calculated. The greenness index is expressed as the percentage of pixels for which the green-to-red ratio exceeds a given threshold. Under normal growth conditions, under salt stress growth conditions, and under reduced nutrient availability growth conditions, the greenness index of plants is measured in the last imaging before flowering. In contrast, under drought stress growth conditions, the greenness index of plants is measured in the first imaging after drought.
Biomass
The term “biomass” as used herein is intended to refer to the total weight of a plant. Within the definition of biomass, a distinction may be made between the biomass of one or more parts of a plant, which may include any one or more of the following:
aboveground parts such as but not limited to shoot biomass, seed biomass, leaf biomass, etc.; aboveground harvestable parts such as but not limited to shoot biomass, seed biomass, leaf biomass, etc.; parts below ground, such as but not limited to root biomass, tubers, bulbs, etc.; harvestable parts below ground, such as but not limited to root biomass, tubers, bulbs, etc.; harvestable parts partially below ground such as but not limited to beets and other hypocotyl areas of a plant, rhizomes, stolons or creeping rootstalks; vegetative biomass such as root biomass, shoot biomass, etc.; reproductive organs; and propagules such as seed.

Marker Assisted Breeding
Such breeding programmes sometimes require introduction of allelic variation by mutagenic treatment of the plants, using for example EMS mutagenesis; alternatively, the programme may start with a collection of allelic variants of so called “natural” origin caused unintentionally. Identification of allelic variants then takes place, for example, by PCR. This is followed by a step for selection of superior allelic variants of the sequence in question and which give increased yield. Selection is typically carried out by monitoring growth performance of plants containing different allelic variants of the sequence in question. Growth performance may be monitored in a greenhouse or in the field. Further optional steps include crossing plants in which the superior allelic variant was identified with another plant. This could be used, for example, to make a combination of interesting phenotypic features.
Use as Probes in (Gene Mapping)
Use of nucleic acids encoding the protein of interest for genetically and physically mapping the genes requires only a nucleic acid sequence of at least 15 nucleotides in length. These nucleic acids may be used as restriction fragment length polymorphism (RFLP) markers. Southern blots (Sambrook J, Fritsch E F and Maniatis T (1989) Molecular Cloning, A Laboratory Manual) of restriction-digested plant genomic DNA may be probed with the nucleic acids encoding the protein of interest. The resulting banding patterns may then be subjected to genetic analyses using computer programs such as MapMaker (Lander et al. (1987) Genomics 1: 174-181) in order to construct a genetic map. In addition, the nucleic acids may be used to probe Southern blots containing restriction endonuclease-treated genomic DNAs of a set of individuals representing parent and progeny of a defined genetic cross. Segregation of the DNA polymorphisms is noted and used to calculate the position of the nucleic acid encoding the protein of interest in the genetic map previously obtained using this population (Botstein et al. (1980) Am. J. Hum. Genet. 32:314-331).
The production and use of plant gene-derived probes for use in genetic mapping is described in Bernatzky and Tanksley (1986) Plant Mol. Biol. Reporter 4: 37-41. Numerous publications describe genetic mapping of specific cDNA clones using the methodology outlined above or variations thereof. For example, F2 intercross populations, backcross populations, randomly mated populations, near isogenic lines, and other sets of individuals may be used for mapping. Such methodologies are well known to those skilled in the art.
The nucleic acid probes may also be used for physical mapping (i.e., placement of sequences on physical maps; see Hoheisel et al. In: Non-mammalian Genomic Analysis: A Practical Guide, Academic press 1996, pp. 319-346, and references cited therein).
In another embodiment, the nucleic acid probes may be used in direct fluorescence in situ hybridisation (FISH) mapping (Trask (1991) Trends Genet. 7:149-154). Although current methods of FISH mapping favour use of large clones (several kb to several hundred kb; see Laan et al. (1995) Genome Res. 5:13-20), improvements in sensitivity may allow performance of FISH mapping using shorter probes.
A variety of nucleic acid amplification-based methods for genetic and physical mapping may be carried out using the nucleic acids. Examples include allele-specific amplification (Kazazian (1989) J. Lab. Clin. Med 11:95-96), polymorphism of PCR-amplified fragments (CAPS; Sheffield et al. (1993) Genomics 16:325-332), allele-specific ligation (Landegren et al. (1988) Science 241:1077-1080), nucleotide extension reactions (Sokolov (1990) Nucleic Acid Res. 18:3671), Radiation Hybrid Mapping (Walter et al. (1997) Nat. Genet. 7:22-28) and Happy Mapping (Dear and Cook (1989) Nucleic Acid Res. 17:6795-6807). For these methods, the sequence of a nucleic acid is used to design and produce primer pairs for use in the amplification reaction or in primer extension reactions. The design of such primers is well known to those skilled in the art. In methods employing PCR-based genetic mapping, it may be necessary to identify DNA sequence differences between the parents of the mapping cross in the region corresponding to the instant nucleic acid sequence. This, however, is generally not necessary for mapping methods.
Plant
The term “plant” as used herein encompasses whole plants, ancestors and progeny of the plants and plant parts, including seeds, shoots, stems, leaves, roots (including tubers), flowers, and tissues and organs, wherein each of the aforementioned comprise the gene/nucleic acid of interest. The term “plant” also encompasses plant cells, suspension cultures, callus tissue, embryos, meristematic regions, gametophytes, sporophytes, pollen and microspores, again wherein each of the aforementioned comprises the gene/nucleic acid of interest.
Plants that are particularly useful in the methods of the invention include all plants which belong to the superfamily Viridiplantae, in particular monocotyledonous and dicotyledonous plants including fodder or forage legumes, ornamental plants, food crops, trees or shrubs selected from the list comprising Acer spp., Actinidia spp., Abelmoschus spp., Agave sisalana, Agropyron spp., Agrostis stolonifera, Allium spp., Amaranthus spp., Ammophila arenaria, Ananas comosus, Annona spp., Apium graveolens, Arachis spp, Artocarpus spp., Asparagus officinalis, Avena spp. (e.g. Avena sativa, Avena fatua, Avena byzantina, Avena fatua var. sativa, Avena hybrida ), Averrhoa carambola, Bambusa sp., Benincasa hispida, Bertholletia excelsea, Beta vulgaris, Brassica spp. (e.g. Brassica napus, Brassica rapa ssp. [canola, oilseed rape, turnip rape]), Cadaba farinosa, Camellia sinensis, Canna indica, Cannabis sativa, Capsicum spp., Carex elata, Carica papaya, Carissa macrocarpa, Carya spp., Carthamus tinctorius, Castanea spp., Ceiba pentandra, Cichorium endivia, Cinnamomum spp., Citrullus lanatus, Citrus spp., Cocos spp., Coffea spp., Colocasia esculenta, Cola spp., Corchorus sp., Coriandrum sativum, Corylus spp., Crataegus spp., Crocus sativus, Cucurbita spp., Cucumis spp., Cynara spp., Daucus carota, Desmodium spp., Dimocarpus longan, Dioscorea spp., Diospyros spp., Echinochloa spp., Elaeis (e.g. Elaeis guineensis, Elaeis oleifera ), Eleusine coracana, Eragrostis tef, Erianthus sp., Eriobotrya japonica, Eucalyptus sp., Eugenia uniflora, Fagopyrum spp., Fagus spp., Festuca arundinacea, Ficus carica, Fortunella spp., Fragaria spp., Ginkgo biloba, Glycine spp. (e.g. Glycine max, Soja hispida or Soja max ), Gossypium hirsutum, Helianthus spp. (e.g. Helianthus annuus ), Hemerocallis fulva, Hibiscus spp., Hordeum spp. (e.g. Hordeum vulgare ), Ipomoea batatas, Juglans spp., Lactuca sativa, Lathyrus spp., Lens culinaris, Linum usitatissimum, Litchi chinensis, Lotus spp., Luffa acutangula, Lupinus spp., Luzula sylvatica, Lycopersicon spp. (e.g. Lycopersicon esculentum, Lycopersicon lycopersicum, Lycopersicon pyriforme ), Macrotyloma spp., Malus spp., Malpighia emarginata, Mammea americana, Mangifera indica, Manihot spp., Manilkara zapota, Medicago sativa, Melilotus spp., Mentha spp., Miscanthus sinensis, Momordica spp., Morus nigra, Musa spp., Nicotiana spp., Olea spp., Opuntia spp., Ornithopus spp., Oryza spp. (e.g. Oryza sativa, Oryza latifolia ), Panicum miliaceum, Panicum virgatum, Passiflora edulis, Pastinaca sativa, Pennisetum sp., Persea spp., Petroselinum crispum, Phalaris arundinacea, Phaseolus spp., Phleum pratense, Phoenix spp., Phragmites australis, Physalis spp., Pinus spp., Pistacia vera, Pisum spp., Poa spp., Populus spp., Prosopis spp., Prunus spp., Psidium spp., Punica granatum, Pyrus communis, Quercus spp., Raphanus sativus, Rheum rhabarbarum, Ribes spp., Ricinus communis, Rubus spp., Saccharum spp., Salix sp., Sambucus spp., Secale cereale, Sesamum spp., Sinapis sp., Solanum spp. (e.g. Solanum tuberosum, Solanum integrifolium or Solanum lycopersicum ), Sorghum bicolor, Spinacia spp., Syzygium spp., Tagetes spp., Tamarindus indica, Theobroma cacao, Trifolium spp., Tripsacum dactyloides, Triticosecale rimpaui, Triticum spp. (e.g. Triticum aestivum, Triticum durum, Triticum turgidum, Triticum hybernum, Triticum macha, Triticum sativum, Triticum monococcum or Triticum vulgare ), Tropaeolum minus, Tropaeolum majus, Vaccinium spp., Vicia spp., Vigna spp., Viola odorata, Vitis spp., Zea mays, Zizania palustris, Ziziphus spp., amongst others.
Control Plant(s)
The choice of suitable control plants is a routine part of an experimental setup and may include corresponding wild type plants or corresponding plants without the gene of interest. The control plant is typically of the same plant species or even of the same variety as the plant to be assessed. The control plant may also be a nullizygote of the plant to be assessed. Nullizygotes (or null control plants) are individuals missing the transgene by segregation. Further, control plants are grown under equal growing conditions to the growing conditions of the plants of the invention, i.e. in the vicinity of, and simultaneously with, the plants of the invention. A “control plant” as used herein refers not only to whole plants, but also to plant parts, including seeds and seed parts.
EXAMPLES
The present invention will now be described with reference to the following examples, which are by way of illustration only. The following examples are not intended to limit the scope of the invention. Unless otherwise indicated, the present invention employs conventional techniques and methods of plant biology, molecular biology, bioinformatics and plant breedings.
DNA manipulation: unless otherwise stated, recombinant DNA techniques are performed according to standard protocols described in (Sambrook (2001) Molecular Cloning: a laboratory manual, 3rd Edition Cold Spring Harbor Laboratory Press, CSH, New York) or in Volumes 1 and 2 of Ausubel et al. (1994), Current Protocols in Molecular Biology, Current Protocols. Standard materials and methods for plant molecular work are described in Plant Molecular Biology Labfax (1993) by R. D. D. Croy, published by BIOS Scientific Publications Ltd (UK) and Blackwell Scientific Publications (UK).
Example 1
GRP Sequences and Homologues Thereof
Table A provides a list of nucleic acid sequences and protein sequences used for generating transgenic plants described in Example 7.














Nucleic acid
Protein

Plant Source
ZM array number
Description
SEQ ID NO:
SEQ ID NO:












Oryza sativa

ZM4s07788
Orysa_auxin-repressed protein
1
2


Oryza sativa

ZM1s68450033
Orysa_CBS domain containing
3
4



membrane protein




Oryza sativa

ZM1s60334585
Orysa_dehydration-induced
5
6



protein ERD15




Oryza sativa

ZM1s57410125
Orysa_LOC_Os01g06640
7
8


Oryza sativa

ZM1s58705134
Orysa_LOC_Os01g63690
9
10


Oryza sativa

ZM1s60103573
Orysa_LOC_Os01g68650
11
12


Oryza sativa

ZM1s57443595
Orysa_LOC_Os01g70860.1
13
14


Oryza sativa

ZM1s60213342
Orysa_LOC_Os02g04120
15
16


Oryza sativa

ZM4s05301
Orysa_LOC_Os02g04230
17
18


Oryza sativa

ZM1s60940818
Orysa_LOC_Os02g06720 for
19
20



downregulation




Oryza sativa

ZM1s60940818
Orysa_LOC_Os02g06725
21
22


Oryza sativa

ZM1s61299604
Orysa_LOC_Os02g07240
23
24


Oryza sativa

ZM4s31512
Orysa_LOC_Os02g15800
25
26


Oryza sativa

ZM1s65223113
Orysa_LOC_Os02g36490
27
28


Oryza sativa

ZM1s62030237
Orysa_LOC_Os02g45170
29
30


Oryza sativa

ZM1s57223022
Orysa_LOC_Os02g48990
31
32


Oryza sativa

ZM1s60187950
Orysa_LOC_Os02g52560
33
34


Oryza sativa

ZM1s62114467
Orysa_LOC_Os02g58730
35
36


Oryza sativa

ZM1s62145107
Orysa_LOC_Os03g09220
37
38


Oryza sativa

ZM1s59517939
Orysa_LOC_Os03g10210
39
40


Oryza sativa

ZM1s59034758
Orysa_LOC_Os03g11770
41
42


Oryza sativa

ZM1s62059350
Orysa_LOC_Os03g17790
43
44


Oryza sativa

ZM1s62187860
Orysa_LOC_Os03g17800
45
46


Oryza sativa

ZM4s07964
Orysa_LOC_Os03g46490
47
48


Oryza sativa

ZM1s62219636
Orysa_LOC_Os03g55670
49
50


Oryza sativa

ZM1s66928877.1
Orysa_LOC_Os03g64219
51
52


Oryza sativa

ZM4s14939
Orysa_LOC_Os04g35790
53
54


Oryza sativa

ZM4s22201
Orysa_LOC_Os05g07070
55
56


Oryza sativa

ZM1s62216784
Orysa_LOC_Os05g40770
57
58


Oryza sativa

ZM1a61978583
Orysa_LOC_Os05g50510
59
60


Oryza sativa

ZM1s62075387
Orysa_LOC_Os06g01440
61
62


Oryza sativa

ZM4s29073
Orysa_LOC_Os06g05120
63
64


Oryza sativa

ZM1s57336820
Orysa_LOC_Os06g06250.2
65
66


Oryza sativa

ZM1s68504043
Orysa_LOC_Os06g11840.1
67
68


Oryza sativa

ZM3s01411
Orysa_LOC_Os07g35004
69
70


Oryza sativa

ZM1s67010779
Orysa_LOC_Os08g01390 for
71
72



downregulation




Oryza sativa

ZM1s57853588
Orysa_LOC_Os08g01760
73
74


Oryza sativa

ZM1s67094909
Orysa_LOC_Os08g26870
75
76


Oryza sativa

ZM1s60496018
Orysa_LOC_Os08g31250
77
78


Oryza sativa

ZM1s57505415
Orysa_LOC_Os08g37070
79
80


Oryza sativa

ZM4s14446
Orysa_LOC_Os08g40030
81
82


Oryza sativa

ZM4s23596
Orysa_LOC_Os08g42400
83
84


Oryza sativa

ZM1s58474578
Orysa_LOC_Os08g44270
85
86


Oryza sativa

ZM3s01404
Orysa_LOC_Os09g11440
87
88


Oryza sativa

ZM1s58561763
Orysa_LOC_Os09g20284
89
90


Oryza sativa

ZM1s62127538
Orysa_LOC_Os10g39130
91
92


Oryza sativa

ZM1a61994414
Orysa_LOC_Os10g40710
93
94


Oryza sativa

ZM1s57236502
Orysa_LOC_Os11g27400
95
96


Oryza sativa

ZM4s20980
Orysa_LOC_Os12g32970
97
98


Oryza sativa

ZM4s14399
Orysa_LOC_Os12g32992
99
100


Oryza sativa

ZM4s27634
Orysa_Os04g42470
101
102


Oryza sativa

ZM1a62119712
Orysa_Os04g54300
103
104


Oryza sativa

ZM1s62025034
Orysa_putative serine/threonine
105
106



protein kinase




Oryza sativa

ZM1s62132790
Orysa_WRKY7 for downregulation
107
108


Zea mays

ZM1s59458241
Zeama_AC214266.3_FGT002
109
110


Zea mays

ZM1s62033152
Zeama_AC214350.3_FGT007
111
112


Zea mays

ZM1s62096975
Zeama_ATACP5; protein serine/
113
114



threonine phosphatase




Zea mays

ZM1s60481108
Zeama_calmodulin binding
115
116


Zea mays

ZM4s37206
Zeama_GRMZM2G000645
117
118


Zea mays

ZM1s57508357
Zeama_GRMZM2G000825
119
120


Zea mays

ZM4s16403
Zeama_GRMZM2G001221
121
122


Zea mays

ZM4s22803
Zeama_GRMZM2G004459
123
124


Zea mays

ZM1s57740507
Zeama_GRMZM2G005040_T01
125
126


Zea mays

ZM4s13892
Zeama_GRMZM2G005260
127
128


Zea mays

ZM1s65250927
Zeama_GRMZM2G005435
129
130


Zea mays

ZM4S19342
Zeama_GRMZM2G007466
131
132


Zea mays

ZM1S62132058
Zeama_GRMZM2G007729
133
134


Zea mays

ZM1s57554822
Zeama_GRMZM2G007899
135
136


Zea mays

ZM1s57259830
Zeama_GRMZM2G008657
137
138


Zea mays

ZM1S61984928
Zeama_GRMZM2G009412
139
140


Zea mays

ZM4s11447
Zeama_GRMZM2G009837_T01
141
142


Zea mays

ZM1s65113928
Zeama_GRMZM2G010356_T01
143
144


Zea mays

ZM1S62273254
Zeama_GRMZM2G011110
145
146


Zea mays

ZM1s57807604
Zeama_GRMZM2G011357_T01
147
148


Zea mays

ZM1s62063823
Zeama_GRMZM2G012224_T01
149
150


Zea mays

ZM1S58972630
Zeama_GRMZM2G013627
151
152


Zea mays

ZM4S02093
Zeama_GRMZM2G013657
153
154


Zea mays

ZM1s58375824
Zeama_GRMZM2G014750
155
156


Zea mays

ZM1S57638002
Zeama_GRMZM2G015433
157
158


Zea mays

ZM1S60466311
Zeama_GRMZM2G018689
159
160


Zea mays

ZM1s57738137
Zeama_GRMZM2G018771
161
162


Zea mays

ZM1s57429621
Zeama_GRMZM2G020150
163
164


Zea mays

ZM4s41243
Zeama_GRMZM2G020281_T03
165
166


Zea mays

ZM1s61998920
Zeama_GRMZM2G021270
167
168


Zea mays

ZM1s62262119
Zeama_GRMZM2G023242
169
170


Zea mays

ZM1S57850754
Zeama_GRMZM2G025812
171
172


Zea mays

ZM1s57263625
Zeama_GRMZM2G027052
173
174


Zea mays

ZM4s45997
Zeama_GRMZM2G028218
175
176


Zea mays

ZM1s60841542
Zeama_GRMZM2G029692
177
178


Zea mays

ZM1s62202989
Zeama_GRMZM2G030284
179
180


Zea mays

ZM1s59258289
Zeama_GRMZM2G031724
181
182


Zea mays

ZM1s55817549
Zeama_GRMZM2G031983_T01
183
184


Zea mays

ZM1s58561763
Zeama_GRMZM2G034152_T01
185
186


Zea mays

ZM3s21693
Zeama_GRMZM2G034157
187
188


Zea mays

ZM1s68299591
Zeama_GRMZM2G036120
189
190


Zea mays

ZM4S19630
Zeama_GRMZM2G036134
191
192


Zea mays

ZM1s60105986
Zeama_GRMZM2G036351
193
194


Zea mays

ZM1s57431009
Zeama_GRMZM2G037001_T01
195
196


Zea mays

ZM4s19587
Zeama_GRMZM2G038066
197
198


Zea mays

ZM1s61992039
Zeama_GRMZM2G040517_T01
199
200


Zea mays

ZM1s60429690
Zeama_GRMZM2G041774
201
202


Zea mays

ZM1s60312671
Zeama_GRMZM2G042895
203
204


Zea mays

ZM1S68765176
Zeama_GRMZM2G043147
205
206


Zea mays

ZM4s39700
Zeama_GRMZM2G043275_T01
207
208


Zea mays

ZM4s13777
Zeama_GRMZM2G044027_T01
209
210


Zea mays

ZM1s58678671
Zeama_GRMZM2G044576_T01
211
212


Zea mays

ZM1s62146990
Zeama_GRMZM2G044851_T01
213
214


Zea mays

ZM1a68352777
Zeama_GRMZM2G047684
215
216


Zea mays

ZM1S55843902
Zeama_GRMZM2G049091
217
218


Zea mays

ZM4s45895
Zeama_GRMZM2G049398_T01
219
220


Zea mays

ZM4s46105
Zeama_GRMZM2G052078
221
222


Zea mays

ZM1s58862414
Zeama_GRMZM2G052509
223
224


Zea mays

ZM1s62012391
Zeama_GRMZM2G053766
225
226


Zea mays

ZM4s14974
Zeama_GRMZM2G055458
227
228


Zea mays

ZM1S60958648
Zeama_GRMZM2G055585
229
230


Zea mays

ZM1s67282786
Zeama_GRMZM2G056038
231
232


Zea mays

ZM1s58335548
Zeama_GRMZM2G056407_T01
233
234


Zea mays

ZM3S00100
Zeama_GRMZM2G056600
235
236


Zea mays

ZM4s03858
Zeama_GRMZM2G057283_T01
237
238


Zea mays

ZM1s62139486
Zeama_GRMZM2G057789
239
240


Zea mays

ZM1s58751265
Zeama_GRMZM2G058690_T02
241
242


Zea mays

ZM4s42869
Zeama_GRMZM2G059580
243
244


Zea mays

ZM1S58075442
Zeama_GRMZM2G059799
245
246


Zea mays

ZM1s62207107
Zeama_GRMZM2G060163
247
248


Zea mays

ZM1s59183793
Zeama_GRMZM2G061745
249
250


Zea mays

ZM1s62167066
Zeama_GRMZM2G061996_T01
251
252


Zea mays

ZM1s57461756
Zeama_GRMZM2G062160
253
254


Zea mays

ZM4s10805
Zeama_GRMZM2G062531
255
256


Zea mays

ZM1s59012895
Zeama_GRMZM2G064096_T01
257
258


Zea mays

ZM4s15692
Zeama_GRMZM2G064960
259
260


Zea mays

ZM4a18634
Zeama_GRMZM2G066528
261
262


Zea mays

ZM4s03279
Zeama_GRMZM2G073755
263
264


Zea mays

ZM4s17107
Zeama_GRMZM2G075942
265
266


Zea mays

ZM1s62055151
Zeama_GRMZM2G077206
267
268


Zea mays

ZM4s24055
Zeama_GRMZM2G077458
269
270


Zea mays

ZM4s13911
Zeama_GRMZM2G078198
271
272


Zea mays

ZM1s57963623
Zeama_GRMZM2G079805
273
274


Zea mays

ZM1s60747775
Zeama_GRMZM2G080295_T01
275
276


Zea mays

ZM1s62026868
Zeama_GRMZM2G082032
277
278


Zea mays

ZM1s62161812
Zeama_GRMZM2G082853
279
280


Zea mays

ZM1s65109710
Zeama_GRMZM2G083068
281
282


Zea mays

ZM4s33018
Zeama_GRMZM2G083935
283
284


Zea mays

ZM1S58460807
Zeama_GRMZM2G084264
285
286


Zea mays

ZM1S60657408
Zeama_GRMZM2G084587
287
288


Zea mays

ZM1s62162828
Zeama_GRMZM2G087103
289
290


Zea mays

ZM1S57495842
Zeama_GRMZM2G087323
291
292


Zea mays

ZM1s58524553
Zeama_GRMZM2G088162
293
294


Zea mays

ZM1S57862466
Zeama_GRMZM2G089259
295
296


Zea mays

ZM1s66890980.1
Zeama_GRMZM2G090487_T01
297
298


Zea mays

ZM1s59343090
Zeama_GRMZM2G090595_T04
299
300


Zea mays

ZM1S59395629
Zeama_GRMZM2G091228
301
302


Zea mays

ZM4s22079
Zeama_GRMZM2G091540
303
304


Zea mays

ZM1s57288195
Zeama_GRMZM2G092137
305
306


Zea mays

ZM1s55864617
Zeama_GRMZM2G093217
307
308


Zea mays

ZM4s17135
Zeama_GRMZM2G093900
309
310


Zea mays

ZM1S57836157
Zeama_GRMZM2G095392
311
312


Zea mays

ZM1s58775973
Zeama_GRMZM2G098046
313
314


Zea mays

ZM1s68295211
Zeama_GRMZM2G100120
315
316


Zea mays

ZM1s61258515
Zeama_GRMZM2G100260_T01
317
318


Zea mays

ZM3s00430
Zeama_GRMZM2G100467_T01
319
320


Zea mays

ZM4s18637
Zeama_GRMZM2G101062
321
322


Zea mays

ZM1s62058690
Zeama_GRMZM2G101125
323
324

Zea mays
ZM1s62258567
Zeama_GRMZM2G101390
325
326


Zea mays

ZM1S65226266
Zeama_GRMZM2G101545
327
328


Zea mays

ZM1s61974300
Zeama_GRMZM2G102347
329
330


Zea mays

ZM3S29285
Zeama_GRMZM2G102356
331
332


Zea mays

ZM4s05881
Zeama_GRMZM2G102786_T01
333
334


Zea mays

ZM4s37616
Zeama_GRMZM2G104610_T01
335
336


Zea mays

ZM1A57699438
Zeama_GRMZM2G105331
337
338


Zea mays

ZM4s21938
Zeama_GRMZM2G105348
339
340


Zea mays

ZM1s58374993
Zeama_GRMZM2G107737_T01
341
342


Zea mays

ZM3s01330
Zeama_GRMZM2G107771_T01
343
344


Zea mays

ZM1S62039207
Zeama_GRMZM2G107839
345
346


Zea mays

ZM4S17089
Zeama_GRMZM2G108115
347
348


Zea mays

ZM4s14568
Zeama_GRMZM2G108716
349
350


Zea mays

ZM4s26880
Zeama_GRMZM2G109130
351
352


Zea mays

ZM1S67201464
Zeama_GRMZM2G110289
353
354


Zea mays

ZM4s18047
Zeama_GRMZM2G111451_T01
355
356


Zea mays

ZM4a14205
Zeama_GRMZM2G112210
357
358


Zea mays

ZM1S58101138
Zeama_GRMZM2G112903
359
360


Zea mays

ZM1s59421232
Zeama_GRMZM2G114048
361
362


Zea mays

ZM1s57921389
Zeama_GRMZM2G114552
363
364


Zea mays

ZM1s62190425
Zeama_GRMZM2G114873
365
366


Zea mays

ZM1s57679770
Zeama_GRMZM2G115975
367
368


Zea mays

ZM1s62204322
Zeama_GRMZM2G116526
369
370


Zea mays

ZM1s57370356
Zeama_GRMZM2G116632_T01
371
372


Zea mays

ZM1S65126433
Zeama_GRMZM2G117989
373
374


Zea mays

ZM4S19741
Zeama_GRMZM2G119906
375
376


Zea mays

ZM1s62126803
Zeama_GRMZM2G121074
377
378


Zea mays

ZM4s11447
Zeama_GRMZM2G122228_T01
379
380


Zea mays

ZM1s61995217
Zeama_GRMZM2G123212
381
382


Zea mays

ZM1s60485681
Zeama_GRMZM2G124863
383
384


Zea mays

ZM4s22440
Zeama_GRMZM2G125923
385
386


Zea mays

ZM4S45457
Zeama_GRMZM2G126397
387
388


Zea mays

ZM1S65239103
Zeama_GRMZM2G126566
389
390


Zea mays

ZM1s65368508
Zeama_GRMZM2G127609
391
392


Zea mays

ZM4s27549
Zeama_GRMZM2G127695
393
394


Zea mays

ZM1s68704853
Zeama_GRMZM2G127850_T01
395
396


Zea mays

ZM4s06440
Zeama_GRMZM2G127893
397
398


Zea mays

ZM1s60510343
Zeama_GRMZM2G132854
399
400


Zea mays

ZM1s57861573
Zeama_GRMZM2G137596_T01
401
402


Zea mays

ZM1S57327061
Zeama_GRMZM2G138131
403
404


Zea mays

ZM1s65129831
Zeama_GRMZM2G139082_T01
405
406


Zea mays

ZM1s58505914
Zeama_GRMZM2G142735
407
408


Zea mays

ZM4s07707
Zeama_GRMZM2G143253_T01
409
410


Zea mays

ZM1s59390494
Zeama_GRMZM2G147623
411
412


Zea mays

ZM1s62038184
Zeama_GRMZM2G152354
413
414


Zea mays

ZM4s04639
Zeama_GRMZM2G152561_T02
415
416


Zea mays

ZM4s17046
Zeama_GRMZM2G152908
417
418


Zea mays

ZM4s29494
Zeama_GRMZM2G154648
419
420


Zea mays

ZM1s57348884
Zeama_GRMZM2G156110_T01
421
422


Zea mays

ZM4s10874
Zeama_GRMZM2G158232
423
424


Zea mays

ZM1s57457322
Zeama_GRMZM2G158313_T01
425
426


Zea mays

ZM1s62093950
Zeama_GRMZM2G159034
427
428


Zea mays

ZM4s19175
Zeama_GRMZM2G161295
429
430


Zea mays

ZM1s57320274
Zeama_GRMZM2G163406
431
432


Zea mays

ZM1s62027902
Zeama_GRMZM2G163409
433
434


Zea mays

ZM1S58592636
Zeama_GRMZM2G163641
435
436


Zea mays

ZM4S06145
Zeama_GRMZM2G164781
437
438


Zea mays

ZM1s57368153
Zeama_GRMZM2G164868
439
440


Zea mays

ZM4s11036
Zeama_GRMZM2G165461
441
442


Zea mays

ZM1s62097506
Zeama_GRMZM2G166946
443
444


Zea mays

ZM1s57441890
Zeama_GRMZM2G168365
445
446


Zea mays

ZM1s58699795
Zeama_GRMZM2G168603_T01
447
448


Zea mays

ZM1s61048230
Zeama_GRMZM2G170400
449
450


Zea mays

ZM4s16359
Zeama_GRMZM2G172657
451
452


Zea mays

ZM1a62154780
Zeama_GRMZM2G174558
453
454


Zea mays

ZM1s62106945
Zeama_GRMZM2G174975
455
456


Zea mays

ZM1s57848822
Zeama_GRMZM2G175280_T02
457
458


Zea mays

ZM1s57305612
Zeama_GRMZM2G176217
459
460


Zea mays

ZM1S68614544
Zeama_GRMZM2G177867
461
462


Zea mays

ZM1s67253381
Zeama_GRMZM2G178815_T01
463
464


Zea mays

ZM4s30153
Zeama_GRMZM2G179679
465
466


Zea mays

ZM1S62018816
Zeama_GRMZM2G180172
467
468


Zea mays

ZM1s66894694
Zeama_GRMZM2G302245_T01
469
470


Zea mays

ZM1a61141890
Zeama_GRMZM2G304362
471
472


Zea mays

ZM4s06217
Zeama_GRMZM2G307119_T01
473
474


Zea mays

ZM1s62016000
Zeama_GRMZM2G310368
475
476


Zea mays

ZM1s60135266
Zeama_GRMZM2G312510
477
478


Zea mays

ZM1S60857688.1
Zeama_GRMZM2G320920
479
480


Zea mays

ZM1s58409566
Zeama_GRMZM2G321023
481
482


Zea mays

ZM1s62083330
Zeama_GRMZM2G327032
483
484


Zea mays

ZM1s61980732
Zeama_GRMZM2G334181_T01
485
486


Zea mays

ZM1s62099803
Zeama_GRMZM2G340130
487
488


Zea mays

ZM1s68469961
Zeama_GRMZM2G341404
489
490


Zea mays

ZM1A57511883
Zeama_GRMZM2G345840
491
492


Zea mays

ZM1s62064657
Zeama_GRMZM2G347043
493
494


Zea mays

ZM1s57766027
Zeama_GRMZM2G347808
495
496


Zea mays

ZM1s57319204
Zeama_GRMZM2G355752
497
498


Zea mays

ZM4s16535
Zeama_GRMZM2G359952
499
500


Zea mays

ZM1s57952044
Zeama_GRMZM2G368698_T02
501
502


Zea mays

ZM4S03593
Zeama_GRMZM2G394410
503
504


Zea mays

ZM1S57374543.1
Zeama_GRMZM2G401521
505
506


Zea mays

ZM1s61979139
Zeama_GRMZM2G402417
507
508


Zea mays

ZM1s62009031
Zeama_GRMZM2G407223
509
510


Zea mays

ZM1S62216882
Zeama_GRMZM2G408598
511
512


Zea mays

ZM1a62058338
Zeama_GRMZM2G411032
513
514


Zea mays

ZM1A57765201
Zeama_GRMZM2G412601
515
516


Zea mays

ZM1s60200790
Zeama_GRMZM2G413337
517
518


Zea mays

ZM1S66879359
Zeama_GRMZM2G416388
519
520


Zea mays

ZM1S58357601
Zeama_GRMZM2G421212
521
522


Zea mays

ZM1s67275443
Zeama_GRMZM2G422240
523
524


Zea mays

ZM1S57790868
Zeama_GRMZM2G438195
525
526


Zea mays

ZM1s62144084
Zeama_GRMZM2G445634
527
528


Zea mays

ZM1s62046845
Zeama_GRMZM2G447984
529
530


Zea mays

ZM1s55843675
Zeama_GRMZM2G450153
531
532


Zea mays

ZM1s62083561
Zeama_GRMZM2G451254
533
534


Zea mays

ZM4s06441
Zeama_GRMZM2G458164
535
536


Zea mays

ZM1s58973247
Zeama_GRMZM2G468756_T01
537
538


Zea mays

ZM1s62187177
Zeama_GRMZM2G476069
539
540


Zea mays

ZM1s57375517
Zeama_GRMZM2G477503
541
542


Zea mays

ZM4s03412
Zeama_GRMZM2G479260
543
544


Zea mays

ZM4s11878
Zeama_GRMZM2G481888_T01
545
546


Zea mays

ZM4s26962
Zeama_GRMZM2G701362
547
548


Zea mays

ZM1s60573546
zeama_heat shock 70 protein
549
550


Zea mays

ZM3s00628
Zeama_Methyl transferase like
551
552


Zea mays

ZM1s61220142
Zeama_nucleic acid binding/
553
554



zinc ion binding




Zea mays

ZM4s15474
Zeama_Pleitropic Drug Resistance
555
556



12 like




Zea mays

ZM1s65094362
Zeama_putative nuclear RNA
557
558



binding protein




Zea mays

ZM4a37469
Zeama_putative zinc-finger protein
559
560


Zea mays

ZM4s40513
Zeama_SYTE like
561
562


Zea mays

ZM1a58376939
Zeama_transferase protein like
563
564


Zea mays

ZM1s61987040
Zeama_unknown protein a
565
566


Zea mays

ZM1s62016945
Zeama_unknown protein b
567
568


Zea mays

ZM1s62111342
Zeama_unknown protein c
569
570


Zea mays

ZM1s61053034
Zeama_Unknown protein d
571
572


Zea mays

ZM1s58196890
Zeama_Unknown protein e
573
574


Zea mays

ZM1s57483885
Zeama_unknown protein f
575
576


Zea mays

ZM4s16739
Zeama_unknown protein g
577
578


Zea mays

ZM1a58376939
Zeama_unknown protein h
579
580


Zea mays

ZM1s55866371
Zeama_ZM1s55866371
581
582


Zea mays

ZM1s55873260
Zeama_ZM1s55873260
583
584


Zea mays

ZM1s58530935
Zeama_ZM1s58530935
585
586


Zea mays

ZM1s59031313
Zeama_ZM1s59031313
587
588


Zea mays

ZM1s61996727
Zeama_ZM1s61996727
589
590


Zea mays

ZM1s65116286
Zeama_ZM1s65116286
591
592


Zea mays

ZM1s62111307
Zm AAA-type ATPase family
593
594



protein-like




Zea mays

ZM1s57394235
Zm ATP-binding protein
595
596


Zea mays

ZM1s58425424
Zm AUXIN RESPONSE
597
598



FACTOR 16




Zea mays

ZM1s62214832
Zm Cyclin A2; 4-like
599
600


Zea mays

ZM1s68382282
Zm embryo-abundant protein-related
601
602


Zea mays

ZM1s57390634
Zm histidine kinase 1
603
604


Zea mays

ZM4s13278
Zm indole-3-acetic acid amido
605
606



synthetase-like 1




Zea mays

ZM1s61040508
Zm indole-3-acetic acid amido
607
608



synthetase-like 2




Zea mays

ZM1s62107983
Zm indoleacetic acid-induced
609
610



protein 13-like 1




Zea mays

ZM1s59209789
Zm indoleacetic acid-induced
611
612



protein 13-like 2




Zea mays

ZM1s58194594
Zm indoleacetic acid-induced
613
614



protein 18




Zea mays

ZM1s57509684
Zm indoleacetic acid-induced
615
616



protein 20-like




Zea mays

ZM3s01845
Zm MYB78 like
617
618


Zea mays

ZM1s62004697
Zm PHYTOCHROME-
619
620



ASSOCIATED PROTEIN 1-like




Zea mays

ZM4s18277
Zm Polygalacturonase inhibiting
621
622



protein 1-like




Zea mays

ZM1s57260204
Zm Proliferating Cellular Nuclear
623
624



Antigen 1




Zea mays

ZM1s60983186
Zm protein kinase-related
625
626


Zea mays

ZM1s60157931
Zm U-box domain-containing protein
627
628


Zea mays

ZM4s09532
Zm unknown protein 1
629
630


Zea mays

ZM1s62122111
Zm unknown protein 2
631
632


Zea mays

ZM1s62075438
Zm unknown protein 3
633
634


Zea mays

ZM1s57691797
Zm unknown protein 4
635
636


Zea mays

ZM1s62132790
Zm WRKY (WRKY DNA-binding
637
638



protein 23-like)




Zea mays

ZM1s60455877
Zm WRKY 40-like
639
640









































































































































































































































































































































































Sequences (full length cDNA, ESTs or genomic) related to those listed in Table A above can be 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, polypeptides encoded by the nucleic acids listed in Table A can be used with the TBLASTN algorithm, with default settings and the filter to ignore low complexity sequences set off. The output of the analysis can be 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 may also be 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.
Sequences have been tentatively assembled and publicly disclosed by research institutions, such as The Institute for Genomic Research (TIGR; beginning with TA). For instance, the Eukaryotic Gene Orthologs (EGO) database may be used to identify such related sequences, either by keyword search or by using the BLAST algorithm with the nucleic acid sequence or polypeptide sequence of interest. Special nucleic acid sequence databases have been created for particular organisms, e.g. for certain prokaryotic organisms, such as by the Joint Genome Institute. Furthermore, access to proprietary databases, has allowed the identification of novel nucleic acid and polypeptide sequences.
Example 2
Alignment of GRP Polypeptide Sequences
Alignment of the polypeptide sequences representing a GRP protein of Table A and its homologues may be performed using the ClustalW 2.0 algorithm of progressive alignment (Thompson et al. (1997) Nucleic Acids Res 25:4876-4882; Chenna et al. (2003). Nucleic Acids Res 31:3497-3500) with standard setting (slow alignment, similarity matrix: Gonnet, gap opening penalty 10, gap extension penalty: 0.2). Minor manual editing may be done to further optimise the alignment. Generation of multiple alignments is well known in the art.
A phylogenetic tree of a GRP polypeptide of Table A and its homologues may be constructed by aligning GRP sequences using, for example, MAFFT (Katoh and Toh (2008)—Briefings in Bioinformatics 9:286-298) with default settings. A neighbour-joining tree is then calculated using, for example, Quick-Tree (Howe et al. (2002), Bioinformatics 18(11): 1546-7), with 100 bootstrap repetitions. The dendrogram is drawn using, for example, Dendroscope (Huson et al. (2007), BMC Bioinformatics 8(1):460). Confidence levels for 100 bootstrap repetitions are usually indicated for major branchings.
Example 3
Calculation of Global Percentage Identity Between Polypeptide Sequences
Global percentages of similarity and identity between full length polypeptide sequences useful in performing the methods of the invention are determined using 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 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, calculates similarity and identity, 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 protein analysis are typically: Scoring matrix: Blosum62, First Gap: 12, Extending Gap: 2.
Like for full length sequences, a MATGAT table based on subsequences of a specific domain, may be generated. Based on a multiple alignment of GRP polypeptides, a skilled person may select conserved sequences and submit as input for a MaTGAT analysis. This approach is useful where overall sequence conservation among GRP proteins is rather low.
Example 4
Identification of Domains Comprised in Polypeptide Sequences Useful in Performing the Methods of the Invention
The Integrated Resource of Protein Families, Domains and Sites (InterPro) database is an integrated interface for the commonly used signature databases for text- and sequence-based searches. The InterPro database combines these databases, which use different methodologies and varying degrees of biological information about well-characterized proteins to derive protein signatures. Collaborating databases include SWISS-PROT, PROSITE, TrEMBL, PRINTS, ProDom and Pfam, Smart and TIGRFAMs. 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.
Results of the InterPro scan (InterPro database, release 34.0) of a selection of GRP polypeptide sequence from Table A are listed hereunder.







Sequence “ZM1S55817549” crc64 checksum: 1DB9E7773AC36AAF length: 361 aa.









InterPro
IPR000679
Zinc finger, GATA-type





Molecular Function: transcription factor activity (GO:0003700), Cellular Component: nucleus (GO:0005634),

Biological Process: regulation of transcription, DNA-dependent (GO:0006355), Molecular Function: zinc ion

binding (GO:0008270), Molecular Function: sequence-specific DNA binding (GO:0043565)








method
AccNumber
shortName
location



HMMPfam
PF00320
GATA
T[191-226] 1.1e−18

HMMSmart
SM00401
no description
T[185-237] 3.3e−19

ProfileScan
PS50114
GATA_ZN_FINGER_2
T[189-221] 12.466

ScanRegExp
PS00344
GATA_ZN_FINGER_1
?[191-216] NA



InterPro
IPR013088
Zinc finger, NHR/GATA-type





Molecular Function: transcription factor activity (GO:0003700), Biological Process: regulation of

transcription, DNA-dependent (GO:0006355), Molecular Function: zinc ion binding (GO:0008270)








method
AccNumber
shortName
location



Gene3D
G3DSA:3.30.50.10
no description
T[185-229] 2.4e−05



InterPro
NULL
NULL



method
AccNumber
shortName
location



HMMPanther
PTHR10071
TRANSCRIPTION FACTOR GATA
T[11-18] 1.7e−05 T[188-221] 1.7e−5



(GATA BINDING FACTOR)


Seg
seg
seg
?[134-139] NA ?[219-236] NA ?[287-302]




NA ?[322-334] NA

superfamily
SSF57716
Glucocorticoid receptor-like
T[80-246] 3.7e−14



(DNA-binding domain)






































































Sequence “ZM1S60958648” crc64 checksum: 10C1558B1D15EA57 length: 426 aa.









InterPro
IPR013781
Glycoside hydrolase, subgroup, catalytic core





Molecular Function: catalytic activity (GO:0003824), Biological Process: carbohydrate metabolic process

(GO:0005975), Molecular Function: cation binding (G0:0043169)








method
AccNumber
shortName
location



Gene3D
G3DSA:3.20.20.80
no description
T[28-410] 2.9e−132



InterPro
IPR017853
Glycoside hydrolase, catalytic core



method
AccNumber
shortName
location



superfamily
SSF51445
(Trans)glycosidases
T[28-400] 7.5e−89



InterPro
NULL
NULL



method
AccNumber
shortName
location



Seg
seg
seg
?[2-14] NA ?[70-83] NA ?[91-96] NA




?[409-425] NA

SignalPHMM
signalp
signal-peptide
?[1-22] NA






















































Sequence “ZM1S60983186” crc64 checksum: E8BAF56AF1838CBC length: 491 aa.









InterPro
IPR000719
Protein kinase, core





Molecular Function: protein kinase activity (GO:0004672), Molecular Function: ATP binding (GO:0005524),

Biological Process: protein amino acid phosphorylation (GO:0006468)








method
AccNumber
shortName
location



BlastProDom
PD000001
Q94GE0_ORYSA Q94GE0;
T[69-313] 3e−134

ProfileScan
PS50011
PROTEIN_KINASE_DOM
T[55-322] 20.160



InterPro
IPR001245
Tyrosine protein kinase





Molecular Function: protein-tyrosine kinase activity (GO:0004713), Molecular Function: ATP binding

(GO:0005524), Biological Process: protein amino acid phosphorylation (GO:0006468)








method
AccNumber
shortName
location



HMMPfam
PF07714
Pkinase_Tyr
T[68-134] 2.1e−06

HMMSmart
SM00219
no description
T[55-307] 2.1e−08



InterPro
IPR002290
Serine/threonine protein kinase





Molecular Function: protein serine/threonine kinase activity (GO:0004674), Molecular Function: ATP binding

(GO:0005524), Biological Process: protein amino acid phosphorylation (GO:0006468)








method
AccNumber
shortName
location



HMMSmart
SM00220
no description
T[55-316] 7.4e−08



InterPro
IPR011009
Protein kinase-like



method
AccNumber
shortName
location



superfamily
SSF56112
Protein kinase-like (PK-like)
T[37-327] 2.2e−43



InterPro
IPR011990
Tetratricopeptide-like helical





Molecular Function: binding (GO:0005488)








method
AccNumber
shortName
location



Gene3D
G3DSA:1.25.40.10
no description
T[378-481] 1.5e−09



InterPro
NULL
NULL



method
AccNumber
shortName
location



Gene3D
G3DSA:1.10.510.10
no description
T[131-335] 7.2e−23

Gene3D
G3DSA:3.30.200.20
no description
T[33-131] 4.8e−11

HMMPanther
PTHR23258
SERINE-THREONINE PROTEIN KINASE, PLANT-TYPE
T[29-484] 4.6e−262

HMMPanther
PTHR23258:SF457
PROTEIN KINASE-LIKE PROTEIN
T[29-484] 4.6e−262

superfamily
SSF48452
TPR-like
T[82-480] 9.5e−10






































































































Sequence “ZM1S62018816” crc64 checksum: DCA5D5DC3B026BD4 length: 426 aa.










InterPro
NULL
NULL




method
AccNumber
shortName
location



Seg
seg
seg
?[112-137] NA ?[248-263] NA

TMHMM
tmhmm
transmembrane_regions
?[315-337] NA
































Sequence “ZM1S62063823” crc64 checksum: 329D087C5DB89A45 length: 390 aa.










InterPro
IPR006946
Protein of unknown function DUF642




method
AccNumber
shortName
location



HMMPfam
PF04862
DUF642
T[64-389] 2.4e−193



InterPro
IPR008979
Galactose-binding like



method
AccNumber
shortName
location



superfamily
SSF49785
Galactose-binding domain-like
T[51-209] 4.2e−07 T[219-388] 0.0019



InterPro
NULL
NULL



method
AccNumber
shortName
location



Seg
seg
seg
?[8-42] NA

SignalPHMM
signalp
signal-peptide
?[1-25] NA












































Sequence “ZM4S45895” crc64 checksum: 9F0BD4B998057088 length: 119 aa.










InterPro
IPR007741
Mitochondrial ribosome









method
AccNumber
shortName
location



HMMPfam
PF05047
L51_S25_CI-B8
T[18-91] 3.2e−24



InterPro
NULL
NULL



method
AccNumber
shortName
location



HMMPanther
PTHR21396
39S RIBOSOMAL PROTEIN L43
T[4-114] 1.2e−48

HMMPanther
PTHR21396:SF2
60S RIBOSOMAL PROTEIN L51
T[4-114] 1.2e−48









































Sequence “ZM1S68704853” crc64 checksum: C1B601532AE00FF3 length: 305 aa.










InterPro
IPR001680
WD40 repeat




method
AccNumber
shortName
location



BlastProDom
PD000018
Q8W403_ORYSA_Q8W403;
T[50-84] 4e−13 T[209-244] 4e−05

FPrintScan
PR00320
GPROTEINBRPT
T[72-86] 8.9e−06 T[182-196] 8.9e−06




T[232-246] 8.9e−06

HMMPfam
PF00400
WD40
T[1-39] 0.0003 T[45-85] 2.3e−10 T[93-133]




1.3e−07 T[140-195] 4.3e−09 T[203-245] 1.5e−08

HMMSmart
SM00320
no description
T[2-39] 0.41 T[44-85] 2.2e−08 T[92-133] 2.7e−05




T[139-195] 0.033 T[202-245] 0.0003 T[254-290] 0.55

ProfileScan
PS50082
WD_REPEATS_2
T[7-37] 8.537 T[51-85] 10.408 T[182-204] 8.704




T[209-244] 10.943

ProfileScan
PS50294
WD_REPEATS_REGION
T[7-299] 27.948



InterPro
IPR011046
WD40 repeat-like



method
AccNumber
shortName
location



superfamily
SSF50978
WD40 repeat-like
T[1-290] 1.9e−50



InterPro
IPR015943
WD40/YVTN repeat-like



method
AccNumber
shortName
location



Gene3D
G3DSA:2.130.10.10
no description
T[8-294] 7.3e−61



InterPro
NULL
NULL



method
AccNumber
shortName
location



HMMPanther
PTHR11024
PROTEIN TRANSPORT PROTEIN SEC13-RELATED
T[7-305] 1.7e−175

HMMPanther
PTHR11024:SF2
PROTEIN TRANSPORT PROTEIN SEC13
T[7-305] 1.7e−175



























































Sequence “ZM4S07964” crc64 checksum: 5F668E8D83CF5645 length: 82 aa.










InterPro
IPR001931
Ribosomal protein S21e






Molecular Function: structural constituent of ribosome (GO:0003735), Cellular Component: intracellular

(GO:0005622), Cellular Component: ribosome (GO:0005840), Biological Process: translation (GO:0006412)








method
AccNumber
shortName
location



BlastProDom
PD006584
RS21_ORYSA_P35687;
T[26-74] 8e−23

HMMPIR
PIRSF002148
Ribosomal protein S21e
T[1-82] 1e−50

HMMPanther
PTHR10442
40S RIBOSOMAL PROTEIN S21E
T[2-82] 4.9e−47

HMMPfam
PF01249
Ribosomal_S21e
T[1-81] 8.5e−60

ScanRegExp
PS00996
RIBOSOMAL_S21E
T[11-19] 8e−5












































Sequence “ZM1S65239103” crc64 checksum: D9F7188DB3E52343 length: 300 aa.










InterPro
IPR001005
SANT, DNA-binding






Molecular Function: DNA binding (GO:0003677)








method
AccNumber
shortName
location



HMMSmart
SM00717
no description
T[13-63] 7.2e-12 T[66-114] 5.6e−15

ProfileScan
PS50090
MYB_3
T[9-61] 15.442 T[62-112] 15.512

ScanRegExp
PS00037
MYB_1
?[17-25] NA

ScanRegExp
PS00334
MYB_2
T[89-112] 8e−5



InterPro
IPR009057
Homeodomain-like



method
AccNumber
shortName
location



superfamily
SSF46689
Homeodomain-like
T[14-84] 9.3e−19 T[86-146] 1e−13



InterPro
IPR012287
Homeodomain-related





Molecular Function: DNA binding (GO:0003677), Biological Process: regulation of transcription (GO:0045449)








method
AccNumber
shortName
location



Gene3D
G3DSA:1.10.10.60
no description
T[12-64] 2.2e−13 T[68-116] 1e−16



InterPro
IPR014778
Myb, DNA-binding



method
AccNumber
shortName
location



HMMPfam
PF00249
Myb_DNA-binding
T[14-61] 4.7e−10 T[67-112] 5.2e−10



InterPro
IPR015495
Myb transcription factor



method
AccNumber
shortName
location



HMMPanther
PTHR10641
MYB-RELATED
T[3-142] 2.5e−97



InterPro
NULL
NULL



method
AccNumber
shortName
location



HMMPanther
PTHR10641:SF30
R2R3-MYB TRANSCRIPTION FACTOR
T[3-142] 2.5e−97

Seg
seg
seg
?[131-143] NA ?[191-236] NA

















































































Sequence “ZM1S59258289” crc64 checksum: E21693C982A6C0C9 length: 338 aa.










InterPro
IPR005123
2OG-Fe(II) oxygenase




method
AccNumber
shortName
location



HMMPfam
PF03171
2OG-FeII_Oxy
T[177-287] 1.5e−33



InterPro
NULL
NULL



method
AccNumber
shortName
location



Gene3D
G3DSA:2.60.120.330
no description
T[29-335] 6.2e−76

HMMPanther
PTHR10209
IRON/ASCORBATE-DEPENDENT
T[39-326] 6.3e−133



OXIDOREDUCTASE FAMILY MEMBER


HMMPanther
PTHR10209:SF8
GIBBERELLIN 2-BETA-DIOXYGENASE 2
T[39-326] 6.3e−133

Seg
seg
seg
?[113-125] NA ?[142-152] NA

superfamily
SSF51197
Clavaminate synthase-like
T[29-329] 1.4e−78










































Sequence ″ZM1A68352777″ crc64 checksum: 0ED63F3B6A2E49FA length: 563 aa.










InterPro
IPR001128
Cytochrome P450




Molecular Function: monooxygenase activity (GO: 0004497), Molecular Function: iron ion binding (GO: 0005506),

Biological Process: electron transport (GO: 0006118), Molecular Function: heme binding (GO: 0020037)






method
AccNumber
shortName
location

FPrintScan
PR00385
P450
T[354-371] 6.5e−14 T[407-418] 6.5e−14



T[484-493] 6.5e−14 T[493-504] 6.5e−14






Gene3D
G3DSA: 1.10.630.10
no description
T[94-548] 1.6e−104

HMMPanther
PTHR19383
CYTOCHROME P450
T[27-73] 5.4e−185 T[94-514] 5.4e−185

HMMPfam
PF00067
p450
T[86-542] 1.4e−59

ScanRegExp
PS00086
CYTOCHROME_P450
?[486-495] NA

superfamily
SSF48264
Cytochrome P450
T[57-544] 2.7e−109

InterPro
IPR002401
Cytochrome P450, E−class, group I




Molecular Function: monooxygenase activity (GO: 0004497), Molecular Function: iron ion binding (GO: 0005506),

Biological Process: electron transport (GO: 0006118), Molecular Function: heme binding (GO: 0020037)






method
AccNumber
shortName
location

FPrintScan
PR00463
EP450I
T[104-123] 2.3e−24 T[128-149] 2.3e−24



T[343-360] 2.3e−24 T[363-389] 2.3e−24 T[483-493] 2.3e−24 T[493-516] 2.3e−24






InterPro
NULL
NULL


method
AccNumber
shortName
location

HMMPanther
PTHR19383: SF174
CYTOCHROME P450, SUBFAMILY 72
T[27-73] 5.4e−185 T[94-514] 5.4e−185

Seg
seg
seg
?[18-30] NA ?[327-335] NA

SignalPHMM
signalp
signal-peptide
?[1-35] NA

TMHMM
tmhmm
transmembrane_regions
?[13-35] NA









































































Sequence ″ZM4S13278″ crc64 checksum: 8FA559338699CF78

length: 610 aa.










InterPro
IPR004993
GH3 auxin-responsive promoter


method
AccNumber
shortName
location

HMMPfam
PF03321
GH3
T[35-584] 0

InterPro
NULL
NULL


method
AccNumber
shortName
location

Seg
seg
seg
?[13-29] NA

































Sequence ″ZM1S61040508″ crc64 checksum: 131843319F9E7FD8 length: 614 aa.










InterPro
IPR004993
GH3 auxin-responsive promoter


method
AccNumber
shortName
location

HMMPfam
PF03321
GH3
T[33-588] 0

InterPro
NULL
NULL


method
AccNumber
shortName
location

Seg
seg
seg
?[72-83] NA ?[386-396] NA
































Sequence ″ZM1S62064657″ crc64 checksum: 0B1A10A3C6485A7F length: 312 aa.










InterPro
IPR003441
No apical meristem (NAM) protein




Molecular Function: DNA binding (G0: 0003677), Biological Process: regulation of transcription (G0: 0045449)






method
AccNumber
shortName
location

HMMPfam
PF02365
NAM
T[17-143] 5.6e−86

ProfileScan
PS51005
NAC
T[17-169] 60.395

superfamily
SSF101941
NAC domain
T[1-170] 1.3e−68

InterPro
NULL
NULL


method
AccNumber
shortName
location

Seg
seg
seg
?[141-158] NA ?[198-211] NA









































Sequence ″ZM4S14568″ crc64 checksum: B6E028AA73574C57 length: 368 aa.










InterPro
IPR001222
Zinc finger, TFIIS-type




Molecular Function: nucleic acid binding (GO: 0003676), Molecular Function: zinc ion binding (GO: 0008270),

Molecular Function: transcription regulator activity (GO: 0030528), Biological Process: regulation of

transcription (GO: 0045449)






method
AccNumber
shortName
location

HMMPfam
PF01096
TFIIS_C
T[328-366] 7.6e−23

HMMSmart
SM00440
no description
T[328-367] 3.5e−22

ProfileScan
PS51133
ZF_TFIIS_2
T[326-366] 14.437

ScanRegExp
PS00466
ZF_TFIIS_1
T[330-365] 8e−5





InterPro
IPR003617
Transcription elongation factor, TFIIS/CRSP70, N-terminal, sub-type



Cellular Component: nucleus (GO: 0005634), Biological Process: transcription (GO: 0006350)






method
AccNumber
shortName
location

HMMSmart
SM00509
no description
T[8-85] 2.3e−12





InterPro
IPR003618
Transcription elongation factor S-II, central region



Biological Process: transcription (GO: 0006350)






method
AccNumber
shortName
location

HMMPfam
PF07500
TFIIS_M
T[189-317] 2.1e−48

HMMSmart
SM00510
no description
T[191-306] 3.2e−23

superfamily
SSF46942
Elongation factor TFIIS domain 2
T[172-299] 4.5e−24

InterPro
IPR006289
Transciption elongation factor, TFIIS




Molecular Function: nucleic acid binding (GO: 0003676), Molecular Function: RNA polymerase II transcription

factor activity (GO: 0003702), Molecular Function: translation elongation factor activity (GO: 0003746),

Molecular Function: protein binding (GO: 0005515), Cellular Component: nucleus (GO: 0005634), Biological

Process: RNA elongation (GO: 0006354), Biological Process: regulation of transcription, DNA-dependent

(GO: 0006355)






method
AccNumber
shortName
location

HMMTigr
TIGR01385
TFSII: transcription elongation factor S-II
T[11-368] 4.4e−101





InterPro
IPR010990
Transcription elongation factor, TFIIS/elongin A/CRSP70, N-terminal



Molecular Function: DNA binding (GO: 0003677), Molecular Function: protein binding (GO: 0005515), Cellular

Component: nucleus (GO: 0005634), Biological Process: transcription (GO: 0006350), Molecular Function:

transcription regulator activity (GO: 0030528)






method
AccNumber
shortName
location

HMMPfam
PF08711
TFIIS
T[1-93] 5.55e−06





superfamily
SSF47676
Conserved domain common to transcription factors TFIIS, elong A, CRSP70T [9-84]



2.5e−10





InterPro
IPR014765
Transcription elongation factor, TFIIS/CRSP70, N-terminal



Molecular Function: DNA binding (GO: 0003677), Molecular Function: protein binding (GO: 0005515), Cellular

Component: nucleus (GO: 0005634), Biological Process: transcription (GO: 0006350), Molecular Function:

transcription regulator activity (GO: 0030528)






method
AccNumber
shortName
location

Gene3D
G3DSA: 1.25.40.260
no description
T[24-100] 3e−07





InterPro
IPR016492
Transcription elongation factor, IIS



Molecular Function: transcription elongation regulator activity (GO: 0003711), Biological Process: regulation

of transcription from RNA polymerase II promoter (GO: 0006357)






method
AccNumber
shortName
location

HMMPIR
PIRSF006704
Transcription elongation factor, IIS type
T[2-368] 1.7e−125

InterPro
NULL
NULL


method
AccNumber
shortName
location

Gene3D
G3DSA: 2.20.25.10
no description
T[299-368] 4.4e−20

HMMPanther
PTHR11477
TRANSCRIPTION ELONGATION FACTOR S-II
T[1-97] 8.4e−51 T[129-367] 8.4e−51

Seg
seg
seg
?[10-23] NA

superfamily
SSF57783
Zinc beta-ribbon
T[305-367] 3.1e−21













































































































































Sequence ″ZM1S62075387″ crc64 checksum: 912650C1CC470182 length: 752 aa.









InterPro
IPR008217
Protein of unknown function DUF125, transmembrane






method
AccNumber
shortName
location

HMMPfam
PF01988
DUF125
T[565-732] 7.099998842111971E−11

























Example 5
Topology Prediction of the GRP Polypeptide Sequences
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. For the sequences predicted to contain an N-terminal presequence a potential cleavage site can also be predicted. TargetP is maintained at the server of the Technical University of Denmark.
A number of parameters must be selected before analysing a sequence, 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).
Results of TargetP 1.1 analysis of a selection of GRP polypeptide sequence from Table A are listed hereunder in Table B.
The “plant” organism group has been selected, no cutoffs defined, and the predicted length of the transit peptide requested.





TABLE B



TargetP 1.1 analysis of some polypeptide sequences listed in Table A,

represented by their identifier. Abbreviations: Len, Length; cTP, Chloroplastic transit

peptide; mTP, Mitochondrial transit peptide, SP, Secretory pathway signal peptide, other,

Other subcellular targeting, Loc, Predicted Location; RC, Reliability class; TPlen, Predicted

transit peptide length.











Name
Len
cTP
mTP
SP
other
Loc
RC
TPlen













ZM1s55817549
361
0.056
0.334
0.056
0.728
—
4
—

cutoff

0.000
0.000
0.000
0.000




ZM1S60958648
426
0.006
0.403
0.716
0.018
S
4
22

cutoff

0.000
0.000
0.000
0.000




ZM1s60983186
491
0.009
0.431
0.084
0.775
—
4
—

cutoff

0.000
0.000
0.000
0.000




ZM1S62018816
426
0.501
0.796
0.002
0.042
M
4
12

cutoff

0.000
0.000
0.000
0.000




ZM1s62063823
390
0.192
0.035
0.646
0.012
S
3
25

cutoff

0.000
0.000
0.000
0.000




ZM4s45895
119
0.026
0.527
0.062
0.138
M
4
102

cutoff

0.000
0.000
0.000
0.000




ZM1s68704853
305
0.079
0.093
0.052
0.919
—
1
—

cutoff

0.000
0.000
0.000
0.000




ZM4s07964
82
0.108
0.285
0.064
0.769
—
3
—

cutoff

0.000
0.000
0.000
0.000




ZM1S65239103
300
0.105
0.058
0.040
0.882
—
2
—

cutoff

0.000
0.000
0.000
0.000




ZM1s59258289
338
0.325
0.231
0.184
0.270
C
5
27

cutoff

0.000
0.000
0.000
0.000




ZM1a68352777
563
0.038
0.249
0.146
0.370
—
5
—

cutoff

0.000
0.000
0.000
0.000




ZM4s13278
610
0.136
0.210
0.181
0.436
—
4
—

cutoff

0.000
0.000
0.000
0.000




ZM1s61040508
614
0.296
0.147
0.165
0.435
—
5
—

cutoff

0.000
0.000
0.000
0.000




ZM1s62064657
312
0.072
0.219
0.049
0.886
—
2
—

cutoff

0.000
0.000
0.000
0.000




ZM4s14568
368
0.131
0.047
0.052
0.913
—
2
—

cutoff

0.000
0.000
0.000
0.000




ZM1s62075387
752
0.160
0.089
0.076
0.895
—
2
—

cutoff

0.000
0.000
0.000
0.000






























































Many other algorithms can be used to perform such analyses, including:
ChloroP 1.1 hosted on the server of the Technical University of Denmark; Protein Prowler Subcellular Localisation Predictor version 1.2 hosted on the server of the Institute for Molecular Bioscience, University of Queensland, Brisbane, Australia; PENCE Proteome Analyst PA-GOSUB 2.5 hosted on the server of the University of

Alberta, Edmonton, Alberta, Canada;
TMHMM, hosted on the server of the Technical University of Denmark PSORT (URL: psort.org) PLOC (Park and Kanehisa, Bioinformatics, 19, 1656-1663, 2003).

Example 6
Cloning of the GRP Encoding Nucleic Acid Sequence
The nucleic acid sequences was amplified by PCR using as template a custom-made Zea mays, Oryza sativa or Arabidopsis thaliana seedlings cDNA libraries. PCR was performed using a commercially available proofreading Taq DNA polymerase in standard conditions, using 200 ng of template in a 50 μl PCR mix. The primers used were designed using standard methods for gene specific amplification and included the AttB sites for Gateway recombination. The amplified PCR fragment was purified also using standard methods. The first step of the Gateway procedure, the BP reaction, was then performed, during which the PCR fragment recombined in vivo with the pDONR201 plasmid to produce, according to the Gateway terminology, an “entry clone”. Plasmid pDONR201 was purchased from Invitrogen, as part of the Gateway® technology.
The entry clone comprising the coding sequences provided in Table A 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. In one set of experiments, the rice GOS2 promoter (SEQ ID NO: 643) for constitutive expression was located upstream of this Gateway cassette. In one embodiment, the Gateway cassette comprising the nucleic acid sequence of interest was designed for overexpressing the nucleic acid sequence of interest; in another embodiment, the Gateway cassette comprising the nucleic acid sequence of interest was designed for decreasing expression of the nucleic acid sequence of interest (as indicated in Table C). In another set of experiments, the CaMV-35S promoter for constitutive expression was located upstream of this Gateway cassette.
After the LR recombination step, the resulting expression vector was transformed into Agrobacterium strain LBA4044 according to methods well known in the art.
Example 7
Plant Transformation
Rice Transformation
The Agrobacterium containing the expression vector was used to transform Oryza sativa plants. Mature dry seeds of the rice japonica cultivar Nipponbare were dehusked. Sterilization was carried out by incubating for one minute in 70% ethanol, followed by 30 to 60 minutes, preferably 30 minutes in sodium hypochlorite solution (depending on the grade of contamination), followed by a 3 to 6 times, preferably 4 time wash with sterile distilled water. The sterile seeds were then germinated on a medium containing 2,4-D (callus induction medium). After incubation in light for 6 days scutellum-derived calli is transformed with Agrobacterium as described herein below.
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 calli were immersed in the suspension for 1 to 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. After washing away the Agrobacterium , the calli were grown on 2,4-D-containing medium for 10 to 14 days (growth time for indica: 3 weeks) under light at 28° C.-32° C. in the presence of a selection agent. During this period, rapidly growing resistant callus developed. After transfer of this material to regeneration media, the embryogenic potential was released and shoots developed in the next four to six 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.
Transformation of rice cultivar indica can also be done in a similar way as give above according to techniques well known to a skilled person.
35 to 90 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 8
Transformation of Other Crops
Corn Transformation
Transformation of maize ( Zea mays ) is performed with a modification of the method described by Ishida et al. (1996) Nature Biotech 14(6): 745-50. Transformation is genotype-dependent in corn and only specific genotypes are amenable to transformation and regeneration. The inbred line A188 (University of Minnesota) or hybrids with A188 as a parent are good sources of donor material for transformation, but other genotypes can be used successfully as well. Ears are harvested from corn plant approximately 11 days after pollination (DAP) when the length of the immature embryo is about 1 to 1.2 mm. Immature embryos are cocultivated with Agrobacterium tumefaciens containing the expression vector, and transgenic plants are recovered through organogenesis. Excised embryos are grown on callus induction medium, then maize regeneration medium, containing the selection agent (for example imidazolinone but various selection markers can be used). The Petri plates are incubated in the light at 25° C. for 2-3 weeks, or until shoots develop. The green shoots are transferred from each embryo to maize rooting medium and incubated at 25° C. for 2-3 weeks, until roots develop. The rooted shoots are transplanted to soil in the greenhouse. T1 seeds are produced from plants that exhibit tolerance to the selection agent and that contain a single copy of the T-DNA insert.
Wheat Transformation
Transformation of wheat is performed with the method described by Ishida et al. (1996) Nature Biotech 14(6): 745-50. The cultivar Bobwhite (available from CIMMYT, Mexico) is commonly used in transformation. Immature embryos are co-cultivated with Agrobacterium tumefaciens containing the expression vector, and transgenic plants are recovered through organogenesis. After incubation with Agrobacterium , the embryos are grown in vitro on callus induction medium, then regeneration medium, containing the selection agent (for example imidazolinone but various selection markers can be used). The Petri plates are incubated in the light at 25° C. for 2-3 weeks, or until shoots develop. The green shoots are transferred from each embryo to rooting medium and incubated at 25° C. for 2-3 weeks, until roots develop. The rooted shoots are transplanted to soil in the greenhouse. T1 seeds are produced from plants that exhibit tolerance to the selection agent and that contain a single copy of the T-DNA insert.
Soybean Transformation
Soybean is transformed according to a modification of the method described in the Texas A&M patent U.S. Pat. No. 5,164,310. Several commercial soybean varieties are amenable to transformation by this method. The cultivar Jack (available from the Illinois Seed foundation) is commonly used for transformation. Soybean seeds are sterilised for in vitro sowing. The hypocotyl, the radicle and one cotyledon are excised from seven-day old young seedlings. The epicotyl and the remaining cotyledon are further grown to develop axillary nodes. These axillary nodes are excised and incubated with Agrobacterium tumefaciens containing the expression vector. After the cocultivation treatment, the explants are washed and transferred to selection media. Regenerated shoots are excised and placed on a shoot elongation medium. Shoots no longer than 1 cm are placed on rooting medium until roots develop. The rooted shoots are transplanted to soil in the greenhouse. T1 seeds are produced from plants that exhibit tolerance to the selection agent and that contain a single copy of the T-DNA insert.
Rapeseed/Canola Transformation
Cotyledonary petioles and hypocotyls of 5-6 day old young seedling are used as explants for tissue culture and transformed according to Babic et al. (1998, Plant Cell Rep 17: 183-188). The commercial cultivar Westar (Agriculture Canada) is the standard variety used for transformation, but other varieties can also be used. Canola seeds are surface-sterilized for in vitro sowing. The cotyledon petiole explants with the cotyledon attached are excised from the in vitro seedlings, and inoculated with Agrobacterium (containing the expression vector) by dipping the cut end of the petiole explant into the bacterial suspension. The explants are then cultured for 2 days on MSBAP-3 medium containing 3 mg/l BAP, 3% sucrose, 0.7 Phytagar at 23° C., 16 hr light. After two days of co-cultivation with Agrobacterium , the petiole explants are transferred to MSBAP-3 medium containing 3 mg/l BAP, cefotaxime, carbenicillin, or timentin (300 mg/l) for 7 days, and then cultured on MSBAP-3 medium with cefotaxime, carbenicillin, or timentin and selection agent until shoot regeneration. When the shoots are 5-10 mm in length, they are cut and transferred to shoot elongation medium (MSBAP-0.5, containing 0.5 mg/l BAP). Shoots of about 2 cm in length are transferred to the rooting medium (MS0) for root induction. The rooted shoots are transplanted to soil in the greenhouse. T1 seeds are produced from plants that exhibit tolerance to the selection agent and that contain a single copy of the T-DNA insert.
Alfalfa Transformation
A regenerating clone of alfalfa ( Medicago sativa ) is transformed using the method of (McKersie et al., 1999 Plant Physiol 119: 839-847). Regeneration and transformation of alfalfa is genotype dependent and therefore a regenerating plant is required. Methods to obtain regenerating plants have been described. For example, these can be selected from the cultivar Rangelander (Agriculture Canada) or any other commercial alfalfa variety as described by Brown 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 Am J Bot 65:654-659). Petiole explants are cocultivated with an overnight culture of Agrobacterium tumefaciens C58C1 pMP90 (McKersie et al., 1999 Plant Physiol 119: 839-847) or LBA4404 containing the expression vector. The explants are cocultivated for 3 d in the dark on SH induction medium containing 288 mg/L Pro, 53 mg/L thioproline, 4.35 g/L K2SO4, and 100 μm acetosyringinone. The explants are washed in half-strength Murashige-Skoog medium (Murashige and Skoog, 1962) and plated on the same SH induction medium without acetosyringinone but with a suitable selection agent and suitable antibiotic to inhibit Agrobacterium growth. After several weeks, somatic embryos are transferred to BOi2Y development medium containing no growth regulators, no antibiotics, and 50 g/L sucrose. Somatic embryos are subsequently germinated on half-strength Murashige-Skoog medium. Rooted seedlings were transplanted into pots and grown in a greenhouse. T1 seeds are produced from plants that exhibit tolerance to the selection agent and that contain a single copy of the T-DNA insert.
Cotton Transformation
Cotton is transformed using Agrobacterium tumefaciens according to the method described in U.S. Pat. No. 5,159,135. Cotton seeds are surface sterilised in 3% sodium hypochlorite solution during 20 minutes and washed in distilled water with 500 μg/ml cefotaxime. The seeds are then transferred to SH-medium with 50 μg/ml benomyl for germination. Hypocotyls of 4 to 6 days old seedlings are removed, cut into 0.5 cm pieces and are placed on 0.8% agar. An Agrobacterium suspension (approx. 108 cells per ml, diluted from an overnight culture transformed with the gene of interest and suitable selection markers) is used for inoculation of the hypocotyl explants. After 3 days at room temperature and lighting, the tissues are transferred to a solid medium (1.6 g/l Gelrite) with Murashige and Skoog salts with B5 vitamins (Gamborg et al., Exp. Cell Res. 50:151-158 (1968)), 0.1 mg/l 2,4-D, 0.1 mg/l 6-furfurylaminopurine and 750 μg/ml MgCL2, and with 50 to 100 μg/ml cefotaxime and 400-500 μg/ml carbenicillin to kill residual bacteria. Individual cell lines are isolated after two to three months (with subcultures every four to six weeks) and are further cultivated on selective medium for tissue amplification (30° C., 16 hr photoperiod). Transformed tissues are subsequently further cultivated on non-selective medium during 2 to 3 months to give rise to somatic embryos. Healthy looking embryos of at least 4 mm length are transferred to tubes with SH medium in fine vermiculite, supplemented with 0.1 mg/l indole acetic acid, 6 furfurylaminopurine and gibberellic acid. The embryos are cultivated at 30° C. with a photoperiod of 16 hrs, and plantlets at the 2 to 3 leaf stage are transferred to pots with vermiculite and nutrients. The plants are hardened and subsequently moved to the greenhouse for further cultivation.
Sugarbeet Transformation
Seeds of sugarbeet ( Beta vulgaris L.) are sterilized in 70% ethanol for one minute followed by 20 min. shaking in 20% Hypochlorite bleach e.g. Clorox® regular bleach (commercially available from Clorox, 1221 Broadway, Oakland, Calif. 94612, USA). Seeds are rinsed with sterile water and air dried followed by plating onto germinating medium (Murashige and Skoog (MS) based medium (Murashige, T., and Skoog., 1962. Physiol. Plant, vol. 15, 473-497) including B5 vitamins (Gamborg et al.; Exp. Cell Res., vol. 50, 151-8.) supplemented with 10 g/l sucrose and 0.8% agar). Hypocotyl tissue is used essentially for the initiation of shoot cultures according to Hussey and Hepher (Hussey, G., and Hepher, A., 1978. Annals of Botany, 42, 477-9) and are maintained on MS based medium supplemented with 30 g/l sucrose plus 0.25 mg/l benzylamino purine and 0.75% agar, pH 5.8 at 23-25° C. with a 16-hour photoperiod. Agrobacterium tumefaciens strain carrying a binary plasmid harbouring a selectable marker gene, for example nptII, is used in transformation experiments. One day before transformation, a liquid LB culture including antibiotics is grown on a shaker (28° C., 150 rpm) until an optical density (O.D.) at 600 nm of ˜1 is reached. Overnight-grown bacterial cultures are centrifuged and resuspended in inoculation medium (O.D.˜1) including Acetosyringone, pH 5.5. Shoot base tissue is cut into slices (1.0 cm×1.0 cm×2.0 mm approximately). Tissue is immersed for 30 s in liquid bacterial inoculation medium. Excess liquid is removed by filter paper blotting. Co-cultivation occurred for 24-72 hours on MS based medium incl. 30 g/l sucrose followed by a non-selective period including MS based medium, 30 g/l sucrose with 1 mg/l BAP to induce shoot development and cefotaxim for eliminating the Agrobacterium . After 3-10 days explants are transferred to similar selective medium harbouring for example kanamycin or G418 (50-100 mg/l genotype dependent). Tissues are transferred to fresh medium every 2-3 weeks to maintain selection pressure. The very rapid initiation of shoots (after 3-4 days) indicates regeneration of existing meristems rather than organogenesis of newly developed transgenic meristems. Small shoots are transferred after several rounds of subculture to root induction medium containing 5 mg/l NAA and kanamycin or G418. Additional steps are taken to reduce the potential of generating transformed plants that are chimeric (partially transgenic). Tissue samples from regenerated shoots are used for DNA analysis. Other transformation methods for sugarbeet are known in the art, for example those by Linsey & Gallois (Linsey, K., and Gallois, P., 1990. Journal of Experimental Botany; vol. 41, No. 226; 529-36) or the methods published in the international application published as WO9623891A.
Sugarcane Transformation
Spindles are isolated from 6-month-old field grown sugarcane plants (Arencibia et al., 1998. Transgenic Research, vol. 7, 213-22; Enriquez-Obregon et al., 1998. Planta, vol. 206, 20-27). Material is sterilized by immersion in a 20% Hypochlorite bleach e.g. Clorox® regular bleach (commercially available from Clorox, 1221 Broadway, Oakland, Calif. 94612, USA) for 20 minutes. Transverse sections around 0.5 cm are placed on the medium in the top-up direction. Plant material is cultivated for 4 weeks on MS (Murashige, T., and Skoog., 1962. Physiol. Plant, vol. 15, 473-497) based medium incl. B5 vitamins (Gamborg, O., et al., 1968. Exp. Cell Res., vol. 50, 151-8) supplemented with 20 g/l sucrose, 500 mg/l casein hydrolysate, 0.8% agar and 5 mg/l 2,4-D at 23° C. in the dark. Cultures are transferred after 4 weeks onto identical fresh medium. Agrobacterium tumefaciens strain carrying a binary plasmid harbouring a selectable marker gene, for example hpt, is used in transformation experiments. One day before transformation, a liquid LB culture including antibiotics is grown on a shaker (28° C., 150 rpm) until an optical density (O.D.) at 600 nm of ˜0.6 is reached. Overnight-grown bacterial cultures are centrifuged and resuspended in MS based inoculation medium (O.D.˜0.4) including acetosyringone, pH 5.5. Sugarcane embryogenic callus pieces (2-4 mm) are isolated based on morphological characteristics as compact structure and yellow colour and dried for 20 min. in the flow hood followed by immersion in a liquid bacterial inoculation medium for 10-20 minutes. Excess liquid is removed by filter paper blotting. Co-cultivation occurred for 3-5 days in the dark on filter paper which is placed on top of MS based medium incl. B5 vitamins containing 1 mg/l 2,4-D. After co-cultivation calli are washed with sterile water followed by a non-selective cultivation period on similar medium containing 500 mg/l cefotaxime for eliminating remaining Agrobacterium cells. After 3-10 days explants are transferred to MS based selective medium incl. B5 vitamins containing 1 mg/l 2,4-D for another 3 weeks harbouring 25 mg/l of hygromycin (genotype dependent). All treatments are made at 23° C. under dark conditions. Resistant calli are further cultivated on medium lacking 2,4-D including 1 mg/l BA and 25 mg/l hygromycin under 16 h light photoperiod resulting in the development of shoot structures. Shoots are isolated and cultivated on selective rooting medium (MS based including, 20 g/l sucrose, 20 mg/l hygromycin and 500 mg/l cefotaxime). Tissue samples from regenerated shoots are used for DNA analysis. Other transformation methods for sugarcane are known in the art, for example from the in-ternational application published as WO2010/151634A and the granted European patent EP1831378.
Example 9
Phenotypic Evaluation Procedure
9.1 Evaluation Setup
35 to 90 independent T0 rice transformants were generated. The primary transformants were transferred from a tissue culture chamber to a greenhouse for growing and harvest of T1 seed. Six events, of which the T1 progeny segregated 3:1 for presence/absence of the transgene, were retained. For each of these events, approximately 10 T1 seedlings containing the transgene (hetero- and homo-zygotes) and approximately 10 T1 seedlings lacking the transgene (nullizygotes) were selected by monitoring visual marker expression. The transgenic plants and the corresponding nullizygotes were grown side-by-side at random positions. Greenhouse conditions were of shorts days (12 hours light), 28° C. in the light and 22° C. in the dark, and a relative humidity of 70%. Plants grown under non-stress conditions were watered at regular intervals to ensure that water and nutrients were not limiting and to satisfy plant needs to complete growth and development, unless they were used in a stress screen.
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.
T1 events can be further evaluated in the T2 generation following the same evaluation procedure as for the T1 generation, e.g. with less events and/or with more individuals per event.
Drought Screen
T1 or T2 plants 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. Soil moisture 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.
Nitrogen Use Efficiency Screen
T1 or T2 plants were grown in potting soil under normal conditions except for the nutrient solution. The pots were watered from transplantation to maturation with a specific nutrient solution containing reduced N nitrogen (N) content, usually between 7 to 8 times less. The rest of the cultivation (plant maturation, seed harvest) was the same as for plants not grown under abiotic stress. Growth and yield parameters were recorded as detailed for growth under normal conditions.
Salt Stress Screen
T1 or T2 plants are grown on a substrate made of coco fibers and particles of baked clay (Argex) (3 to 1 ratio). A normal nutrient solution is used during the first two weeks after transplanting the plantlets in the greenhouse. After the first two weeks, 25 mM of salt (NaCl) is added to the nutrient solution, until the plants are harvested. Growth and yield parameters are 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.
9.3 Parameters Measured
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 as described in WO2010/031780. These measurements were used to determine different parameters.
Biomass-Related Parameter Measurement
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.
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. In other words, the root/shoot index is defined as the ratio of the rapidity of root growth to the rapidity of shoot growth in the period of active growth of root and shoot. Root biomass can be determined using a method as described in WO 2006/029987.
Parameters Related to Development Time
The early vigour is the plant 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.
AreaEmer is an indication of quick early development when this value is decreased compared to control plants. It is the ratio (expressed in %) between the time a plant needs to make 30% of the final biomass and the time needs to make 90% of its final biomass.
The “time to flower” or “flowering time” of the plant can be determined using the method as described in WO 2007/093444.
Seed-Related Parameter Measurements
The mature primary panicles were harvested, counted, bagged, barcode-labelled and then dried for three days in an oven at 37° C. The panicles were then threshed and all the seeds were collected and counted. The seeds are usually covered by a dry outer covering, the husk. The filled husks (herein also named filled florets) 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 total number of seeds was determined by counting the number of filled husks that remained after the separation step. The total seed weight (Seed Yield in Table C) was measured by weighing all filled husks harvested from a plant.
The total number of seeds (or florets) per plant was determined by counting the number of husks (whether filled or not) harvested from a plant.
Thousand Kernel Weight (TKW) is extrapolated from the number of seeds counted and their total weight.
The Harvest Index (HI) in the present invention is defined as the ratio between the total seed weight and the above ground area (mm 2 ), multiplied by a factor 10 6 .
The number of flowers per panicle as defined in the present invention is the ratio between the total number of seeds over the number of mature primary panicles.
The “seed fill rate” or “seed filling rate” as defined in the present invention is the proportion (expressed as a %) of the number of filled seeds (i.e. florets containing seeds) over the total number of seeds (i.e. total number of florets). In other words, the seed filling rate is the percentage of florets that are filled with seed.
Example 10
Results of the Phenotypic Evaluation of the Transgenic Plants
The results of the evaluation of transgenic rice plants in the T1 generation and expressing a nucleic acid encoding a GRP polypeptide as given in Table A are presented below in Table C. In a number of cases the gene of interest was downregulated, indicated in the comumn “expression”. In the Yield screen, plants were grown under optimal conditions, in the Nitrogen screen, plants were grown under reduced nutrient availability as described above, in the Drought screen, plants were subjected to reduced water availability, as described above.





TABLE C



Data summary for transgenic T1 rice plants; for each parameter, the phenotype is indicated as follows: ++++: at least 4 positive

events with a positive difference >5%, p < 0.2 for any of the parameters indicated, +++: 3 positive events with a positive difference >5%,

p < 0.1, ++: 2 positive events with a positive difference >10%, p < 0.1.










Protein






Identifier
SEQ ID NO:
Phenotype ++++
Phenotype +++
Phenotype ++
Screen
Expression











Orysa_CBS domain
4


Flowers per panicle
Yield
downregulation

containing membrane







protein







Orysa_LOC_Os01g06640
8

Seed yield
Biomass, Number of
Yield
downregulation





flowers, Fillrate,







Harvest index; Seed







size (TKW)



Orysa_LOC_Os01g63690
10

Seed yield,

Yield
downregulation




Fillrate, Harvest







index, Number of







filled seeds




Orysa_LOC_Os02g36490
28


Harvest index
Yield
downregulation

Orysa_LOC_Os02g52560
34


Fillrate, Harvest index
Yield
downregulation

Orysa_LOC_Os03g 10210
40

Fillrate

Yield
downregulation

Orysa_LOC_Os03g 17790
44


Biomass
Yield
downregulation

Orysa_LOC_Os03g17800
46

Seed yield,
Fillrate, Harvest Index,
Yield
downregulation




number of filled
Flowers per panicle






seeds




Orysa_LOC_Os03g46490
48
Flowers per

Seed Yield, Number of
Yield
downregulation



panicle

flowers



Orysa_LOC_Os03g55670
50


Fillrate
Yield
downregulation

Orysa_LOC_Os04g35790
54


Fillrate, Harvest index
Yield
downregulation

Orysa_LOC_Os05g07070
56


Seed yield, Fillrate,
Yield
downregulation





number of filled seeds



Orysa_LOC_Os05g40770
58

Harvest Index
Seed yield, Fillrate,
yield
downregulation





Seed size (TKW),







Number of filled seeds



Orysa_LOC_Os06g01440
62
Seed Yield,


yield
downregulation



Fillrate, Harvest







Index, Number of







filled seeds





Orysa_LOC_Os06g06250.2
66


Fillrate
Yield
downregulation

Orysa_LOC_Os08g01760
74


Seed yield, Number of
Yield
downregulation





flowers, Fillrate,







Harvest index, Number







of panicles



Orysa_LOC_Os08g26870
76


Fillrate, Harvest index
Yield
downregulation

Orysa_LOC_Os10g39130
92


Number of flowers
Nitrogen
downregulation

Orysa_WRKY7
108


Seed yield, Fillrate,
Drought
downregulation





Harvest index, Number







of panicles, Number of







filled seeds



Zeama_GRMZM2G005260
128


Fillrate
Drought
normal

Zeama_GRMZM2G005435
130


Seed siz (TKW)
Drought
normal

Zeama_GRMZM2G010356_
144


Fillrate
Yield
normal

T01







Zeama_GRMZM2G012224_
150
Fillrate


Nitrogen
normal

T01







Zeama_GRMZM2G013627
152


Seed yield, Fillrate,
Drought
normal





Harvest index, Number







of panicles, Number of







filled seeds



Zeama_GRMZM2G014750
156


Seed yield, Number of
Drought
normal





flowers, Fillrate,







Harvest index, Flowers







per panicle



Zeama_GRMZM2G015433
158

Biomass

Nitrogen
normal

Zeama_GRMZM2G020281_
166


Seed yield, Fillrate,
yield
normal

T03



Harvest index, Seed







size (TKW), Number of







filled seeds



Zeama_GRMZM2G028218
176


Seed size (TKW)
Yield
normal

Zeama_GRMZM2G031724
182
Root Shoot Index


Drought
normal

Zeama_GRMZM2G031983_
184
Seed size (TKW)
Fillrate
Harvest Index
Yield
normal

T01







Zeama_GRMZM2G037001_
196


Harvest index, Seed
Yield
normal

T01



size (TKW), Number of







Panicles



Zeama_GRMZM2G040517_
200


Seed yield, Harvest
Nitrogen
normal

T01



index, Number of filled







seeds



Zeama_GRMZM2G043147
206


Seed yield, Fillrate,
Drought
normal





Harvest index, Number







of filled seeds



Zeama_GRMZM2G044027_
210

Biomass,
Seed yield, number of
Yield
normal

T01


Number of
panicles, flowers per






flowers
panicle



Zeama_GRMZM2G047684
216
Root Shoot Index


Nitrogen
normal

Zeama_GRMZM2G049398_
220
Fillrate, Harvest

Seed Yield, number of
Yield
normal

T01

index

filled seeds



Zeama_GRMZM2G053766
226

Number of

Drought
normal




Panicles




Zeama_GRMZM2G055585
230
Fillrate
Harvest Index
Seed Yield
Drought
normal

Zeama_GRMZM2G058690_
242

Biomass
Number of panicles
Yield
normal

T02







Zeama_GRMZM2G059580
244


Fillrate
Drought
normal

Zeama_GRMZM2G059799
246


Flowers per panicle
Drought
normal

Zeama_GRMZM2G061745
250


Seed yield, Fillrate,
Drought
normal





Number of filled seeds



Zeama_GRMZM2G061996_
252


Seed yield, Fillrate,
Yield
normal

T01



Harvest index, Number







of filled seeds



Zeama_GRMZM2G062160
254

Fillrate, Number
Seed yield, Harvest
Drought
normal




of filled seeds
index



Zeama_GRMZM2G062531
256

Flowers per

Drought
normal




panicle




Zeama_GRMZM2G077458
270


Seed yield, Fillrate,
Drought
normal





number of filled seeds



Zeama_GRMZM2G087323
292


Seed yield, Number of
Drought
normal





filled seeds



Zeama_GRMZM2G092137
306


Seed size (TKW)
Nitrogen
normal

Zeama_GRMZM2G101390
326

Biomass

Nitrogen
normal

Zeama_GRMZM2G102356
332


Fillrate, Number of
Drought
normal





panicles



Zeama_GRMZM2G104610_
336


Fillrate
Yield
normal

T01







Zeama_GRMZM2G105331
338


Number of panicles
Drought
normal

Zeama_GRMZM2G105348
340

Number of filled
Seed yield, fillrate,
Drought
normal




seeds
harvest index



Zeama_GRMZM2G107839
346


Seed yield, Fillrate,
Drought
normal





Harvest index



Zeama_GRMZM2G108716
350
Seed Yield,


Drought
normal



Fillrate, Harvest







Index, Number of







filled seeds





Zeama_GRMZM2G110289
354


Biomass, Seed yield
Nitrogen
normal

Zeama_GRMZM2G112210
358


Number of panicles
Drought
normal

Zeama_GRMZM2G115975
368


Seed yield, Fillrate,
Drought
normal





Harvest index



Zeama_GRMZM2G116632_
372


Fillrate
Yield
normal

T01







Zeama_GRMZM2G119906
376


Seed size (TKW)
Nitrogen
normal

Zeama_GRMZM2G126397
388


Seed yield, Fillrate
Drought
normal

Zeama_GRMZM2G126566
390
Flowers per


Drought
normal



panicle





Zeama_GRMZM2G127850_
396
fillrate, harvest

Seed size (TKW), seed
yield
normal

T01

index, number of

yield





filled seeds





Zeama_GRMZM2G127893
398


Seed yield, Fillrate,
Drought
normal





Harvest index, number







of filled seeds



Zeama_GRMZM2G137596_
402


Emergence Vigor,
Yield
normal

T01



Flowers per panicle



Zeama_GRMZM2G156110_
422


Emergence Vigor,
Yield
normal

T01



Seed yield, Fillrate,







Harvest index, Seed







size (TKW), Number of







filled seeds



Zeama_GRMZM2G159034
428


Seed yield, Fillrate,
Drought
normal





Harvest index, Number







of filled seeds



Zeama_GRMZM2G161295
430


Flowers per panicle
Nitrogen
normal

Zeama_GRMZM2G163406
432


Fillrate, Number of
Drought
normal





panicles



Zeama_GRMZM2G174558
454

Flowers per
Root Shoot Index
Nitrogen
normal




panicle




Zeama_GRMZM2G178815_
464

Fillrate
Seed yield, Harvest
Nitrogen
normal

T01



index



Zeama_GRMZM2G180172
468
Biomass


Nitrogen
normal

Zeama_GRMZM2G302245_
470

Seed size (TKW)

Yield
normal

T01







Zeama_GRMZM2G334181_
486

Fillrate
Seed yield, Harvest
yield
normal

T01



index



Zeama_GRMZM2G340130
488

Seed size (TKW)

Nitrogen
normal

Zeama_GRMZM2G347043
494
Seed size (TKW)


Nitrogen
normal

Zeama_GRMZM2G355752
498


Fillrate
Drought
normal

Zeama_GRMZM2G359952
500

Flowers per

Nitrogen
normal




panicle




Zeama_GRMZM2G401521
506

Biomass
Seed yield, Fillrate,
Nitrogen
normal





number of filled seeds



Zeama_GRMZM2G413337
518


Flowers per panicle
Nitrogen
normal

Zeama_GRMZM2G447984
530


Harvest index, Number
Nitrogen
normal





of panicles



Zeama_GRMZM2G479260
544


Seed yield, Fillrate
Drought
normal

Zeama_GRMZM2G481888_
546


Biomass, RootMax,
Yield
normal

T01



Fillrate



Zeama_nucleic acid binding
554

Fillrate
Harvest Index
Yield
normal

/zinc ion binding







Zeama_putative zinc-finger
560


Biomass, Fillrate,
Yield
normal

protein



Number of filled seeds



Zeama_SYTE like
562


Harvest index
Drought
normal

Zeama_unknown protein a
566


Seed size (TKW)
Drought
normal

Zeama_unknown protein b
568


Emergence Vigor,
Drought
normal





Fillrate, Seed size







(TKW)



Zeama_Unknown protein e
574


Seed yield, Fillrate
Drought
normal

Zeama_unknown protein g
578


Flowers per panicle
Drought
normal

Zeama_ZM1s61996727
590

Biomass
RootMax
Nitrogen
normal

Zm ATP-binding protein
596


Seed yield, Fillrate,
Drought
normal





Harvest index, Number







of filled seeds



Zm embryo-abundant
602

Seed size (TKW)
Seed yield, Fillrate,
Drought
normal

protein-related



Harvest index, Number







of filled seeds



Zm indole-3-acetic acid
606
Root Shoot Index


Drought
normal

amido synthetase-like 1







Zm indole-3-acetic acid
608
Root Shoot Index


Drought
normal

amido synthetase-like 2







Zm indoleacetic acid-
610


Seed size (TKW)
Drought
normal

induced protein 13-like 1







Zm indoleacetic acid-
612


Seed yield, Fillrate,
Drought
normal

induced protein 13-like 2



Harvest index, Number







of filled seeds



Zm indoleacetic acid-
614


Fillrate, Seed size
Drought
normal

induced protein 18



(TKW)



Zm indoleacetic acid-
616


Seed yield, Harvest
Drought
normal

induced protein 20-like



index



Zm PHYTOCHROME-
620


Biomass, seed yield,
Drought
normal

ASSOCIATED PROTEIN 1-



seed size (TKW)



like







Zm protein kinase-related
626
Fillrate, Seed
Seed Yield
Harvest Index
Yield
normal



size (TKW)





Zm protein kinase-related
626
Seed size (TKW)
Seed Yield
Fillrate, Harvest Index,
Drought
normal





number of filled seeds






































































































































































































































Example 11
Phenotypical Analysis of Arabidopsis Transformed with GRP Encoding Sequences
In order to analyse the potential role of selected genes in root development, a detailed analysis of the root system architecture was performed on transgenic Arabidopsis lines expressing the maize transcripts.
Entry clones were prepared by cloning the Zea mais coding sequence in pDONRTM221 or PDONRTM/Zeo as described in Example 7. Ectopic over-expression in Arabidopsis was obtained by cloning the coding sequence in pK7WG2 destination vectors, under control of the 35S promoter (Karimi et al., Trends in plant science 7(5): 193-195, 2002), or in a destination vector with the UAS promoter, which was transformed by floral dip (Clough and Bent, The Plant journal 16(6): 735-743, 1998) in Arabidopsis thaliana (L.) Heyhn. plants, ecotype Col0. In subsequent generations single-insert and homozygous lines were selected by growth on selective medium. For xylem-pole pericycle specific expression, vectors were prepared by cloning the required fragments into pH7m24GW or pB7m24GW (Karimi et al., Plant physiology, 145, 1144-1154, 2007; Hilson, Trends in plant science, 11, 133-141, 2006) which were transformed into plants from the J0121 GAL4 enhancer-trap line (Laplaze et al., 2005).
For phenotypic analyses Arabidopsis thaliana seeds (7 to 15 plants) were sown on solid 0.5×MS medium in square Petri dishes in near-vertical position and after 48h stratification at 4° C., incubated in a growth chamber at 22° C. under continuous light conditions (110 μE·m −2 ·s −1 photosynthetically active radiation, supplied by cool-white fluorescent tungsten tubes; Osram) till 10 days after germination (DAG). Lines were not grouped per construct but analysed randomly, depending on seed availability and capacity. In each experiment a Col0 was included as a reference, which was grown for one generation under the same conditions as the transgenic lines.
The number of emerged lateral roots (LR) was counted and a high resolution scan was made from the plates with a flatbed scanner (Epson Perfection V700 Photo). Scans were analysed with ImageJ (http://rsbweb.nih.gov/) and WinRHIZO (Regent Instruments) software, which was used to measure the total length of the root system. Through image analyses, primary root length (PR) was measured (expressed in cm) and size of the naked zone (in cm), which is the distance between the root tip and the youngest emerged lateral. The lateral root density (LRD) was calculated by dividing the number of laterals by the length of the primary root minus the naked zone. Average values were calculated per line and are represented as percentages compared to the non-transformed Col0 control. Data are presented in Table D and Table E.





TABLE D



Data summary for transgenic Arabidopsis plants screend for lateral root initiation

on the primary root; for each parameter the increase is shown as: + = 0-10%, ++ = 10-20%,

+++ = 20-30%, ++++ = >30%; for the scores between brackets the p-value was ≦0.05,

otherwise the p-value was ≦0.01.









Identifier
Expression
Line nr
PR
LR
LRD
Total Root Length



ZM1s60983186
35S-up
2
++
+++
(+)


ZM1s68382282
35S-up
1
(++)
++




35S-up
2
(++)
(+++)




35S-up
3


(+)



35S-up
4


+


ZM1s57394235
35S-up
1


(++)



35S-up
3
+





35S-up
4
++




ZM1s62016945
35S-up
1
++
++++
(++++)



35S-up
2

(++++)
(++++)



35S-up
4


++



35S-up
5
+++
+++



ZM1s58194594
UAS-up
1

++



ZM1s62122111
35S-up
4



(+++)


35S-up
5

(++)

(++++)

ZM4s18277
UAS-up
1
(++)

++


ZM1s61987040
35S-up
2


++



35S-up
3


+++


ZM1s62111342
35S-up
3



(++)


35S-up
5



(++)

ZM3s00628
35S-up
1
+++
++++
++
(++++)


35S-up













































In addition, the Arabidopsis knock out line SALK — 022198 (downregulation of the Ara orthologue of ZM1s62132790 (At5g64810, SEQ ID NO: 641) showed improved root growth: the primary root length was increased with 17.5%, the lateral root number was increased with 16.0% and the total root length was 26.8% increased.





TABLE E



Data summary for transgenic Arabidopsis plants screend for lateral root initiation

on the adventitious root; for each parameter the increase is shown as: + = 0-10%, ++ = 10-

20%, +++ = 20-30%, ++++ = >30%; for the scores between brackets the p-value was

≦0.05, otherwise the p-value was ≦0.01. For the adventitious roots, calculations for the LRD

were made with or without taking the naked zone into account (respectively indicated by

+NZ or −NZ).















LRD
LRD
Total Root

Identifier
Expression
Line nr
PR
LR
−NZ
+NZ
Length












ZM4s18047
35S-up
1
++
+++





35S-up
4
(+)
(++)




ZM1s61992039
35S-up
1

(++)

(++)



35S-up
4

+++
++
++



35S-up
5
++
+++

(++)


ZM4s07707
35S-up
2

(++)
(+)
(+)



35S-up
3
++
+++
++
++



35S-up
4
++
+++

(+)



35S-up
5
++
+++
(+)
(+)


ZM1s57368153
35S-up
3
++






35S-up
4
++
(++)


(++++)

ZM1s62202989
35S-up
1


(+)




35S-up
2
++
++





35S-up
4
++
(++)




ZM1s57508357
35S-up
1


+++
+++



35S-up
2


++
+++



35S-up
3


++
++



35S-up
4


(+)
+


ZM1s62258567
35S-up
1
+






35S-up
4
++
++
(+)

+++


35S-up
5


(+)



ZM1a62154780
35S-up
1
+++
++++
+++
++
(++++)


35S-up
2
++++
++++
++
+++
(++++)


35S-up
3
(+)
+++
++
++



35S-up
4
++
++++
+++
+++



35S-up
5
+++
++++
+++
+++
++++


35S-up
6

(++)
(+)
(++)



35S-up
7
++
+++
(++)
++



35S-up
9
++
++++
++
++



35S-up
10

(++)
(+)
(+)


ZM3s01330
35S-up
1

(+)





35S-up
3
(+)






35S-up
5
(+)
(++)


(+++)

ZM4s29494
35S-up
3


+




35S-up
4


+



ZM1s58530935
35S-up
5
++
+++




ZM1s62083330
35S-up
5
(++)
(++)


(++++)

ZM4s16359
35S-up
1


(+)




35S-up
2
++
++





35S-up
5
(+)



++

ZM1s61996727
35S-up
3


++
(++)



35S-up
4


++
++



35S-up
5


(++)
++