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WO2015056130A1 - Plantes présentant une biomasse et/ou une teneur en sucre accrues et leur procédé de production - Google Patents

Plantes présentant une biomasse et/ou une teneur en sucre accrues et leur procédé de production Download PDF

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WO2015056130A1
WO2015056130A1 PCT/IB2014/065099 IB2014065099W WO2015056130A1 WO 2015056130 A1 WO2015056130 A1 WO 2015056130A1 IB 2014065099 W IB2014065099 W IB 2014065099W WO 2015056130 A1 WO2015056130 A1 WO 2015056130A1
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plant
nucleic acid
plants
polypeptide
seq
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Janneke Hendriks
Jerome Martin
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BASF China Co Ltd
BASF Plant Science Co GmbH
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BASF China Co Ltd
BASF Plant Science Co GmbH
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8221—Transit peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C12N15/8245—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving modified carbohydrate or sugar alcohol metabolism, e.g. starch biosynthesis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/93—Ligases (6)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y603/00—Ligases forming carbon-nitrogen bonds (6.3)
    • C12Y603/01—Acid-ammonia (or amine)ligases (amide synthases)(6.3.1)
    • C12Y603/01002—Glutamate-ammonia ligase (6.3.1.2)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/08—Fusion polypeptide containing a localisation/targetting motif containing a chloroplast localisation signal
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

  • the present invention relates generally to the field of plant molecular biology and concerns a method for enhancing one or more yield-related traits in plants by modulating expression in a plant of a nucleic acid encoding a POI (Protein Of Interest) polypeptide.
  • the present invention also concerns plants having modulated expression of a nucleic acid encoding a POI polypeptide, which plants have one or more one or more enhanced yield-related traits relative to corresponding wild type plants or other control plants.
  • the invention also provides constructs useful in the methods uses, plants, harvestable parts and products of the invention of the invention.
  • Such technology has the capacity to deliver crops or plants having various improved economic, agronomic or horticultural traits.
  • a trait of 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 vig- our may also be important factors in determining yield. Optimizing the abovementioned factors may therefore contribute to increasing crop yield.
  • Seed yield is an 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, assimi- lates the metabolic precursors of carbohydrates, oils and proteins and synthesizes them into storage macromolecules to fill out the grain.
  • 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, nutrient deficiency, 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 possi- ble.
  • Crop yield may therefore be increased by optimising one of the above-mentioned factors.
  • Glutamine synthetase has been studied in a number of animals and plants, humans and micro-organism.
  • the baker's yeast gene YPR035W is known as GLN 1 in scientific community and is known to act as Glutamine synthetase (GS, enzyme class EC 6.3.1.2).
  • GS enzyme class EC 6.3.1.2
  • three different classes of GS are known (http://pfam.sanger.ac.uk/family/PF03951 ), with YPR035W belonging to Class II:
  • GSI I Class II enzymes
  • GSIII Class III enzymes
  • GSII targeted to the chloroplasts typically one GSII gene in a given species, no isoenzymes
  • GSII targeted to the cytoplasm typically several isozymes.
  • the yeast gene YPR035W has been disclosed in the international patent application PCT/EP2006/060588 published as WO2006/092449 to increase a num- ber of metabolites when expressed in plants without any targeting, i.e. cytoplasmic localisation.
  • EP 06127389.2 published as EP 1777296 disclosed that expression of YPR035W with plastid targeting resulted in increases of raffinose and myo-inositol and palmitic acid in the plants.
  • the modification of certain yield traits may be favoured over others.
  • 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.
  • the present invention concerns a method for enhancing one or more yield-related traits in plants by increasing the expression in a plant of a nucleic acid encoding a POI polypeptide and targeting of the POI polypeptide to the plastid(s).
  • the present invention also concerns plants having increased expression of a nucleic acid encoding a POI polypeptide and plastid localisation of the POI polypeptide, which plants have one or more enhanced yield- related traits compared with control plants.
  • the invention also provides hitherto unknown constructs comprising POI-encoding nucleic acids, useful in performing the methods of the invention.
  • a preferred embodiment is a method for enhancing one or more yield-related traits in a plant relative to control plants, comprising the steps of increasing the expression, preferably by recombinant methods, in a plant of a nucleic acid encoding a POI polypeptide preferably said nucleic acid is exogenous, wherein preferably the expression is under the control of a promoter sequence operably linked to the nucleic acid encoding the POI polypeptide, and growing the plant, wherein the POI polypeptide is targeted to the plastid(s).
  • inventive methods comprise increasing the expression in a plant of a nucleic acid encoding a POI polypeptide and targeting of the POI polypeptide to the plastid(s) and thereby enhancing one or more yield-related traits of said plant compared to the control plant.
  • the term "thereby enhancing" is to be understood to include direct effects of increasing the expression of the POI polypeptide as well as indirect effects as long as the increased expression of the POI polypeptide encoding nucleic acid and targeting of the POI polypeptide to the plastid(s) results in an enhancement of at least one of the yield-related traits.
  • transcription factor A may increase transcription of another transcription factor B that in turn controls the expression of a number of genes of a given pathway leading to enhanced biomass or seed yield.
  • transcription factor A does not directly enhance the expression of the genes of the pathway leading to enhanced yield-related traits, increased expression of A is the cause for the effect of enhanced yield-related-trait(s).
  • an expression cassette and a vector construct comprising a nucleic acid encoding a POI polypeptide, operably linked to a beneficial promoter sequence and a plastid targeting sequence.
  • transgenic plants transformed with one or more expression cassettes of the invention, and thus, expressing in a particular way the nucleic acids encoding a POI protein, wherein the plants have one or more enhanced yield-related trait.
  • Harvestable parts of the transgenic plants of the present invention and products derived from the transgenic plants and their harvestable parts are also part of the present invention.
  • Fig. 1 shows the vector VC-MME489-1 QCZ shown in SEQ I D NO: 50, used for the creation of a vector for plastid targeted expression according to Example 6.
  • Fig. 2 shows the vector shown in SEQ ID NO: 51 , used for the cloning of the YPR35W with plastid targeted expression of the YPR035W protein according to Example 6.
  • Fig. 3 represents a multiple alignment of various GS polypeptides using ClustalW (see example 2 for details).
  • the single letter code for amino acids is used.
  • White letters on black background indicate identical amino acids among the various protein sequences, white letters on grey background represent highly conserved amino acid substitutions.
  • These alignments can be used for defining further motifs or signature sequences, when using conserved amino acids, i.e. those identical in the aligned sequences and / or those highly conserved.
  • YPR035W_S.cerevisiae is used to indicate the GS polypeptide of SEQ ID NO:2.
  • Fig. 4 shows phylogenetic tree of GS polypeptides, as given by the guide tree of the ClustalW software.
  • SEQ ID NO 2 is clustered the closest with two other GS polypeptides from baker's yeast, then with one from S.arboricola.
  • YPR035W_S.cerevisiae is used to indicate the GS polypeptide of SEQ ID NO:2.
  • the other sequences are identified by their short name.
  • Table A provides the details for each sequence such as organism and SEQ ID NO.
  • Fig. 5 shows the NEEDLE results for sequence identity analysis of Example 3.
  • YPR035W_S.cerevisiae is used to indicate the GS polypeptide of SEQ ID NO:2.
  • Fig. 6 provides tables I and I I showing the relations of the different SEQ ID NOs. to the lead sequence.
  • YPR035W represents the GS sequences of SEQ ID NO: 1 & 2.
  • Fig. 7 provides tables III and IV showing the primers for amplification of the YPR35W nucleic acid and the consensus and pattern sequences.
  • YPR035W represents the GS sequences of SEQ I D NO: 1 & 2.
  • the present invention shows that increasing expression in a plant of a nucleic acid encoding a POI polypeptide in combination with a plastid targeting of the resulting POI polypep- tide gives plants having enhanced one or more enhanced yield-related traits relative to control plants.
  • the present invention provides a method for enhancing one or more yield-related traits in plants relative to control plants, comprising increasing expression in a plant of a nucleic acid encoding a POI polypeptide and targeting the POI polypeptide to plastid(s), and optionally selecting for plants having one or more enhanced yield-related traits.
  • the present invention provides a method for producing plants having one or more enhanced yield-related traits relative to control plants, wherein said method comprises the steps of increasing expression in said plant of a nucleic acid encoding a POI polypeptide as described herein and targeting the POI polypeptide to plastid(s), and optionally selecting for plants having one or more enhanced yield-related traits.
  • a preferred method for increasing expression of a nucleic acid encoding a POI polypeptide and targeting of the POI polypeptide to the plastid(s) is by introducing and expressing in a plant a nucleic acid encoding a POI polypeptide with artificial plastid targeting.
  • any reference hereinafter to a "protein useful in the methods of the invention” is taken to mean a POI polypeptide as defined herein.
  • Any reference hereinafter to a “nucleic acid use- ful in the methods of the invention” is taken to mean a nucleic acid capable of encoding such a POI polypeptide.
  • any reference to a protein or nucleic acid "useful in the methods of the invention” is to be understood to mean proteins or nucleic acids "useful in the methods, constructs, plants, harvestable parts and products of the invention”.
  • the nucleic acid to be introduced into a plant is any nucleic acid encoding the type of protein which will now be described, hereafter also named "POI nucleic acid” or "POI gene”.
  • a "POI polypeptide” as defined herein refers to any polypeptide being a Glutamine synthetase enzyme (enzyme class EC 6.3.1.2; abbreviated throughout this application as GS), preferably comprising the PFAM domains PF03951 and / or PF00120 using program "hmmscan" from the HMMer 3.0 software collection to search the high quality section "PFAM -A" of Pfam release 27.0 of the Welcome Trust SANGER Institute, Hinxton, England, UK (http://pfam.sanger.ac.uk/).
  • the GS comprises any, preferably all the domains as listed in table B1 when the sotware InterProScan ((see Zdobnov E.M.
  • GS polypeptide is grouped in the class II of Glutamine synthetases when using the methods of the art.
  • GS poylpeptides from non-plant sources, preferably from fungal sources, and more preferably from Saccharomycetales and most preferably from the family of Saccharomycetaceae.
  • the GS polypeptide is not targeted to plastid in its natural form, for example when analysed with the software TargetP 1.1 as described in example 5, and said GS polypeptide is then operably linked to a plastid targeting signal by recombinant means.
  • plastid targeting by recombinant means and artificial plastid targeting are used interchangeably, and both indicate a non-naturally occurring combination of the GS polypeptide and a transit peptide for plastid import, in that the GS polypeptide is fused, typically at its N-terminal end, to a transit peptide for plastid import not normally operably linked to the GS polypeptide and allowing for the import of the GS polypeptide into one or more plastids of the plant cell.
  • a method for improving yield-related traits as provided herein in plants relative to control plants comprising increasing expression in a plant of a nucleic acid encoding a GS polypeptide as defined herein, wherein the GS poly- peptide has an artificial plastid targeting.
  • said one or more enhanced yield- related traits comprise increased yield relative to control plants, and preferably comprise increased biomass and/or increased seed yield relative to control plants, and more preferably comprise increased aboveground biomass and/or increased below-ground biomass, and/or increased sugar yield (as harvestable sugar per plant, per fresh weight, per dry weight and/or per area) relative to control plants.
  • Increased sugar yield may be due to increased sugar content and/or increased sugar concentration per plant, per fresh weight, per dry weight and/or per area.
  • the sugar yield of only the harvestable parts, more preferably the aboveground harvestable parts optionally excluding seed and/or the below-ground harvestable parts is increased.
  • the increased sugar yield is an increased yield of sucrose, and/or hexose sugar(s), more preferably glucose and/or fructose.
  • the sugar yield is meant to be the yield of aldohexoses, preferably the yield of glucose and/or fructose, more preferably the yield of glucose.
  • the sugar content is meant to be the content of aldohexoses, preferably the content of glucose and/or fructose, more preferably the yield of glucose.
  • the above-ground biomass and the yield of glucose are increased com- pared to control plants under non-stress conditions.
  • nucleic acid sequences employed in the methods, constructs, plants, harvestable parts and products of the invention are provided.
  • nucleic acid molecule selected from the group consisting of:
  • nucleic acid encoding a GS polypeptide having in increasing order of preference at least 50%, 51 %, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%,
  • nucleic acid molecule which hybridizes with a nucleic acid molecule of (i) to (iii) un- der high stringency hybridization conditions and preferably confers one or more enhanced yield-related traits relative to control plants wherein the encoded polypeptide has substantially the same biological activity as the polypeptide of SEQ ID NO: 2;
  • nucleic acid molecule encoding a polypeptide selected from the group consisting of:
  • an amino acid sequence having, in increasing order of preference, at least 50%, 51 %, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence represented by SEQ
  • polypeptide has substantially the same biological activity as the polypep- tide of SEQ ID NO: 2;
  • nucleic acid sequences encode a GS polypeptide as defined herein and operably linked to the GS polypeptide a plastid targeting sequence, wherein the plastid targeting sequence is not naturally in operable linkage to the GS polypeptide.
  • nucleic acid molecules useful in the methods of the invention are those listed in Tables I as lead or homologue, or those encoding the protein sequences listed in tables II as lead or homologues, or those comprising the consensus sequence and the patterns shown in table IV.
  • the terms "GS encoding nucleic acid”, “GS nucleic acid”, “GS gene”, “GS nucleotide sequence” and “GS encoding nucleotide sequence” are used interchangeably herein.
  • polypeptide comprises one or more motifs and/or domains as defined elsewhere herein.
  • Motifs 1 to 8 were derived 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, California, 1994), see example 4 for details. 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.
  • the GS polypeptide as used herein comprises at least one of the motifs GS pattern 1 (SEQ ID NO: 42) to GS pattern 8 (SEQ ID NO: 49) as defined herein below, wherein in the letter-numbers combination
  • -x(a,b)- of any motif the letter x stands for Xaa , i.e. any amino acid, and the integer numbers a and b give the minimum and the maximum number of Xaa that may be found after the amino acid preceding the x.
  • S-x(0,3)-P indicates that following the amino acid Serine either one, two or three amino acids of any choice may be included before a Proline resi- due, or that no amino acid is to be found between the Serine and the Proline residue of this motif.
  • any amino acid residue(s) replacing -x may be identical to or different from the amino acid residue preceding or succeeding it, or any other amino acid inserted instead of the -x at the same or any other position.
  • Residues within square brackets represent alternatives, e.g the pattern Y-x(21 ,23)-[FW] means that a conserved tyrosine is separated by minimum 21 and maximum 23 amino acid residues from either a phenylalanine or tryptophane.
  • the GS polypeptide comprises
  • Motif 1 SEQ ID NO: 42
  • the GS polypeptide comprises in increasing order of preference, at least 2, at least 3, at least 4, at least 5 motifs in addition to the consensus sequence as defined above. Additionally or alternatively, the GS protein has in increasing order of preference at least 25%, 26%, 27%, 28%, 29%, 30%, 31 %, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41 %, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51 %, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%,
  • sequence identity level is determined by comparison of the polypeptide sequences over the entire length of the sequence of SEQ ID NO: 2.
  • sequence identity is determined by comparison of a nucleic acid sequence to the sequence encoding the mature protein in SEQ ID NO: 1.
  • sequence identity level of a nucleic acid sequence is determined by comparison of the nucleic acid sequence over the entire length of the coding sequence of the sequence of SEQ ID NO: 1.
  • sequence identity level is determined by comparison of one or more conserved domains or motifs in SEQ ID NO: 2 with corresponding conserved domains or motifs in other GS polypeptides. Compared to overall sequence identity, the sequence identity will generally be higher when only conserved domains or motifs are considered.
  • GS polypeptide comprises a conserved domain (or motif) with at least 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the conserved domain (or motif, respectively) starting with the amino acids and ending with the amino acid in SEQ ID NO:2 as shown below in table B1 and B2, respectively.
  • domain domain
  • signature and “motif” are defined in the "definitions” section herein.
  • polypeptide sequence which when used in the construction of a phylogenet- ic tree, such as the one depicted in Figure 4, clusters with the group of GS polypeptides comprising the amino acid sequence represented by SEQ ID NO: 2 rather than with any other group.
  • polypeptides of the invention when used in the construction of a phylogenetic tree cluster not more than 4, 3, or 2 hierarchical branch points away from the amino acid sequence of SEQ ID NO: 2.
  • nucleic acid sequences encoding GS polypeptides Another function of the nucleic acid sequences encoding GS polypeptides is to confer information for synthesis of the GS 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 the nucleic acid sequence represented by SEQ ID NO: 1 , encoding the polypeptide sequence of SEQ ID NO: 2.
  • performance of the invention is not restricted to these sequences; the methods of the invention may advantageously be performed using any GS-encoding nucleic acid or GS polypeptide as defined herein.
  • the term "GS" or “GS polypeptide” as used herein also intends to include homologues as defined hereunder of SEQ ID NO: 2.
  • nucleic acids encoding GS polypeptides are given in Table A of the Examples section herein. Such nucleic acids are useful in performing the methods of the invention.
  • the amino acid sequences given in Table A of the Examples section are example sequences of orthologues and paralogues of the GS polypeptide represented by SEQ ID NO: 2, the terms "orthologues” and “paralogues” being as defined herein. Further orthologues and paralogues may readily be identified by performing a so-called reciprocal blast search as described in the definitions section; where the query sequence is SEQ ID NO: 1 or SEQ ID NO: 2, the second BLAST (back-BLAST) would be against Saccharomyces cerevisiae sequences.
  • nucleic acid or a polypeptide sequence originating not from higher plants is used in the methods of the invention or the expression construct useful in the methods of the invention.
  • a nucleic acid or a polypeptide sequence of plant origin is used in the methods, constructs, plants, harvestable parts and products of the invention because said nucleic acid and polypeptides has the characteristic of a codon usage optimised for expression in plants, and of the use of amino acids and regulatory sites common in plants, respectively, but wherein the plant sequence does not naturally result in the polypeptide to be targeted to the plastid(s).
  • a sequence of fungal origin is used, since fungus are eukaryotes but have no plastids and hence the GS sequences from fungus have no natural plastid tar- geting sequence.
  • the fungus of origin may be any fungus, but preferably those fungi of the Saccharomycetales and most preferably from the family of Saccharomycetaceae.
  • a nucleic acid sequence originating not from higher plants but artificially altered to have the codon usage of higher plants is used in the expression construct useful in the methods of the invention.
  • any reference to one or more enhanced yield-related trait(s) is meant to exclude the restoration of the expression and / or activity of the GS polypeptide in a plant in which the expression and / or the activity of the GS polypeptide has been reduced or disabled when compared to the original wildtype plant or original variety.
  • the overexpression of the GS polypeptide in a knock-out mutant variety of a plant, wherein said GS polypeptide or an orthologue or paralogue has been knocked-out is not considered enhancing one or more yield-related trait(s) within the meaning of the current invention, when the expression level and /or the level of biological activity and / or the enzymatic activity level of the GS polypeptide is substantially the same as in the control plant, i.e. the non-mutant wildtype plant.
  • 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 "homo- logue” and “derivative” being as defined herein.
  • Also useful in the methods, constructs, plants, harvestable parts and products 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 prac- tising the methods of the invention are variants in which codon usage is optimised or in which miRNA target sites are removed.
  • nucleic acid variants useful in practising the methods of the invention include portions of nucleic acids encoding GS polypeptides, nucleic acids hybridising to nucleic acids encoding GS polypeptides, splice variants of nucleic acids encoding GS polypeptides, allelic variants of nucleic acids encoding GS polypeptides and variants of nucleic acids encoding GS polypeptides obtained by gene shuffling.
  • the terms hybridising sequence, splice variant, allelic variant and gene shuffling are as described herein.
  • Nucleic acids encoding GS 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.
  • a method for enhancing one or more yield-related traits in plants comprising introducing, preferably by recombinant methods, 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
  • Portions useful in the methods, constructs, plants, harvestable parts and products of the invention encode a GS 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.
  • 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.
  • the portion is at least 800, 850, 900, 950, 1000, 1050, 1 107, 1 1 10, 1 1 13, 1 1 16, 1 1 19 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.
  • the portion is a portion of the nucleic acid of SEQ ID NO: 1.
  • the portion encodes a fragment of an amino acid sequence which comprises motifs 1 to 8, and/or has biological activity of a glutamine synthetase without natural plastid targeting, and/or has at least 80% sequence identity to SEQ ID NO: 2.
  • nucleic acid variant useful in the methods, constructs, plants, harvestable parts and products of the invention is a nucleic acid capable of hybridising, under reduced stringency conditions, preferably under stringent conditions, with a nucleic acid encoding a GS polypeptide as defined herein, or with a portion as defined herein.
  • a method for enhancing one or more yield-related traits in plants comprising introducing, preferably by recombinant methods, 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 the complement of 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, constructs, plants, harvestable parts and products of the invention encode a GS polypeptide as defined herein, having substantially the same biological activity as the amino acid sequences given in Table A of the Examples section.
  • 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.
  • the hybridising sequence is capable of hybridising to the complement of a nucleic acid encoding the polypeptide as represented by SEQ ID NO: 2 or to a portion thereof.
  • the hybridization conditions are of medium stringency, preferably of high stringency, as defined herein.
  • the hybridising sequence encodes a polypeptide with an amino acid sequence which in the absence of an artificial plastid targeting sequence is not targeted to the plas- tid(s), and/ or comprises motifs 1 to 8, and/or has biological activity of glutamine synthetase, and/or has at least 80% sequence identity to SEQ ID NO: 2.
  • a method for enhancing one or more yield-related traits in plants comprising introducing, preferably by recombinant methods, 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, pa- ralogue 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 represented by SEQ ID NO: 1 , or a splice variant of a nucleic acid encoding an orthologue or paralogue of SEQ ID NO: 2.
  • the amino acid sequence encoded by the splice variant in the absence of an artificial plastid targeting sequence is not targeted to the plastid(s), and/ or comprises motifs 1 to 8, and/or has biological activity of glutamine synthetase, and/or has at least 80% sequence identity to SEQ ID NO: 2.
  • the amino acid sequence encoded by the allelic variant in the absence of an artificial plastid targeting sequence is not targeted to the plastid(s), and/ or comprises motifs 1 to 8, and/or has biological activity of glutamine synthetase, and/or has at least 80% se- quence identity to SEQ ID NO: 2.
  • polypeptide sequences useful in the methods, constructs, plants, harvestable parts and products of the invention have substitutions, deletions and/or insertions compared to the sequence of SEQ ID NO: 2, wherein the amino acid substitutions, insertions and/or deletions may range from 1 to 10 amino acids each.
  • the amino acid sequence encoded by the variant nucleic acid obtained by gene shuffling in the absence of an artificial plastid targeting sequence is not targeted to the plas- tid(s), and/ or comprises motifs 1 to 8, and/or has biological activity of glutamine synthetase, and/or has at least 80% sequence identity to SEQ ID NO: 2.
  • nucleic acid variants may also be obtained by site-directed mutagenesis.
  • 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.).
  • GS polypeptides differing from the sequence of SEQ ID NO: 2 by one or several amino acids (substitution ⁇ ), 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 GS 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.
  • the GS polypeptide- encoding nucleic acid is from a fungus, more preferably fungi of the Saccharomycetales and even more preferably from the family of Saccharomycetaceae and most preferably from Saccharomyces cerevisiae.
  • nucleic acid sequences coding for the proteins as shown in table II, column 4 (SEQ ID NO: 2), and its homologs as disclosed in table I, columns 5 can be joined to a nucleic acid sequence encoding a transit peptide.
  • This nucleic acid sequence encoding a transit peptide generally ensures transport of the protein to the respective organelle, especially the plastid.
  • the nucleic acid sequence of the gene to be expressed and the nucleic acid sequence encoding the transit peptide are operably linked. Therefore the trans- it peptide is fused in frame to the nucleic acid sequence coding for GS protein, e.g. SEQ ID NO: 2 or any of its homologs SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 and 38.
  • organelle shall mean preferably “plastid” (throughout the specification the "plural” shall comprise the “singular” and vice versa).
  • plastid according to the invention are intended to include various forms of plastids including pro- plastids, chloroplasts, chromoplasts, gerontoplasts, leucoplasts, amyloplasts, elaioplasts and etioplasts, preferably chloroplasts. They all have as a common ancestor the aforementioned proplasts.
  • Transit peptide sequences which are used in the inventive process and which form part of the inventive nucleic acid sequences are generally enriched in hydroxylated amino acid residues (serine and threonine), with these two residues generally constituting 20 to 35 % of the total. They often have an amino-terminal region empty of Gly, Pro, and charged residues. Furthermore they have a number of small hydrophobic amino acids such as valine and alanine and generally acidic amino acids are lacking. In addition they generally have a middle region rich in Ser, Thr, Lys and Arg. Overall they have very often a net positive charge.
  • nucleic acid sequences coding for the transit peptides may be chemically synthesized either in part or wholly according to structure of transit peptide sequences disclosed in the prior art.
  • Said natural or chemically synthesized sequences can be directly linked to the sequences encoding the mature protein or via a linker nucleic acid sequence, which may be typically less than 500 base pairs, preferably less than 450, 400, 350, 300, 250 or 200 base pairs, more preferably less than 150, 100, 90, 80, 70, 60, 50, 40 or 30 base pairs and most preferably less than 25, 20, 15, 12, 9, 6 or 3 base pairs in length and are in frame to the coding sequence.
  • nucleic acid sequences encoding transit peptides may comprise sequences derived from more than one biological and/or chemical source and may include a nucleic acid sequence derived from the amino-terminal region of the mature protein, which in its native state is linked to the transit peptide.
  • said amino-terminal region of the mature protein is typically less than 150 amino acids, preferably less than 140, 130, 120, 1 10, 100 or 90 ami- no acids, more preferably less than 80, 70, 60, 50, 40, 35, 30, 25 or 20 amino acids and most preferably less than 19, 18, 17, 16, 15, 14, 13, 12, 1 1 or 10 amino acids in length. But even shorter or longer stretches are also possible.
  • target sequences which facilitate the transport of proteins to other cell compartments such as the vacuole, endoplasmic reticulum, Golgi complex, glyoxysomes, peroxisomes or mitochondria may be also part of the inventive nucleic acid sequence.
  • the proteins translated from said inventive nucleic acid sequences are a kind of fusion proteins that means the nucleic acid sequences encoding the transit peptide, for example the ones shown in table 0, for example the last one of the table, are joint to any of the nucleic acid sequences shown in table I, columns 4 and 5.
  • the person skilled in the art is able to join said sequences in a functional manner.
  • the transit peptide part is cleaved off from the protein part shown in table II, columns 4 and 5 during the transport preferably into the plastids.
  • all products of the cleavage of the preferred transit peptide shown in the line 18 of table 0 have preferably the N-terminal amino acid sequences QIA CSS or QIA EFQLTT in front of the start methionine of the protein mentioned in table II, columns 4 and 5.
  • Other short amino acid sequences of an range of 1 to 20 amino acids preferable 2 to 15 amino acids, more preferable 3 to 10 amino acids most preferably 4 to 8 amino acids are also possible in front of the start methionine of the protein mentioned in table II, columns 4 and 5.
  • nucleic acids of the invention can directly be introduced into the plastid genome. Therefore in a preferred embodiment the nucleic acid sequences shown in table I, columns 4 and 5 are directly introduced and expressed in plastids.
  • the term "introduced” means the insertion of a nucleic acid sequence into the organism by means of a transfection, transduction or preferably by transformation.
  • a plastid such as a chloroplast
  • a plastid has been "transformed” by an exogenous (preferably foreign) nucleic acid sequence if nucleic acid sequence has been introduced into the plastid that means that this sequence has crossed the membrane or the membranes of the plastid.
  • the foreign DNA may be integrated (covalently linked) into plastid DNA making up the ge- nome of the plastid, or it may remain not integrated (e.g., by including a chloroplast origin of replication).
  • "Stably" integrated DNA sequences are those, which are inherited through plastid replication, thereby transferring new plastids, with the features of the integrated DNA sequence to the progeny.
  • a preferred method is the transformation of microspore-derived hypocotyl or cotyledonary tissue (which are green and thus contain numerous plastids) leaf tissue and afterwards the regeneration of shoots from said transformed plant material on selective medium.
  • methods for the transformation bombarding of the plant material or the use of independently replicating shuttle vectors are well known by the skilled worker. But also a PEG-mediated transformation of the plastids or Ag- robacterium transformation with binary vectors is possible.
  • Useful markers for the transformation of plastids are positive selection markers for example the chloramphenicol-, streptomycin-, kanamycin-, neomycin-, amikamycin-, spectinomycin-, triazine- and/or lincomycin- tolerance genes.
  • an activity disclosed herein as being conferred by a polypeptide shown in table II is increase or generated by linking the polypeptide disclosed in table II or a polypeptide conferring the same said activity with an targeting signal as herein described.
  • the polypeptide described can be linked to the targeting signal of the Spinach FNR (SEQ ID NO: 57).
  • the method of the invention for producing a transgenic plant with increased yield as compared to a corresponding, e.g. non-transformed, wild type plant comprising transforming a plant cell or a plant cell nucleus or a plant tissue with the mentioned nucleic acid molecule, said nucleic acid molecule selected from said mentioned group encodes for a polypeptide conferring said activity being linked to a targeting signal as mentioned herein.
  • Reporter genes are for example ⁇ -galactosidase-, p-glucuronidase-(GUS), alkaline phosphatase- and/or green-fluorescent protein-genes (GFP).
  • GUS p-glucuronidase
  • GFP green-fluorescent protein-genes
  • a further embodiment of the invention relates to the use of so called "chloroplast localization sequences", in which a first RNA sequence or molecule is capable of transporting or “chaperoning" a second RNA sequence, such as a RNA sequence transcribed from the sequences depicted in table I, columns 4 and 5 or a sequence encoding a protein, as depicted in table II, columns 4 and 5, from an external environment inside a cell or outside a plastid into a chloroplast.
  • the chloroplast localization signal is substantially similar or complementary to a complete or intact viroid sequence.
  • the chloroplast localization signal may be encoded by a DNA sequence, which is transcribed into the chloroplast localization RNA.
  • viroid refers to a naturally occurring single stranded RNA molecule (Flores, C. R. Acad Sci III. 324 (10), 943 (2001 )). Viroids usually contain about 200-500 nu- cleotides and generally exist as circular molecules. Examples of viroids that contain chloroplast localization signals include but are not limited to ASBVd, PLMVd, CChMVd and ELVd.
  • the viroid sequence or a functional part of it can be fused to the sequences depicted in table I or a sequence encoding a protein, as depicted in table II in such a manner that the viroid sequence transports a sequence transcribed from a sequence as depicted in table I, or a sequence encoding a protein as depicted in table II into the chloroplasts.
  • a preferred embodiment uses a modified ASBVd (Navarro et al., Virology. 268 (1 ), 218 (2000)).
  • the protein to be expressed in the plastids such as the proteins depicted in table II, columns 4 and 5, are encoded by different nucleic acids.
  • WO 2004/040973 teaches a method, which relates to the translocation of an RNA corresponding to a gene or gene fragment into the chloroplast by means of a chloroplast localization sequence.
  • the genes, which should be expressed in the plant or plants cells, are split into nucleic acid fragments, which are introduced into different compartments in the plant e.g. the nucleus, the plastids and/or mitochondria.
  • the chloroplast contains a ribozyme fused at one end to an RNA encoding a fragment of a protein used in the inventive process such that the ribozyme can trans-splice the translocated fusion RNA to the RNA encoding the gene fragment to form and as the case may be reunite the nucleic acid fragments to an intact mRNA encoding a functional protein for ex- ample as disclosed in table II, columns 4 and 5.
  • nucleic acid sequences as shown in table I, col- umns 4 and 5, used in the inventive process are transformed into plastids, which are metabolic active.
  • plastids should preferably maintain at a high copy number in the plant or plant tissue of interest, most preferably the chloroplasts found in green plant tissues, such as leaves or cotyledons or in seeds.
  • nucleic acid sequences as shown in table I, columns 4 and 5 are introduced into an expression cassette using a preferably a promoter and terminator, which are active in plastids preferably a chloroplast promoter.
  • promoters include the psbA promoter from the gene from spinach or pea, the rbcL promoter, and the atpB promoter from corn.
  • inventive methods for enhancing one or more yield-related traits in plants as described herein comprising introducing, preferably by recombinant methods, and expressing in a plant the nucleic acid(s) as defined herein with artificial plastid targeting, and preferably the further step of growing the plants and optionally the step of harvesting the plants or part(s) thereof.
  • the present invention extends to recombinant chromosomal DNA comprising a nucleic acid sequence useful in the methods of the invention, wherein said nucleic acid sequence comprises a nucleic acid encoding a GS polypeptide as defined herein operably linked to a plastid targeting sequence by recombinant means and this nucleic acid sequence is present in the chromosomal DNA as a result of recombinant methods, but is not in its natural genetic environment.
  • the recombinant chromosomal DNA of the invention is comprised in a plant cell.
  • DNA comprised within a cell is better protected from degradation, damage and/or breakdown than a bare nucleic acid sequence.
  • a DNA construct comprised in a host cell for example a plant cell.
  • the invention relates to compositions comprising the recombinant chromosomal DNA of the invention and/or the construct of the invention, and a host cell, preferably a plant cell, wherein the recombinant chromosomal DNA and/or the con- struct are comprised within the host cell, preferably within a plant cell or a host cell with a cell wall.
  • said composition comprises dead host cells, living host cells or a mixture of dead and living host cells, wherein the recombinant chromosomal DNA and/or the construct of the invention may be located in dead host cells and/or living host cell.
  • the composition may comprise further host cells that do not comprise the recombinant chromosomal DNA of the invention or the construct of the invention.
  • the compositions of the invention may be used in processes of multiplying or distributing the recombinant chromosomal DNA and/or the construct of the invention, and or alternatively to protect the recombinant chromosomal DNA and/or the construct of the invention from breakdown and/or degradation as explained herein above.
  • the recombinant chromosomal DNA of the invention and/or the construct of the invention can be used as a quality marker of the compositions of the invention, as an indicator of origin and/or as an indication of producer.
  • the methods of the present invention may be performed under non-stress conditions.
  • 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.
  • the above-ground biomass and the yield of glucose are increased compared to control plants under non-stress conditions.
  • the methods of the present invention may be performed under stress conditions, preferably under abiotic stress conditions.
  • the methods of the present invention may be performed under stress condi- tions such as drought to give plants having increased yield, preferably increased above- ground biomass and sugar yield, relative to control plants.
  • the methods of the present invention may be performed under stress conditions such as nutrient deficiency to give plants having increased yield, preferably increased above-ground biomass and sugar 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.
  • the methods of the present invention may be performed under stress conditions such as salt stress to give plants having increased yield, preferably in- creased above-ground biomass and sugar yield, relative to control plants.
  • salt- stress is not restricted to common salt (NaCI), but may be any one or more of: NaCI, KCI, LiCI, MgCI2, CaCI2, amongst others.
  • 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, preferably increased above-ground biomass and sugar yield, relative to control plants.
  • the methods of the invention are performed using plants in need of increased abiotic stress-tolerance for example tolerance to drought, salinity and/or cold or hot temperatures and/or nutrient use due to one or more nutrient deficiency such as nitrogen deficiency.
  • Performance of the methods of the invention gives plants having one or more enhanced yield-related traits.
  • performance of the methods of the invention gives plants having increased biomass and/or increased sugar yield, especially increased above-ground biomass and increased hexose yield, preferably glucose yield, relative to control plants.
  • Yield means increased above-ground biomass and increased hexose yield, preferably glucose yield, relative to control plants.
  • sugar yield especially increased above-ground biomass and increased hexose yield, preferably glucose yield
  • the present invention thus provides a method for increasing biomass and/or increased sugar yield of plants relative to control plants, especially increased above-ground biomass and / or increased hexose yield, preferably glucose yield, relative to control plants which method comprises increasing expression in a plant of a nucleic acid encoding a GS polypeptide operably linked to a plastid targeting sequence by recombinant means as defined herein.
  • a preferred embodiment of the present invention are methods for simultaneously increasing biomass and increased sugar yield of plants relative to control plants, especially increased above-ground biomass and increased hexose yield, preferably glucose yield, relative to control plants which method comprises increasing expression in a plant of a nucleic acid encoding a GS polypeptide operably linked to a plastid targeting sequence by recombinant means
  • the methods of the invention involves the step of recombinant introduction into a plant or plant cell or plant part of the nucleic acid sequence that comprises a nucleic acid encoding a GS polypeptide as defined herein operably linked to a plastid targeting se- quence by recombinant means.
  • performance of the methods of the invention gives plants having an increased growth rate relative to control plants. Therefore, according to the present invention, there is provided a method for increasing the growth rate of plants, which method comprises increasing expression in a plant of a nucleic acid encoding a GS polypeptide as defined herein.
  • Performance of the methods of the invention results in plants having increased above- ground biomass, in particular stem biomass relative to the aboveground biomass, and in particular stem biomass of control plants, and/or increased root biomass relative to the root biomass of control plants and/or increased beet biomass relative to the beet biomass of control plants.
  • the sugar content in particular the content of sucrose and/or hexose sugars, more preferably the glucose content
  • the sugar content in the above-ground parts particularly stem (in particular of sugar cane plants) and/or in the be- lowground parts, in particular in roots including taproots and tubers, and/or in beets (in particular in sugar beets) is increased relative to the sugar content ((in particular the content of sucrose and/or hexose sugars, more preferably the glucose content) in corresponding part(s) of the control plant.
  • the sugar content in particular the content of su- erase and /or hexose sugars, more preferably the glucose content
  • the sugar content in the above-ground parts preferably excluding seed and particularly in stem (in particular of sugar cane plants) and simultaneously the aboveground biomass, preferably excluding seed, in particular stem biomass (in particular of sugar cane plants) is increased by the methods of the invention relative control plants.
  • Performance of the methods of the invention gives plants grown under non-stress conditions or under mild drought conditions increased yield-related traits, preferably increased above-ground biomass and/or sugar yield as defined herein, relative to control plants grown under comparable conditions. Therefore, according to the present invention, there is pro- vided a method for increasing yield-related traits, preferably increased above-ground biomass and/or sugar yield as defined herein, in plants grown under non-stress conditions or under mild drought conditions, which method comprises increasing expression in a plant of a nucleic acid encoding a GS polypeptide as defined herein operably linked to a plastid targeting sequence by recombinant means.
  • Performance of the methods of the invention gives plants grown under conditions of drought, increased yield-related traits, preferably increased above-ground biomass and/or sugar yield as defined herein, 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 increasing expression in a plant of a nucleic acid encoding a GS polypeptide as defined herein operably linked to a plastid targeting sequence by recombinant means.
  • 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, preferably increased above-ground biomass and / or sugar yield as defined herein, relative to control plants grown under comparable conditions. Therefore, according to the present invention, there is provided a method for increasing yield-related traits, preferably increased above-ground biomass and / or sugar yield as defined herein, in plants grown under conditions of nutrient deficiency, which method comprises increasing expression in a plant of a nucleic acid encoding a GS polypeptide as defined herein operably linked to a plastid tar- geting sequence by recombinant means.
  • Performance of the methods of the invention gives plants grown under conditions of salt stress, increased yield-related traits, preferably increased above-ground biomass and / or sugar yield as defined herein, relative to control plants grown under comparable conditions. Therefore, according to the present invention, there is provided a method for increasing yield-related traits, preferably increased above-ground biomass and / or sugar yield as defined herein, in plants grown under conditions of salt stress, which method comprises increasing expression in a plant of a nucleic acid encoding a GS polypeptide as defined herein operably linked to a plastid targeting sequence by recombinant means.
  • root biomass is increased, preferably beet and/or taproot biomass, more preferably in sugar beet plants, and optionally seed yield and/or above ground biomass are not increased.
  • above ground biomass is increased, preferably stem, stalk and/or sett biomass, more preferably in Poaceae, even more preferably in a Saccharum species, most preferably in sugarcane, and optionally seed yield, below-ground biomass and/or root growth is not increased.
  • the total harvestable sugar preferably glucose, fructose and/or sucrose, more preferably glucose
  • the invention also provides genetic constructs and vectors to facilitate introduction and/or expression in plants of nucleic acids encoding a GS polypeptide as defined herein operably linked to a plastid targeting sequence by recombinant means.
  • 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:
  • the nucleic acid encoding a GS polypeptide is as defined above.
  • control sequence and "termination sequence” are as defined herein.
  • the genetic construct of the invention is a plant expression construct, i.e. a genetic construct that allows for the expression of the nucleic acid encoding a GS polypeptide in a plant, plant cell or plant tissue after the construct has been introduced into this plant, plant cell or plant tissue, preferably by recombinant means.
  • the plant expression construct may for example comprise said nucleic acid encoding a GS polypeptide as defined herein operably linked to a plastid targeting sequence by recombinant means in functional linkage to a promoter and optionally other control sequences controlling the expression of said nucleic acid in one or more plant cells, wherein the promoter and optional the other control sequences are not natively found in functional linkage to said nucleic acid.
  • control sequence(s) including the promoter result in overexpression of said nucleic acid when the construct of the invention has been introduced into a plant, plant cell or plant tissue.
  • the genetic construct of the invention may be comprised in a host cell - for example a 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.
  • the invention furthermore provides plants or host cells transformed with a construct as described above.
  • the invention provides plants transformed with a construct as described above, which plants have increased yield-related traits as described herein.
  • the genetic construct of the invention confers increased yield or yield- related trait(s), preferably above ground biomass and sugar yield, to a plant when it has been introduced into said plant, which plant expresses the nucleic acid encoding the GS polypeptide with artificial plastid targeting comprised in the genetic construct and preferably resulting in increased abundance of the GS polypeptide, preferably in the plastid(s).
  • the genetic construct of the invention confers increased yield or yield- related trait(s) to a plant comprising plant cells in which the construct has been introduced, which plant cells express the nucleic acid encoding the GS polypeptide with artificial plastid targeting comprised in the genetic construct and preferably resulting in increased abundance of the GS polypeptide, preferably in the plastid(s).
  • the promoter in such a genetic construct may be a promoter not native to the nucleic acid described above, i.e. a promoter different from the promoter regulating the expression of the GS nucleic acid in its native surrounding.
  • nucleic acid encoding a GS polypeptide as defined herein operably linked to a plastid targeting sequence by recombinant means is in functional link- age to a promoter resulting in the expression of the GS nucleic acid in aboveground bio- mass preferably the leaves and shoot, more preferably the stem, of monocot plants, preferably Poaceae plants, more preferably Saccharum species plants.
  • the expression cassette or the genetic construct of the invention may be comprised in a host cell, plant cell, seed, agricultural product or plant.
  • sequence of interest is operably linked to one or more control sequences (at least to a promoter).
  • any type of promoter 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.
  • the constitutive promoter is preferably a ubiquitous constitutive promoter of medium to high strength.
  • the constitutive promoter is represented by a nucleic acid sequence substantially similar to the "super promoter" as disclosed in WO 95/14098, most preferably the constitutive promoter is the "super promoter". See the "Definitions” section herein for further examples of constitutive promoters.
  • Yet another embodiment relates to genetic constructs useful in the methods, vector con- structs, plants, harvestable parts and products of the invention wherein the genetic construct comprises the GS nucleic acid of the invention functionally linked to a promoter as disclosed herein above and further functionally linked to one or more of
  • NEENAs nucleic acid expression enhancing nucleic acids
  • a preferred embodiment of the invention relates to a nucleic acid molecule useful in the methods, constructs, plants, harvestable parts and products of the invention and encoding a GS polypeptide as defined herein operably linked to a plastid targeting sequence by recombinant means, under the control of a promoter as described herein above, wherein the NEENA, RENA and/or the promoter is heterologous to the GS nucleic acid molecule of the invention.
  • one or more terminator sequences may be used in the construct introduced into a plant.
  • terminator sequences may be suitable for use in performing the invention.
  • a preferred method for increasing expression of a nucleic acid encoding a GS polypeptide is by introducing, preferably by recombinant methods, and expressing in a plant a nucleic acid encoding a GS polypeptide as defined herein operably linked to a plastid targeting sequence by recombinant means; however the effects of performing the method, i.e. enhancing one or more 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 one or more enhanced yield-related traits relative to control plants, comprising introduction and expression in a plant of any nucleic acid encoding a GS polypeptide as defined herein op- erably linked to a plastid targeting sequence by recombinant means as defined herein.
  • the present invention provides a method for the production of transgenic plants having one or more enhanced yield-related traits, particularly increased biomass and sugar yield, which method comprises:
  • the introduction of the GS polypeptide-encoding nucleic acid is by recombinant methods.
  • the nucleic acid of (i) may be any of the nucleic acids capable of encoding a GS polypeptide with artificial plastid targeting as defined herein.
  • the nucleic acid encoding the GS polypeptide with artificial plastid targeting and to be introduced into the plant is an isolated nucleic acid or is comprised in a genetic construct as described herein.
  • the plant cell transformed by the method according to the invention is regenerable into a transformed plant.
  • 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.
  • the plant cells of the invention are plant cells that do not sustain themselves in an autotrophic way.
  • One example are plant cells that do not sustain themselves through photosynthesis by synthesizing carbohydrate and protein from such inorganic substances as water, carbon dioxide and mineral salt.
  • the invention relates to transgenic plant cells and / or transgenic plant parts of the invention wherein said plant cells and / or plant parts are non- propagatable.
  • the invention relates to dead plant cells comprising the construct, recombinant chromosomal DNA and / or polynucleotide and / or polypeptide of the invention. These dead cells can not be used to regenerate a plant and are not photosynthetically active.
  • 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.
  • transformation is described in more detail in the "definitions” section herein.
  • the methods of the invention are methods for the production of a transgenic Poaceae plant, preferably a Saccharum species plant, a transgenic part thereof, or a transgenic plant cell thereof, having one or more enhanced yield-related traits, preferably increased above-ground biomass and/or sugar yield, more preferably increased stem bio- mass and / or increased content of sucrose, fructose, glucose or a combination thereof, relative to control plants, comprises the steps of
  • 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 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 GS polypeptide with artificial plastid targeting 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 substantially the same genotypic and/or phenotypic characteristic(s) as those produced by the parent in the methods according to the invention.
  • the invention extends to seeds recombinantly comprising the expression cassettes of the invention, the genetic constructs of the invention, or the nucleic acids encoding the GS and/or the GS polypeptides with artificial plastid targeting as described above.
  • a plant grown from the seed of the invention will also show enhanced yield-related traits.
  • the invention also includes host cells containing an isolated nucleic acid encoding a GS polypeptide with artificial plastid targeting as defined above.
  • 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.
  • the plant cells of the invention overexpress the nucleic acid molecule of the invention.
  • the invention relates to a transgenic pollen grain comprising the construct of the invention and/or a haploid derivate of the plant cell of the invention.
  • the pollen grain of the invention can not be used to regenerate an intact plant without adding further genetic material and/or is not capable of photosynthesis, said pollen grain of the invention may have uses in introducing the enhanced yield-related trait into another plant by fertilizing an egg cell of the other plant using a live pollen grain of the invention, producing a seed from the fertilized egg cell and growing a plant from the resulting seed. Further pollen grains find use as marker of geographical and/or temporal origin.
  • Plants that are particularly useful in the methods of the invention include all plants which belong to the superfamily Viridiplantae, in particular monocoty- ledonous and dicotyledonous plants including fodder or forage legumes, ornamental plants, food crops, trees or shrubs.
  • the plant is a crop plant.
  • crop plants include but are not limited to chicory, carrot, cassava, trefoil, soybean, beet, sugar beet, sunflower, canola, alfalfa, rapeseed, linseed, cotton, tomato, potato, Stevia species such as but not limited to Stevia rebaudiana and tobacco.
  • the plant is a monocotyledonous plant. Examples of monocotyledonous plants include sugarcane.
  • the plant is a cereal.
  • cereals examples include rice, maize, wheat, barley, millet, rye, triticale, sorghum, emmer, spelt, einkorn, teff, milo and oats.
  • the plants of the invention or 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.
  • 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 harvestable parts of a plant such as, but not limited to seeds, leaves, fruits, flowers, stems, setts, sugarcane gems, roots, rhizomes, tubers and bulbs, which harvestable parts comprise a recombinant nucleic acid encoding a GS polypeptide with artificial plastid targeting as defined herein.
  • the invention furthermore relates to products derived or produced, preferably directly derived or directly produced, from one or more harvestable part(s) of such a plant, such as dry pellets, pulp pellets, pressed stems, setts, sugarcane gems, meal or powders, fibres, cloth, paper or cardboard containing fibres produced by the plants of the invention, oil, fat and fatty acids, carbohydrates - including starches, paper or cardboard containing carbohydrates produced by the plants of the invention -, sap, juice, molasses, syrup, chaff or proteins.
  • Preferred carbohydrates are starches, cellulose, molasses, syrup and / or sugars, preferably sucrose.
  • Also preferred products are residual dry fibers, e.g., of the stem (like bagasse from sugar cane after cane juice removal), molasses, syrups and / or filtercake, preferably from sugarcane and / or sugar beet.
  • Said products can be agricultural products.
  • the product comprises a recombinant nucleic acid encoding a GS polypeptide and/or a recombinant GS polypeptide for example as an indicator of the particular quality of the product.
  • the invention in another embodiment relates to anti-counterfeit milled seed, milled stem and/or milled root having as an indication of origin and/or as an indication of producer a plant cell of the invention and/or the construct of the invention, wherein milled root preferably is milled beet, more preferably milled sugar beet.
  • 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 stem, sett, sugarcane gem, root, beet and/or seeds.
  • 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.
  • Another embodiment of the invention relates to a method for producing feedstuff for bioreactors, fermentation processes or biogas plants, comprising a) growing the plants of the invention, b) removing the harvestable parts as described herein from the plants and c) producing feedstuff for bioreactors, fermentation processes or biogas plants.
  • the method of the invention is a method for producing alcohol(s) from plant material comprising a) growing the plants of the invention, b) removing the harvestable parts as described herein from the plants and c) optionally producing feedstuff for fermentation process, and d) - following step b) or c) - producing one or more alcohol(s) from said feedstuff or harvestable parts, preferably by using microorganisms such as fungi, algae, bacteria or yeasts, or cell cultures.
  • microorganisms such as fungi, algae, bacteria or yeasts, or cell cultures.
  • a typical example would be the production of ethanol using carbohydrate containing harvestable parts, for example corn seed, sugarcane stem parts or beet parts of sugar beet.
  • the product is produced from the stem of the transgenic plant.
  • the product is produced from the root, preferable taproot and/or beet of the plant.
  • the method of the invention is a method for the production of one or more polymers comprising a) growing the plants of the invention, b) removing the harvestable parts as described herein from the plants and c) producing one or more monomers from the harvestable parts, optionally involving intermediate products, d) producing one or more polymer(s) by reacting at least one of said monomers with other monomers or reacting said monomer(s) with each other.
  • the method of the invention is a method for the production of a pharmaceutical compound comprising a) growing the plants of the invention, b) removing the harvestable parts as described herein from the plants and c) producing one or more monomers from the harvestable parts, optionally involving interme- diate products, d) producing a pharmaceutical compound from the harvestable parts and/or intermediate products.
  • the method of the invention is a method for the production of one or more chemicals comprising a) growing the plants of the invention, b) removing the harvestable parts as described herein from the plants and c) producing one or more chemical building blocks such as but not limited to Acetate, Pyruvate, lactate, fatty acids, sugars, amino acids, nucleotides, carotenoids, terpenoids or steroids from the harvestable parts, optionally involving intermediate products, d) producing one or more chemicals) by reacting at least one of said building blocks with other building block or reacting said building block(s) with each other.
  • chemical building blocks such as but not limited to Acetate, Pyruvate, lactate, fatty acids, sugars, amino acids, nucleotides, carotenoids, terpenoids or steroids
  • the present invention is also directed to a product obtained by a method for manufacturing a product, as described herein.
  • the products produced by the manufacturing methods of the invention are plant products such as, but not limited to, a foodstuff, feedstuff, a food supplement, feed supplement, fibre, cosmetic or pharmaceutical.
  • the methods for production are used to make agricultural products such as, but not limited to, fibres, plant extracts, meal or presscake and other leftover material after one or more extraction processes, flour, proteins, amino acids, carbohydrates, fats, oils, polymers, vitamins, and the like.
  • Preferred carbohydrates are sugars, preferably su- erase.
  • the agricultural product is selected from the group consisting of 1 ) fibres, 2) timber, 3) plant extracts, 4) meal or presscake or other leftover material after one or more extraction processes, 5) flour, 6) proteins, 7) carbohydrates, 8) fats, 9) oils, 10) polymers e.g. cellulose, starch, lignin, lignocellulose, and 1 1) combinations and/or mixtures of any of 1 ) to 10).
  • the product or agricultural product does generally not comprise living plant cells, does comprise the expression cassette, genetic construct, protein and/or polynucleotide as described herein.
  • the polynucleotides and/or the polypeptides and/or the constructs of the invention are comprised in an agricultural product.
  • 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.
  • 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.
  • propagules of the plants of the invention such as but not limited to setts or gems of sugarcane, and / or
  • propagules of the plants of the invention such as but not limited to setts or gems of sugarcane, and / or
  • the protective covering is any kind of repository which allows safe-keeping of the material according to points 1 to 4 above.
  • the protective covering can be re-usable and / or re-sealable.
  • the protective covering can be of one-way nature and /or biodegradable.
  • the protective covering is a commercial package. More preferably, the protective covering is testa.
  • nucleic acids encoding GS polypeptides as described herein may find use in breeding programmes in which a DNA marker is identified which may be genetically linked to a GS polypeptide-encoding gene.
  • the nucleic acids/genes, or the GS 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 one or more enhanced yield-related traits as defined herein in the methods of the invention.
  • allelic variants of a GS polypeptide-encoding nucleic acid/gene may find use in marker-assisted breeding programmes.
  • Nucleic acids encoding GS 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.
  • the biomass, preferably above-ground biomass, and the content and /or yield of sugars, preferably hexoses, more preferably glucose is increased simulta- neously in the plants of the invention.
  • the biomass, preferably above-ground biomass, of the plant, preferably the green part excluding seeds is increased in the plant or part thereof and in the same plant or a part thereof the sugar, preferably hexose, more preferably glucose, content and/ or yield is increased compared to a control plant or the respective parts thereof.
  • the part of the plant of the invention with increased biomass, preferably above-ground biomass, and the part of the plant of the invention with increased sugars, preferably hexoses, more preferably glucose content and / or sugar, preferably hexose, more preferably glucose yield may be different parts, overlapping parts or identical parts of the plant of the invention. In a more preferred embodiment of the invention these parts are at least overlapping or substantially identical, further preferably largely identical or identical.
  • the total storage carbohydrate content of the plants of the invention, or parts thereof and in particular of the harvestable parts of the plant(s) is increased compared to control plant(s) and the corresponding plant parts of the control plants.
  • the total storage carbohydrate content and the content of individual groups or species of carbohydrates may be measured in a number of ways known in the art.
  • the international application published as WO2006066969 discloses in paragraphs [79] to [117] a method to determine the total storage carbohydrate content of sugarcane, including fructan content.
  • the following method can be used for sugar content analysis:
  • the stalk discs are first comminuted in a Waring blender (from Waring, New Hartford, Connecticut, USA). The sugars are extracted by shaking for one hour at 95°C in 10 mM sodium phosphate buffer pH 7.0. Thereafter, the solids are removed by filtration through a 30 ⁇ sieve. The resulting solution is subsequently employed for the sugar determination (see herein below).
  • a Waring blender from Waring, New Hartford, Connecticut, USA.
  • the sugars are extracted by shaking for one hour at 95°C in 10 mM sodium phosphate buffer pH 7.0. Thereafter, the solids are removed by filtration through a 30 ⁇ sieve. The resulting solution is subsequently employed for the sugar determination (see herein below).
  • the glucose, fructose and sucrose contents in the extract obtained in accordance with the sugar extraction method described above is determined photometrically in an enzyme as- say via the conversion of NAD+ (nicotinamide adenine dinucleotide) into NADH (reduced nicotinamide adenine dinucleotide).
  • NAD+ nicotinamide adenine dinucleotide
  • NADH reduced nicotinamide adenine dinucleotide
  • the glucose-6-phosphate is subsequently oxidized by the enzyme glucose-6-phosphate dehydrogenase to give 6- phosphogluconate.
  • NAD+ is reduced to give NADH, and the amount of NADH formed is determined photometrically.
  • the ratio between the NADH formed and the glucose present in the extract is 1 :1 , so that the glucose content can be calculated from the NADH content using the molar absorption coefficient of NADH (at 340 nm 6.2 per mmol and per cm lightpath).
  • fructose-6- phosphate which has likewise formed in the solution, is converted by the enzyme phos- phoglucoisomerase to give glucose-6-phosphate which, in turn, is oxidized to give 6- phosphogluconate.
  • the ratio between fructose and the amount of NADH formed is 1 :1.
  • sucrose present in the extract is cleaved by the enzyme sucrase (Megazyme) to give glucose and fructose.
  • the glucose and fructose molecules liberated are then converted with the abovementioned enzymes in the NAD+-dependent reaction to give 6-phosphogluconate.
  • the conversion of one sucrose molecule into 6- phosphogluconate results in two NADH molecules.
  • the amount of NADH formed is likewise determined photometrically and used for calculating the sucrose content, using the molar absorption coefficient of NADH.
  • transgenic sugarcane plants may be analysed using any method known in the art for example but not limited to:
  • the storage carbohydrate content of sugar beet may be determined by any of methods described for sugarcane above with adaptations to sugar beet.
  • transgenic sugar beet plants may be analysed for biomass or their sugar content or other phenotypic parameters using any method known in the art for example but not limited to:
  • the present invention relates to the following specific embodiments, wherein the expression "as defined in claim/item X” is meant to direct the artisan to apply the definition as disclosed in item/claim X.
  • a nucleic acid as defined in item 1 has to be understood such that the definition of the nucleic acid as in item 1 is to be applied to the nucleic acid.
  • the term "as defined in item” or “as defined in claim” may be replaced with the corresponding definition of that item or claim, respectively:
  • a method for increasing biomass and/or sugar content and/or sugar yield in plants relative to control plants comprising increasing expression in a plant of a nucleic acid encoding a glutamine synthetase (GS) polypeptide with artificial plastid targeting, wherein said GS polypeptide comprises the PFAM domains PF03951 and / or PF00120 using program "hmmscan" from the HMMer 3.0 software collection to search the high quality section "PFAM -A" of Pfam release 27.0.
  • GS glutamine synthetase
  • Method according to embodiment 1 wherein said increased expression is effected by introducing and expressing in a plant said nucleic acid encoding said GS polypeptide with artificial plastid targeting.
  • Method according to embodiment 1 or 2 wherein said increased biomass is increased above-ground biomass relative to control plants, and preferably is above-ground biomass of the green part excluding seed, further preferably is the stem biomass relative to control plants.
  • Method according to embodiment 1 or 3 wherein said increased sugar content and/or sugar yield is effected by increasing sucrose and/or hexose sugars in plants compared to control plants.
  • Method according to embodiment 1 or 4 wherein said increased sugar content and/or sugar yield is effected by increasing one or more aldohexose, preferably glucose in plants compared to control plants.
  • nucleic acid encoding a GS poylpeptide from a non-plant source preferably from fungal sources, and more preferably from Saccharomycetales and most preferably from the family of Sac- charomycetaceae.
  • nucleic acid encoding a GS encodes any one of the polypeptides listed in Table A or is a portion of such a nucleic acid, or a nucleic acid capable of hybridising with a complementary sequence of such a nucleic acid.
  • nucleic acid se- quence encodes an orthologue or paralogue of any of the polypeptides given in Table A.
  • nucleic acid sequence is selected from the group of nucleic acid sequences consisting of:
  • nucleic acid encoding a GS polypeptide having in increasing order of preference at least 50%, 51 %, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence represented by SEQ ID NO: 2 and additionally or alternatively comprising one or more motifs having in increasing order of preference at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the
  • nucleic acid molecule which hybridizes with a nucleic acid molecule of (i) to (iii) under high stringency hybridization conditions and wherein the encoded polypeptide has substantially the same biological activity as the polypeptide of SEQ ID NO: 2;
  • nucleic acid molecule comprising the consensus sequence of SEQ ID NO: 41 and encoding for a glutamine synthetase polypeptide
  • an amino acid sequence having, in increasing order of preference, at least 50%, 51 %, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence represented by SEQ ID NO: 2, and additionally or alternatively comprising one or more motifs having in increasing order of preference at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any one or more of the amino acid
  • Method according to any one of embodiments 1 to 13, wherein said nucleic acid is operably linked to a constitutive promoter, preferably to a medium strength constitutive promoter.
  • Plant, or part thereof, or plant cell obtainable by a method according to any one of embodiments 1 to 14, wherein said plant, plant part or plant cell comprises a recombinant nucleic acid encoding a GS polypeptide as defined in any of embodiments 1 and 8 to 13.
  • plants having increased biomass and/or sugar content and/or sugar yield preferably increased above-ground biomass and sucrose content, glucose content and/or fructose content and/or sucrose yield, glucose yield and/or fructose yield relative to control plants, and more preferably increased glucose content and/or glucose yield and/or increased above-ground biomass relative to control plants.
  • Method for the production of a transgenic plant having increased biomass and/or sugar content and/or sugar yield compared to control plants, above-ground biomass and sucrose content, glucose content and/or fructose content and/or sucrose yield, glucose yield and/or fructose yield relative to control plants, and more preferably increased glucose content and/or glucose yield and/or increased above-ground biomass relative to control plants, comprising:
  • transgenic plant or part thereof according to any of the previous em- bodiments or a transgenic plant cell derived therefrom, wherein said plant is a crop plant, such as beet, sugar beet 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.
  • a crop plant such as beet, sugar beet or alfalfa
  • a monocotyledonous plant such as sugarcane
  • a cereal such as rice, maize, wheat, barley, millet, rye, triticale, sorghum, emmer, spelt, einkorn, teff, milo or oats.
  • nucleic acid encoding a GS polypeptide as defined in any of embodiments 1 and 8 to 14 for increased biomass, preferably increased above-ground biomass relative to control plants, and preferably comprise above-ground biomass of the green part excluding seed, further preferably of the stem biomass relative to control plants.
  • a method for manufacturing a product comprising the steps of growing the plants ac- cording to embodiment 15 or 17 and producing said product from or by said plants; or parts thereof, including seeds, wherein the product comprises the nucleic acid encoding the GS polypeptide with artificial plastid targeting as defined in claims 1 or 8 to 13 and / or the GS polypeptide as defined in claims 1 or 8 to 13.
  • Composition comprising a Recombinant plant chromosomal DNA comprising the construct as defined in embodiment 16 and increased sugar content compared to a control composition comprising chromosomal DNA of a control plant.
  • a method for producing a transgenic propagule comprising the steps of (i) introducing into a plant the nucleic acid encoding an GS as defined in any of embodiments 1 and 8 to 14 or the construct as defined in embodiment 16; (ii) selecting a transgenic plant having increased biomass, preferably above-ground biomass and/or increased sugar content, preferably sucrose and/or hexoses content, more preferably glucose content, produced by comparing said transgenic plant with a control plant; (iii) growing the transgenic plant to produce a transgenic propagule, wherein the transgenic propagule comprises the nucleic acid or the construct.
  • Construct according to embodiment 16 preferably a plant expression construct com- prised in a host cell, preferably in a plant cell, more preferably in a crop plant cell.
  • a protective covering comprising
  • nucleic acid encoding the polypeptides as defined in any of embodiments 1 , 8 to 15and / or the polypeptides as defined in any of embodiments 1 , 8 to 15 and / or the constructs of embodiment 16 comprised in an agricultural product, and / or
  • reference to “a cell” can mean that at least one cell can be uti- lized.
  • 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.
  • the term “comprising” also encompasses the term “consisting of.
  • peptides amino acids in a polymeric form of any length, linked together by peptide bonds, unless mentioned herein otherwise.
  • nucleic acid sequence(s) refers to nucleotides, either ribonucleotides or deoxyribonucleotides or a combination of both, in a polymeric un- branched form of any length.
  • nucleotide refers to a nucleic acid building block consisting of a nucleobase, a pentose and at least one phosphate group.
  • nucleotide includes a nukleo- sidmonophosphate, nukleosiddiphosphate, and nukleosidtriphosphate.
  • 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 substantially the same and functional activity as the unmodified protein from which they are derived.
  • “Homologues” of a gene encompass nucleic acid sequences with nucleotide substitutions, deletions and/or insertions relative to the unmodified gene in question and having substantially the same activity and/or functional properties as the unmodified gene from which they are derived, or encoding polypeptides having substantially the same biological and/or functional activity as the polypeptide encoded by the unmodified nucleic acid sequence
  • Orthologues and paralogues are two different forms of homologues and encompass evolutionary concepts used to describe the ancestral relationships of genes or proteins. Paralogues are genes or proteins within the same species that have originated through duplication of an ancestral gene; orthologues are genes or proteins 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 or a removal of one or more nucleotides from a nucleic acid.
  • insertion refers to one or more amino acid residues being introduced into a predeter- mined site in a protein or to one or more nucleotides being introduced into a predetermined site in a nucleic acid sequence.
  • insertions may comprise N-terminal and/or C-terminal fusions as well as intra-sequence insertions of single or multiple amino acids.
  • insertions within the amino acid sequence will be smaller than N- or C- terminal fusions, of the order of about 1 to 10 residues.
  • 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 transcriptional activator as used in the yeast two-hybrid system
  • phage coat proteins phage coat proteins
  • glutathione S-transferase-tag glutathione S-transferase-tag
  • protein A maltose-binding protein
  • dihydrofolate reductase dihydrofolate reductase
  • 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 a-helical structures or ⁇ -sheet structures).
  • Amino acid substitutions are typically of single residues, but may be clustered depending upon functional constraints placed upon the polypeptide.
  • the amino acid substitutions are preferably conservative amino acid substitutions. Conservative substitution tables are well known in the art (see for ex- ample Creighton (1984) Proteins. W.H. Freeman and Company (Eds) and Table 1 below).
  • 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.
  • substitution mutations at predetermined sites in DNA are well known to those skilled in the art and include M 13 mutagenesis, T7-Gen in vitro mutagenesis (USB, Cleveland, OH), QuickChange Site Directed mutagenesis (Stratagene, San Diego, CA), 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” 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.
  • 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.
  • derivatives also include fusions of the naturally-occurring form of the protein with tagging peptides such as FLAG, HIS6 or thi- oredoxin (for a review of tagging peptides, see Terpe, Appl. Microbiol. Biotechnol. 60, 523- 533,
  • the term "functional fragment” refers to any nucleic acid or protein which comprises merely a part of the fulllength nucleic acid or fulllength protein, respectively, but still provides substantially the same function e.g. enhanced yield-related trait(s) when overexpressed or repressed in a plant respectively.
  • substantially the same functional activity or substantially the same function means that any homologue and/or fragment provide increased / enhanced yield-related trait(s) when expressed in a plant.
  • substantially the same functional activity or substantially the same function means at least 50%, at least 60%, at least 70%, at least 80 %, at least 90 %, at least 95%, at least 98 %, at least 99% or 100% or higher increased / enhanced yield-related trait(s) compared with the functional activity provided by the exogenous expression of the full- length POI encoding nucleotide sequence or the POI amino acid sequence.
  • 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 amino acid or nucleic acid sequences. For amino acid sequences 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).
  • 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).
  • Homo- logues 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 deter- mined 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.
  • 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.
  • Smith-Waterman algorithm is particularly useful (Smith TF, Waterman MS (1981) J. Mol. Biol 147(1 ); 195-7).
  • 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.
  • Computation of the E-value is well known in the art.
  • 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.
  • a “transit peptide” (or transit signal, signal peptide, signal sequence) is a short (3-60 amino acids long) peptide chain that directs the transport of a protein, preferably to organelles within the cell or to certain subcellular locations or for the secretion of a protein.
  • Transit peptides may also be called transit signal, signal peptide, signal sequence, targeting signals, or (subcellular) localization signals.
  • hybridisation 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 pro- cess 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).
  • 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.
  • 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 Tm 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.
  • 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:
  • T m 81.5°C + 16.6xlogi 0 [Na + ] a + 0.41x%[G/C b ] - 500x[L c ]- 1 - 0.61x% formamide
  • T m 79.8°C+ 18.5 (logi 0 [Na + ] a ) + 0.58 (%G/C b ) + 1 1.8 (%G/C b ) 2 - 820/L c
  • T m 22 + 1.46 (l 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 L length of duplex in base pairs.
  • 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.
  • 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%).
  • annealing temperature for example from 68°C to 42°C
  • formamide concentration for example from 50% to 0%
  • hybridisation typically also depends on the function of post-hybridisation washes.
  • 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.
  • 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.
  • typical high stringency hybridisation conditions for DNA hybrids longer than 50 nucleotides encompass hybridisation at 65°C in 1x SSC or at 42°C in 1x SSC and 50% formamide, followed by washing at 65°C in 0.3x SSC.
  • Examples of medium stringency hy- bridisation conditions for DNA hybrids longer than 50 nucleotides encompass hybridisation at 50°C in 4x SSC or at 40°C in 6x SSC and 50% formamide, followed by washing at 50°C in 2x 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 xSSC is 0.15M NaCI and 15mM sodium citrate; the hybridisation solution and wash solutions may additionally include 5x Denhardt's reagent, 0.5-1.0% SDS, 100 ⁇ g/ml denatured, fragmented salmon sperm DNA, 0.5% sodium pyrophosphate.
  • high stringency conditions mean hybridisation at 65°C in 0.1x SSC comprising 0.1 SDS and optionally 5x Denhardt's reagent, 100 ⁇ g/ml denatured, fragmented salmon sperm DNA, 0.5% sodium pyrophosphate, followed by the washing at 65°C in 0.3x SSC.
  • splice variant 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 variants are alternative forms of a given gene, located at substantially 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.
  • nucleic acid and / or protein refers to the nucleic acid and / or protein in question as found in a plant in its natural form (i.e., without there being any human intervention like recombinant DNA technology), but also refers to that same gene (or a substantially homologous nucleic acid/gene) in an isolated form subsequently (re) in transduced into a plant (a transgene).
  • 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.
  • exogenous nucleic acid or gene refers to a nucleic acid that has been introduced in a plant by means of recombinant DNA technology.
  • An "exogenous" nucleic acid can either not occur in this plant in its natural form, be different from the nucleic acid in question as found in the plant in its natural form, or can be identical to a nucleic acid found in the plant in its natural form, but not integrated within its natural genetic environment. The corresponding meaning of "exogenous” is applied in the context of protein expression.
  • a transgenic plant containing a transgene i.e., an exogenous nucleic acid
  • a transgenic plant according to the present invention includes an exogenous POI nucleic acid integrated at any genetic loci and optionally the plant may also include the endogenous gene within the natural genetic background.
  • 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): 1 151 -4; US patents 5,81 1 ,238 and 6,395,547).
  • “Expression cassette” as used herein is DNA capable of being expressed in a host cell or in an in-vitro expression system.
  • the DNA, part of the DNA or the arrangement of the genetic elements forming the expression cassette is artificial.
  • the skilled artisan is well aware of the genetic elements that must be present in the expression cassette in order to be successfully expressed.
  • the expression cassette comprises a sequence of interest to be expressed 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 trans- lational enhancers, one or more NEENA as described herein, and/or one or more RENA as described herein.
  • 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 for increased expression/overexpression.
  • 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 expression cassette may be integrated into the genome of a host cell and replicated together with the genome of said host cell- Construct / genetic construct
  • 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 construct / genetic construct is an expression construct and comprises one or more expression cassettes that may lead to overexpression (overexpression construct) or reduced expression of a gene of interest.
  • a construct may consist of an expression cassette.
  • the sequence(s) of interest is/are 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, one or more NEENA as described herein, and/or one or more RENA as described herein.
  • terminator and enhancer sequences 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 for increased expression/overexpression.
  • 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.
  • an origin of replication sequence that is required for maintenance and/or replication in a specific cell type.
  • Preferred origins of replication include, but are not limited to, the f 1 -ori and colE1.
  • the genetic con- struct 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.
  • DNA such as but, not limited to plasmids or viral DNA
  • a vector may be a construct or may comprise at least one construct.
  • a vector may replicate without integrating into the genome of a host cell, e.g. a plasmid vector in a bacterial host cell, or it may integrate part or all of its DNA into the genome of the host cell and thus lead to replication and expression of its DNA.
  • 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 vector comprises at least one expression cassette.
  • the one or more se- quence(s) 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, one or more NEENA as described herein and/or one or more RENA as described herein.
  • 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 ele- ments. Such sequences would be known or may readily be obtained by a person skilled in the art.
  • regulatory element means of effecting expression of the sequences to which they are associated.
  • promoter or "promoter sequence” 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 transcrip- tion of an operably linked nucleic acid.
  • 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 develop- mental and/or external stimuli, or in a tissue-specific manner.
  • additional regulatory elements i.e. upstream activating sequences, enhancers and silencers
  • 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.
  • 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.
  • the nucleic acid molecule must, as described herein, 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.
  • 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).
  • 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).
  • weak promoter is intended a promoter that drives expression of a coding sequence at a low level.
  • 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.
  • 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.
  • “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 or “functionally linked” is used interchangeably and, 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 direct transcription of the gene of interest.
  • the term "functional linkage” or “functionally linked” with respect to regulatory elements is to be understood as meaning, for example, the sequential arrangement of a regulatory ele- ment (e.g. a promoter) with a nucleic acid sequence to be expressed and, if appropriate, further regulatory elements (such as e.g., a terminator, NEENA as described herein or a RENA as described herein) in such a way that each of the regulatory elements can fulfil its intended function to allow, modify, facilitate or otherwise influence expression of said nucleic acid sequence.
  • a regulatory ele- ment e.g. a promoter
  • further regulatory elements such as e.g., a terminator, NEENA as described herein or a RENA as described herein
  • operble linkage or “operably linked” may be used.
  • the expression may result, depending on the arrangement of the nucleic acid sequences, in sense or antisense RNA.
  • Genetic control sequences such as, for example, enhancer se- quences, can also exert their function on the target sequence from positions which are further away, or indeed from other DNA molecules.
  • Preferred arrangements are those in which the nucleic acid sequence to be expressed recombinantly is positioned behind the sequence acting as promoter, so that the two sequences are linked covalently to each other.
  • the distance between the promoter sequence and the nucleic acid sequence to be expressed recombinantly is preferably less than 200 base pairs, especially preferably less than 100 base pairs, very especially preferably less than 50 base pairs.
  • the nucleic acid sequence to be transcribed is located behind the promoter in such a way that the transcription start is identical with the desired beginning of the RNA of the invention.
  • Functional linkage, and an expression construct can be generated by means of customary recombination and cloning techniques as described (e.g., in Maniatis T, Fritsch EF and Sambrook J (1989) Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor (NY); Silhavy et al. (1984) Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor (NY); Ausubel et al. (1987) Current Protocols in Molecular Biology, Greene Publishing Assoc.
  • sequences which, for example, act as a linker with specific cleavage sites for restriction enzymes, or as a signal peptide, may also be positioned between the two sequences.
  • the insertion of sequences may also lead to the expression of fusion proteins.
  • the expression construct consisting of a linkage of a regulatory region for example a promoter and nucleic acid sequence to be expressed, can exist in a vector-integrated form and be inserted into a plant genome, for example by transformation.
  • 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.
  • 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 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 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.
  • 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”.
  • root-specific promoters examples are listed in Table 2b below:
  • ALF5 (Arabidopsis) Diener et al. (2001 , Plant Cell 13: 1625)
  • NRT2;1 Np N. plumbagini- Quesada et al. (1997, Plant Mol. Biol. 34:265)
  • 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 Bio- technol. J. 2, 1 13-125, 2004), which disclosure is incorporated by reference herein as if fully set forth.
  • a-amylase (Amy32b) Lanahan et al, Plant Cell 4:203-21 1 , 1992; Skriver et al,
  • 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.
  • green tissue-specific promoters which may be used to perform the methods of the invention are shown in Table 2g below.
  • 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.
  • terminal 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”, “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.
  • selectable marker genes include genes conferring resistance to antibiotics (such as nptll that phosphorylates neomycin and kanamycin, or hpt, phosphorylating hygromycin, or genes conferring resistance to, for example, bleomycin, streptomycin, tetracyclin, chloramphenicol, ampicillin, gentamycin, geneticin (G418), spec- tinomycin 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 xy- lose isomerase for the utilisation of xylose, or antinutritive markers such as the resistance to 2-deoxyglucose).
  • antibiotics such as npt
  • Visual marker genes results in the formation of colour (for example ⁇ -glucuronidase, GUS or ⁇ -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).
  • colour for example ⁇ -glucuronidase, GUS or ⁇ -galactosidase with its coloured substrates, for example X-Gal
  • luminescence such as the luciferin/luceferase system
  • fluorescence Green Fluorescent Protein
  • nucleic acid molecules encoding a selectable marker can be introduced into a host cell on the same vector that comprises the sequence encoding the polypeptides of the invention or used in the methods of the invention, or else in a separate vector. Cells which have been stably transfected with the introduced nucleic acid can be identified for example by selection (for example, cells which have integrated the selectable marker survive whereas the other cells die).
  • the 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 vec- tor 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.
  • the transformants In case of transformation with Agro- bacteria, 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.
  • 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 (ap- prox. 10%), the transposon jumps out of the genome of the host cell once transformation has taken place successfully and is lost.
  • the transposon jumps to a different location.
  • the marker gene must be eliminated by performing crosses.
  • techniques were developed which make possible, or facilitate, the detection of such events.
  • a further advantageous method relies on what is known as re- combination systems; whose advantage is that elimination by crossing can be dispensed with.
  • the best-known system of this type is what is known as the Cre/lox system. Cre1 is a recombinase that removes the sequences located between the loxP sequences. If the marker gene is integrated between the loxP sequences, it is removed once transformation has taken place successfully, by expression of the recombinase.
  • transgenic means with regard to, for example, a nucleic acid sequence, an expression cassette, genetic construct or a vector comprising the nucleic acid sequence or an organism transformed with the nu- cleic acid sequences, expression cassettes or vectors according to the invention, all those constructions brought about by recombinant methods in which either
  • 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.
  • 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 protein useful in the methods of the present invention, as defined above - becomes a recombinant expression cassette when this expression cassette is not integrated in the natural genetic environment but in a different genetic environment as a result of an isolation of said expression cassette from its natural genetic environment and re-insertion at a different genetic environment.
  • isolated nucle- ic acid or isolated polypeptide
  • 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 or cellular environment, respectively, and/or that has been modified by recombinant methods.
  • An isolated nucleic acid sequence or isolated nucleic acid molecule is one that is not in its native surrounding or its native nucleic acid neighbourhood, yet it is physically and functionally connected to other nucleic acid sequences or nucleic acid molecules and is found as part of a nucleic acid construct, vector sequence or chromosome.
  • 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.
  • 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.
  • the term "transgenic" relating to an organisms e.g. transgenic plant refers to an organism, e.g., a plant, plant cell, callus, plant tissue, or plant part that exogenously contains the nucleic acid, construct, vector or expression cassette described herein or a part thereof which is preferably introduced by processes that are not essentially biological, preferably by Agrobacteria-mediated transformation or particle bombardment.
  • a transgenic plant for the purposes of the invention is thus understood as meaning, as above, that the nucleic acids described herein are not present in, or not originating from the genome of said plant, or are present in the genome of said plant but not at their natural genetic environment in the genome of said plant, it being possible for the nucleic acids to be expressed homolo- gously or heterologously
  • 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 ex- pression may be of any kind of expression of a structural RNA (rRNA, tRNA) or mRNA with subsequent translation.
  • the original unmodulated expression may also be absence of any expression.
  • modulating the activity or the term “modulating expression” with respect to the proteins or nucleic acids used in the methods, constructs, expression cassettes, vectors, plants, seeds, host cells and uses of the invention shall mean any change of the expression of the inventive nucleic acid sequences or encoded proteins which leads to increased or decreased yield-related traits in 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 means the transcription of a specific gene or specific genes or specific genetic construct.
  • 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.
  • expression or “gene expression” can also include the translation of the mRNA and therewith the synthesis of the encoded protein, i.e., protein expression. I ncreased expression/overexpression
  • increased expression means any form of expression that is additional to the original wild-type expression level.
  • the original wild-type expression level might also be zero, i.e. absence of expression or immeasurable expression.
  • Reference herein to "increased expression”, “enhanced expression” or “overexpression” is taken to mean an increase in gene expression and/or, as far as referring to polypeptides, increased polypeptide levels and/or increased polypeptide activity, relative to control plants.
  • the increase in expression, polypeptide levels or polypeptide activity is in increasing order of preference at least 10%, 20%, 30%, 40% or 50%, 60%, 70%, 80%, 85%, 90%, or 100% or even more compared to that of control plants.
  • the increase in expression may be in increasing order of preference at least 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, 2000%, 3000%, 4000% or 5000% or even more compared to that of control plants.
  • polypeptide levels or polypeptide activity of the sequence in question and/or the recombinant gene is under the control of strong regulatory element(s) the increase in expression, polypeptide levels or polypeptide activity may be at least 100 times, 200 times, 300 times, 400 times, 500 times, 600 times, 700 times, 800 times, 900 times, 1000 times, 2000 times, 3000 times, 5000 times, 10 000 times, 20 000 times, 50 000 times, 100 000 times or even more compared to that of control plants.
  • 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 increase expression of a nucleic acid encoding the polypeptide of interest.
  • endogenous promoters may be altered in vivo by mutation, deletion, and/or substitution (see, Kmiec, US 5,565,350; Zarling et al., W09322443), 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.
  • polypeptide expression 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 oc- topine 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.
  • UTR 5' untranslated region
  • nucleic acid encoding this polypeptide is overexpressed in sense orientation with a polyad- enylation signal.
  • Introns or other enhancing elements may be used in addition to a promoter suitable for driving expression with the intended expression pattern.
  • overexpression of the same nucleic acid sequence as antisense construct will not result in increased expression of the protein, but decreased expression of the protein.
  • 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 compared to that of control plants.
  • 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, 1 1 , 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.
  • the stretch of substantially contiguous nucleotides is capable of forming hydrogen bonds with the target gene (either sense or anti- sense 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.
  • a preferred method for the reduction or substantial elimination of endogenous gene expression is by introducing, preferably by recombinant methods, 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).
  • 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
  • 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.
  • MAR matrix attachment region fragment
  • 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).
  • RISC RNA-induced silencing complex
  • the RISC further cleaves the mRNA transcripts, thereby substantially reducing the number of mRNA transcripts to be translated into polypeptides.
  • 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.
  • RNA-mediated silencing of gene expression is triggered in a plant by a double stranded RNA sequence (dsRNA) that is substantially similar to the target endog- enous gene.
  • dsRNA double stranded RNA sequence
  • 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.
  • RISC RNA- induced silencing complex
  • the double stranded RNA sequence corresponds to a target gene.
  • 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.
  • 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.
  • 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).
  • the antisense oligonucleotide sequence may be complementary to the region surrounding the translation start site of an mRNA transcript encoding a polypeptide.
  • 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.
  • an antisense nucleic acid 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 se- quences, e.g., phosphorothioate derivatives and acridine substituted nucleotides may be used.
  • modified nucleotides that may be used to generate the antisense nucleic acid sequences are well known in the art.
  • nucleotide modifications include methyla- tion, cyclization and 'caps' and substitution of one or more of the naturally occurring nucleotides with an analogue such as inosine.
  • 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).
  • 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.
  • 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 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.
  • antisense nucleic acid sequences can be modified to target selected cells and then administered sys- temically.
  • 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.
  • 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 ⁇ -units, the strands run paral- lei 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.
  • 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.
  • ribozymes e.g., hammerhead ribozymes (described in Haselhoff and Gerlach (1988) Nature 334, 585-591) can be used to catalyti- cally 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. Patent No. 4,987,071 ; and Cech et al. U.S. Patent No. 5,1 16,742).
  • 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 , 141 1 -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 97/381
  • 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).
  • insertion mutagenesis for example, T-DNA insertion or transposon insertion
  • 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 mu- tation on an isolated gene/nucleic acid subsequently introduced into a plant.
  • the reduction or substantial elimination may be caused by a non-functional polypeptide.
  • the polypeptide may bind to various interacting proteins; one or more mutation(s) and/or truncation ⁇ ) 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.
  • the regulatory region of the gene e.g., the promoter and/or enhancers
  • 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.
  • natural variants may also be used for example, to perform homologous recombination.
  • 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.
  • amiRNAs 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, 1 121-1 133, 2006).
  • the gene silencing techniques used for reducing expression in a plant of an endogenous gene requires the use of nucleic acid sequences from monocotyle- donous plants for transformation of monocotyledonous plants, and from dicotyledonous plants for transformation of dicotyledonous plants.
  • a nucleic acid sequence from any given plant species is introduced into that same species.
  • a nucleic acid sequence from rice is transformed into a rice plant.
  • 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 homol- ogy 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.
  • 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 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.
  • tissue targets include leaf disks, pollen, embryos, cotyledons, hy- pocotyls, megagametophytes, callus tissue, existing meristematic tissue (e.g., apical meri- stem, axillary buds, and root meristems), and induced meristem tissue (e.g., cotyledon me- ristem 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. Alterna- tively, 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.
  • 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.
  • Transformation of plant species is now a fairly routine technique.
  • 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.
  • Transgenic plants including transgenic crop plants, are preferably produced via Agrobacterium-med ⁇ ated transformation.
  • An advantageous transformation method is the transformation in planta.
  • agrobacteria 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).
  • 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) 871 1 ).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the plant material obtained in the transformation is, as a rule, subjected to selective conditions so that transformed plants can be distinguished from un- transformed plants.
  • 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.
  • the transformed plants are screened for the presence of a selectable marker such as the ones described herein.
  • putatively transformed plants may also be evalu- ated, for instance using Southern analysis, for the presence of the gene of interest, copy number and/or genomic organisation.
  • 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.
  • 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).
  • a plant, plant part, seed or plant cell transformed with - or interchangeably transformed by - a construct or transformed with or by a nucleic acid is to be understood as meaning a plant, plant part, seed or plant cell that carries said construct or said nucleic acid as a transgene due the result of an introduction of this construct or this nucleic acid by biotechnological means.
  • the plant, plant part, seed or plant cell therefore comprises this recombinant construct or this recombinant nucleic acid.
  • null-segregant any plant, plant part, seed or plant cell that no longer contains said recombinant construct or said recombinant nucleic acid after introduction in the past, is termed null-segregant, nullizygote or null control, but is not considered a plant, plant part, seed or plant cell transformed with said construct or with said nucleic acid within the meaning of this application.
  • T-DNA activation tagging 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.
  • a promoter may also be a translation enhancer or an intron
  • 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 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 expres- sion, 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.
  • 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.
  • 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 increased yield in rice), Water Use Efficiency (WUE), Nitrogen Use Efficiency (NUE), etc.
  • WUE Water Use Efficiency
  • NUE Nitrogen Use Efficiency
  • the term “one or more yield-related traits” is to be understood to refer to one yield-related trait, or two, or three, or four, or five, or six or seven or eight or nine or ten, or more than ten yield-related traits of one plant compared with a control plant.
  • Reference herein to "enhanced yield-related trait” is taken to mean an increase relative to control plants in a yield-related trait, for instance in early vigour and/or in biomass, of a whole plant or of one or more parts of a plant, which may include (i) aboveground parts, preferably aboveground harvestable parts, and/or (ii) parts below ground, preferably har- vestable parts below ground.
  • harvestable parts are roots such as taproots, stems, beets, tubers, leaves, flowers or seeds.
  • the tolerance of and / or the resistance to one or more agrochemicals by a plant is not considered a yield-related trait within the meaning of this term of the present application.
  • An altered tolerance of and / or the resistance to one or more agrochemicals by a plant, e.g. improved herbicide tolerance, is not an "enhanced yield-related trait" as used throughout this application.
  • 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.
  • 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 spike- lets encloses two fertile florets, one of them will usually mature into a maize kernel once fertilized.
  • a yield increase in maize may be manifested as one or more of the follow- ing: 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.
  • 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.
  • 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 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.
  • 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 grow- ing 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.
  • 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.
  • Biotic stress is understood as the negative impact done to plants by other living organisms, such as bacteria, viruses, fungi, nematodes, insects, other animals or other plants.
  • Biotic stresses are typically those stresses caused by pathogens, such as bacteria, viruses, fungi, plants, nematodes and insects, or other animals, which may result in negative effects on plant growth and/ or yield.
  • Abiotic stress is understood as the negative impact of non-living factors on the living plant in a specific environment.
  • Abiotic stresses or environmental stresses may be due to drought or excess water, anaerobic stress, salt stress, chemical toxicity, oxidative stress and hot, cold or freezing temperatures.
  • the "abiotic stress” may be an osmotic stress caused by a water stress, 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.
  • non-stress conditions 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 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. Increase/Improve/Enhance
  • the terms “increase”, “improve” or “enhance” in the context of a yield-related trait 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% increase in the yield-related trait(s) (such as but not limited to more yield and/or growth) in comparison to control plants as defined herein.
  • Increased seed yield may manifest itself as one or more of the following:
  • total seed weight 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;
  • TKW thousand kernel weight
  • 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.
  • 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 as used herein is intended to refer to the total weight of a plant or plant part. Total weight can be measured as dry weight, fresh weight or wet weight. 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, stem biomass, setts 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.
  • propagules such as seed.
  • any reference to "root” as biomass or as harvestable parts or as organ e.g. of increased sugar content is to be understood as a reference to harvestable parts partly inserted in or in physical contact with the ground such as but not limited to beets and other hypocotyl areas of a plant, rhizomes, stolons or creeping root- stalks, but not including leaves, as well as harvestable parts belowground, such as but not limited to root, taproot, tubers or bulbs.
  • aboveground parts or aboveground harvestable parts or above- ground biomass are to be understood as aboveground vegetative biomass not including seeds and/or fruits.
  • Such breeding programmes sometimes require introduction of allelic variation by mutagenic treatment of the plants, using for example EMS mutagenesis; alternatively, the programme may start with a collection of allelic variants of so called "natural" origin caused unintentionally. Identification of allelic variants then takes place, for example, by PCR. This is followed by a step for selection of superior allelic variants of the sequence in question and which give increased yield. Selection is typically carried out by monitoring growth performance of plants containing different allelic variants of the sequence in question. Growth performance may be monitored in a greenhouse or in the field. Further optional steps include crossing plants in which the superior allelic variant was identified with another plant. This could be used, for example, to make a combination of interesting phenotypic features.
  • nucleic acids encoding 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 EF 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.
  • MapMaker Large et al. (1987) Genomics 1 : 174-181
  • 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 nucleic acid probes may be used in direct fluorescence in situ hybridisation (FISH) mapping (Trask (1991 ) Trends Genet. 7:149-154).
  • FISH direct fluorescence in situ hybridisation
  • nucleic acid amplification-based methods for genetic and physical mapping may be carried out using the nucleic acids. Examples include allele-specific amplification (Kaza- zian (1989) J. Lab. Clin. Med 1 1 :95-96), polymorphism of PCR-amplified fragments (CAPS; Sheffield et al. (1993) Genomics 16:325-332), allele-specific ligation (Landegren et al.
  • nucleic Acid Res. 17:6795-6807 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.
  • 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.
  • 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 si- salana, Agropyron spp., Agrostis stolonifera, Allium spp., Amaranthus spp., Ammophila arenaria, Ananas comosus, Annona spp., Apium graveolens, Arachis spp, Artocarpus spp., Asparagus officinalis, Avena spp.
  • Avena sativa e.g. Avena sativa, Avena fatua, Avena byzantina, Avena fatua var. sativa, Avena hybrida
  • Averrhoa carambola e.g. Bambusa sp.
  • Benincasa hispida Bertholletia excelsea
  • Beta vulgaris Brassica spp.
  • Brassica napus e.g. Brassica napus, Brassica rapa ssp.
  • Glycine spp. e.g. Glycine max, Soja hispida or Soja max
  • Helianthus an- nuus Hemerocallis fulva
  • Hibiscus spp. Hordeum spp. (e.g. Hordeum vulgare), Ipomoea batatas, Juglans spp., Lactuca sativa, L a thyrus spp., /.ens culinaris, Linum usitatissimum, Litchi chinensis, Lotus spp., /.u/fa acutangula, Lupinus spp., Luzula sylvatica, Lycopersicon spp. (e.g.
  • control plants are 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.
  • 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.
  • Propagation material is any kind of organ, tissue, or cell of a plant capable of developing into a complete plant.
  • Propagation material can be based on vegetative reproduction (also known as vegetative propagation, vegetative multiplication, or vegetative cloning) or sexual reproduction.
  • Propagation material can therefore be seeds or parts of the non-reproductive organs, like stem or leave.
  • suitable propagation material can also be sections of the stem, i.e., stem cuttings (like setts or sugarcane gems).
  • Non-propagative material is any kind of organ, tissue, or cell of a plant not capable of developing into a complete plant; e. g., dead cells cannot be used to regenerate a plant.
  • a “stalk” is the stem of a plant belonging the Poaceae, and is also known as the “millable cane”. In the context of poaceae “stalk”, “stem”, “shoot”, or “tiller” are used interchangeably.
  • a “sett” is a section of the stem of a plant from the Poaceae, which is suitable to be used as propagation material. Synonymous expressions to “sett” are “seed-cane”, “stem cutting”, “section of the stalk”, and “seed piece”.
  • “Gem” or “sugarcane gem” is a part of the sugarcane stem that is cut, often in a round or oval shape with respect to the surface of the them stem, and contains part of a node of the stem, preferably with a meristem, and is suitable for regeneration of a sugarcane plant.
  • the plants used in the described experiments are used because Arabidopsis, tobacco, rice and corn plants are model plants for the testing of transgenes. They are widely used in the art for the relative ease of testing while having a good transferability of the results to other plants used in agriculture, such as but not limited to maize, wheat, rice, soybean, cotton, oilseed rape including canola, sugarcane, sugar beet and alfalfa, or other di- cot or monocot crops.
  • the present invention employs conventional techniques and methods of plant biology, molecular biology, bioinformatics and plant breedings.
  • Example 1 Identification of sequences related to SEQ ID NO: 1 and SEQ ID NO: 2
  • Sequences (full length cDNA, ESTs or genomic) related to SEQ ID NO: 1 and SEQ ID NO: 2 were identified amongst those maintained in the Entrez Nucleotides database at the National Center for Biotechnology Information (NCBI) using database sequence search tools, such as the Basic Local Alignment Tool (BLAST) (Altschul et al. (1990) J. Mol. Biol.
  • BLAST Basic Local Alignment Tool
  • 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.
  • the polypeptide encoded by the nucleic acid of SEQ I D NO: 1 was used for the TBLASTN algorithm, with default settings and the filter to ignore low complexity sequences set off.
  • the output of the analysis was viewed by pairwise comparison, and ranked according to the probability score (E-value), where the score reflect the probability that a particular alignment occurs by chance (the lower the E-value, the more significant the hit). In addition to E-values, comparisons were also scored by percentage identity.
  • Percent- age identity refers to the number of identical nucleotides (or amino acids) between the two compared nucleic acid (or polypeptide) sequences over a particular length.
  • the default parameters may be adjusted to modify the stringency of the search. For example the E-value may be increased to show less stringent matches. This way, short nearly exact matches may be identified.
  • Table A provides a list of nucleic acid sequences related to SEQ ID NO: 1 and SEQ ID NO: 2.
  • Eukaryotic Gene Orthologs EGO
  • TIGR The Institute for Genomic Research
  • EGO Eukaryotic Gene Orthologs
  • Special nucleic acid sequence databases have been created for particular organisms, e.g. for certain prokaryotic organisms, such as by the Joint Genome Institute.
  • access to proprietary databases has allowed the identification of novel nucleic acid and polypeptide sequences.
  • 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 were determined usingthe program "needle” from the EMBOSS software collection (The European Molecular Biology Open Software Suite ; http://www.ebi.ac.uk/Tools/psa/).
  • 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, PRO- SITE, 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 (the Welcome Trust SANGER Institute, Hinxton, England, UK
  • HMMER is a collection profile hidden Markov methods for protein sequence analysis developed by Sean Eddy and coworkers (HMMER web server: interactive sequence similarity searching R.D. Finn, J.
  • N-terminal domain (aa residues 22 to 101 of the YPR035W protein): Pfam entry PF03951 "Glutamine synthetase, beta-Grasp domain"; and
  • C-terminal domain (aa residues 107 to 352 YPR035W protein): Pfam entry PF00120 "Glutamine synthetase, catalytic domain”.
  • PROSITE PS00180 "Glutamine synthetase signature 1"
  • superfamily SSF54368 "Glutamine synthetase, N-terminal domain”
  • a GS polypeptide comprises a conserved domain (or motif) with 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 a conserved domain from the start amino acid to the end amino acid of said domain in in SEQ ID NO:2 as listed in table B1.
  • Pratt Prosite patterns for conserved domains were generated with the software tool Pratt version 2.1 or manually.
  • Pratt was developed by Inge Jonassen, Dept. of Informatics, University of Bergen, Norway and is described by Jonassen et al. (I.Jonassen, J.F.Collins and
  • the presence of motivs, given in the PROSITE pattern format, within a given polypeptide sequence can be identified with progam Fuzzpro, as implemented in the "The European Molecular Biology Open Software Suite” (EMBOSS), version 6.3.1.2 (Trends in Genetics 16 (6), 276 (2000)).
  • the consensus sequence of SEQ I D NO: 41 was derived using the multiple sequence alignments.
  • the consensus sequence was derived from a multiple alignment of the sequences as listed in table II .
  • the letters represent the one letter amino acid code and indicate that the amino acids are conserved in at least 80% of the aligned proteins, whereas the letter X stands for amino acids, which are not conserved in at least 80% of the aligned sequences.
  • the consensus sequence starts with the first conserved amino acid in the alignment, and ends with the last conserved amino acid in the alignment of the investigated sequences. The number of given X indicates the distances between conserved
  • Y-x(21 ,23)-F conserved tyrosine and phenylalanine residues in the alignment are separated from each other by minimum 21 and maximum 23 amino acid residues in the alignment of all investigated sequences.
  • conserved domains were identified from all sequences and are described using a subset of the standard Prosite notation, e.g the pattern Y-x(21 ,23)-[FW] means that a conserved tyrosine is separated by minimum 21 and maximum 23 amino acid residues from either a phenylalanine or trypto- phane. Patterns had to match at least 80% of the investigated proteins.
  • Table B2 Protein patterns (in prosite annotation) and boundaries of the respective pattern matches to the YPR035W protein (SEQ ID NO: 2): pattern Pattern-sequence Start End mis ⁇
  • a GS polypeptide comprises a motif with 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 of the six conserved motifs contained in SEQ ID NO: 2 as shown by their starting and end positions in table B2.
  • 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 was used as implemented in thecommercial software package "metalife” /version 5.3), but the algorithm is also publicly available: TargetP is maintained at the server of the Technical University of Denmark (see http://www.cbs.dtu.dk/services/TargetP/ & "Locating proteins in the cell using TargetP, SignalP, and related tools", Olof Emanuelsson, Soren Brunak, Gun- nar von Heijne, Henrik Nielsen, Nature Protocols 2, 953-971 (2007)) and also Swiss Institute of Bioinformatics (http://expasy.org/tools/).
  • 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).
  • TargetP 1.1 analysis of the polypeptide sequence as represented by SEQ ID NO: 2 and the other GS polypeptides as listed in table A showed that these are not naturally targeted to plastids.
  • 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;
  • the sequence as shown in SEQ ID NO: 1 was amplified by PCR as described in the protocol of the Pfu Ultra, Pfu Turbo or Herculase DNA polymerase (Stratagene).
  • the composition for the protocol of the Pfu Ultra, Pfu Turbo or Herculase DNA polymerase was as fol- lows: 1 x PCR buffer (Stratagene), 0.2 mM of each dNTP, 100 ng genomic DNA of Saccha- romyces cerevisiae (strain S288C; Research Genetics, Inc., now Invitrogen), 50 pmol forward primer, 50 pmol reverse primer, with or without 1 M Betaine, 2.5 u Pfu Ultra, Pfu Turbo or Herculase DNA polymerase.
  • the amplification cycles were as follows:
  • ORF specific primer pairs for the genes to be expressed are shown in table III , column 5.
  • the following adapter sequences were added to the Saccharomyces cerevisiae ORF specific primers (see table III) for cloning purposes:
  • Table V Overview of the different vectors used in the cloning process and their SEQ IDs (column A), their vector names (column B), the promoter contained for expression of the ORFs (column C), the additional artificial targeting sequence column D), the adapter sequence (column E), the expression type conferred by the promoter mentioned in column B
  • genomic DNA was extracted from leaves of 4 weeks old S. oleracea plants (DNeasy Plant Mini Kit, Qi- agen, Hilden). The gDNA was used as the template for a PCR.
  • the coding sequence is interrupted by an intronic sequence from bp 274 to bp 350: gcataaacttatcttcatagttgccactccaatttgctccttgaatctcctccacccaatacataatccactcctccatcaccc acttcactactaaatcaaacttaactctgttttttctctctcctctttcatttctttattcttccaatcatcgtactccgccatgaccac cgctgtttctttcccctaccaaaccacctctctctcgcccctaccaaaccacctctctctctccc
  • the PCR-product representing the amplified ORF with the respective adapter sequences and the vector DNA were treated with T4 DNA polymerase according to the stand- ard protocol (MBI Fermentas) to produce single stranded overhangs with the parameters 1 unit T4 DNA polymerase at 37°C for 2-10 minutes for the vector and 1 -2 u T4 DNA polymerase at 15-17°C for 10-60 minutes for the PCR product representing SEQ ID NO: 1.
  • reaction was stopped by addition of high-salt buffer and purified over QIAquick or Nu- cleoSpin Extract II columns following the standard protocol (Qiagen or Macherey-Nagel).
  • the ligated constructs were transformed in the same reaction vessel by addition of competent E. coli cells (strain DH5alpha) and incubation for 20 minutes at 1 °C followed by a heat shock for 90 seconds at 42°C and cooling to 1 -4°C. Then, complete medium (SOC) was added and the mixture was incubated for 45 minutes at 37°C. The entire mixture was subsequently plated onto an agar plate with 0.05 mg/ml kanamycin and incubated overnight at 37°C. The outcome of the cloning step was verified by amplification with the aid of primers which bind upstream and downstream of the integration site, thus allowing the amplification of the insertion. The amplifications were carried out as described in the protocol of Taq DNA pol- ymerase (Gibco-BRL).
  • the amplification cycles were as follows:
  • the plasmid preparation was carried out as specified in the Qiaprep or NucleoSpin Multi-96 Plus standard protocol (Qiagen or Macherey-Nagel).
  • the agrobacteria that contains the plasmid construct were then used for the transformation of plants.
  • a colony was picked from the agar plate with the aid of a pipette tip and taken up in 3 ml of liquid TB medium, which also contained suitable antibiotics as described above.
  • the preculture was grown for 48 hours at 28°C and 120 rpm.
  • dishes Piki Saat 80, green, provided with a screen bottom, 30 x 20 x 4.5 cm, from Wiesauplast, Kunststofftechnik, Germany
  • the dishes were watered overnight with 0.05% Proplant solution (Chimac-Apriphar, Belgium).
  • A. thali- ana C24 seeds (Nottingham Arabidopsis Stock Centre, UK ; NASC Stock N906) were scattered over the dish, approximately 1 000 seeds per dish.
  • the dishes were covered with a hood and placed in the stratification facility (8 h, 1 10 mol/m2s1 , 22°C; 16 h, dark, 6°C). After 5 days, the dishes were placed into the short-day controlled environment chamber (8 h, 130 ⁇ / ⁇ 25 ⁇ , 22°C; 16 h, dark, 20°C), where they remained for approximately 10 days until the first true leaves had formed.
  • the seedlings were transferred into pots containing the same substrate (Teku pots, 7 cm, LC series, manufactured by Poppelmann GmbH & Co, Germany). Five plants were pricked out into each pot. The pots were then returned into the short-day controlled environment chamber for the plant to continue growing.
  • the plants were transferred into the greenhouse cabinet (supplementary illu- mination, 16 h, 340 E/m2s, 22°C; 8 h, dark, 20°C), where they were allowed to grow for further 17 days.
  • the plants were subsequently placed for 18 hours into a humid chamber. Thereafter, the pots were returned to the greenhouse for the plants to continue growing. The plants remained in the greenhouse for another 10 weeks until the seeds were ready for harvesting.
  • the harvested seeds were planted in the greenhouse and subjected to a spray selection or else first sterilized and then grown on agar plates supplemented with the respective selection agent. Since the vector contained the bar gene as the resistance marker, plantlets were sprayed four times at an interval of 2 to 3 days with 0.02 % BASTA® and transformed plants were allowed to set seeds. The seeds of the transgenic A. thaliana plants were stored in the freezer (at -20°C).
  • the Agrobacterium containing the expression vector is used to transform Oryza sativa plants.
  • Mature dry seeds of the rice japonica cultivar Nipponbare are dehusked. Sterilization is carried out by incubating for one minute in 70% ethanol, followed by 30 minutes in 0.2% HgC , followed by a 6 times 15 minutes wash with sterile distilled water. The sterile seeds are then germinated on a medium containing 2,4-D (callus induction medium). After incubation in the dark for four weeks, embryogenic, scutellum-derived calli are excised and propagated on the same medium. After two weeks, the calli are multiplied or propagated by subculture on the same medium for another 2 weeks. Embryogenic callus pieces are sub- cultured on fresh medium 3 days before co-cultivation (to boost cell division activity).
  • Agrobacterium strain LBA4404 containing the expression vector is used for co-cultivation.
  • Agrobacterium is inoculated on AB medium with the appropriate antibiotics and cultured for 3 days at 28°C.
  • the bacteria are then collected and suspended in liquid co-cultivation medium to a density (OD6 00 ) of about 1.
  • the suspension is then transferred to a Petri dish and the calli immersed in the suspension for 15 minutes.
  • the callus tissues are then blotted dry on a filter paper and transferred to solidified, co-cultivation medium and incubated for 3 days in the dark at 25°C.
  • Co-cultivated calli are grown on 2,4-D-containing medium for 4 weeks in the dark at 28°C in the presence of a selection agent.
  • TO rice transformants Approximately 35 to 90 independent TO rice transformants are generated for one construct.
  • the primary transformants are 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 are kept for harvest of T1 seed. Seeds are then harvested three to five months after transplanting. The method yielded single locus transformants at a rate of over 50 % (Aldemita and Hodges1996, Chan et al. 1993, Hiei et al. 1994).
  • Transformation of maize 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 suc- cessfully as well.
  • Ears are harvested from corn plant approximately 1 1 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.
  • 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, Apdo. Postal 6- 641 06600 Mexico, D.F., 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 is transformed according to a modification of the method described in the Texas A&M patent US 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.
  • 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. Plants 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.
  • 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 (6- Benzylamino-'purine), 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.
  • MSBAP-3 medium containing 3 mg/l BAP, 3 % sucrose, 0.7 % Phytagar at 23 °C, 16 hr light.
  • MSBAP-3 medium containing 3 mg/l BAP (6- Benzylamino-'purine), cefotaxime, carbenicillin, or timentin (300 mg/l) for
  • the shoots 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.
  • MSBAP-0.5 shoot elongation medium
  • MS0 rooting medium
  • T1 seeds are produced from plants that exhibit tolerance to the selection agent and that contain a single copy of the T-DNA insert.
  • a regenerating clone of alfalfa (Medicago sativa) is transformed using the method of (McKersie et al., 1999 Plant Physiol 1 19: 839-847). Regeneration and transformation of alfalfa is genotype dependent and therefore a regenerating plant is required. Methods to obtain regenerating plants have been described. For example, these can be selected from the cultivar Rangelander (Agriculture Canada) or any other commercial alfalfa variety as described by Brown DCW and A Atanassov (1985. Plant Cell Tissue Organ Culture 4: 1 1 1 - 1 12).
  • 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 1 19: 839-847) or LBA4404 containing the expression vector. The ex- plants 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 K2S04, and 100 ⁇ acetosyringinone.
  • the explants are washed in half-strength Murashige-Skoog medium (Murashige and Skoog, 1962) and plat- ed 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 is transformed using Agrobacterium tumefaciens according to the method described in US 5, 159,135. Cotton seeds are surface sterilised in 3% sodium hypochlorite solution during 20 minutes and washed in distilled water with 500 ⁇ g/ml cefotaxime. The seeds are then transferred to SH-medium with 50 ⁇ g/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.
  • 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 ⁇ g/ml MgCL2, and with 50 to 100 ⁇ g/ml cefotaxime and 400- 500 ⁇ g/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 selec- tive 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 furfu- rylaminopurine 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.
  • 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, CA 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.
  • 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 30g/l sucrose plus 0,25mg/l benzylamino purine and 0,75% agar, pH 5,8 at 23-25°C with a 16- hour photoperiod.
  • a liquid LB culture including antibiotics is grown on a shaker (28°C, 150rpm) 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.
  • Plant base tissue is cut into slices (1.0 cm x 1.0 cm x 2.0 mm approximately). Tissue is immersed for 30s 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.
  • 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 W09623891A.
  • B5 vitamins (Gamborg, O., et al., 1968. Exp. Cell Res., vol. 50, 151 -8) supplemented with 20g/l sucrose, 500 mg/l casein hydroly- sate, 0,8% agar and 5mg/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, 150rpm) until an optical density (O.D.) at 600 nm of -0,6 is reached.
  • O.D. optical density
  • 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.
  • the induction of callus and the transformation of sugarcane can be carried out by the method of Snyman et al. (Snyman et al., 1996, S. Afr. J. Bot 62, 151 -154).
  • the construct can be cotransformed with the vector pEmuKN, which expressed the npt[pi] gene (Beck et al. Gene 19, 1982, 327-336; Gen-Bank Accession No. V00618) under the control of the pEmu promoter (Last et al. (1991 ) Theor. Appl. Genet. 81 , 581 -588). Plants are regenerated by the method of Snyman et al. 2001 (Acta Horticulturae 560, (2001), 105-108).
  • the latter were grown uniformly in a specific culture facility.
  • the GS-90 substrate as the compost mixture was introduced into the potting machine (Laible System GmbH, Singen, Germany) and filled into the pots. Thereafter, 35 pots were combined in one dish and treated with Previcur.
  • Previcur 25 ml of Previcur were taken up in 10 1 of tap water. This amount was sufficient for the treatment of approximately 200 pots.
  • the pots were placed into the Previcur solution and additionally irrigated overhead with tap water without Previcur. They were used within four days.
  • the seeds which had been stored in the refrigerator (at -20°C), were removed from the Eppendorf tubes with the aid of a toothpick and transferred into the pots with the compost. In total, approximately 5 to 12 seeds were distributed in the middle of the pot.
  • the dishes with the pots were covered with matching plastic hoods and placed into the stratification chamber for 4 days in the dark at 4°C.
  • the humidity was approximately 90%.
  • the test plants were grown for 22 to 23 days at a 16-h-light, 8-h-dark rhythm at 20°C, an atmospheric humidity of 60% and a CO2 concentration of approximately 400 ppm.
  • the light sources used were Powerstar HQI-T 250 W/D Daylight lamps from Osram, which generate a light resembling the solar color spectrum with a light intensity of approximately 220 ⁇ ⁇ 2 s _1 .
  • transgenic plants were depending on the used resistance marker.
  • bar gene as the resistance marker plantlets were sprayed three times at days 8-10 after sowing with 0.02 % BASTA®, (Glufosinate ammonium; Bayer CropScience, Germany,
  • the plants, which had grown best in the center of the pot were considered the target plants.
  • the plants received overhead irrigation with distilled water (onto the compost) and bottom irrigation into the placement grooves. Once the grown plants had reached the age of 23 or 24 days, they were harvested.
  • the aluminum rack with the plant samples in the extraction sleeves was placed into the pre- cooled (-40°C) lyophilization facility.
  • the initial temperature during the main drying phase was-35°C and the pressure was 0.120 mbar.
  • the parameters were altered following a pressure and temperature program.
  • the final temperature after 12 hours was +30°C and the final pressure was 0.001 to 0.004 mbar.
  • the system was flushed with air (dried via a drying tube) or argon.
  • the extraction sleeves with the lyophilized plant material were transferred into the 5 ml extraction cartridges of the ASE device (Accelerated Solvent Extractor ASE 200 with Solvent Controller and AutoASE software (prodced by DIONEX, available from Thermo Fisher Scientific Inc.; 81 Wyman Street; Waltham, MA 02454; USA)).
  • ASE device Accelerated Solvent Extractor ASE 200 with Solvent Controller and AutoASE software (prodced by DIONEX, available from Thermo Fisher Scientific Inc.; 81 Wyman Street; Waltham, MA 02454; USA)
  • the 24 sample positions of an ASE device (Accelerated Solvent Extractor ASE 200 with Solvent Controller and AutoASE software (DIONEX)) were filled with plant samples, including some samples for testing quality control.
  • the two solvent mixtures were extracted into the same glass tubes (centrifuge tubes, 50 ml, equipped with screw cap and pierceable septum for the ASE (DION EX)).
  • the solution was treated with commercial available internal standards, such as ribitol, L- glycine-2,2-d2, L-alanine-2,3,3,3-d4, methionine-d3, and a-methylglucopyranoside and methyl nonadecanoate, methyl undecanoate, methyl tridecanoate, methyl pentadecanoate, methyl nonacosanoate.
  • commercial available internal standards such as ribitol, L- glycine-2,2-d2, L-alanine-2,3,3,3-d4, methionine-d3, and a-methylglucopyranoside and methyl nonadecanoate, methyl undecanoate, methyl tridecanoate, methyl pentadecanoate, methyl nonacosanoate.
  • the total extract was treated with 8 ml of water.
  • the solid residue of the plant sample and the extraction sleeve were discarded.
  • the lipid extract, which had been evaporated to dryness was taken up in mobile phase.
  • the HPLC was run with gradient elution.
  • the LC part was carried out on a commercially available LCMS system from Agilent Technologies, USA.
  • polar extracts 10 ⁇ are injected into the system at a flow rate of 200 ⁇ / ⁇ .
  • the separation column (Reversed Phase C18) was maintained at 15 °C during chromatography.
  • lipid extracts 5 ⁇ are injected into the system at a flow rate of 200 ⁇ /min.
  • the separation column (Reversed Phase C18) was maintained at 30°C. HPLC was performed with gradient elution.
  • the methoximation of the carbonyl groups was carried out by reaction with methoxyamine hydrochloride (5 mg/ml in pyridine, 100 Dl for 1.5 hours at 60°C) in a tightly sealed vessel. 20 ⁇ of a solution of odd-numbered, straight-chain fatty acids (solution of each 0.3 mg/ml of fatty acids from 7 to 25 carbon atoms and each 0.6 mg/ml of fatty acids with 27, 29 and 31 carbon atoms in 3/7 (v/v) pyridine/toluene) were added as time standards.
  • the methoximation of the carbonyl groups was carried out by reaction with methoxyamine hydrochloride (5 mg/ml in pyridine, 50 Dl for 1.5 hours at 60°C) in a tightly sealed vessel. 10 ⁇ of a solution of odd-numbered, straight-chain fatty acids (solution of each 0.3 mg/ml of fatty acids from 7 to 25 carbon atoms and each 0.6 mg/ml of fatty acids with 27, 29 and 31 carbon atoms in 3/7 (v/v) pyridine/toluene were added as time standards.
  • the GC-MS systems consist of an Agilent 6890 GC coupled to an Agilent 5973 MSD.
  • the autosamplers were CompiPal or GCPal from CTC.
  • CTC CTC-Chip
  • For the analysis usual commercial capillary separation columns (30 m x 0,25 mm x 0,25 ⁇ ) with different poly-methyl-siloxane stationary phases containing 0 % up to 35% of aromatic moieties, depending on the analysed sample materials and fractions from the phase separation step, were used (for example: DB-1 ms, HP-5ms, DB-XLB, DB-35ms, Agilent Technologies).
  • the samples were measured in individual series of 20 to 21 plant samples each (also referred to as sequences), each sequence containing at least 5 wild-type plants as controls.
  • the peak area of each analyte was divided by the peak area of the respective internal standard.
  • the data were standardized for the fresh weight established for the plant sample, respectively.
  • the values calculated thus were related to the wild-type control group by being divided by the mean of the corresponding data of the wild-type control group of the same sequence.
  • the values obtained were referred to as ratio_by_weight, they are comparable between sequences and indicate how much the analyte concentration in the mutant differs in relation to the wild-type control.
  • Appropriate controls were done before to proof that the vector and transformation procedure itself has no significant influence on the metabolic composition of the plants. Therefore the described changes in comparison with wildtypes were caused by the introduced genes. 8 independent lines were analyzed for each construct, with at least 14 samples (individual plants) per construct.
  • Example 10 Results of the phenotypic evaluation of the transgenic plants
  • Table D1 increase in glucose (median ratio_by_weight) in transgenic A. thaliana expressing YPR035W with plastid targeting.
  • the grown plants were harvested by measuring the fresh weight of the aerial part (rosette) of the plant.
  • Two successive experiments were conducted. In the first experiment, at least one individual of each transformed event and 8 events per transformed construct was tested. In the second experiment at least four sibling events were put through a confirmation screen according to the same experimental procedures. Individuals of each event were grown, treated and measured as before. Biomass increase was calculated as ratio of the median of the weights for transgenic plants compared to median of the weights of wild type control plants from the same experiment, grown in the same culture facility as the transformed plants and harvested on the same day. The results can be seen from the Table D2 (below).
  • Table D2 Biomass production of transgenic A. thaliana overexpressing YPR035W with plastid targeting developed under ambient growth conditions.
  • Glutamine synthetase is a well-studied enzyme and assays to measure the activity of this enzyme are known, for example from Liaw SH, Pan C, Eisenberg D (Jun 1993). "Feedback inhibition of fully unadenylylated glutamine synthetase from Salmonella typhimurium by glycine, alanine, and serine". Proc. Natl. Acad. Sci. USA 90 (1 1 ): 4996-5000. Methods to identify and subcellular localise glutamine synthetase or other proteins are also known in the art, e.g. from Tkach et al,. : Nat Cell Biol. 2012 September; 14(9): 966-976.

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Abstract

La présente invention concerne un procédé d'amélioration de diverses caractéristiques associées au rendement présentant un intérêt économique chez des plantes et un procédé qui permet d'améliorer, chez des plantes, une ou plusieurs caractéristiques associées au rendement, grâce à la modulation spécifique de l'expression, chez une plante, d'un acide nucléique codant pour un polypeptide POI (protéine d'intérêt). La présente invention concerne également des plantes chez lesquelles l'expression d'un acide nucléique codant pour un polypeptide POI est spécifiquement modulée, et qui présentent une ou plusieurs caractéristiques associées au rendement améliorées par rapport à des plantes témoins. L'invention concerne également des constructions génétiques, utiles pour la mise en œuvre des procédés selon l'invention.
PCT/IB2014/065099 2013-10-14 2014-10-07 Plantes présentant une biomasse et/ou une teneur en sucre accrues et leur procédé de production Ceased WO2015056130A1 (fr)

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CN102197137A (zh) * 2008-10-30 2011-09-21 先锋国际良种公司 对谷氨酰胺合成酶(gs)进行操作以改善高等植物中的氮利用效率和籽粒产量
WO2012143877A2 (fr) * 2011-04-19 2012-10-26 Council Of Scientific & Industrial Research Construction d'expression et procédé pour améliorer le carbone, l'azote, la biomasse et le rendement de plantes
US8343764B2 (en) * 2005-05-10 2013-01-01 Monsanto Technology Llc Genes encoding glutamine synthetase and uses for plant improvement

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US8343764B2 (en) * 2005-05-10 2013-01-01 Monsanto Technology Llc Genes encoding glutamine synthetase and uses for plant improvement
CN102197137A (zh) * 2008-10-30 2011-09-21 先锋国际良种公司 对谷氨酰胺合成酶(gs)进行操作以改善高等植物中的氮利用效率和籽粒产量
WO2012143877A2 (fr) * 2011-04-19 2012-10-26 Council Of Scientific & Industrial Research Construction d'expression et procédé pour améliorer le carbone, l'azote, la biomasse et le rendement de plantes

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DATABASE GENBANK 6 September 2013 (2013-09-06), BUSSEY, H. ET AL.: "Saccharomyces cerevisiae S288c glutamate--ammonia ligase", accession no. M_0014184132.2 *

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CN108667786A (zh) * 2017-04-02 2018-10-16 田雪松 一种信息交互方法和系统
CN108667786B (zh) * 2017-04-02 2020-12-01 北京拓思德科技有限公司 一种信息交互方法和系统

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