WO2016050512A1 - Procédés et moyens pour augmenter la tolérance au stress et la biomasse chez des plantes - Google Patents

Procédés et moyens pour augmenter la tolérance au stress et la biomasse chez des plantes Download PDF

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WO2016050512A1
WO2016050512A1 PCT/EP2015/071214 EP2015071214W WO2016050512A1 WO 2016050512 A1 WO2016050512 A1 WO 2016050512A1 EP 2015071214 W EP2015071214 W EP 2015071214W WO 2016050512 A1 WO2016050512 A1 WO 2016050512A1
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seq
plant
protein
gene
expression
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Simon Barak
Vanessa RANSBOTYN
Matthew Hannah
Christoph VERDUYN
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Bayer Cropscience Nv
B.G. Negev Technologies And Applications Ltd., At Ben-Gurion University
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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
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • 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
    • C12N15/8218—Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]

Definitions

  • the present invention relates generally to the field of plant molecular biology and concerns a method for improving plant tolerance to stress conditions or for increasing plant growth rate or biomass. More specifically, the present invention concerns a method for increasing stress tolerance and/or growth rate and/or biomass comprising modulating the expression of any of the new abiotic stress regulator genes identified herein, as well as chimeric genes, nucleic acids and polypeptides or mutant alleles leading to reduced protein expression of said genes. Also provided are plants or part thereof modulated according to the invention having an increased stress tolerance and/or growth rate and/or biomass.
  • NGS network-guided genetic screen
  • the NGGS approach has not only reduced the number of mutants to be screened but several reports have demonstrated the possibility of improving the frequency of desired phenotypes (Lan (2007) supra; Bassel (201 1 ), supra; Mutwil (2010), supra; Lee (2010) supra; Lee 2001 , supra).
  • diverse Arabidopsis thaliana functional genomics datasets (co-expression, protein-protein interactions, phylogenetic profiles etc.) were integrated to generate a probabilistic functional gene network - AraNet (Lee (2010), supra). This network was employed to generate a list of new candidate seed pigmentation genes and the resulting mutant screen showed a 10-fold increase in the hit rate (-15%) of desired phenotypes compared to screening random insertion mutants.
  • the invention describes a method for increasing the tolerance to stress conditions of a plant, plant part, plant cell or seed or for increasing growth rate and/or biomass of a plant, plant part, plant cell or seed, comprising the step of modulating the expression and/or activity of a protein having the activity of a protein having the amino acid sequence of SEQ ID NO. 2., SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID NO. 14, SEQ ID NO. 16, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22, SEQ ID NO. 24, SEQ ID NO. 26, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO.
  • said modulating the expression and/or activity of said protein comprises expressing in said plant, plant part, plant cell or seed a chimeric gene comprising the following operably linked elements:
  • SEQ ID NO. 48 SEQ ID NO. 50, SEQ ID NO. 52, SEQ ID NO. 54, SEQ ID NO. 56, SEQ ID NO. 58, SEQ ID NO. 60, SEQ ID NO. 62, SEQ ID NO. 64, SEQ ID NO. 66, SEQ ID NO. 68, SEQ ID NO. 70, SEQ ID NO. 72, SEQ ID NO. 74, SEQ ID NO. 76, SEQ ID NO. 78 or SEQ ID NO. 80, SEQ ID NO. 82, SEQ ID NO. 84, SEQ ID NO. 86, SEQ ID NO. 88, SEQ ID NO. 90.
  • said modulating the expression and/or activity of a protein comprises decreasing the expression and/or activity of a protein having the activity of a protein having the amino acid sequence of SEQ ID NO. 2., SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID NO. 14, SEQ ID NO. 16, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22, SEQ ID NO. 24, SEQ ID NO. 26, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 34, SEQ ID NO. 36, SEQ ID NO. 38, SEQ ID NO. 40, SEQ ID NO.
  • SEQ ID NO. 44 SEQ ID NO. 46, SEQ ID NO. 48, SEQ ID NO. 50, SEQ ID NO. 52, SEQ ID NO. 54, SEQ ID NO. 56, SEQ ID NO. 58, SEQ ID NO. 60, SEQ ID NO. 62, SEQ ID NO. 64.
  • said RNA molecule decreases the expression and/or activity of a protein having the activity of a protein having the amino acid sequence of SEQ ID NO. 2., SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID NO. 14, SEQ ID NO. 16, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22, SEQ ID NO. 24, SEQ ID NO. 26, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 34, SEQ ID NO. 36, SEQ ID NO. 38, SEQ ID NO. 40, SEQ ID NO. 42, SEQ ID NO. 44, SEQ ID NO. 46, SEQ ID NO. 48, SEQ ID NO. 50, SEQ ID NO. 52, SEQ ID NO. 54, SEQ ID NO. 56, SEQ ID NO. 58, SEQ ID NO. 60, SEQ ID NO. 62, SEQ ID NO. 64.
  • said RNA molecule comprises at least 21 nucleotides having at least 76% sequence identity to the nucleotide sequence of a gene present in said plant, plant part, plant cell or seed, said gene encoding a protein having the activity of a protein having the amino acid sequence of SEQ ID NO. 2., SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID NO. 14, SEQ ID NO. 16, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22, SEQ ID NO. 24, SEQ ID NO. 26, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 34, SEQ ID NO. 36, SEQ ID NO.
  • said RNA molecule comprises at least 21 nucleotides having at least 76% sequence identity to the complement of the nucleotide sequence of a gene present in said plant, plant part, plant cell or seed, said gene encoding a protein having the activity of a protein having the amino acid sequence of SEQ ID NO. 2., SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID NO. 14, SEQ ID NO. 16, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22, SEQ ID NO. 24, SEQ ID NO. 26, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 34, SEQ ID NO.
  • SEQ ID NO. 38 SEQ ID NO. 40, SEQ ID NO. 42, SEQ ID NO. 44, SEQ ID NO. 46, SEQ ID NO. 48, SEQ ID NO. 50, SEQ ID NO. 52, SEQ ID NO. 54, SEQ ID NO. 56, SEQ ID NO. 58, SEQ ID NO. 60, SEQ ID NO. 62, SEQ ID NO. 64.
  • said RNA molecule comprises a sense region comprising a nucleotide sequence of at least 21 nucleotides having at least 76% sequence identity to the nucleotide sequence of a gene present in said plant, plant part, plant cell or seed, said gene encoding a protein having the activity of a protein having the amino acid sequence of SEQ ID NO. 2., SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID NO. 14, SEQ ID NO. 16, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22, SEQ ID NO. 24, SEQ ID NO. 26, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO.
  • SEQ ID NO. 36 SEQ ID NO. 38, SEQ ID NO. 40, SEQ ID NO. 42, SEQ ID NO. 44, SEQ ID NO. 46, SEQ ID NO. 48, SEQ ID NO. 50, SEQ ID NO. 52, SEQ ID NO. 54, SEQ ID NO. 56, SEQ ID NO. 58, SEQ ID NO. 60, SEQ ID NO. 62, SEQ ID NO. 64 and an antisense region comprising a nucleotide sequence of at least 21 nucleotides having at least 76% sequence identity to the complement of the nucleotide sequence of said gene, wherein said sense and antisense region are capable of forming a double stranded RNA region comprising said at least 21 nucleotides.
  • said RNA region comprises at least 21 nucleotides having at least 76% sequence identity to a nucleotide sequence encoding a protein having at least 70% sequence identity to a protein having the amino acid sequence of SEQ ID NO. 2., SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID NO. 14, SEQ ID NO. 16, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22, SEQ ID NO. 24, SEQ ID NO. 26, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 34, SEQ ID NO. 36, SEQ ID NO. 38, SEQ ID NO. 40, SEQ ID NO.
  • SEQ ID NO. 44 SEQ ID NO. 46, SEQ ID NO. 48, SEQ ID NO. 50, SEQ ID NO. 52, SEQ ID NO. 54, SEQ ID NO. 56, SEQ ID NO. 58, SEQ ID NO. 60, SEQ ID NO. 62, SEQ ID NO. 64 and/or at least 21 nucleotides having at least 76% sequence identity to the complement of said nucleotide sequence having at least 70% sequence identity to SEQ ID NO. 2., SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID NO. 14, SEQ ID NO. 16, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22, SEQ ID NO.
  • SEQ ID NO. 26 SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 34, SEQ ID NO. 36, SEQ ID NO. 38, SEQ ID NO. 40, SEQ ID NO. 42, SEQ ID NO. 44, SEQ ID NO. 46, SEQ ID NO. 48, SEQ ID NO. 50, SEQ ID NO. 52, SEQ ID NO. 54, SEQ ID NO. 56, SEQ ID NO. 58, SEQ ID NO. 60, SEQ ID NO. 62, SEQ ID NO. 64.
  • said RNA region comprises at least 21 nucleotides having at least 76% sequence identity to any one of SEQ ID NO. 1 , SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9, SEQ ID NO. 1 1 , SEQ ID NO. 13, SEQ ID NO. 15, SEQ ID NO. 17, SEQ ID NO. 19, SEQ ID NO. 21 , SEQ ID NO. 23, SEQ ID NO. 25, SEQ ID NO. 27, SEQ ID NO. 29, SEQ ID NO. 31 , SEQ ID NO. 33 SEQ ID NO. 35, SEQ ID NO. 37, SEQ ID NO. 39, SEQ ID NO. 41 , SEQ ID NO. 43, SEQ ID NO. 45, SEQ ID NO.
  • SEQ ID NO. 49 SEQ ID NO. 51 , SEQ ID NO. 53, SEQ ID NO. 55, SEQ ID NO. 57, SEQ ID NO. 59, SEQ ID NO. 61 , SEQ ID NO. 63, and/or at least 21 nucleotides having at least 76% sequence identity to the complement of any one of SEQ ID NO. 1 , SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9, SEQ ID NO. 1 1 , SEQ ID NO. 13, SEQ ID NO. 15, SEQ ID NO. 17, SEQ ID NO. 19, SEQ ID NO. 21 , SEQ ID NO. 23, SEQ ID NO. 25, SEQ ID NO. 27, SEQ ID NO. 29, SEQ ID NO.
  • SEQ ID NO. 33 SEQ ID NO. 35, SEQ ID NO. 37, SEQ ID NO. 39, SEQ ID NO. 41 , SEQ ID NO. 43, SEQ ID NO. 45, SEQ ID NO. 47, SEQ ID NO. 49, SEQ ID NO. 51 , SEQ ID NO. 53, SEQ ID NO. 55, SEQ ID NO. 57, SEQ ID NO. 59, SEQ ID NO. 61 , SEQ ID NO. 63.
  • said decreasing the expression and/or activity comprises introducing into said plant, plant part, plant cell or seed a mutant or knock-out allele of a gene encoding a protein having the activity of a protein having the amino acid sequence of SEQ ID NO. 2., SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID NO. 14, SEQ ID NO. 16, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22, SEQ ID NO. 24, SEQ ID NO. 26, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 34, SEQ ID NO. 36, SEQ ID NO. 38, SEQ ID NO. 40, SEQ ID NO.
  • SEQ ID NO. 44 SEQ ID NO. 46, SEQ ID NO. 48, SEQ ID NO. 50, SEQ ID NO. 52, SEQ ID NO. 54, SEQ ID NO. 56, SEQ ID NO. 58, SEQ ID NO. 60, SEQ ID NO. 62, SEQ ID NO. 64.
  • said stress condition is selected from salt stress, osmotic stress, ABA treatment, heat stress, drought stress.
  • the invention also provides chimeric gene as described in any of the above methods.
  • the invention also provides a mutant allele or knock-out allele of a gene encoding a protein having the activity of a protein having the amino acid sequence of SEQ ID NO. 2., SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID NO. 14, SEQ ID NO. 16, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22, SEQ ID NO. 24, SEQ ID NO. 26, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 34, SEQ ID NO. 36, SEQ ID NO. 38, SEQ ID NO. 40, SEQ ID NO. 42, SEQ ID NO. 44, SEQ ID NO. 46, SEQ ID NO. 48, SEQ ID NO. 50, SEQ ID NO. 52, SEQ ID NO. 54, SEQ ID NO. 56, SEQ ID NO. 58.
  • the invention also provides a plant, plant part or plant cell comprising the chimeric gene or the mutant or knockout allele as described above.
  • the invention also provides the use of the chimeric gene or the mutant allele as described above to produce a plant, plant part, plant cell or seed with increased stress tolerance and/or increased biomass.
  • FIG. 1 Hierarchical clustering of MST-scored gene expression in response to abiotic stresses demonstrates clustering of MST genes by stress. Hierarchical average linkage clustering was performed on the log2 signals from 6,025 probes designed for 6,005 MST-scored genes using an Arabidopsis ATH1 stress response microarray compendium. Close-up of heat (red) and osmotic and salt (turquoise) clades is shown. (B to D) Examples of organ-specific abiotic stress-responsive gene expression as revealed by hierarchical clustering and principal component analysis.
  • ABA abscisic acid
  • C cold stress
  • D drought
  • H heat
  • HH high light
  • Os osmotic stress
  • Ox oxidative, Oz
  • S salt
  • UV UV
  • U/V-B root
  • S shoot
  • Ro rosette leaves
  • Se seedling
  • cc cell culture.
  • Each array can be identified by its T-number that can be traced in Table S1.
  • D and E Principal component analysis of MST-scored gene expression from cold (D) and osmotic stress microarrays (E). Each spot on the PCA plot represents one array. Organs, cell culture are color-coded according to the legend.
  • FIG. 2 Examples of organ-specific abiotic stress-responsive gene expression as revealed by hierarchical clustering and principal component analysis.
  • A, C Hierarchical average linkage clustering was performed on the log2 fold-change values from 6,025 probes designed for 6,005 MST-scored genes using Arabidopsis ATH1 cold and osmotic stress response microarrays.
  • BP values are shown at branches. Os, osmotic stress; C, cold stress, R, root; S, shoot; Ro, rosette leaves; Se, seedling; cc, cell culture.
  • Each array can be identified by its T-number that can be traced in Table S1 .
  • B, D Principal component analysis (PCA) of MST-scored gene expression from osmotic and cold stress microarrays, respectively. Each spot on the PCA plot represents one array. Organs, cell culture are color-coded according to the legend.
  • FIG. 3 Distribution of correlation coefficients for MSTR genes associated with stress-related GO terms.
  • A, C Spearman rank correlation coefficients (p ⁇ 0.01 ) were computed for MSTR genes associated with abiotic stress-specific parent and children GO biological process terms, using the root microarray compendium.
  • Figure 4 Effect of Spearman rank correlation coefficient threshold on size and homogeneity of modules from the root MSTR co-expression network.
  • A Module size.
  • FIG. 5 Use of MSTR gene co-expression networks for gene discovery based on guilt-by-association.
  • A MSTR network modules are associated with specific abiotic stresses. Each graph represents one MSTR gene module. The average log2 fold-change expression of all genes in each module was calculated and plotted with Expander software [58]. Data are mean ⁇ S.D.
  • B First neighbors of the stress transcriptional regulator gene, DREB2A in the root network (r > ⁇ 0.85). Red edges, positive correlation; Blue edges, negative correlation; Genes in red, described in the literature as regulating one or more abiotic stress responses; Genes in black, candidate MSTR genes. The size of the node is proportional to the gene's MST score.
  • (D) Germination of a T-DNA insertion mutant of At3g57540 associated with the ABA-related module is insensitive to ABA compared to WT (Col-8). The ABA-insensitive mutant abil (also present in the module) was used as a positive control. Data are mean ⁇ S.D. (n 3). Each replicate plate contained ca. 40 seeds. Data are representative of similar results from two independent experiments where germination index of the mutant was significantly different (p ⁇ 0.05, linear mixed models) in response to 2 ⁇ ABA (Table 5).
  • FIG. 6 Discovery of novel MSTR genes regulating the response to salt and osmotic stresses.
  • B Enlargement of the three regions in module 2 showing the first neighbors of tested genes (green circles) ERF4A (At3g15210), HSFA4A (At4g18880) and NAC032 (At1 g77450).
  • FIG. 7 Discovery of novel MSTR genes regulating the response to heat stress.
  • C, D Cotyledon survival phenotypes of T-DNA insertion lines of At1 g77450 and At1 g73805 and their wild type control (Col 8) in response to heat stress (45 °C for 90 min and 150 min, red and green lines, respectively, and then returned to 22 °C for 5 d) Blue lines, control MS plates (22 °C); solid lines, wild-type (WT); broken lines, mutant.
  • FIG. 8 (A, B) The MST score can be employed to prioritize candidate genes only when used in combination with co-expression network analysis.
  • A Receiver-Operator Characteristic (ROC) curves were used to measure true- positive phenotype rate (sensitivity; TP/(TP+FN)) versus false-positive rate (1 -specificity; FP/(FP+TN)) as a function of MST score.
  • B Area under the ROC curve (AUC). The higher the value of the AUC, the better the performance of the MST score. P-values above each bar show that only the AUC for MSTR genes in the network is significantly (p ⁇ 0.05, asymptotic p-value for ROC curves) higher than random expectation.
  • C Receiver-Operator Characteristic (ROC) curves were used to measure true-positive phenotype rate (sensitivity; TP/(TP+FN)) versus false-positive rate (1 -specificity; FP/(FP+TN)) as a function of MST score.
  • D Area under the ROC curve (AUC). The higher the value of the AUC, the better the performance of the MST score. P-values above each bar show that the AUC for the combined MSTR genes within and outside the network is significantly (p ⁇ 0.05) higher than random expectation. Results show that also for the combined set of network and non-network MSTR genes, the magnitude of the MST score can be employed to prioritize candidate genes only when used in combination with co-expression network analysis.
  • Figure 9 Analysis of global network properties, (a) Shoot and root network degree distribution, (b) Shoot and root median clustering coefficient.
  • the present invention has thus identified novel polypeptides and polynucleotides which can be used to modulate stress tolerance and/or biomass of a plant, plant part, plant cell or seed.
  • novel polypeptides and polynucleotides which can be used to modulate stress tolerance and/or biomass of a plant, plant part, plant cell or seed.
  • stress tolerance and/or biomass of a plant, plant part, plant cell or seed can be increased by decreasing the transcript and/or protein expression of the genes or functional homologues thereof of which the corresponding T-DNA insertion mutants were found to displays increased stress tolerance and/or growth rate and/or biomass in at least one of the below described assays (see e.g. Example 1 , Table 4, Example 7) when compared to control plants.
  • stress tolerance and/or growth rate and/or biomass of a plant, plant part, plant cell or seed can be increased by increasing the transcript and/or protein expression of the genes or functional homologues thereof of which the corresponding T-DNA insertion mutant were found to display decreased stress tolerance (i.e. increased stress sensitivity) and/or growth rate and/or biomass in at least one of the below described assays (see e.g. Example 1 , Table 4, Example 7) when compared to control plants.
  • the invention describes a method for increasing the tolerance to stress conditions of a plant, plant part, plant cell or seed or for increasing biomass of a plant, plant part, plant cell or seed, comprising the step of modulating the expression and/or activity of a protein having the activity of a protein having the amino acid sequence of SEQ ID NO. 2., SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID NO. 14, SEQ ID NO. 16, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22, SEQ ID NO. 24, SEQ ID NO. 26, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO.
  • this step is followed by the selection of a plant, plant part, plant cell or seed that has increased stress tolerance and/or increased biomass and/or increased growth rate as compared to control plants.
  • a protein having the activity of a protein having the amino acid sequence of SEQ ID NO. X relates to a protein having the amino acid sequence of any of the above cited SEQ ID NOs, but also to functional variants of such proteins, e.g. proteins having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of the amino acid sequences cited above and having the same function and/or activity.
  • a protein having the activity of a protein having the amino acid sequence of any of the above amino acid sequences can be encoded by the nucleic acid sequence SEQ ID NO. 1 ., SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9, SEQ ID NO. 1 1 , SEQ ID NO. 13, SEQ ID NO. 15, SEQ ID NO. 17, SEQ ID NO. 19, SEQ ID NO. 21 , SEQ ID NO. 23, SEQ ID NO. 25, SEQ ID NO. 27, SEQ ID NO. 29, SEQ ID NO. 31 , SEQ ID NO. 33 , SEQ ID NO. 35, SEQ ID NO. 37, SEQ ID NO. 39, SEQ ID NO. 41 , SEQ ID NO.
  • SEQ ID NO. 45 SEQ ID NO. 47, SEQ ID NO. 49, SEQ ID NO. 51 , SEQ ID NO. 53, SEQ ID NO. 55, SEQ ID NO. 57, SEQ ID NO. 59, SEQ ID NO. 61 , SEQ ID NO. 63, SEQ ID NO. 65, SEQ ID NO. 67, SEQ ID NO. 69, SEQ ID NO. 71 , SEQ ID NO. 73, SEQ ID NO. 75, SEQ ID NO. 77, SEQ ID NO. 79, SEQ ID NO. 81 , SEQ ID NO. 83, SEQ ID NO. 85, SEQ ID NO. 87, SEQ ID NO. 89.
  • sequence identity of two related nucleotide or amino acid sequences, expressed as a percentage, refers to the number of positions in the two optimally aligned sequences which have identical residues (x100) divided by the number of positions compared.
  • a gap i.e., a position in an alignment where a residue is present in one sequence but not in the other, is regarded as a position with non-identical residues.
  • the "optimal alignment” of two sequences is found by aligning the two sequences over the entire length according to the Needleman and Wunsch global alignment algorithm (Needleman and Wunsch, 1970, J Mol Biol 48(3):443-53) in The European Molecular Biology Open Software Suite (EMBOSS, Rice et al.
  • BLAST Altschul et al. (1990), Journal of Molecular Biology, 215, 403410), which stands for Basic Local Alignment Search Tool or ClustalW (Thompson et al. (1994), Nucleic Acid Res., 22, 4673-4680) or any other suitable program which is suitable to generate sequence alignments.
  • Homologous sequences as described above can comprise orthologous or paralogous sequences.
  • Several different methods are known by those of skill in the art for identifying and defining these functionally homologous sequences. Three general methods for defining orthologous and paralogous genes are described; an ortholog, paralog or homolog may be identified by one or more of the methods described below.
  • Orthologs and paralogs are evolutionarily related genes that have similar sequence and similar functions. Orthologs are structurally related genes in different species that are derived by a speciation event. Paralogs are structurally related genes within a single species that are derived by a duplication event.
  • gene duplication may result in two copies of a particular gene, giving rise to two or more genes with similar sequence and often similar function known as paralogs.
  • a paralog is therefore a similar gene formed by duplication within the same species.
  • Paralogs typically cluster together or in the same clade (a group of similar genes) when a gene family phylogeny is analyzed using programs such as CLUSTAL (Thompson, et ah, (1994) Nucleic Acids Res. 22:4673-4680; Higgins, et al, (1996) Methods Enzymol. 266:383402). Groups of similar genes can also be identified with pair- wise BLAST analysis (Feng and Doolittle, (1987) J. Mol. Evol.
  • Orthologous sequences can also be identified by a reciprocal BLAST strategy. Once an orthologous sequence has been identified, the function of the ortholog can be deduced from the identified function of the reference sequence.
  • Orthologous genes from different organisms have highly conserved functions, and very often essentially identical functions (Lee, et al, (2002) Genome Res. 12:493-502; Remm, et al, (2001 ) J. Mol. Biol. 314:1041 -1052). Paralogous genes, which have diverged through gene duplication, may retain similar functions of the encoded proteins. In such cases, paralogs can be used interchangeably with respect to certain embodiments of the instant invention (for example, transgenic expression of a coding sequence).
  • Functional variants or homologues of the sequences disclosed herein may also be identified and isolated by hybridization under stringent conditions using as probes identified nucleotide sequences or fragments thereof.
  • homologous sequences from other monocot species than the specific sequences disclosed herein are said to be substantially identical or essentially similar if they can be detected by hybridization under stringent, preferably highly stringent conditions.
  • Stringent conditions are sequence dependent and will be different in different circumstances.
  • stringent conditions are selected to be about 5°C lower than the thermal melting point (Tm) for the specific sequences at a defined ionic strength and pH.
  • Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe.
  • Stringent conditions will be chosen in which the salt concentration is about 0.02 molar at pH 7 and the temperature is at least 60°C. Lowering the salt concentration and/or increasing the temperature increases stringency.
  • Stringent conditions for RNA-DNA hybridizations are for example those which include at least one wash in 0.2X SSC at 63°C for 20min, or equivalent conditions.
  • High stringency conditions can be provided, for example, by hybridization at 65°C in an aqueous solution containing 6x SSC (20x SSC contains 3.0 M NaCI, 0.3 M Na-citrate, pH 7.0), 5x Denhardfs (100X Denhardt's contains 2% Ficoll, 2% Polyvinyl pyrollidone, 2% Bovine Serum Albumin), 0.5% sodium dodecyl sulphate (SDS), and 20 ⁇ glm ⁇ denaturated carrier DNA (single-stranded fish sperm DNA, with an average length of 120 - 3000 nucleotides) as non- specific competitor. Following hybridization, high stringency washing may be done in several steps, with a final wash (about 30 min) at the hybridization temperature in 0.2-0.1 ⁇ SSC, 0.1 % SDS.
  • Moderate stringency conditions refers to conditions equivalent to hybridization in the above described solution but at about 60-62°C. Moderate stringency washing may be done at the hybridization temperature in 1x SSC, 0.1 % SDS.
  • Low stringency refers to conditions equivalent to hybridization in the above described solution at about 50- 52°C. Low stringency washing may be done at the hybridization temperature in 2x SSC, 0.1 % SDS. See also Sambrook et al. (1989) and Sambrook and Russell (2001).
  • sequences corresponding to variants or homologues of the sequences disclosed herein may also be obtained by DNA amplification (PCR) using oligonucleotides specific for said sequences as primers, such as but not limited to oligonucleotides comprising or consisting of about 20 to about 50 consecutive nucleotides from the presently disclosed sequences and their complement.
  • PCR DNA amplification
  • oligonucleotides specific for said sequences as primers such as but not limited to oligonucleotides comprising or consisting of about 20 to about 50 consecutive nucleotides from the presently disclosed sequences and their complement.
  • the modulating the expression and/or activity of a protein as described above comprises expressing in said plant, plant part, plant cell or seed a chimeric gene comprising the following operably linked elements:
  • SEQ ID NO. 48 SEQ ID NO. 50, SEQ ID NO. 52, SEQ ID NO. 54, SEQ ID NO. 56, SEQ ID NO. 58, SEQ ID NO. 60, SEQ ID NO. 62, SEQ ID NO. 64, SEQ ID NO. 66, SEQ ID NO. 68, SEQ ID NO. 70, SEQ ID NO. 72, SEQ ID NO. 74, SEQ ID NO. 76, SEQ ID NO. 78 or SEQ ID NO. 80, SEQ ID NO. 82, SEQ ID NO. 84, SEQ ID NO. 86, SEQ ID NO. 88, SEQ ID NO. 90.
  • a chimeric gene refers to a gene that is made up of heterologous elements that are operably linked to enable expression of the gene, whereby that combination is not normally found in nature.
  • heterologous refers to the relationship between two or more nucleic acid or protein sequences that are derived from different sources.
  • a promoter is heterologous with respect to an operably linked nucleic acid sequence, such as a coding sequence, if such a combination is not normally found in nature.
  • a particular sequence may be "heterologous” with respect to a cell or organism into which it is inserted (i.e. does not naturally occur in that particular cell or organism).
  • operably linked means that said elements of the chimeric gene are linked to one another in such a way that their function is coordinated and allows expression of the coding sequence, i.e. they are functionally linked.
  • a promoter is functionally linked to another nucleotide sequence when it is capable of ensuring transcription and ultimately expression of said other nucleotide sequence.
  • Two proteins encoding nucleotide sequences e.g. a transit peptide encoding nucleic acid sequence and a nucleic acid sequence encoding a protein according to the invention, are functionally or operably linked to each other if they are connected in such a way that a fusion protein of first and second protein or polypeptide can be formed.
  • a gene e.g. the chimeric gene of the invention, is said to be expressed when it leads to the formation of an expression product.
  • An expression product denotes an intermediate or end product arising from the transcription and optionally translation of the nucleic acid, DNA or RNA, coding for such product, e. g. the second nucleic acid described herein.
  • a DNA sequence under control of regulatory regions, particularly the promoter is transcribed into an RNA molecule.
  • An RNA molecule may either itself form an expression product or be an intermediate product when it is capable of being translated into a peptide or protein.
  • a gene is said to encode an RNA molecule as expression product when the RNA as the end product of the expression of the gene is, e.
  • RNA expression products include inhibitory RNA such as e. g. sense RNA (co-suppression), antisense RNA, ribozymes, miRNA or siRNA, but also mRNA, rRNA and tRNA.
  • a gene is said to encode a protein as expression product when the end product of the expression of the gene is a protein or peptide.
  • promoters may be used to promote the transcription of the nucleic acid of the invention, i.e. the nucleic acid which when transcribed yields an RNA molecule that modulates the expression and /or activity of a protein having the activity of a protein having any of the above amino acid sequences
  • promoters include for example constitutive promoters, inducible promoters (e.g. stress-inducible promoters, drought-inducible promoters, hormone-inducible promoters, chemical-inducible promoters, etc.), tissue-specific promoters, developmental ⁇ regulated promoters and the like.
  • a plant expressible promoter can be a constitutive promoter, i.e. a promoter capable of directing high levels of expression in most cell types (in a spatio-temporal independent manner).
  • plant expressible constitutive promoters include promoters of bacterial origin, such as the octopine synthase (OCS) and nopaline synthase (NOS) promoters from Agrobacterium, but also promoters of viral origin, such as that of the cauliflower mosaic virus (CaMV) 35S transcript (Hapster et al., 1988, Mol. Gen. Genet. 212: 182-190) or 19S RNAs genes (Odell et al., 1985, Nature.
  • CCS octopine synthase
  • NOS nopaline synthase
  • promoters of plant origin mention will be made of the promoters of the plant ribulose-biscarboxylase/oxygenase (Rubisco) small subunit promoter (US 4,962,028; W099/25842) from zea mays and sunflower, the promoter of the Arabidopsis thaliana histone H4 gene (Chaboute et al., 1987), the ubiquitin promoters (Holtorf et al., 1995, Plant Mol. Biol.
  • Rubisco ribulose-biscarboxylase/oxygenase
  • a variety of plant gene promoters that regulate gene expression in response to environmental, hormonal, chemical, developmental signals, and in a tissue-active manner can be used for expression of a sequence in plants.
  • Choice of a promoter is based largely on the phenotype of interest and is determined by such factors as tissue (e.g., seed, fruit, root, pollen, vascular tissue, flower, carpel, etc.), inducibility (e.g., in response to wounding, heat, cold, drought, light, pathogens, etc.), timing, developmental stage, and the like.
  • Additional promoters that can be used to practice this invention are those that elicit expression in response to stresses, such as the RD29 promoters that are activated in response to drought, low temperature, salt stress, or exposure to ABA (Yamaguchi-Shinozaki et al., 2004, Plant Cell, Vol. 6, 251 -264; WO12/101 1 18), but also promoters that are induced in response to heat (e.g., see Ainley et al. (1993) Plant Mol. Biol. 22: 13-23), light (e.g., the pea rbcS-3A promoter, Kuhlemeier et al.
  • stresses such as the RD29 promoters that are activated in response to drought, low temperature, salt stress, or exposure to ABA (Yamaguchi-Shinozaki et al., 2004, Plant Cell, Vol. 6, 251 -264; WO12/101 1 18), but also promoters that are induced in response to heat (e.g., see
  • timing of the expression can be controlled by using promoters such as those acting at senescence (e.g., see Gan and Amasino (1995) Science 270: 1986-1988); or late seed development (e.g., see Odell et al. (1994) Plant Physiol. 106: 447458).
  • promoters such as those acting at senescence (e.g., see Gan and Amasino (1995) Science 270: 1986-1988); or late seed development (e.g., see Odell et al. (1994) Plant Physiol. 106: 447458).
  • salt-inducible promoters such as the salt-inducible NHX1 promoter of rice landrace Pokkali (PKN) (Jahan et al., 6 th International Rice Genetics symposium, 2009, poster abstract P4-37), the salt inducible promoter of the vacuolar ⁇ -pyrophosphatase from Thellungiella halophila (TsVP1 ) (Sun et al., BMC Plant Biology 2010, 10:90), the salt-inducible promoter of the Citrus sinensis gene encoding phospholipid hydroperoxide isoform gpxl (Avsian-Kretchmer et al., Plant Physiology July 2004 vol. 135, p1685-1696).
  • PPN salt-inducible NHX1 promoter of rice landrace Pokkali
  • TsVP1 Thellungiella halophila
  • TsVP1 Thellungiella halophila
  • tissue-specific and/or developmental stage-specific promoters are used, e.g., promoter that can promote transcription only within a certain time frame of developmental stage within that tissue. See, e.g., Blazquez (1998) Plant Cell 10:791 -800, characterizing the Arabidopsis LEAFY gene promoter. See also Cardon (1997) Plant J 12:367-77 , describing the transcription factor SPL3, which recognizes a conserved sequence motif in the promoter region of the A. thaliana floral meristem identity gene API; and Mandel (1995) Plant Molecular Biology, Vol. 29, pp 995-1004, describing the meristem promoter elF4.
  • Tissue specific promoters which are active throughout the life cycle of a particular tissue can be used.
  • the nucleic acids of the invention are operably linked to a promoter active primarily only in cotton fiber cells
  • the nucleic acids of the invention are operably linked to a promoter active primarily during the stages of cotton fiber cell elongation, e.g., as described by Rinehart (1996) supra.
  • the nucleic acids can be operably linked to the Fbl2A gene promoter to be preferentially expressed in cotton fiber cells (Ibid) . See also, John (1997) Proc. Natl. Acad. Sci. USA 89:5769-5773; John, et al., U.S. Patent Nos.
  • Root- specific promoters may also be used to express the nucleic acids of the invention.
  • Examples of root-specific promoters include the promoter from the alcohol dehydrogenase gene (DeLisle (1990) Int. Rev. Cytol. 123:39-60) and promoters such as those disclosed in U.S. Pat. Nos. 5,618,988, 5,837,848 and 5,905,186.
  • a leaf-specific promoter see, e.g., Busk (1997) Plant J. 1 1 :1285 1295, describing a leaf-specific promoter in maize
  • the ORF 13 promoter from Agrobacterium rhizogenes which exhibits high activity in roots,
  • a tomato promoter active during fruit ripening, senescence and abscission of leaves a guard-cell preferential promoter e.g. as described in PCT/EP12/065608, and, to a lesser extent, of flowers can be used (see, e.g., Blume (1997) Plant J. 12:731 746); a pistil-specific promoter from the potato SK2 gene (see, e.g., Ficker (1997) Plant Mol. Biol.
  • the Blec4 gene from pea which is active in epidermal tissue of vegetative and floral shoot apices of transgenic alfalfa making it a useful tool to target the expression of foreign genes to the epidermal layer of actively growing shoots or fibers
  • the ovule-specific BELI gene see, e.g., Reiser (1995) Cell 83:735-742, GenBank No. U39944)
  • the promoter in Klee, U.S. Patent No. 5,589,583, describing a plant promoter region is capable of conferring high levels of transcription in meristematic tissue and/or rapidly dividing cells.
  • tissue specific promoters that may be used according to the invention include: seed-specific promoters (such as the napin, phaseolin or DC3 promoter described in U .S. Pat. No. 5,773,697), fruit-specific promoters that are active during fruit ripening (such as the dru 1 promoter (U.S. Pat. No. 5,783,393), or the 2AI 1 promoter (e.g., see U.S. Pat. No. 4,943,674) and the tomato polygalacturonase promoter (e.g., see Bird et al. (1988) Plant Mol. Biol. 1 1 : 651 -662), flower- specific promoters (e.g., see Kaiser et al. (1995) Plant Mol. Biol.
  • seed-specific promoters such as the napin, phaseolin or DC3 promoter described in U .S. Pat. No. 5,773,697
  • fruit-specific promoters that are active during fruit ripening such as the dru 1 promoter (
  • pollen-active promoters such as PTA29, PTA26 and PTAI 3 (e.g., see U.S. Pat. No. 5,792,929) and as described in e.g. Baerson et al. (1994 Plant Mol. Biol. 26: 1947-1959), promoters active in vascular tissue (e.g., see Ringli and Keller (1998) Plant Mol. Biol. 37: 977-988), carpels (e.g., see Oh l et al. (1990) Plant Cell 2:), pollen and ovules (e.g., see Baerson et al. (1993) Plant Mol. Biol. 22: 255- 267).
  • PTA29 e.g., see U.S. Pat. No. 5,792,929
  • promoters active in vascular tissue e.g., see Ringli and Keller (1998) Plant Mol. Biol. 37: 977-988
  • carpels e.g., see Oh l et al. (1990
  • plant promoters which are inducible upon exposure to plant hormones, such as auxins, are used to express the nucleic acids used to practice the invention.
  • the invention can use the auxin- response elements El promoter fragment (AuxREs) in the soybean ⁇ Glycine max L.) (Liu (1997) Plant Physiol. 1 15:397- 407); the auxin-responsive Arabidopsis GST6 promoter (also responsive to salicylic acid and hydrogen peroxide) (Chen (1996) Plant J. 10: 955-966); the auxin-inducible parC promoter from tobacco (Sakai (1996) 37:906-913); a plant biotin response element (Streit (1997) Mol. Plant Microbe Interact.
  • ABA abscisic acid
  • Further hormone inducible promoters include auxin-inducible promoters (such as that described in van der Kop et al. (1999) Plant Mol. Biol. 39: 979-990 or Baumann et al., (1999) Plant Cell 1 1 : 323-334), cytokinin-inducible promoter (e.g., see Guevara-Garcia (1998) Plant Mol. Biol. 38: 743-753), promoters responsive to gibberellin (e.g., see Shi et al. (1998) Plant Mol. Biol.
  • nucleic acids used to practice the invention can also be operably linked to plant promoters which are inducible upon exposure to chemicals reagents which can be applied to the plant, such as herbicides or antibiotics.
  • plant promoters which are inducible upon exposure to chemicals reagents which can be applied to the plant, such as herbicides or antibiotics.
  • the maize ln2-2 promoter activated by benzenesulfonamide herbicide safeners, can be used (De Veylder (1997) Plant Cell Physiol. 38:568-577); application of different herbicide safeners induces distinct gene expression patterns, including expression in the root, hydathodes, and the shoot apical meristem.
  • Coding sequence can be under the control of, e.g., a tetracycline-inducible promoter, e.g. , as described with transgenic tobacco plants containing the Avena sativa L. (oat) arginine decarboxylase gene (Masgrau (1997) Plant J. 1 1 :465 73); or, a salicylic acid-responsive element (Stange (1997) Plant J. 1 1 :1315-1324).
  • a tetracycline-inducible promoter e.g.
  • hormone- or pesticide induced promoters i.e., promoter responsive to a chemical which can be applied to the transgenic plant in the field
  • expression of a polypeptide of the invention can be induced at a particular stage of development of the plant.
  • Use may also be made of the estrogen-inducible expression system as described in US patent 6,784,340 and Zuo et al. (2000, Plant J. 24: 265-273) to drive the expression of the nucleic acids used to practice the invention.
  • a promoter may be used whose host range is limited to target plant species, such as corn, rice, barley, wheat, potato or other crops, inducible at any stage of development of the crop.
  • tissue-specific plant promoter may drive expression of operably linked sequences in tissues other than the target tissue.
  • a tissue-specific promoter that drives expression preferentially in the target tissue or cell type, but may also lead to some expression in other tissues as well, is used.
  • promoter use may also be made, in combination with the promoter, of other regulatory sequences, which are located between the promoter and the coding sequence, such as transcription activators ("enhancers"), for instance the translation activator of the tobacco mosaic virus (TMV) described in Application WO 87/07644, or of the tobacco etch virus (TEV) described by Carrington & Freed 1990, J. Virol. 64: 1590-1597, for example.
  • transcription activators for instance the translation activator of the tobacco mosaic virus (TMV) described in Application WO 87/07644, or of the tobacco etch virus (TEV) described by Carrington & Freed 1990, J. Virol. 64: 1590-1597, for example.
  • Introns are intervening sequences present in the pre-mRNA but absent in the mature RNA following excision by a precise splicing mechanism.
  • the ability of natural introns to enhance gene expression, a process referred to as intron-mediated enhancement (IME) has been known in various organisms, including mammals, insects, nematodes and plants (WO 07/098042, p1 1 -12).
  • IME is generally described as a posttranscriptional mechanism leading to increased gene expression by stabilization of the transcript.
  • the intron is required to be positioned between the promoter and the coding sequence in the normal orientation.
  • introns have also been described to affect translation, to function as promoters or as position and orientation independent transcriptional enhancers (Chaubet-Gigot et al., 2001 , Plant Mol Biol. 45(1 ):17-30, p27-28).
  • genes containing such introns include the 5' introns from the rice actin 1 gene (see US5641876), the rice actin 2 gene, the maize sucrose synthase gene (Clancy and Hannah, 2002, Plant Physiol. 130(2):918-29), the maize alcohol dehydrogenase-1 (Adh-1 ) and Bronze-1 genes (Callis et al. 1987 Genes Dev.
  • Suitable regulatory sequences include 5' UTRs.
  • a 5' UTR also referred to as a leader sequence, is a particular region of a messenger RNA (mRNA) located between the transcription start site and the start codon of the coding region. It is involved in mRNA stability and translation efficiency.
  • mRNA messenger RNA
  • the 5' untranslated leader of a petunia chlorophyll a/b binding protein gene downstream of the 35S transcription start site can be utilized to augment steady-state levels of reporter gene expression (Harpster et al., 1988, Mol Gen Genet. 212(1 ):182-90).
  • WO95/006742 describes the use of 5' non-translated leader sequences derived from genes coding for heat shock proteins to increase transgene expression.
  • the chimeric gene may also comprise a 3' end region, i.e. a transcription termination or polyadenylation sequence, operable in plant cells.
  • a transcription termination or polyadenylation sequence use may be made of any corresponding sequence of bacterial origin, such as for example the nos terminator of Agrobacterium tumefaciens, of viral origin, such as for example the CaMV 35S terminator, or of plant origin, such as for example a histone terminator as described in published Patent Application EP 0 633 317 A1.
  • the polyadenylation region can be derived from the natural gene, from a variety of other plant genes, or from T-DNA.
  • the 3' end sequence to be added may be derived from, for example, the nopaline synthase or octopine synthase genes, or alternatively from another plant gene, or less preferably from any other eukaryotic gene.
  • Another measure to increase the expression of the nucleic acid of the invention that may be applied is optimizing the coding region for expression in the target organism, which may include adapting the codon usage , CG content, and elimination of unwanted nucleotide sequences (e.g. premature polyadenylation signals, cryptic intron splice sites, ATTTA pentamers, CCAAT box sequences, sequences that effect pre-mRNA splicing by secondary RNA structure formation such as long CG or AT stretches).
  • unwanted nucleotide sequences e.g. premature polyadenylation signals, cryptic intron splice sites, ATTTA pentamers, CCAAT box sequences, sequences that effect pre-mRNA splicing by secondary RNA structure formation such as long CG or AT stretches.
  • Stress conditions refers e.g. to stress imposed by the application of chemical compounds (e.g., herbicides, fungicides, insecticides, plant growth regulators, adjuvants, fertilizers), exposure to abiotic stress (e.g., drought, waterlogging, submergence, high light conditions, high UV radiation, increased hydrogen peroxide levels, extreme (high or low) temperatures, ozone and other atmospheric pollutants, soil salinity or heavy metals, hypoxia, anoxia, osmotic stress, oxidative stress, low nutrient levels such as nitrogen or phosphorus etc.) or biotic stress (e.g., pathogen or pest infection including infection by fungi, viruses, bacteria, insects, nematodes, mycoplasms and mycoplasma like organisms, etc.). Stress may also be imposed by hormones such as ABA or compounds influencing hormone activity.
  • chemical compounds e.g., herbicides, fungicides, insecticides, plant growth regulators, adjuvants, fertilizers
  • Drought, salinity, extreme temperatures, high light stress and oxidative stress are known to be interconnected and may induce growth and cellular damage through similar mechanisms.
  • Rabbani et al. Plant Physiol (2003) 133: 1755- 1767
  • osmotic stress For example, drought and/or salinisation are manifested primarily as osmotic stress, resulting in the disruption of homeostasis and ion distribution in the cell.
  • Oxidative stress which frequently accompanies high or low temperature, salinity or drought stress, may cause denaturing of functional and structural proteins.
  • these diverse environmental stresses often activate similar cell signalling pathways and cellular responses, such as the production of stress proteins, up- regulation of anti-oxidants, accumulation of compatible solutes and growth arrest.
  • a "control plant” as used herein is generally a plant of the same species which has wild-type levels of a protein having the activity of a protein having the amino acid sequence of SEQ ID NO. 2., SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID NO. 14, SEQ ID NO. 16, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22, SEQ ID NO. 24, SEQ ID NO. 26, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 34, SEQ ID NO. 36, SEQ ID NO. 38, SEQ ID NO. 40, SEQ ID NO. 42, SEQ ID NO. 44, SEQ ID NO.
  • SEQ ID NO. 48 SEQ ID NO. 50, SEQ ID NO. 52, SEQ ID NO. 54, SEQ ID NO. 56, SEQ ID NO. 58, SEQ ID NO. 60, SEQ ID NO. 62, SEQ ID NO. 64, SEQ ID NO. 66, SEQ ID NO. 68, SEQ ID NO. 70, SEQ ID NO. 72, SEQ ID NO. 74, SEQ ID NO. 76, SEQ ID NO. 78 or SEQ ID NO. 80, SEQ ID NO. 82, SEQ ID NO. 84, SEQ ID NO. 86, SEQ ID NO. 88, SEQ ID NO. 90.
  • Wild-type levels of a protein as used herein refers to the typical levels of said protein in a plant as it most commonly occurs in nature. Said control plant has thus not been provided either with a chimeric gene according to the invention nor with a mutant allele according to the invention (as described further below).
  • Stress tolerance may also be apparent as the higher chlorophyll content, higher germination rates, higher photosynthesis and lower chlorophyll fluorescence under stress conditions in stress tolerant plants compared to control plants under the same conditions.
  • stress tolerance it is also not required that the plant be grown continuously under the adverse conditions for the stress tolerance to become apparent.
  • the difference in stress tolerance between a plant or plant cell produced according to the invention and a control plant or plant cell will become apparent even when only a relatively short period of adverse conditions is encountered during growth.
  • Yield or biomass refers to seed number/weight, fruit number/weight, fresh weight, dry weight, leaf number/area, plant height, branching, boll number/size, fiber length, seed oil content, seed protein content, seed carbohydrate content.
  • An increased growth rate as used herein refers to a period of increased growth or allocation to one or more of these cells or tissues that comprise the aforementioned plant organs.
  • An increase in biomass or yield or growth can be an increase of at least 2%, or at least 5%, or at least 10%, or at least 15%, or at least 20%, or at least 25%, or at least 30%, or at least 40% , or at least 50%. Said increase is an increase with respect to biomass or yield or growth of control plants.
  • a plant made according to the invention expressing a nucleic acid which when transcribed yields an RNA molecule that modulates the expression and/or activity of a protein having the activity of a protein having the amino acid sequence of SEQ ID NO. 2., SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID NO. 14, SEQ ID NO. 16, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22, SEQ ID NO. 24, SEQ ID NO. 26, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 34, SEQ ID NO. 36, SEQ ID NO. 38, SEQ ID NO. 40, SEQ ID NO.
  • SEQ ID NO. 44 SEQ ID NO. 46, SEQ ID NO. 48, SEQ ID NO. 50, SEQ ID NO. 52, SEQ ID NO. 54, SEQ ID NO. 56, SEQ ID NO. 58, SEQ ID NO. 60, SEQ ID NO. 62, SEQ ID NO. 64, SEQ ID NO. 66, SEQ ID NO. 68, SEQ ID NO. 70, SEQ ID NO. 72, SEQ ID NO. 74, SEQ ID NO. 76, SEQ ID NO. 78 or SEQ ID NO. 80, SEQ ID NO. 82, SEQ ID NO. 84, SEQ ID NO. 86, SEQ ID NO. 88, SEQ ID NO.
  • phenotypes when compared to control plants, especially under adverse conditions (stress conditions), but also under control conditions, as described above, including but not limited to: increased overall plant yield, increased root mass, increased root length, increased leaf size, increased ear size, increased seed size, increased endosperm size, improved standability, alterations in the relative size of embryos and endosperms leading to changes in the relative levels of protein, oil and/or starch in the seeds, altered floral development, changes in leaf number, altered leaf surface, altered vasculature, altered intemodes, alterations in leaf senescence, absence of tassels, absence of functional pollen bearing tassels, or increased plant size when compared to a non-modified plant under normal growth conditions or under adverse conditions, such as water limiting conditions.
  • stress tolerance and/or growth rate and/or biomass of a plant, plant part, plant cell or seed is increased by decreasing the transcript and/or protein expression of the genes or functional homologues thereof of which the corresponding T-DNA insertion mutants were found to display increased stress tolerance and/or growth rate or biomass in at least one of the below described assays when compared to control plants (i.e. increased stress sensitivity) and/or growth rate and/or biomass in at least one of the below described assays (e.g. the stress conditions as listed in Table 4).
  • the modulating the expression and/or activity a protein as described above comprises decreasing the expression and/or activity in said plant, plant part, plant cell or seed of a protein having the activity of a protein having the amino acid sequence of SEQ ID NO. 2., SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID NO. 14, SEQ ID NO. 16, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22, SEQ ID NO. 24, SEQ ID NO. 26, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 34, SEQ ID NO. 36, SEQ ID NO. 38, SEQ ID NO. 40, SEQ ID NO.
  • SEQ ID NO. 44 SEQ ID NO. 46, SEQ ID NO. 48, SEQ ID NO. 50, SEQ ID NO. 52, SEQ ID NO. 54, SEQ ID NO. 56, SEQ ID NO. 58, SEQ ID NO. 60, SEQ ID NO. 62, SEQ ID NO. 64.
  • RNA molecule that results in a decreased expression and/or activity of a protein having the activity of a protein having any of the above amino acid sequences can be an RNA encoding a protein which inhibits expression and/or activity of said protein. Further, said RNA molecule can also be an RNA molecule which inhibits expression of a gene which is an activator of expression and/or activity of said protein. Said RNA molecule may also be an RNA molecule that directly inhibits expression and/or activity of a gene present in said plant, plant part, plant cell or seed encoding said protein, such as an RNA which mediates silencing of said gene.
  • Decreasing the expression and/or activity of a protein having the activity of a protein having any of the above amino acid sequences can be decreasing the amount of functional protein produced.
  • Said decrease can be a decrease with at least 30%, 40%, 50%, 60% , 70%, 80%, 90%, 95% or 100% (i.e. no functional protein is produced by the cell) as compared to the amount of functional protein produced by a cell with wild type expression levels and activity of said protein.
  • Said decrease in expression and/or activity can be a constitutive decrease in the amount of functional protein produced.
  • Said decrease can also be a temporal/inducible decrease in the amount of functional protein produced.
  • silencing RNA refers to any RNA molecule, which upon introduction into a plant cell, reduces the expression of a target gene.
  • silencing RNA may e.g.
  • antisense RNA whereby the RNA molecule comprises a sequence of at least 20 consecutive nucleotides having 95% sequence identity to the complement of the sequence of the target nucleic acid, preferably the coding sequence of the target gene.
  • antisense RNA may also be directed to regulatory sequences of target genes, including the promoter sequences and transcription termination and polyadenylation signals.
  • Silencing RNA further includes so-called “sense RNA” whereby the RNA molecule comprises a sequence of at least 20 consecutive nucleotides having 95% sequence identity to the sequence of the target nucleic acid.
  • silencing RNA may be "unpolyadenylated RNA" comprising at least 20 consecutive nucleotides having 95% sequence identity to the complement of the sequence of the target nucleic acid, such as described in WO01 /12824 or US6423885 (both documents herein incorporated by reference).
  • RNA molecules as described in WO03/076619 (herein incorporated by reference) comprising at least 20 consecutive nucleotides having at least 95%, at least 96%, at least 97% at least 98%, at least 99% or 100% sequence identity to the sequence of the target nucleic acid or the complement thereof, and further comprising a largely-double stranded region as described in WO03/076619 (including largely double stranded regions comprising a nuclear localization signal from a viroid of the Potato spindle tuber viroid-type or comprising CUG trinucleotide repeats).
  • Silencing RNA may also be double stranded RNA comprising a sense and antisense strand as herein defined, wherein the sense and antisense strand are capable of base-pairing with each other to form a double stranded RNA region (preferably the said at least 20 consecutive nucleotides of the sense and antisense RNA are complementary to each other).
  • the sense and antisense region may also be present within one RNA molecule such that a hairpin RNA (hpRNA) can be formed when the sense and antisense region form a double stranded RNA region.
  • hpRNA hairpin RNA
  • the hpRNA may be classified as long hpRNA, having long, sense and antisense regions which can be largely complementary, but need not be entirely complementary (typically larger than about 200 bp, ranging between 200-1000 bp). hpRNA can also be rather small ranging in size from about 30 to about 42 bp, but not much longer than 94 bp (see WO04/073390, herein incorporated by reference).
  • An ihpRNA is an intron-containing hairpin RNA, which has the same general structure as an hpRNA, but the RNA molecule additionally comprises an intron in the loop of the hairpin that is capable of being spliced in the cell in which the ihpRNA is expressed.
  • an intron minimizes the size of the loop in the hairpin RNA molecule following splicing, and this increases the efficiency of interference. See, for example, Smith et al (2000) Nature 407:319-320. In fact, Smith et al, show 100% suppression of endogenous gene expression using ihpRNA-mediated interference.
  • the intron is the ADHI intron 1 . Methods for using ihpRNA interference to inhibit the expression of endogenous plant genes are described, for example, in Smith et al, (2000) Nature 407:319-320; Waterhouse and Helliwell, (2003) Nat. Rev. Genet.
  • the chimeric gene for hpRNA interference may also be designed such that the sense sequence and the antisense sequence do not correspond to an endogenous RNA.
  • the sense and antisense sequence flank a loop sequence that comprises a nucleotide sequence corresponding to all or part of the endogenous messenger RNA of the target gene present in the plant. Thus, it is the loop region that determines the specificity of the RNA interference. See, for example, WO0200904 herein incorporated by reference.
  • a silencing RNA may also encode an artificial micro-RNA (miRNA) molecule as described e.g. in Schwab et al. 2006 (Plant Cell 18: 1 121 -1 133), WO2005/052170, WO2005/047505 or US 2005/0144667, or ta-siRNAs as described in WO2006/074400 (all documents incorporated herein by reference).
  • MiRNAs are about 21 nucleotides in length and in plants up to 5 mismatches to their target sequence are allowed (Schwab et al. 2006, supra).
  • amplicon chimeric genes can be used.
  • Amplicon chimeric genes according to the invention comprise a plant virus-derived sequence that contains all or part of the target gene but generally not all of the genes of the native virus.
  • the viral sequences present in the transcription product of the chimeric gene allow the transcription product to direct its own replication.
  • the transcripts produced by the amplicon may be either sense or antisense relative to the target sequence.
  • the nucleic acid expressed by the chimeric gene of the invention is catalytic RNA or has ribozyme activity specific for the target sequence.
  • the polynucleotide causes the degradation of the endogenous messenger RNA transcribed from the target gene/sequence, resulting in reduced expression of the protein present in the plant. This method is described, for example, in US4987071 , herein incorporated by reference.
  • the nucleic acid expressed by the chimeric gene of the invention encodes a zinc finger protein that binds to the gene encoding said protein, resulting in reduced expression of the target gene.
  • the zinc finger protein binds to a regulatory region of said gene, thereby reducing gene transcription.
  • the zinc finger protein binds to the coding region thereby preventing its transcription.
  • the nucleic acid expressed by the chimeric gene of the invention encodes a TALE protein that binds to a gene encoding said protein, resulting in reduced expression of the gene.
  • the TALE protein binds to a regulatory region of said gene, thereby reducing gene transcription.
  • the TALE protein binds to the coding region thereby preventing its transcription.
  • the nucleic acid expressed by the chimeric gene of the invention encodes a CAS9- based repressor - guide RNA complex that binds to a gene encoding said protein, resulting in reduced expression of the gene.
  • said complex binds to a regulatory region of said gene, thereby reducing gene transcription.
  • the complex binds to the coding region thereby preventing its transcription.
  • CRISPRi CRISPR/CAS system for use in targeted gene repression
  • the nucleic acid expressed by the chimeric gene of the invention encodes a nuclease, e.g. a meganuclease, zinc finger nuclease, TALEN, or CRISPR/CAS nuclease that specifically inactivates the endogenous target gene by recognizing and cleaving a sequence specific for said endogenous target gene.
  • a nuclease e.g. a meganuclease, zinc finger nuclease, TALEN, or CRISPR/CAS nuclease that specifically inactivates the endogenous target gene by recognizing and cleaving a sequence specific for said endogenous target gene.
  • TALEN zinc finger nuclease
  • CRISPR/CAS nuclease that specifically inactivates the endogenous target gene by recognizing and cleaving a sequence specific for said endogenous target gene.
  • Chimeric genes encoding such nuclease can be removed afterwards by segregation.
  • RNA molecule which is translated into a polypeptide
  • the polypeptide is capable of reducing the expression and/or activity of said protein directly, i.e. the RNA molecule encodes an inhibitory protein or polypeptides.
  • such an inhibitory protein or polypeptide can be an antibody (including a nanobody etc) that binds to the target protein present in the plant and reduces the activity thereof.
  • the binding of the antibody results in increased turnover of the antibody complex by cellular quality control mechanisms.
  • inhibitory protein or polypeptide may also be a dominant negative protein or protein fragment of the target protein.
  • decreasing the expression and/or activity of a protein having the activity of a protein having any of the above amino acid sequences can be achieved by contacting the plant or plant cell with molecules interfering with the function of the endogenous protein present in the plant, e. g. by triggering aggregation of the target protein (interferor peptides) as e.g. described in WO2007/071789 and WO2008/148751 .
  • decreasing the expression and/or activity the target protein according to the invention can be achieved by contacting the plant or plant cell can with so-called alphabodies specific for said protein present in the plant, i.e. non-natural proteinaceous molecules that can antagonize protein function, as e.g. described in WO2009/030780, WO2010/066740 and WO2012/092970.
  • decreasing the expression and/or activity of protein having the activity of a protein having any of the above amino acid sequences can be achieved by inhibition of the expression of said protein present in the plant. Inhibition of the expression of said protein can be induced at the desired moment using a spray (systemic application) with inhibitory nucleic acids, such as RNA or DNA molecules that function in RNA-mediated gene silencing (similar to the above described molecules), as e.g. described in WO201 1/1 12570 (incorporated herein by reference).
  • inhibitory nucleic acids such as RNA or DNA molecules that function in RNA-mediated gene silencing (similar to the above described molecules), as e.g. described in WO201 1/1 12570 (incorporated herein by reference).
  • said RNA is a silencing RNA molecule, said molecule comprising: a. at least 21 nucleotides having at least 76% sequence identity to the nucleotide sequence of a gene present in said plant, plant part, plant cell or seed encoding a protein having the activity of a protein having the amino acid sequence of SEQ ID NO. 2., SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID NO. 14, SEQ ID NO. 16, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22, SEQ ID NO. 24, SEQ ID NO. 26, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO.
  • SEQ ID NO. 34 SEQ ID NO. 36, SEQ ID NO. 38, SEQ ID NO. 40, SEQ ID NO. 42, SEQ ID NO. 44, SEQ ID NO. 46, SEQ ID NO. 48, SEQ ID NO. 50, SEQ ID NO. 52, SEQ ID NO. 54, SEQ ID NO. 56, SEQ ID NO. 58, SEQ ID NO. 60, SEQ ID NO. 62, SEQ ID NO. 64 present in said plant;
  • SEQ ID NO. 2 amino acid sequence of SEQ ID NO. 2., SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID NO. 14, SEQ ID NO. 16, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22, SEQ ID NO. 24, SEQ ID NO. 26, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 34, SEQ ID NO. 36, SEQ ID NO. 38, SEQ ID NO.
  • SEQ ID NO. 42 SEQ ID NO. 44, SEQ ID NO. 46, SEQ ID NO. 48, SEQ ID NO. 50, SEQ ID NO. 52, SEQ ID NO. 54, SEQ ID NO. 56, SEQ ID NO. 58, SEQ ID NO. 60, SEQ ID NO. 62, SEQ ID NO. 64 present in said plant; or
  • a sense region comprising a nucleotide sequence of at least 21 nucleotides having at least 76% sequence identity to the nucleotide sequence of a gene present in said plant, plant part, plant cell or seed encoding a protein having the activity of a protein having the amino acid sequence of SEQ ID NO. 2., SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID NO. 14, SEQ ID NO. 16, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22, SEQ ID NO. 24, SEQ ID NO. 26, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 34, SEQ ID NO.
  • SEQ ID NO. 38 SEQ ID NO. 40, SEQ ID NO. 42, SEQ ID NO. 44, SEQ ID NO. 46, SEQ ID NO. 48, SEQ ID NO. 50, SEQ ID NO. 52, SEQ ID NO. 54, SEQ ID NO. 56, SEQ ID NO. 58, SEQ ID NO. 60, SEQ ID NO. 62, SEQ ID NO. 64 and an antisense region comprising a nucleotide sequence of at least 21 nucleotides having at least 76% sequence identity to the complement of the nucleotide sequence of said gene present in said plant, wherein said sense and antisense region are capable of forming a double stranded RNA region comprising said at least 21 nucleotides.
  • At least 76% sequence identity in this respect can be at least 80% sequence identity (e.g. 4 mismatches over 21 nt), at least 85% sequence identity (e.g. 3 mismatches over 21 nt), at least 90% sequence identity (e.g. 2 mismatches over 21 nt), at least 95% sequence identity (e.g. 1 mismatch over 21 nt) or 100% sequence identity (no mismatches).
  • a protein having the activity of a protein having the amino acid sequence of SEQ ID NO. X relates to a protein having the amino acid sequence of any of the above cited SEQ ID NOs, but also to functional variants of such proteins, e.g. proteins having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of the amino acid sequences cited above and having the same function and/or activity.
  • the gene present in said plant, plant part, plant cell or seed encodes a protein having the amino acid sequence of SEQ ID NO.
  • SEQ ID NO. 4 SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID NO. 14, SEQ ID NO. 16, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22, SEQ ID NO. 24, SEQ ID NO. 26, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 34, SEQ ID NO. 36, SEQ ID NO. 38, SEQ ID NO. 40, SEQ ID NO. 42, SEQ ID NO. 44, SEQ ID NO. 46, SEQ ID NO. 48, SEQ ID NO. 50, SEQ ID NO. 52, SEQ ID NO. 54, SEQ ID NO. 56, SEQ ID NO. 58, SEQ ID NO.
  • the inhibitory RNA molecule may comprise at least 21 nucleotides having at least 76% sequence identity to a nucleotide sequence encoding a protein having the amino acid sequence of SEQ ID NO. 2., SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID NO. 14, SEQ ID NO. 16, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22, SEQ ID NO. 24, SEQ ID NO. 26, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 34, SEQ ID NO. 36, SEQ ID NO. 38, SEQ ID NO.
  • SEQ ID NO. 42 SEQ ID NO. 44, SEQ ID NO. 46, SEQ ID NO. 48, SEQ ID NO. 50, SEQ ID NO. 52, SEQ ID NO. 54, SEQ ID NO. 56, SEQ ID NO. 58, SEQ ID NO. 60, SEQ ID NO. 62, SEQ ID NO. 64 and/or to the complement of such a nucleotide sequence.
  • the gene present in said plant, plant part, plant cell or seed encoding a protein having the activity of a protein having any of the above the amino acid sequences can have the nucleic acid sequence of SEQ ID NO. 1 , SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9, SEQ ID NO. 1 1 , SEQ ID NO. 13, SEQ ID NO. 15, SEQ ID NO. 17, SEQ ID NO. 19, SEQ ID NO. 21 , SEQ ID NO. 23, SEQ ID NO. 25, SEQ ID NO. 27, SEQ ID NO. 29, SEQ ID NO. 31 , SEQ ID NO. 33, SEQ ID NO. 35, SEQ ID NO. 37, SEQ ID NO.
  • the inhibitory RNA molecule may comprise at least 21 nucleotides having at least 76% sequence identity to the nucleic acid sequence of SEQ ID NO. 1 , SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9, SEQ ID NO. 1 1 , SEQ ID NO. 13, SEQ ID NO. 15, SEQ ID NO. 17, SEQ ID NO.
  • SEQ ID NO. 21 SEQ ID NO. 23, SEQ ID NO. 25, SEQ ID NO. 27, SEQ ID NO. 29, SEQ ID NO. 31 , SEQ ID NO. 33, SEQ ID NO. 35, SEQ ID NO. 37, SEQ ID NO. 39, SEQ ID NO. 41 , SEQ ID NO. 43, SEQ ID NO. 45, SEQ ID NO. 47, SEQ ID NO. 49, SEQ ID NO. 51 , SEQ ID NO. 53, SEQ ID NO. 55, SEQ ID NO. 57, SEQ ID NO. 59, SEQ ID NO. 61 , SEQ ID NO. 63 and/or to the complement of such a nucleic acid sequence.
  • methods according the invention are provided wherein reducing the expression and/activity of a protein having the activity of a protein having the amino acid sequence of SEQ ID NO. 2., SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID NO. 14, SEQ ID NO. 16, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22, SEQ ID NO. 24, SEQ ID NO. 26, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 34, SEQ ID NO. 36, SEQ ID NO. 38, SEQ ID NO. 40, SEQ ID NO. 42, SEQ ID NO. 44, SEQ ID NO.
  • SEQ ID NO. 48 comprises the step of introducing a knock-out allele of an endogenous gene encoding such a protein.
  • a "knock-out allele” as used herein is an allele which is mutated as compared to the wild type allele (i.e. it is a mutant allele) and encodes a non-functional protein (i.e. a protein having no activity) or results in a significantly reduced amount of protein (by for example a mutation in a regulatory region such as the promoter), or which encodes a protein with significantly reduced activity.
  • Said "knock-out allele” can be a mutant allele, which may encode no protein, or which may encode a non-functional protein or a protein with significantly reduced function, such as a mutant protein or a truncated protein.
  • the allele may also be referred to as an inactivated allele.
  • Said "significantly reduced amount of protein” can be a reduction in the amount (or levels) of protein produced by the cell comprising a knock-out allele by at least 30%, 40% , 50%, 60%, 70%, 80%, 90%, 95% or 100% (i.e. no protein is produced by the allele) as compared to the amount of the protein produced by the corresponding wild-type allele.
  • the amount or level of a transcript e.g. an mRNA
  • encoding a protein or the amount of level of a protein itself can be measured according to various methods known in the art such as (quantitative) RT-PCR, northern blotting, microarray analysis, western blotting, ELISA and the like.
  • a "significantly reduced activity” can be a reduction in the activity of the protein produced by the cell comprising a knock-out allele of said protein by at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% (i.e. no protein activity) as compared to the activity of the corresponding wild-type allele.
  • a wild-type allele refers to a typical form of an allele as it most commonly occurs in nature, such as the alleles as represented by the nucleic acid sequence described in this application, e.g. of SEQ ID NO. 2., SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID NO. 14, SEQ ID NO. 16, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22, SEQ ID NO. 24, SEQ ID NO. 26, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 34, SEQ ID NO. 36, SEQ ID NO. 38, SEQ ID NO. 40, SEQ ID NO.
  • SEQ ID NO. 44 SEQ ID NO. 46, SEQ ID NO. 48, SEQ ID NO. 50, SEQ ID NO. 52, SEQ ID NO. 54, SEQ ID NO. 56, SEQ ID NO. 58, SEQ ID NO. 60, SEQ ID NO. 62, SEQ ID NO. 64, SEQ ID NO. 66, SEQ ID NO. 68, SEQ ID NO. 70, SEQ ID NO. 72, SEQ ID NO. 74, SEQ ID NO. 76, SEQ ID NO. 78 or SEQ ID NO. 80, SEQ ID NO. 82, SEQ ID NO. 84, SEQ ID NO. 86, SEQ ID NO. 88, SEQ ID NO. 90.
  • any mutation in the wild type nucleic acid sequences which results in a protein comprising at least one amino acid insertion, deletion and/or substitution relative to the wild type protein can lead to significantly reduced or no biological activity. It is, however, understood that certain mutations in the protein are more likely to result in a complete abolishment of the biological activity of the protein, such as mutations whereby significant portions of the functional domains are lacking.
  • a “mutant gene” or a “mutant allele” refers to a gene or allele comprising one or more mutations, such as a “missense mutation”, a “nonsense mutation” or “STOP codon mutation” (including a mutation resulting in no functional protein ("knock-out allele of a gene"), an "insertion mutation”, a “deletion mutation” or a “frameshift mutation” (the latter two including one or more mutations resulting in a "knock-out allele”) with respect to the corresponding wild-type gene or allele, such as such as the genes and alleles as described in this application, e.g. as represented by SEQ ID NO. 2., SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO.
  • SEQ ID NO. 8 SEQ ID NO. 10
  • SEQ ID NO. 64 SEQ ID NO. 66, SEQ ID NO. 68, SEQ ID NO. 70, SEQ ID NO. 72, SEQ ID NO. 74, SEQ ID NO. 76, SEQ ID NO. 78 or SEQ ID NO. 80, SEQ ID NO. 82, SEQ ID NO. 84, SEQ ID NO. 86, SEQ ID NO. 88, SEQ ID NO. 90.
  • a nonsense mutation in an allele is a mutation in an allele whereby one or more translation stop codons are introduced into the coding DNA and the corresponding mRNA sequence of the corresponding wild type allele.
  • Translation stop codons are TGA (UGA in the mRNA), TAA (UAA) and TAG (UAG).
  • a full knockout mutant allele may comprise a nonsense mutation wherein an in-frame stop codon is introduced in the coding sequence by a single nucleotide substitution, such as the mutation of CAG to TAG, TGG to TAG, TGG to TGA, or CAA to TAA.
  • a full knockout mutant allele may comprise a nonsense mutation wherein an in-frame stop codon is introduced in the coding sequence by double nucleotide substitutions, such as the mutation of CAG to TAA, TGG to TAA, or CGG to TAG or TGA.
  • a full knockout mutant allele may further comprise a nonsense mutation wherein an in-frame stop codon is introduced in the coding sequence by triple nucleotide substitutions, such as the mutation of CGG to TAA.
  • the truncated protein lacks the amino acids encoded by the coding DNA downstream of the mutation (i.e. the C-terminal part of the protein) and maintains the amino acids encoded by the coding DNA upstream of the mutation (i.e. the N-terminal part of the protein).
  • a missense mutation in an allele is any mutation (deletion, insertion or substitution) in an allele whereby one or more codons are changed in the coding DNA and the corresponding mRNA sequence of the corresponding wild type allele, resulting in the substitution of one or more amino acids in the wild type protein for one or more other amino acids in the mutant protein.
  • a frameshift mutation in an allele is a mutation (deletion, insertion, duplication of one or more nucleotides, and the like) in an allele that results in the nucleic acid sequence being translated in a different frame downstream of the mutation.
  • An "insertion mutation” is present if one or more melons have been added in the coding sequence of the nucleic acid resulting in the presence of one or more amino acids in the translated protein, whereas a “deletion mutation” is present if one or more codons have been deleted in the coding sequence of the nucleic acid resulting in the deletion of one or more amino acids in the translated protein.
  • Said mutant allele can be introduced into said plant e. g. through mutagenesis.
  • mutagenic agent such as a chemical substance (such as ethylmethylsulfonate (EMS), ethylnitrosourea (ENU), etc.) or ionizing radiation (neutrons (such as in fast neutron mutagenesis, etc.), alpha rays, gamma rays (such as that supplied by a Cobalt 60 source), X-rays, UV-radiation, etc.), T-DNA insertion mutagenesis (Azpiroz-Leehan et al.
  • the desired mutagenesis of one or more genes or alleles may be accomplished by one of the above methods. While mutations created by irradiation are often large deletions or other gross lesions such as translocations or complex rearrangements, mutations created by chemical mutagens are often more discrete lesions such as point mutations.
  • EMS alkylates guanine bases which results in base mispairing: an alkylated guanine will pair with a thymine base, resulting primarily in G/C to A/T transitions.
  • plants are regenerated from the treated cells using known techniques. For instance, the resulting seeds may be planted in accordance with conventional growing procedures and following pollination seed is formed on the plants. Additional seed that is formed as a result of such pollination in the present or a subsequent generation may be harvested and screened for the presence of mutant alleles.
  • DeleteageneTM Delete-a-gene; Li et al., 2001 , Plant J 27: 235-242
  • PCR polymerase chain reaction
  • Said mutant allele can also be introduced via gene targeting techniques.
  • gene targeting refers herein to directed gene modification that uses mechanisms such as double stranded DNA break repair via non-homologous end-joining, homologous recombination, mismatch repair or site-directed mutagenesis.
  • the method can be used to replace, insert and delete endogenous sequences or sequences previously introduced in plant cells. Methods for gene targeting can be found in, for example, WO 2006/105946 or WO2009/002150.
  • Double stranded DNA breaks can be induced in a targeted manner using custom designed sequence specific nucleases and nuclease systems such as meganucleases/homing endonucleases, zinc finger nucleases, TALENs or CRISPR/CAS (for a review see Gaj et al., 2013, Trends Biotechnol 31 : 397405).
  • sequence specific nucleases and nuclease systems such as meganucleases/homing endonucleases, zinc finger nucleases, TALENs or CRISPR/CAS (for a review see Gaj et al., 2013, Trends Biotechnol 31 : 397405).
  • such techniques can also be used to delete entire genes encoding proteins having the activity of a protein having any of the above amino acid sequences.
  • Said mutant allele can also be introduced through introgression of a mutant allele into said plant.
  • stress tolerance and/or biomass of a plant, plant part, plant cell or seed is increased by decreasing the expression of two or more of the genes or functional homologues thereof of which the corresponding T- DNA insertion mutants were found to display increased stress tolerance and/or biomass compared to control plants.
  • the modulating the expression and/or activity comprises decreasing the expression and/or activity of two or more (e.g. three, four, five etc) proteins having the activity of a protein having the amino acid sequence of SEQ ID NO. 2., SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO.
  • SEQ ID NO. 14 SEQ ID NO. 16, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22, SEQ ID NO. 24, SEQ ID NO. 26, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 34, SEQ ID NO. 36, SEQ ID NO. 38, SEQ ID NO. 40, SEQ ID NO. 42, SEQ ID NO. 44, SEQ ID NO. 46, SEQ ID NO. 48, SEQ ID NO. 50, SEQ ID NO. 52, SEQ ID NO. 54, SEQ ID NO. 56, SEQ ID NO. 58, SEQ ID NO. 60, SEQ ID NO. 62, SEQ ID NO. 64, as described above.
  • stress tolerance and/or biomass of a plant, plant part, plant cell or seed is increased by decreasing the expression of one or more of the genes or functional homologues thereof of which the corresponding T- DNA insertion mutants were found to display increased stress tolerance and/or biomass in at least two of the below described assays when compared to control plants.
  • stress tolerance and/or biomass of a plant, plant part, plant cell or seed is increased by decreasing the expression of one or more the genes or functional homologues thereof of which the corresponding T-DNA insertion mutants were found to display increased stress tolerance and/or biomass in at least three or more of the below described assays when compared to control plants.
  • stress tolerance and/or growth rate and/or biomass of a plant, plant part, plant cell or seed is increased by increasing the expression of the genes or functional homologues thereof of which the corresponding T-DNA insertion mutants were found to display decreased stress tolerance (i.e. increased stress sensitivity) and/or growth rate and/or biomass in at least one of the below described assays (e.g. the stress conditions as listed in Table 4) when compared to control plants.
  • the modulating the expression and/or activity comprises increasing the expression and/or activity in said plant, plant part, plant cell or seed of a protein having the activity of a protein having the amino acid sequence of SEQ ID NO. 66, SEQ ID NO. 68, SEQ ID NO.
  • SEQ ID NO. 70 SEQ ID NO. 72, SEQ ID NO. 74, SEQ ID NO. 76, SEQ ID NO. 78 or SEQ ID NO. 80, SEQ ID NO. 82, SEQ ID NO. 84, SEQ ID NO. 86, SEQ ID NO. 88, SEQ ID NO. 90.
  • the expression of any of the above proteins is increased if the transcript or protein level is statistically higher than the transcript or protein level of said protein in a plant that has not been modified to increase the expression of that protein.
  • the transcript or protein level of the protein may be increased by more than 5%, more than 10%, more than 20%, more than 50%, more than 100%, more than 200% or even more when compared to the mRNA or protein level of the same gene in a plant that is not a mutant or that has not been modified to increase the expression of that protein.
  • Expression of a transcript (e.g. an mRNA) or a protein can be measured according to various methods known in the art such as (quantitative) RT-PCR, northern blotting, microarray analysis, western blotting, ELISA and the like.
  • increasing the expression and/or activity of the protein having the activity of the protein of any of the above amino acid sequences involves expressing a nucleic acid which encodes protein having the activity of a protein having the amino acid sequence of SEQ ID NO. 66, SEQ ID NO. 68, SEQ ID NO. 70, SEQ ID NO. 72, SEQ ID NO. 74, SEQ ID NO. 76, SEQ ID NO. 78 or SEQ ID NO. 80, SEQ ID NO. 82, SEQ ID NO. 84, SEQ ID NO. 86, SEQ ID NO. 88, SEQ ID NO. 90.
  • increasing the expression and/or activity of the protein having the activity of the protein of any of the above amino acid sequences involves expressing a nucleic acid nucleic acid encoding a protein having the amino acid sequence of SEQ ID NO. 66, SEQ ID NO. 68, SEQ ID NO. 70, SEQ ID NO. 72, SEQ ID NO. 74, SEQ ID NO. 76, SEQ ID NO. 78 or SEQ ID NO. 80, SEQ ID NO. 82, SEQ ID NO. 84, SEQ ID NO. 86, SEQ ID NO. 88, SEQ ID NO. 90.
  • Such proteins may be encoded by a nucleic acid comprising the nucleotide sequence of SEQ ID NO. 65, SEQ ID NO. 67, SEQ ID NO. 69, SEQ ID NO. 71 , SEQ ID NO. 73, SEQ ID NO. 75, SEQ ID NO. 77, SEQ ID NO. 79, SEQ ID NO. 81 , SEQ ID NO. 83, SEQ ID NO. 85, SEQ ID NO. 87, SEQ ID NO. 89.
  • increasing the expression and/or activity of the protein having the activity of the protein of any of the above amino acid sequences involves expressing a nucleic acid comprising the nucleotide sequence of SEQ ID NO. 65, SEQ ID NO. 67, SEQ ID NO. 69, SEQ ID NO. 71 , SEQ ID NO. 73, SEQ ID NO.
  • stress tolerance and/or growth rate and/or biomass of a plant, plant part, plant cell or seed is increased by increasing the expression of two or more of the genes or functional homologues thereof of which the corresponding T-DNA insertion mutants were found to display decreased stress tolerance and/or biomass compared to control plants.
  • the modulating the expression and/or activity comprises increasing the expression and/or activity of two or more (e.g. three, four, five etc.) proteins having the activity of a protein having the amino acid sequence of SEQ ID NO. 66, SEQ ID NO. 68, SEQ ID NO. 70, SEQ ID NO. 72, SEQ ID NO. 74, SEQ ID NO.
  • stress tolerance and/or growth rate and/or biomass of a plant, plant part, plant cell or seed is increased by increasing the expression of one or more of the genes or functional homologues thereof of which the corresponding T-DNA insertion mutants were found to display decreased stress tolerance and/or biomass in at least two of the below described assays when compared to control plants.
  • stress tolerance and/or growth rate and/or biomass of a plant, plant part, plant cell or seed is increased by increasing the expression of one or more of the genes or functional homologues thereof of which the corresponding T-DNA insertion mutants were found to display decreased stress tolerance and/or biomass in at least three or at least four of the below described assays when compared to control plants.
  • a chimeric gene encoding such a protein.
  • Such a chimeric gene can comprise the following operably linked elements:
  • Said chimeric gene can comprise any of the promoters, 3' end regions and additional elements as described above.
  • increasing the expression and/or activity of the protein having the activity of the protein of any of the above amino acid sequences can be achieved by modifying the endogenous gene(s) said protein. This can be done through, for example, T-DNA activation tagging, mutagenesis (e.g. EMS mutagenesis) or by targeted genome engineering technologies. Using such technologies for example, the endogenous promoter can be modified such that it drives higher levels of expression, or the endogenous promoter can be replaced with a stronger promoter, or mutations can be introduced into the coding region that enhance mRNA stability, translation efficiency, protein activity and/or stability, similar to the above described methods for enhancing the expression of the introduced chimeric gene.
  • mutagenesis e.g. EMS mutagenesis
  • the endogenous promoter can be modified such that it drives higher levels of expression, or the endogenous promoter can be replaced with a stronger promoter, or mutations can be introduced into the coding region that enhance mRNA stability, translation efficiency, protein activity and/or stability,
  • T-DNA activation tagging is a method to activate endogenous genes by random insertion of a T-DNA carrying promoter or enhancer elements, which can cause transcriptional activation of flanking plant genes.
  • the method can consist of generating a large number of transformed plants or plant cells using a specialized T-DNA construct, followed by selection for the desired phenotype.
  • Targeted genome engineering refers to generating intended and directed modifications into the genome. Such intended modifications can be insertions at specific genomic locations, deletions of specific endogenous sequences, and replacements of endogenous sequences. Targeted genome engineering can be based on homologous recombination. Targeted genome engineering to increase the functional expression of a gene can consist of insertion of a promoter, stronger than the endogenous promoter, in front of the coding sequence, or insertion of an enhancer to increase promoter activity. Such techniques can also be applied e.g. to insert elements increasing RNA stability or enhancing translation of the encoded mRNA, or modify the coding sequence to enhance translation, protein stability and activity, similar to the above described methods for enhancing the expression of the introduced chimeric gene.
  • the invention also provides chimeric genes comprising a nucleic acid which when transcribed yields an RNA molecule that modulates the expression and/or activity of a protein having the activity of a protein having the amino acid sequence of SEQ ID NO. 2., SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID NO. 14, SEQ ID NO. 16, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22, SEQ ID NO. 24, SEQ ID NO. 26, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 34, SEQ ID NO. 36, SEQ ID NO. 38, SEQ ID NO. 40, SEQ ID NO.
  • Nucleic acids used to practice the invention can be expressed by introduction into a plant cell by any means.
  • nucleic acids or expression constructs can be introduced into the genome of a desired plant host, or, the nucleic acids or chimeric genes can be episomes.
  • "Introducing” in connection with the present application relates to the placing of genetic information in a plant cell or plant by artificial means, such as transformation. This can be effected by any method known in the art for introducing RNA or DNA into plant cells, tissues, protoplasts or whole plants. In addition to artificial introduction as described above, “introducing” also comprises introgressing genes or alleles as defined further below.
  • Transformation means introducing a nucleotide sequence into a plant in a manner to cause stable or transient expression of the sequence. Transformation and regeneration of both monocotyledonous and dicotyledonous plant cells is now routine, and the selection of the most appropriate transformation technique will be determined by the practitioner. The choice of method will vary with the type of plant to be transformed; those skilled in the art will recognize the suitability of particular methods for given plant types. Suitable methods can include, but are not limited to: electroporation of plant protoplasts; liposome-mediated transformation; polyethylene glycol (PEG) mediated transformation; transformation using viruses; micro-injection of plant cells; micro-projectile bombardment of plant cells; vacuum infiltration; and Agrobacterium- mediated transformation.
  • PEG polyethylene glycol
  • the invention uses Agrobacterium tumefaciens mediated transformation.
  • Other bacteria capable of transferring nucleic acid molecules into plant cells may be used, such as certain soil bacteria of the order of the Rhizobiales, e.g. Rhizobiaceae (e.g. Rhizobium spp., Sinorhizobium spp., Agrobacterium spp); Phyllobacteriaceae (e.g. Mesorhizobium spp., Phyllobacterium spp.); Brucellaceae (e.g. Ochrobactrum spp.); Bradyrhizobiaceae (e.g.
  • Bradyrhizobium spp. Bradyrhizobium spp.
  • Xanthobacteraceae e.g. Azorhizobium spp.
  • Agrobacterium spp. Rhizobium spp.
  • Sinorhizobium spp. Mesorhizobium spp.
  • Phyllobacterium spp. Ochrobactrum spp.
  • Bradyrhizobium spp. examples of which include Ochrobactrum sp., Rhizobium sp., Mesorhizobium loti, Sinorhizobium meliloti.
  • Rhizobia include R. leguminosarum bv, trifolii, R.
  • leguminosarum bv,phaseoli and Rhizobium leguminosarum, bv, viciae US Patent 7,888,552.
  • Other bacteria that can be employed to carry out the invention which are capable of transforming plants cells and induce the incorporation of foreign DNA into the plant genome are bacteria of the genera Azobacter (aerobic), Closterium (strictly anaerobic), Klebsiella (optionally aerobic), and Rhodospirillum (anaerobic, photosynthetically active).
  • Rhizobiaceae members such as Rhizobium trifolii, Rhizobium leguminosarum and Phyllobacterium myrsinacearum, while Rhizobium sp. NGR234, Sinorhizobium meliloti and Mesorhizobium loti could indeed be modified to mediate gene transfer to a number of diverse plants (Broothaerts et al., 2005, Nature, 433:629-633).
  • making transgenic plants or seeds comprises incorporating sequences used to practice the invention and, in one aspect (optionally), marker genes into a target expression construct (e.g., a plasmid), along with positioning of the promoter and the terminator sequences.
  • a target expression construct e.g., a plasmid
  • This can involve transferring the modified gene into the plant through a suitable method.
  • a construct may be introduced directly into the genomic DNA of the plant cell using techniques such as electroporation and microinjection of plant cell protoplasts, or the constructs can be introduced directly to plant tissue using ballistic methods, such as DNA particle bombardment. For example, see, e.g., Christou (1997) Plant Mol. Biol. 35:197-203; Pawlowski (1996) Mol. Biotechnol.
  • protoplasts can be immobilized and injected with a nucleic acid, e.g., an expression construct.
  • a nucleic acid e.g., an expression construct.
  • plant regeneration from protoplasts is not easy with cereals, plant regeneration is possible in legumes using somatic embryogenesis from protoplast derived callus.
  • Organized tissues can be transformed with naked DNA using gene gun technique, where DNA is coated on tungsten microprojectiles, shot 1 /100th the size of cells, which carry the DNA deep into cells and organelles. Transformed tissue is then induced to regenerate, usually by somatic embryogenesis. This technique has been successful in several cereal species including maize and rice.
  • a third step can involve selection and regeneration of whole plants capable of transmitting the incorporated target gene to the next generation.
  • Such regeneration techniques rely on manipulation of certain phytohormones in a tissue culture growth medium, typically relying on a biocide and/or herbicide marker that has been introduced together with the desired nucleotide sequences. Plant regeneration from cultured protoplasts is described in Evans et al., Protoplasts Isolation and Culture, Handbook of Plant Cell Culture, pp. 124-176, MacMillilan Publishing Company, New York, 1983; and Binding, Regeneration of Plants, Plant Protoplasts, pp. 21 -73, CRC Press, Boca Raton, 1985.
  • Regeneration can also be obtained from plant callus, explants, organs, or parts thereof. Such regeneration techniques are described generally in Klee (1987) Ann. Rev. of Plant Phys. 38:467486. To obtain whole plants from transgenic tissues such as immature embryos, they can be grown under controlled environmental conditions in a series of media containing nutrients and hormones, a process known as tissue culture. Once whole plants are generated and produce seed, evaluation of the progeny begins.
  • Viral transformation may also be used for transient or stable expression of a gene, depending on the nature of the virus genome.
  • the desired genetic material is packaged into a suitable plant virus and the modified virus is allowed to infect the plant.
  • the progeny of the infected plants is virus free and also free of the inserted gene.
  • Suitable methods for viral transformation are described or further detailed e. g. in WO 90/12107, WO 03/052108 or WO 2005/098004.
  • the chimeric gene after the chimeric gene is stably incorporated in transgenic plants, it can be introduced into other plants by sexual crossing or introgression, as can a mutant allele according to the invention. Any of a number of standard breeding techniques can be used, depending upon the species to be crossed. Since transgenic expression of the nucleic acids of the invention leads to phenotypic changes, plants comprising the recombinant nucleic acids of the invention can be sexually crossed with a second plant to obtain a final product. Thus, the seed of the invention can be derived from a cross between two transgenic plants of the invention, or a cross between a transgenic or mutant plant of the invention and another plant.
  • the desired effects can be enhanced when both parental plants express the chimeric genes or mutant alleles of the invention.
  • the desired effects can be passed to future plant generations by standard propagation means.
  • the modified trait can be any of those traits described above.
  • confirmation that the modified trait is due to changes in expression levels or activity of the transgenic or mutated polypeptide or nucleic acid can be determined by analyzing mRNA expression using Northern blots, RT-PCR or microarrays, or protein expression using immunoblots or Western blots or gel shift assays.
  • “Introgressing” means the integration of a gene or allele in a plant's genome by natural means, i.e. by crossing a plant comprising the chimeric gene or mutant allele described herein with a plant not comprising said chimeric gene or mutant allele.
  • the offspring can be selected for those comprising the chimeric gene or mutant allele.
  • nucleic acids and polypeptides used to practice this invention can be expressed in or inserted in any plant cell, organ, seed or tissue, including differentiated and undifferentiated tissues or plants, including but not limited to roots, stems, shoots, cotyledons, epicotyl, hypocotyl, leaves, pollen, seeds, tumor tissue and various forms of cells in culture such as single cells, protoplast, embryos, and callus tissue.
  • the plant tissue may be in plants or in organ, tissue or cell culture.
  • the invention further provides plants, plant cells, organs, seeds or tissues that have been modified so as to have a modulated expression and/or activity of a protein having the activity of a protein having the amino acid sequence of SEQ ID NO. 2., SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID NO. 14, SEQ ID NO. 16, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22, SEQ ID NO. 24, SEQ ID NO. 26, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 34, SEQ ID NO. 36, SEQ ID NO. 38, SEQ ID NO. 40, SEQ ID NO. 42, SEQ ID NO.
  • the invention provides plants, plant cells, organs, seeds or tissues that have been modified so as to have a reduced expression and/or activity of a protein having the activity of a protein having the amino acid sequence of SEQ ID NO. 2., SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID NO. 14, SEQ ID NO. 16, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22, SEQ ID NO. 24, SEQ ID NO. 26, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 34, SEQ ID NO. 36, SEQ ID NO. 38, SEQ ID NO. 40, SEQ ID NO. 42, SEQ ID NO.
  • the invention further provides plants, plant cells, organs, seeds or tissues that have been modified so as to have an increased expression and/or activity of a protein having the activity of a protein having the amino acid sequence of SEQ ID NO. 66, SEQ ID NO. 68, SEQ ID NO. 70, SEQ ID NO. 72, SEQ ID NO. 74, SEQ ID NO. 76, SEQ ID NO. 78 or SEQ ID NO.
  • transgenic or mutant plants or parts thereof such as plant cells, organs, seeds or tissues, comprising and expressing the nucleic acids (chimeric genes or mutant alleles) used to practice this invention resulting in a modulated expression and/or activity of a protein having the activity of a protein having the amino acid sequence of any of the above sequences; for example, the invention provides plants, e.g., transgenic or mutant plants or parts thereof, such as, plant cells, organs, seeds or tissues that show improved tolerance to abiotic stress conditions, such as drought stress, osmotic stress, salt stress, heat stress, or that show reduced ABA sensitivity; thus, the invention provides stress-tolerant plants, plant cells, organs, seeds or tissues (e.g., crops).
  • abiotic stress conditions such as drought stress, osmotic stress, salt stress, heat stress, or that show reduced ABA sensitivity
  • the invention also provides plants, e.g., transgenic or mutant plants or parts thereof such as plant cells, organs, seeds or tissues that show improved growth under control conditions; thus, the invention provides plants, plant cells, organs, seeds or tissues (e.g., crops) with increased biomass and/or yield and/or growth rate.
  • the invention further provides plants, e.g., transgenic or mutant plants or parts thereof such as plant cells, organs, seeds or tissues that show improved growth under limiting water conditions; thus, the invention provides drought-tolerant plants, plant cells, organs, seeds or tissues (e.g., crops).
  • a mutant plant refers to a plant comprising a mutant allele according to the invention.
  • the plant, plant part, plant organs and plant cell of the invention comprising a nucleic acid (chimeric gene or mutant allele) used to practice this invention can be dicotyledonous (a dicot) or monocotyledonous (a monocot).
  • monocots comprising a nucleic acid of this invention e.g., as monocot transgenic plants of the invention, are grasses, such as meadow grass (blue grass, Poa), forage grass such as festuca, lolium, temperate grass, such as Agrostis, and cereals, e.g., wheat, oats, rye, barley, rice, sorghum, and maize (corn).
  • dicots comprising a nucleic acid of this invention, e.g., as dicot transgenic plants of the invention, are cotton, tobacco, legumes, such as lupins, potato, sugar beet, pea, bean and soybean, and cruciferous plants (family Brassicaceae), such as cauliflower, rape seed, and the closely related model organism Arabidopsis thaliana.
  • dicot transgenic plants of the invention are cotton, tobacco, legumes, such as lupins, potato, sugar beet, pea, bean and soybean, and cruciferous plants (family Brassicaceae), such as cauliflower, rape seed, and the closely related model organism Arabidopsis thaliana.
  • plant or plant cell comprising a nucleic acid of this invention include a broad range of plants, including, but not limited to, species from the genera Anacardium, Arachis, Asparagus, Atropa, Avena, Brassica, Citrus, Citrullus, Capsicum, Carthamus, Cocos, Cojfea, Cucumis, Cucurbita, Daucus, Elaeis, Fragaria, Glycine, Gossypium, Helianthus, Heterocallis, Hordeum, Hyoscyamus, Lactuca, Linum, Lolium, Lupinus, Lycopersicon, Malus, Manihot, Majorana, Medicago, Nicotiana, Olea, Oryza, Panieum, Pannisetum, Persea, Phaseolus, Pistachia, Pisum, Pyrus, Prunus, Raphanus, Ricinus, Secale, Senecio,
  • the invention furthermore provides propagating material created from the plant of plants cells of the invention.
  • the creation of propagating material relates to any means known in the art to produce further plants, plant parts or seeds and includes inter alia vegetative reproduction methods (e.g. air or ground layering, division, (bud) grafting, micropropagation, stolons or runners, storage organs such as bulbs, corms, tubers and rhizomes, striking or cutting, twin- scaling), sexual reproduction (crossing with another plant) and asexual reproduction (e.g. apomixis, somatic hybridization).
  • vegetative reproduction methods e.g. air or ground layering, division, (bud) grafting, micropropagation, stolons or runners, storage organs such as bulbs, corms, tubers and rhizomes, striking or cutting, twin- scaling
  • sexual reproduction crossing with another plant
  • asexual reproduction e.g. apomixis, somatic hybridization
  • the plant cell described herein is a non-propagating plant cell, or a plant cell that cannot be regenerated into a plant, or a plant cell that cannot maintain its life by synthesizing carbohydrate and protein from the inorganics, such as water, carbon dioxide, and inorganic salt, through photosynthesis.
  • the invention also provides the use of the chimeric gene or mutant allele according to the invention to produce a plant, plant part, plant cell or seed with increased stress tolerance or increased biomass. Also provided is the use of the plant according to the invention to produce a population of plants, such as crop plants with increased stress tolerance or increased biomass (e.g. increased yield).
  • a nucleic acid or polynucleotide can be DNA or RNA, single- or double-stranded. Nucleic acids can be synthesized chemically or produced by biological expression in vitro or even in vivo. Nucleic acids can be chemically synthesized using appropriately protected ribonucleoside phosphoramidites and a conventional DNA/RNA synthesizer.
  • DNA includes cDNA and genomic DNA.
  • protein or "polypeptide” as used herein describe a group of molecules consisting of more than 30 amino acids, whereas the term “peptide” describes molecules consisting of up to 30 amino acids. Proteins and peptides may further form dimers, trimers and higher oligomers, i.e. consisting of more than one (poly)peptide molecule. Protein or peptide molecules forming such dimers, trimers etc. may be identical or non-identical. The corresponding higher order structures are, consequently, termed homo- or heterodimers, homo- or heterotrimers etc.
  • protein and “peptide” also refer to naturally modified proteins or peptides wherein the modification is effected e.g. by glycosylation, acetylation, phosphorylation and the like. Such modifications are well known in the art.
  • nucleic acid or protein comprising a sequence of nucleotides or amino acids, may comprise more nucleotides or amino acids than the actually cited ones, i.e., be embedded in a larger nucleic acid or protein.
  • a chimeric gene comprising a nucleic acid which is functionally or structurally defined, may comprise additional DNA regions etc.
  • Example 1 Experimen tal methods Preparation ofmicroarray datasets.
  • MST scores were calculated as described below in Example 2. Genes with an MST score ⁇ 6 (differentially expressed in less than three stresses) were removed and the remaining 6,005 genes were ranked based on their MST score. To compile a list of putative MSTR genes, the AGRIS (4), DATF (5) and PlantP (Gribskov M, et al. (2001 ) Nucleic Acids Res 29(1): 11 1 -113) databases were used to identify genes encoding regulatory proteins such as transcription factors, kinases and phosphatases. Other types of regulatory genes were extracted based on their MAPMAN bin code obtained with ROBIN. In total, 1 ,845 putative MSTR genes were identified representing -8% of the all non-control array identifiers on the ATHI chip.
  • HCA Hierarchical clustering
  • PCA principal component analysis
  • Time points from the same stress treatment present in more than one experiment and/or from the same experiment were merged by their median, irrespective of different cell types from the same tissue (e.g. root collumella cell and root stele cell) or treatment level (e.g. heat 37 °C and 40 °C). This is because they grouped closely together in the HCA and PCA. Furthermore, outlier datasets from the cell culture experiment and one of the UV experiments that were located on isolated clades, were discarded.
  • tissue e.g. root collumella cell and root stele cell
  • treatment level e.g. heat 37 °C and 40 °C
  • R software version 2.15.1
  • the TANGO software within the Expander software package was used. TANGO performs hypergeometric enrichment tests corrected for multiple hypothesis testing by bootstrapping and estimating the empirical p-value distribution for the evaluated sets.
  • Discovery rate was calculated as number of positive (stress phenotype) genes divided by number of genes tested. The probability of getting at least this number of positives was computed using the binomial distribution test with a probability of observing a positive gene set to 0.013, a value obtained by calculating the median discovery rate of several different large-scale screens of insertion mutants (Budziszewski GJ, et al. (2001 ) Genetics 159(4): 1765-1778; Rama Devi S, et al. (2006) Plant J 47(4): 652-663; Koiwa H,et al. (2006) J Exp Bot 57(5): 1 1 19-1 128; Dobritsa AA, et al.
  • T-DNA insertion lines were indeed homozygous for the T-DNA insert.
  • 10 lines that exhibited a stress phenotype were selected and then tested using gene-specific primers and a T-DNA-specific primer (Alonso et al., supra).
  • Nine lines were found to be homozygote and one line was heterozygote (Table 1 ). All the homozygote lines exhibited a stress phenotype while the heterozygote showed no significant difference to wild-type.
  • Abiotic stress assays were conducted according to (Kant P, et al (2007) Plant Physiol 145(3): 814-830) with some modifications. Approximately 40 to 50 surface-sterilized seeds were sown on plates containing MS salts, pH 5.8, 0.5 g L-1 MES, 0.8% (w/v) agar and either 2% (w/v) sucrose (heat stress assays) or no sucrose (salt and osmotic assays). Seeds were stratified at 4 °C for 4 d in the dark before being placed in a growth room at 22 °C, 50% relative humidity, and a photoperiod of 16 h light (150 ⁇ photons m-2s-1 )/8 h dark.
  • MS medium was supplemented with various concentrations of NaCI, mannitol or ABA, respectively.
  • Germination emergence of radicals
  • cotyledon emergence green, fully-open cotyledons
  • Germination was expressed either as percentage germination or germination index (a measure of germination rate) (Chiapusio G, et al. (1997) J Chem Ecol 23(1 1 ): 2445-2453). Three replicate plates were used per treatment.
  • Basal thermotolerance assays were performed essentially according to (Bolle C (2009) ed Pfannschmidt T (Springer, Jena) 479: pp 1 -25). Seeds were allowed to germinate and seedlings were grown for 5 d after stratification in the growth room. Seedlings were heat-treated at 45 °C for differing periods of time and then returned to the growth room. Untreated seedlings left in the growth room were used as a control. Four plates were used per treatment, and survival (i.e. presence of green true leaves) was recorded daily for 5 d, at which point, survival had stabilized. At least two independent experiments for each stress assay were performed on all lines with different batches of seeds for several lines tested to remove any variability due to growth conditions of parental plants.
  • MST score 2A + ⁇ (B/B C )
  • A is the number of distinct stresses in which a gene is differentially regulated
  • Bi/Bc estimates the repeatability of the differential expression per stress, summed over the 10 stresses.
  • a score of 1 was given for each stress type such that the A value of a gene that was up-/down-regulated under cold, salt and drought conditions would be 3;
  • B is the number of independent experiments of same stress in which gene expression is affected;
  • B c is the total number of experiments, above the first experiment, available for that stress.
  • Bi/Bc is summed over the 10 stress treatments.
  • the 2:1 ratio between the A and B gives weight to the effect on gene expression of different stresses while the B value represents a bonus for repeatability of microarray results.
  • MST Multiple Stress
  • MST Multiple Stress Regulatory
  • MST score 2A + ⁇ (B/B C )
  • A is the number of stress types in which a gene is differentially regulated. A score of 1 was given for each stress type such that the A value of a gene that was up-/down-regulated under cold, salt and drought conditions would be 3; B, is the number of independent experiments of same stress in which gene expression is affected; B c is the total number of experiments, above the first experiment, available for that stress.
  • the well-characterized DREB2A abiotic stress transcriptional regulator (Liu Q, et al. (1998) Plant Cell 10(8): 1391 -1406, Nakashima K, et al. (2000) Plant Mol Biol 42(4): 657-665) obtained a high MST score of 24.4 whereas the housekeeping gene UBQ10 obtained an MST score of zero.
  • the microarray compendium comprises diverse experiments from multiple stresses, different organs, developmental stages and even cell culture, which could lead to false positive associations between gene pairs.
  • we performed hierarchical clustering of MST gene expression over the whole microarray compendium see Example 1 .
  • This analysis revealed that MST genes clustered by stress ( Figure 1 ).
  • This analysis further showed that experiments using cell culture clustered separately from shoots and roots and these experiments were therefore removed from the analysis. In light of these data, we decided to generate separate co-expression networks for shoots and roots.
  • Example 4 Construction of MSTR gene co-expression networks, iden tifica tion of functional modules and selection of new candida te MSTR genes for abiotic stress screening
  • the modules possessed a higher median homogeneity score than networks generated with lower r-values ( Figures 4A and B). Similar results were obtained for the shoot network (not shown). Based on this analysis, we selected r ⁇ 0.85, which yielded 10 and 15 shoot and root modules, respectively (Table 3).
  • the shoot MSTR network comprised 26 connected components with the largest connected component containing 70 nodes (genes). Together, the shoot network comprised 535 edges linking 236 nodes.
  • the root MSTR network comprised 39 connected components with the largest connected component containing 66 nodes. Together, the root network comprised 382 edges linking 253 nodes.
  • analyses of global network properties showed that the networks are small world and scale-free (Ravasz et al Methods Mol.Biol. 541 , 145-160, 2009) ( Figure 9). Overall the modules comprised 360 distinct genes, a manageable number for selecting candidate genes for mutant screening.
  • At3g57540 seedlings showed an ABA insensitive phenotype and only displayed 52% reduction in germination, an even stronger phenotype than the classic ABA insensitive mutant, abi1 (Koornneef et al., 1989, Plant Physiol 90: 463469) that showed a 65% decrease in germination.
  • abi1 Zaornneef et al., 1989, Plant Physiol 90: 463469
  • Example 5 Mutan t screen of candida te Arabidopsis MSTR genes reveals previous ⁇ ' uniden tified ab iotic stress reg ula tors
  • AtERF4 is a negative regulator of ABA and ethylene responsive gene expression while ovenexpnession of BrERF4 from Brassica rapa in transgenic Arabidopsis generates stress tolerant plants (Yang Z, et al. (2005) Plant Mol. Biol. 58(4): 585-596; Seo, Y-J, et al.
  • HSFA4A is a principal candidate H 2 0 2 sensor during oxidative stress (Davletova S, et al. (2005) Plant Cell 17(1): 268-281 ; Miller G, Mittler R (2006) Ann Bot 98(2): 279-288; Scarped TE, et al. (2008) Plant Signal Behav 3(10): 856-857);
  • NAC032 is a member of a large family of plant NAC genes, many of which have been shown to be involved in regulating abiotic stress responses (Nakashima K,et al. (2012; Biochim Biophys Acta 1819(2): 97-103).
  • ROC curves that measure true-positive phenotype rate versus false-positive rate as a function of MST score.
  • the performance can be assessed by calculating the area under the curve (AUC), which is equal to the probability that the
  • MST score will rank a randomly chosen positive phenotype higher than a randomly chosen negative one.
  • Figure 8C-D For analyses of non-network and network MSTR genes together, see Figure 8C-D.
  • Phenopsis automated watering and measurements.
  • sorbitol 100mM
  • 14cm gridded petri dishes containing half MS medium supplemented with 0.8% agar
  • Seeds are sterilized using a vapor-phase method (for 1 h) and sown the day after on control and sorbitol- supplemented plates.
  • the gridded plate is split into four to allow the sowing of four different lines. Eight seeds per lines are thus sown in each plate and this is replicated 10 times. In total 80 plants, for each line, are sown either on control or on sorbitol-supplemented medium.
  • the plates are then stored in the dark at 4°C for three days. Following the stratification, plates are transferred to the growth chamber (22°C, 16/8 light/dark) and arranged following a randomized block design. To measure growth rate and biomass parameters, pictures are taken at 11 , 14, 16 and 18 day. The average area of the plants is measured and compared (at each time point) to that of the Col8 WT. The area and growth rate results are presented as percentage difference.
  • the herein identified sequences or fragments thereof are cloned into a vector for downregulation of the endogenous gene as described elsewhere in this application (e.g. sense, antisense, hairpin or miRNA suppression) and transformed into plants, also as described elsewhere in this application. Downregulation of expression in the transformed plants is confirmed on the RNA and/or protein level. Plants are subjected to abiotic stress assays as described above. Plants are selected that have increased stress tolerance and/or that show increase growth rate or biomass as compared to control plants.
  • At3g33066a 98 94.3 0.608 97.5 94.3 0.658 5 c
  • At3g57540a 100 100 1.000 100 98 0.594

Abstract

La présente invention concerne un procédé pour augmenter la tolérance au stress et/ou la biomasse qui consiste à moduler l'expression de l'un quelconque des nouveaux gènes régulateurs du stress abiotique identifiés dans la description, ainsi que des gènes chimères, des acides nucléiques et des polypeptides codant pour lesdits gènes. L'invention concerne également des plantes ou une partie de ces dernières modulées selon l'invention, présentant une tolérance au stress et/ou une biomasse augmentée.
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