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WO2018078390A1 - Compositions et procédés pour augmenter la tolérance au stress abiotique - Google Patents

Compositions et procédés pour augmenter la tolérance au stress abiotique Download PDF

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WO2018078390A1
WO2018078390A1 PCT/GB2017/053253 GB2017053253W WO2018078390A1 WO 2018078390 A1 WO2018078390 A1 WO 2018078390A1 GB 2017053253 W GB2017053253 W GB 2017053253W WO 2018078390 A1 WO2018078390 A1 WO 2018078390A1
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plant
nucleotide sequence
seq
sequence
nucleic acid
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Inventor
Qi Xie
Jian LV
Gang Li
Ran XIA
Yongshen SHANG
Jiang Li
Pingsha HU
Michael Nuccio
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Institute of Genetics and Developmental Biology of CAS
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Institute of Genetics and Developmental Biology of CAS
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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
    • C12N15/8273—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 for drought, cold, salt resistance
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • 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

Definitions

  • the present invention relates to compositions and methods for enhancing abiotic stress tolerance in plants.
  • Drought, salinity, and temperature extremes are all abiotic stresses which affect the normal growth and development of plants and limit crop yields. Identifying genes that improve abiotic stress tolerances could lead to more efficient crop production.
  • a breeding program which selects and produces plants with improved abiotic stress tolerance is one approach; however, the complexity of plant stress adaptation makes breeding for abiotic stress tolerance complicated. Therefore, identification of a gene from any plant species which confers abiotic stress tolerance(s) and introduction of that gene into an important crop species is a powerful approach toward increasing abiotic stress tolerances in that crop species.
  • the present invention provides abiotic stress tolerant plants and/or plant parts, as well as methods and compositions for identifying, selecting and/or producing abiotic stress tolerant plants and/or plant parts.
  • Some embodiments provide abiotic stress tolerant plants and/or plant parts, which are cold tolerant and/or are tolerant to at least two abiotic stresses, as well as methods and compositions for identifying, selecting and/or producing abiotic stress tolerant (e.g., cold tolerant) plants and/or plant parts.
  • the at least two abiotic stresses that a plant and/or plant part are tolerant to does not include salt stress, but the plant and/or plant part may be salt stress tolerant and tolerant to at least two different abiotic stresses.
  • plants and/or plant parts having increased yield and/or increased seed germination under abiotic stress conditions such as, e.g., cold stress conditions
  • abiotic stress conditions such as, e.g., cold stress conditions
  • methods and compositions for identifying, selecting and/or producing plants and/or plant parts having increased yield and/or increased seed germination under abiotic stress conditions such as, e.g., cold stress conditions.
  • the present invention provides an expression cassette, vector, transgenic bacterium, plant and/or plant part that comprises a promoter operably linked to an exogenous nucleic acid comprising one or more of the nucleotide sequences of any one of SEQ ID NOs: 1 to 3, 6 ,7, or 14 to 18, one or more of the nucleotide sequences that encode a polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 4, 5, 8, 9, 10, 11, 12, 13, 19, 20, 21, 22, or 23, one or more nucleotide sequences that are at least 70% identical to the nucleotide sequence of any one of SEQ ID NOs: 1 to 3, 6 ,7, or 14 to 18, one or more nucleotide sequences that encode a polypeptide comprising an amino acid sequence that is at least 70% identical to the amino acid sequence of any one of SEQ ID NOs: 4, 5, 19, 20, 21, 22, or 23, one or more nucleotide sequences that are complementary to one of the aforementioned nucleotide sequences
  • the present invention provides a method of identifying a plant and/or plant part having enhanced cold stress tolerance and/or enhanced tolerance to at least two abiotic stresses, the method comprising detecting, in a plant and/or plant part, one or more nucleic acids that comprise one or more of the nucleotide sequences of any one of SEQ ID NOs: 1 to 3, 6 ,7, or 14 to 18, one or more nucleotide sequences that encode a polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 4, 5, 8, 9, 10, 1 1, 12, 13, 19, 20, 21, 22, or 23, one or more nucleotide sequences that are at least 70% identical to the nucleotide sequence of any one of SEQ ID NOs: 1 to 3, 6 ,7, or 14 to 18, one or more nucleotide sequences that encode a polypeptide comprising an amino acid sequence that is at least 70% identical to the amino acid sequence of any one of SEQ ID NOs: 4, 5, 19, 20, 21, 22, or 23, one or more nucleot
  • the present invention provides a method of producing a plant having enhanced cold stress tolerance and/or having enhanced abiotic stress tolerance to at least two abiotic stresses as compared to a control plant or plant part.
  • this method comprises introducing an exogenous nucleic acid encoding a polypeptide comprising a C2 domain capable of binding calcium (e.g., a C2 domain that binds calcium) into a plant part.
  • this exogenous nucleic acid comprises a nucleotide sequence that encodes a polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 4, 5, 8, 9, 10, 11, 12, 13, 19, 20, 21, 22, or 23, or the exogenous nucleic acid encodes a polypeptide comprising an amino acid sequence that is at least 70% identical to SEQ ID NO:4, or the exogenous nucleic acid encodes a polypeptide comprising an amino acid sequence that is SEQ ID NO:4, or one or more nucleotide sequences that are complementary to one of the aforementioned nucleotide sequences, or one or more nucleotide sequences that specifically hybridize to any one of the aforementioned nucleotide sequences under stringent hybridization conditions, and/or a functional fragment of one or more of the aforementioned nucleotide sequences.
  • the plant part having enhanced cold stress tolerance and/or having enhanced abiotic stress tolerance to at least two abiotic stresses may be grown into a plant that expresses the exogenous nucleic acid.
  • the resulting plant may also have enhanced cold stress tolerance and/or enhanced abiotic stress tolerance to at least two abiotic stresses as compared to a control plant that has not been transformed with the exogenous nucleic acid.
  • the present invention provides a method of enhancing cold stress tolerance and/or abiotic stress tolerance to at least two abiotic stresses in a plant, as compared to a control plant or plant part.
  • This method comprises expressing in the plant an exogenous nucleic acid, which in some embodiments comprises a nucleotide sequence of any one of SEQ ID NOs: 1 to 3, 6 ,7, or 14 to 18; in some embodiments comprises a nucleotide sequence that is at least 70% identical to the nucleotide sequence of any one of SEQ ID NOs: 1 to 3, 6 ,7, or 14 to 18; in some embodiments comprises a nucleotide sequence that encodes a polypeptide comprising a C2 domain capable of binding calcium, wherein the amino acid sequence of the polypeptide comprises any one of SEQ ID NOs: 4, 5, 8, 9, 10, 11, 12, 13, 19, 20, 21, 22, or 23; in some embodiments comprises a nucleotide sequence that encodes a polypeptide comprising a C2 domain capable of binding
  • this exogenous nucleic acid results in enhanced cold stress tolerance and/or enhanced abiotic stress tolerance to at least two abiotic stresses in a plant, as compared to a control plant or plant part.
  • this method of enhancing cold stress tolerance and/or enhancing abiotic stress tolerance to at least two abiotic stresses further comprises introducing the exogenous nucleic acid into the plant.
  • this method of enhancing cold stress tolerance and/or enhancing abiotic stress tolerance to at least two abiotic stresses further comprises introducing the exogenous nucleic acid into a plant part and producing the plant from the plant part.
  • the present invention provides a method of identifying a plant or plant part having enhanced cold stress tolerance and/or enhanced abiotic stress tolerance to at least two abiotic stresses as compared to a control plant or plant part.
  • This method comprises detecting in a plant part an exogenous nucleic acid, which in some embodiments comprises a nucleotide sequence of any one of SEQ ID NOs: 1 to 3, 6 ,7, or 14 to 18; in some embodiments comprises a nucleotide sequence that encodes a polypeptide comprising a C2 domain capable of binding calcium, wherein the amino acid sequence of the polypeptide comprises any one of SEQ ID NOs: 4, 5, 8, 9, 10, 11, 12, 13, 19, 20, 21, 22, or 23; in some embodiments comprises a nucleotide sequence that is at least 70% identical to the nucleotide sequence of any one of SEQ ID NOs: 1 to 3, 6 ,7, or 14 to 18; in some embodiments comprises a nucleotide sequence that encodes a polypeptide comprising a
  • exogenous nucleic acid thereby identifies a plant or plant part having enhanced cold stress and/or enhanced abiotic stress tolerance to at least two abiotic stresses.
  • the exogenous nucleic acid or an informative fragment thereof is detected in an amplification product from a nucleic acid sample from the plant or plant part.
  • the present invention provides a method of producing a plant having enhanced cold stress and/or enhanced abiotic stress tolerance to at least two abiotic stresses as compared to a control plant or plant part, whereby following the identification of a plant or plant part having enhanced cold tolerance and/or abiotic stress tolerance as described above, a plant is produced from the plant part, thereby producing a plant having enhanced cold stress tolerance and/or enhanced abiotic stress tolerance to at least two abiotic stresses as compared to a control plant.
  • the present invention provides a method of producing a plant having enhanced cold stress tolerance and/or enhanced abiotic stress tolerance to at least two abiotic stresses as compared to a control plant or plant part using breeding techniques.
  • This method comprises crossing a first parent plant with a second parent plant, wherein the first parent plant comprises within its genome an exogenous nucleic acid, which in some embodiments comprises a nucleotide sequence of any one of SEQ ID NOs: 1 to 3, 6 ,7, or 14 to 18; in some embodiments comprises a nucleotide sequence that is at least 70% identical to the nucleotide sequence of any one of SEQ ID NOs: 1 to 3, 6 ,7, or 14 to 18; in some embodiments comprises a nucleotide sequence that encodes a polypeptide comprising a C2 domain capable of binding calcium, wherein the amino acid sequence of the polypeptide comprises any one of SEQ ID NOs: 4, 5, 8, 9, 10, 11, 12, 13, 19, 20, 21, 22, or 23; in some embodiments comprises a nucleo
  • the cross produces a progeny generation comprising at least one plant that possesses the exogenous nucleic acid within its genome and that exhibits enhanced cold stress tolerance and/or enhanced abiotic stress tolerance to at least two abiotic stresses, as compared to a control plant.
  • the present invention provides a nonnaturally occurring nucleic acid that is a monocot codon optimized nucleotide sequence that encodes a polypeptide that comprises an eight stranded anti-parallel ⁇ -sandwich, that comprises a C2 domain, and/or that binds calcium.
  • the nonnaturally occurring nucleic acid comprises a monocot codon-optimized nucleotide sequence, such as, for example, a nucleotide sequence that is codon-optimized for expression in maize.
  • the nonnaturally occurring nucleic acid comprises a monocot codon-optimized nucleotide sequence encoding a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:4. In some embodiments, the nonnaturally occurring nucleic acid comprises a monocot codon-optimized nucleotide sequence that is at least 70% identical to the nucleotide sequence of SEQ ID NO:7. In some embodiments, the nonnaturally occurring nucleic acid is isolated.
  • the present invention provides nonnaturally occurring nucleic acids comprising the nucleotide sequence set forth in SEQ ID NO:7, a monocot codon-optimized nucleotide sequence that encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:4, a monocot codon-optimized nucleotide sequence that is at least 70% identical to the nucleotide sequence set forth in SEQ ID NO: 7, a monocot codon-optimized nucleotide sequence that encodes a polypeptide comprising an amino acid sequence that is at least 70% identical to the amino acid sequence of SEQ ID NO:4, a nucleotide sequence that is complementary to one or more of the aforementioned nucleotide sequences, a nucleotide sequence that specifically hybridizes to one or more of the aforementioned nucleotide sequences under stringent hybridization conditions, and/or a functional fragment of one or more of the aforementioned nucleotide sequences.
  • the present invention provides an expression cassette or vector comprising a promoter operably linked to an exogenous nucleic acid sequence, which in some embodiments comprises a nucleotide sequence of any one of SEQ ID NOs: 1 to 3, 6 ,7, or 14 to 18; in some embodiments comprises a nucleotide sequence that is at least 70% identical to the nucleotide sequence of any one of SEQ ID NOs: 1, 3, 6, 7, or 14 to 18; in some embodiments comprises a nucleotide sequence that encodes a polypeptide comprising a C2 domain capable of binding calcium, wherein the amino acid sequence of the polypeptide comprises any one of SEQ ID NOs: 4, 5, 8, 9, 10, 11, 12, 13, 19, 20, 21, 22, or 23; in some embodiments comprises a nucleotide sequence that encodes a polypeptide comprising a C2 domain capable of binding calcium, wherein the amino acid sequence of the polypeptide is at least 70% identical to the amino acid sequence of any one of SEQ ID NOs: 4, 5, 19, 20, 21,
  • the present invention provides a transgenic plant comprising the expression cassette, wherein the plant has enhanced cold stress tolerance and/or enhanced abiotic stress tolerance to at least two abiotic stresses compared to a plant lacking the expression cassette when grown in similar conditions.
  • the present invention provides a transgenic plant comprising an exogenous nucleic acid sequence which confers enhanced cold stress tolerance and/or enhanced abiotic stress tolerance to at least two abiotic stresses.
  • the exogenous nucleic acid sequence comprises a nucleotide sequence of any one of SEQ ID NOs: 1 to 3, 6, 7, or 14 to 18; in some embodiments comprises a nucleotide sequence that is at least 70% identical to the nucleotide sequence of any one of SEQ ID NOs: 1, 3, 6, 7, or 14 to 18; in some embodiments the exogenous nucleic acid sequence comprises a nucleotide sequence that encodes a polypeptide comprising a C2 domain capable of binding calcium, wherein the amino acid sequence of the polypeptide comprises any one of SEQ ID NOs: 4, 5, 8, 9, 10, 11, 12, 13, 19, 20, 21, 22, or 23; in some embodiments the exogenous nucleic acid sequence comprises a nucleotide sequence that encodes a polypeptide comprising a C2 domain capable
  • the transgenic plant is a dicotyledonous plant.
  • the transgenic plant is selected from the group consisting of Brassica ssp, millet, switchgrass, maize, sorghum, wheat, oat, turf grass, pasture grass, papaya, flax, peppers, potato, sunflower, tomato, crucifers, soybean, common bean, lotus, grape, peach, cacao, cotton, rice, soybean, sugarcane, sugar beet, tobacco, barley, cassava, cucumber, watermelon, melon, orange, Clementine, castor bean, and grapevine.
  • the transgenic plant is not Thellungiella salsuginea (previously referred to as T. halophila) and/or the transgenic plant is not Arabidopsis thaliana.
  • Fig. 1 provides a phylogenic tree showing the relationships between ThST03 (also referred to herein as TsST03) and its orthologs in other plant species.
  • ThTS03 homologous protein sequences were retrieved by using ThTS03 peptide sequence for BLASTP search against all C2 domain proteins in an internal database with a cutoff E value less than 10. Sequences were aligned by Clustalx with a gap open penalty of 10 and a gap extension penalty of 0.2. The aligned peptides were used for phylogenetic construction by the UPGMA method using the software program MEGA6.
  • the UPGMA tree was constructed based on the Poisson correction distance with 5,000 bootstrap replicates. No sequence was selected as an outgroup. The number on each node represents a measure of support for the node. For example, 95 means the same node is recovered through 95 of 100 iterations during the bootstrap resampling analysis. The genetic distance is indicated on the bottom ruler.
  • Fig. 7 is a graph showing the survival rate percentage of plants watered with NaCl- containing nutrient solution.
  • the concentration of NaCl was increased by 50 mM every 3 days to the final 200 mM.
  • Data presented as mean ⁇ s.d. (n 30 seedlings, with four biological replicates).
  • Fig. 8 is a graph showing the survival rate percentage of plants treated with 14 days water withholding and subsequent 4 days of rehydration recovery.
  • Fig. 9 is an illustration of a conservative sequence alignment of the C2 domains from Thellungiella salsuginea (ST03) (SEQ ID NO:44), Arabidopsis (At3g55470, SEQ ID NO:45, and At2g63220, SEQ ID NO:48) and pumpkin (Cmppl6-1, SEQ ID NO:46, and Cmppl6-2, SEQ ID NO:47; Xoconostle-Cazares et al, 1999. Science 288: 94-98). Amino acid residues that form the Ca 2+ binding sites of the C2 domains in Cmppl6-1 and 16-2 are indicated by pentagrams.
  • Fig. 10 illustrates the results obtained from Ca 2+ -dependent phospholipid binding assays.
  • panel (i) is an image of an SDS-PAGE gel showing the results after the GST-TsST03 (also referred to herein as GST-ST03) fusion protein was incubated with liposomes (25%
  • Fig. 10 panel (ii) is an image of an SDS-PAGE gel showing the results after the GST-ST03 (mCBS) fusion protein was used in phospholipid binding assays to determine the Ca 2+ -dependent phospholipid binding ability.
  • Fig. 11 is a graph showing the fresh weight for seedlings three days after being transferred to hydroponic medium to which 120 mM NaCl along with either 0, 1, 5, or 10 mM calcium nitrate was added. For fresh weight determination, the same number of seedlings were weighed at one time. Data are means of three independent assays.
  • Fig. 12 is a graph showing germination rate percentages for wild-type, 35S-ST03 and
  • Fig. 13 is a graph showing post-germination phenotype percentages for wild-type, 35S- ST03 and 35S-ST03 (ACBS) transgenic lines under NaCl treatments. Data are means of three independent assays.
  • Fig. 14 is a graph showing seedling root length for seedlings grown at different Ca 2+ concentrations under salt stress. Wild-type, P 3 ss-'ST03 and P 3 ss-'ST03(mCBS) transgenic line seedlings were grown for 3 days in half-strength MS medium and then transferred to one-tenth MS medium at the Ca 2+ concentrations indicated and lOOmM NaCl. Data are means of three independent assays.
  • Fig. 15 is a graph showing ion leakage percentages for wild-type, P 3 ss-'ST03 and P35S-'ST03 (mCBS) transgenic lines.
  • One-week-old seedlings grown in half-strength MS medium without NaCl were transferred to one-tenth MS medium plus the indicated Ca 2+ concentrations and 150mM NaCl, and after 12 h electrolyte leakage was determined.
  • Data are means of three independent assays. P values are significantly different between the wild type and P 3 ss-'ST03 transgenic line.
  • Fig. 16 is a graph showing survival rate percentages for Pubi-'Myc-ST03 transgenic rice under NaCl under salt stress. 14-day-old seedlings of two transgenic lines and wild-type control were grown in Hoagland' s hydroponical medium were treated with 100 mM NaCl for 1 week and rehydrated.
  • Fig. 17 is a polypeptide alignment of ThST03 (SEQ ID NO:4) and its orthologs from Brassica oleracea (cabbage) (SEQ ID NO:50), Brassica rapa (mustard) (SEQ ID NO:49), and Arabidopsis (SEQ ID NO:5). Sequences are at least 80% identical to the consensus sequence (SEQ ID NO: 8).
  • Fig. 18 is a polypeptide alignment of ST03 orthologs from Cajanus cajan (pigeon pea)
  • Sequences are at least 74% identical to the consensus sequence (SEQ ID NO: 9).
  • Fig. 19 is a polypeptide alignment of ST03 orthologs from Cucumis melo (melon) (SEQ ID NO:22) and Cucumis sativus (cucumber) (SEQ ID NO:55). Sequences are at least 85% identical to the consensus sequence (SEQ ID NO: 10).
  • Fig. 20 is a polypeptide alignment of ST03 orthologs from Vitis vinifera (grape) (SEQ ID NO:57), Citrus Clementina (clementine) (SEQ ID NO:23), and Citrus sinensis (orange) (SEQ ID NO:56). Sequences are at least 70% identical to the consensus sequence (SEQ ID NO: 11).
  • Fig. 21 is a polypeptide alignment of ST03 orthologs from Solanum lycopersicum
  • Fig. 22 is a polypeptide alignment of ST03 orthologs from Zea mays (corn) (SEQ ID NO: 19) and Oryza sativa (rice) (SEQ ID NO: 60). Sequences are at least 71% identical to the consensus sequence (SEQ ID NO: 13).
  • Fig. 23 illustrates that transgenic Arabidopsis expressing TsST03-OX have increased drought tolerance.
  • Wild type plants (Col-I) and two transgenic lines (20.3 and 16.2) were grown under the same condition in soil (Fig 23 A).
  • Drought treatment was applied to soil-grown 3- week-old TsST03 and control plant lines to assess whether the TsST03 gene also confers drought tolerance.
  • Both TsST03 and control plants became wilted following withholding of water for 14 days (Fig 23B).
  • Fig 23D Importantly, within 4 days of re-watering, TsST03 plants had recovered, whereas most control plants failed to recover from this stress treatment (Fig 23D).
  • Survival rate for wild type plant is 32%, but 86% and 92% respectively for the two trangenic lines (Fig 23C).
  • no evident phenotypic differences were observed for plants grown in the presence of an adequate water supply.
  • Fig. 24 illustrates that transgenic Arabidopsis expressing TsST03-OX have increased salt tolerance.
  • P35S:Myc-TsST03 construct were used (TsST03/Col and Col/TsST03), with wild-type self- grafted plants (Col/Col) being used as the control.
  • These grafting experiments were performed with 4-week-old plants soon after bolting by using stem grafting techniques, and the grafted plants were subjected to salt stress 2 weeks after grafting.
  • 3 weeks after NaCl treatment allografted seedlings, in which the wild-type plant acts as scion and TsST03 transgenic plant as stock and that in opposite direction, have a significantly higher survival rate than the self-grafted wild-type plants; note the strong growth inhibition showed by both the scion and the stock of wild-type self-grafted plants. This result demonstrated that TsST03 moves long-distance through the phloem to contribute the salt tolerance in recipient part of plants.
  • Fig. 25 shows the response of Arabidopsis transformed with ST03-OE to heat stress.
  • Control plants Col-0
  • plants overexpressing WT-TsST03 P35S:TsST03
  • plants overexpressing of calcium binding domain mutated ST03 gene P35S:TsST03 (mCBS)
  • mCBS calcium binding domain mutated ST03 gene
  • Fig. 26 shows an increased dehydration tolerance in TsST03 transgenic rice.
  • Control (WT) and TsST03 transgenic rice (231-4) were grown in MS medium contaning 20% PEG6000 for 10 days and then moved to MS medium for recovery for one week. The results show that TsST03 transgenic rice has increased dehydration tolerance.
  • Fig. 27 shows an increased drought tolerance in TsST03 transgenic rice.
  • drought treatment was applied at two different growth stages. At the young seedling stage (4-week-old plants having 5 leaves), imposing a 14 day drought treatment caused leaf rolling and wilting phenotypes, with wild-type plants exhibiting the most sever symptoms. After re-watering for 14 days, wild-type plants failed to recover and died. In contrast, most of the TsST03 transgenic lines recovered.
  • TsST03 transgenic plants displayed only mild symptomsm Five days after re-watering, TsST03 transgenic plant lines exhibited a high survival rate (80%-90%), whereas the corresponding survival rate for the wild-type control was very low (15%). Taken together, these studies indicate that over-expression of TsST03 can improve drought tolerance in rice.
  • the present invention provides compositions and methods for identifying, selecting and/or producing plants and/or plant parts having enhanced abiotic stress tolerance, as well as plants and/or plant parts identified, selected and/or produced using compositions and methods of the present invention. Some embodiments provide compositions and methods for identifying, selecting and/or producing plants and/or plant parts having enhanced abiotic stress tolerance, as well as plants and/or plant parts identified, selected and/or produced using compositions and methods of the present invention. In some embodiments, provided are compositions and methods for identifying, selecting and/or producing plants and/or plant parts having enhanced abiotic stress tolerance to at least two abiotic stresses and/or enhanced cold stress tolerance, as well as plants and/or plant parts identified, selected and/or produced using compositions and methods of the present invention.
  • the at least two abiotic stresses that a plant and/or plant part are tolerant to does not include salt stress, but the plant and/or plant part may be salt stress tolerant and tolerant to at least two different abiotic stresses (e.g., cold stress and drought stress).
  • a plant and/or plant part may be tolerant to at least three abiotic stresses (e.g., salt stress, cold stress, and drought stress).
  • an endogenous nucleic acid can mean one endogenous nucleic acid or a plurality of endogenous nucleic acids.
  • a given composition is described as comprising "about 50% X,” it is to be understood that, in some embodiments, the composition comprises
  • 50% X whilst in other embodiments it may comprise anywhere from 40% to 60% X (i.e., 50% ⁇ 10%).
  • backcross and “backcrossing” refer to the process whereby a progeny plant is crossed back to one of its parents.
  • the "donor” parent refers to the parental plant with the desired allele or locus to be introgressed.
  • the “recipient” parent (used one or more times) or “recurrent” parent (used two or more times) refers to the parental plant into which the gene or locus is being introgressed.
  • the initial cross gives rise to the Fl generation.
  • BC1 refers to the second use of the recurrent parent
  • BC2 refers to the third use of the recurrent parent, and so on.
  • cross refers to the fusion of gametes to produce progeny (e.g., cells, seeds or plants).
  • progeny e.g., cells, seeds or plants.
  • the term encompasses both sexual crosses (e.g., the pollination of one plant by another or the combination of protoplasts from two distinct plants via protoplast fusion) and selfing (e.g., self-pollination wherein the pollen and ovule are from the same plant).
  • the terms “cultivar” and “variety” refer to a group of similar plants that by structural or genetic features and/or performance can be distinguished from other
  • the terms “decrease,” “decreases,” “decreasing” and similar terms refer to a reduction of at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or more.
  • the reduction results in no or essentially no activity (i.e., an insignificant or undetectable amount of activity).
  • abiotic stress and “abiotic stress conditions” refer to nonliving factors that negatively affect a plant's ability to grow, reproduce and/or survive (e.g., drought, flooding, extreme temperatures (either cold or heat), extreme light conditions, extreme osmotic pressures, extreme salt concentrations, high winds, and poor edaphic conditions (e.g., extreme soil pH, nutrient-deficient soil, compacted soil, etc.)).
  • abiotic stress tolerance and “abiotic stress tolerant” refer to a plant's ability to endure and/or thrive under abiotic stress conditions (e.g., drought stress conditions, osmotic stress conditions, salt stress conditions and/or temperature stress conditions).
  • abiotic stress conditions e.g., drought stress conditions, osmotic stress conditions, salt stress conditions and/or temperature stress conditions.
  • a plant and/or plant part can have enhanced stress tolerance to cold stress and/or to at least two abiotic stresses, and that the enhanced stress tolerance to cold stress and/or to at least two abiotic stresses may be achieved in a crop plant (e.g., a monocot or a dicot).
  • a plant or plant part may experience an abiotic stress or stresses that is stressful enough to inhibit or alter the ability of the plant or plant part to grow, reproduce, and/or survive when compared to conditions at which the plant or plant part exhibits normal growth and/or development.
  • a plant or plant part may experience an abiotic stress or stresses that is/are perceived by the plant or plant part.
  • a plant or plant part may perceive cold stress through a cell membrane receptor in the plant or plant part, which may signal cold-responsive genes and/or transcription factors to be turned on to mediate the cold stress.
  • an abiotic stress or stresses may be determined in a plant or plant part by detecting the transcription of one or more abiotic stress- responsive genes and/or transcription factors.
  • an abiotic stress or stresses may be determined in a plant or plant part by detecting physiological, biochemical, metabolic and/or molecular changes within the plant or plant part.
  • conditions that may be stressful for one plant or plant part e.g., a temperature at which the plant or plant part experiences cold stress
  • parameters for any given abiotic stress may be species specific and even variety specific, and, therefore, may vary widely according to the species/variety exposed to the abiotic stress or stresses.
  • one species may experience cold stress at a temperature of 15°C, another species may not be impacted until at least 10°C, and the like.
  • a plant or plant part may experience cold stress at a temperature of about 15°C or less, such as, for example, at a temperature of about 15°C, 14°C, 13°C, 12°C, 1 1°C, 10°C, 9°C, 8°C, 7°C, 6°C, 5°C, 4°C, 3°C, 2°C, 1°C, 0°C, -FC, -2°C, -3°C, -4°C, -5°C, -6°C, -7°C, -8°C, - 9°C, -10°C, or less.
  • a “cold tolerant” and/or “cold stress tolerant” plant and/or plant part may also be referred to as a "temperature stress tolerant” or “abiotic stress tolerant” plant and/or plant part because cold stress is a temperature stress, which is an abiotic stress.
  • the term “drought resistance” or “drought tolerant”, including any other grammatical variations, refers to the ability of a plant to recover from periods of drought stress (i.e., little or no water for a period of days).
  • the drought stress will be at least 5 days and can be as long as, for example, 18 to 20 days or more (e.g., at least 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20 days) depending on, for example, the plant species.
  • salt tolerant As used herein, the term “salt tolerant”, “salinity tolerance”, or “salt stress tolerant”, including any other grammatical variations, refers to the relative ability of a plant to survive in conditions where the salt concentration is significantly higher than what is a typical native environment for the plant. The ability of a plant to tolerate salt is determined by its ability to retain or acquire water, protect chloroplast functions, and/or maintain ion homeostasis. High salinity can lead to adverse effects on germination, plant vigor, and/or crop yield.
  • heat stress is due to an environmental condition which has a relatively significant increased temperature, for example a very high temperature for a short period of time or a moderately high temperature for a longer period of time, compared to the typical native environment in which a given plant lives. Transitory or constantly high temperatures can affect plant growth and development and impact crop yield.
  • the term "enhanced abiotic stress tolerance” and grammatical variations thereof refers to an improvement in the ability of a plant and/or plant part to grow, reproduce and/or survive under abiotic stress conditions, as compared to one or more controls (e.g., a native plant/plant part of the same species). "Enhanced” may refer to any improvement in a plant's or plant part's ability to thrive and/or endure when grown under stress conditions, including, but not limited to, cold stress conditions.
  • enhanced abiotic stress tolerance is evidenced by increased seed germination, decreased water loss, decreased accumulation of one or more reactive oxygen species, decreased accumulation of one or more salts, increased salt excretion, increased accumulation of one or more dehydrins, improved root architecture, improved osmotic pressure regulation, increased accumulation of one or more late
  • a plant or plant part that exhibits enhanced abiotic stress tolerance as compared to a control plant or plant part may be designated as "abiotic stress tolerant.
  • the improvement in an abiotic stress tolerance trait may include, but is not limited to, increased seed germination, increased yield, increased seedling growth, decreased chlorosis, increased leaf expansion, decreased wilting, decreased necrosis, increased reproductive development, and/or decreased cell and/or organelle membrane damage.
  • a plant or plant part that exhibits an improvement in one or more abiotic stress tolerance traits as compared to a control plant (e.g., one or both of its parents) when each is grown under the same or substantially the same abiotic stress conditions displays enhanced abiotic stress tolerance and may be designated as "abiotic stress tolerant.
  • the improvement in an abiotic stress tolerance trait may include, but is not limited to, increased seed germination, decreased water loss, decreased accumulation of one or more reactive oxygen species, decreased accumulation of one or more salts, increased salt excretion, increased accumulation of one or more dehydrins, improved root architecture, improved osmotic pressure regulation, increased accumulation of one or more late embryogenesis abundant proteins, increased survival rate, increased growth rate, increased height, increased chlorophyll content, increased sugar concentration and/or availability, increased yield stability, and/or increased yield (e.g., increased biomass, increased seed yield, increased GSC, increased YGSMN, increased GMSTP, increased GWTPN, increased PYREC, decreased YRED, and/or decreased PB).
  • increased seed yield e.g., increased seed yield, increased GSC, increased YGSMN, increased GMSTP, increased GWTPN, increased PYREC, decreased YRED, and/or decreased PB.
  • a plant or plant part that exhibits an improvement in one or more abiotic stress tolerance traits as compared to a control plant or plant part (e.g., one or both of its parents) when each is grown under the same or substantially the same conditions where one or more abiotic stresses are present displays enhanced abiotic stress tolerance and may be designated as "abiotic stress tolerant.”
  • the plant or plant part may exhibit an improved abiotic tolerance trait as compared to the control plant or plant part and/or the plant or plant part may exhibit an abiotic stress tolerance trait that is absent in the control plant or plant part.
  • a plant may have enhanced abiotic stress tolerance to more than one abiotic stress, at least two abiotic stresses, or enhanced tolerance to multiple abiotic stresses (e.g., three or more).
  • a plant may have enhanced stress tolerance to at least two abiotic stresses, wherein the abiotic stresses are selected from the group comprising drought stress, flooding stress, osmotic stress, oxidative stress, light stress, cold stress, heat stress, flooding stress, and edaphic stresses (including extreme soil pH, nutrient-deficient soil, compact soil, etc.).
  • a plant may have enhanced abiotic stress tolerance to any combination of at least two abiotic stresses, including for example drought stress and light stress; drought stress, light stress, and heat stress; drought stress and cold stress; drought stress, cold stress, and salt stress; drought stress and heat stress; drought stress, heat stress, and salt stress; drought stress, heat stress, cold stress, and salt stress; drought stress, heat stress, cold stress, and salt stress; drought stress, heat stress, cold stress, salt stress, and light stress; drought stress, heat stress, cold stress, salt stress, light stress, and osmotic stress; drought stress, heat stress, cold stress, salt stress, light stress, and osmotic stress; drought stress, heat stress, cold stress, salt stress, light stress, osmotic stress, and an edaphic stress, drought stress, heat stress, cold stress, salt stress, light stress, osmotic stress, an edaphic stress, and oxidative stress.
  • drought stress and light stress including for example drought stress and light stress; drought stress, light stress, and
  • the at least one or at least two abiotic stresses does not include salt stress, but the plant and/or plant part may be salt stress tolerant and tolerant to one, two, or more different abiotic stresses, such as, for example, cold stress, drought stress, flooding stress, osmotic stress, oxidative stress, light stress, heat stress, flooding stress, and edaphic stresses.
  • the term "expression cassette” refers to a nucleic acid capable of directing expression of a particular nucleotide sequence in a host cell.
  • the expression cassette may be chimeric, meaning that at least one of its components is heterologous with respect to at least one of its other components.
  • the expression cassette may also be one that is naturally occurring but has been obtained in a recombinant form useful for heterologous expression.
  • the expression cassette is heterologous with respect to the host (i.e., one or more of the nucleic acid sequences in the expression cassette do(es) not occur naturally in the host cell and must have been introduced into the host cell or an ancestor of the host cell by a transformation event or transformation followed by traditional breeding).
  • exogenous nucleic acid refers to a nucleic acid that is not in the natural genetic background of the cell/organism in which it resides.
  • an exogenous nucleic acid may also be referred to as a nonnaturally occurring nucleic acid.
  • the exogenous nucleic acid comprises one or more nucleic acid sequences that are not found in the natural genetic background of the cell/organism.
  • the exogenous nucleic acid comprises one or more additional copies of a nucleic acid that is endogenous to the cell/organism. These additional copies may be at a genomic location or genomic locations that differ from that of the endogenous copy or copies.
  • heterologous refers to a nucleotide/polypeptide that originates from a foreign species, or, if from the same species, is substantially modified from its native form in composition and/or genomic locus by deliberate human intervention.
  • expression refers to transcription and/or translation of the sequences.
  • fragment refers to a nucleic acid that is reduced in length relative to a reference nucleic acid and that comprises, consists essentially of and/or consists of a nucleotide sequence of contiguous nucleotides identical or almost identical (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical) to a corresponding portion of the reference nucleic acid.
  • a nucleic acid fragment may be, where appropriate, included in a larger polynucleotide of which it is a constituent.
  • the nucleic acid fragment comprises, consists essentially of or consists of at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 300, 350, 400, 450, 500, 600, 700, 800 or more consecutive nucleotides.
  • the nucleic acid fragment comprises, consists essentially of or consists of less than about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 300, 350, 400, 450 500, 600, 700, or 800 consecutive nucleotides.
  • fragment refers to a polypeptide that is reduced in length relative to a reference polypeptide and that comprises, consists essentially of and/or consists of an amino acid sequence of contiguous amino acids identical or almost identical (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical) to a corresponding portion of the reference polypeptide.
  • a polypeptide fragment may be, where appropriate, included in a larger polypeptide of which it is a constituent.
  • the polypeptide fragment comprises, consists essentially of or consists of at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 300, 350, 400, 450, 500, or more consecutive amino acids.
  • the polypeptide fragment comprises, consists essentially of or consists of less than about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 300, 350, 400, 450 or 500 consecutive amino acids.
  • the term "functional fragment” refers to nucleic acid that encodes a functional fragment of a polypeptide.
  • the term "functional fragment” refers to polypeptide fragment that retains at least about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or more of at least one biological activity of the full-length polypeptide (e.g., enzymatic activity). In some embodiments, the functional fragment actually has a higher level of at least one biological activity of the full-length polypeptide.
  • Polypeptides and fragments of the invention can be modified for in vivo use by the addition, at the amino- and/or carboxyl-terminal ends, of a blocking agent to facilitate survival of the relevant polypeptide in vivo.
  • a blocking agent to facilitate survival of the relevant polypeptide in vivo.
  • Such blocking agents can include, without limitation, additional related or unrelated peptide sequences that can be attached to the amino and/or carboxyl terminal residues of the peptide to be administered.
  • one or more non-naturally occurring amino acids such as D-alanine, can be added to the termini.
  • blocking agents such as pyroglutamic acid or other molecules known in the art can be attached to the amino and/or carboxyl terminal residues, or the amino group at the amino terminus or carboxyl group at the carboxyl terminus can be replaced with a different moiety.
  • the peptide terminus can be modified, e.g., by acetylation of the N-terminus and/or amidation of the C-terminus.
  • the peptides can be covalently or noncovalently coupled to pharmaceutically acceptable "carrier" proteins prior to administration.
  • the term "germplasm” refers to genetic material of or from an individual plant, a group of plants (e.g., a plant line, variety or family), or a clone derived from a plant line, variety, species, or culture.
  • the genetic material can be part of a cell, tissue or organism, or can be isolated from a cell, tissue or organism.
  • the terms “increase,” “increases,” “increasing” and similar terms refer to an elevation of at least about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 75%, 100%, 125%, 150%, 175%, 200%, 350%, 300%, 350%, 400%, 450%, 500% or more.
  • an informative fragment refers to a nucleotide sequence comprising a fragment of a larger nucleotide sequence, wherein the fragment allows for the identification of one or more alleles within the larger nucleotide sequence.
  • an informative fragment of the nucleotide sequence of SEQ ID NO: 1 comprises a fragment of the nucleotide sequence of SEQ ID NO: 1 and allows for the identification of one or more alleles located within the portion of the nucleotide sequence corresponding to that fragment of SEQ ID NO: l .
  • isolated refers to a nucleic acid, polynucleotide or polypeptide that, by the hand of man, exists apart from its native environment and is therefore not a product of nature.
  • the nucleic acid, polynucleotide or polypeptide exists in a purified form that is substantially free of cellular material, viral material, culture medium (when produced by recombinant DNA techniques), or chemical precursors or other chemicals (when chemically synthesized).
  • An "isolated fragment” is a fragment of a polynucleotide or polypeptide that is not naturally occurring as a fragment and would not be found in the natural state. "Isolated” does not mean that the preparation is technically pure (homogeneous), but rather that it is sufficiently pure to provide the polynucleotide or polypeptide in a form in which it can be used for the intended purpose.
  • the composition comprising the polynucleotide or polypeptide is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more pure.
  • isolated refers to a cell that, by the hand of man, exists apart from its native environment and is therefore not a product of nature.
  • the cell is separated from other components with which it is normally associated in its natural state.
  • an isolated plant cell may be a plant cell in culture medium and/or a plant cell in a suitable carrier.
  • the composition comprising the cell is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more pure.
  • nonfunctional fragment refers to nucleic acid that encodes a nonfunctional fragment of a polypeptide.
  • nonfunctional fragment refers to polypeptide fragment that exhibits none or essentially none (i.e., less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less) of the biological activities of the full-length polypeptide.
  • nonnaturally occurring refers to nucleic acids, proteins, plants, plant parts, bacteria, viruses or algae that do not naturally exist in nature.
  • a nonnaturally occurring nucleic acid does not naturally exist in nature in that it is not in the natural genetic background of the cell/organism in which it resides.
  • a plant, plant part, bacteria, virus and/or algae of the present invention comprising the nonnaturally occurring nucleic acid may also be nonnaturally occurring and/or may express a nonnaturally occurring protein.
  • a nonnaturally occurring nucleic acid, protein, plant, plant part, bacteria, virus, and/or algae of the present invention may comprise any suitable variation(s) from their closest naturally occurring counterparts.
  • nonnaturally occurring nucleic acids of the present invention may comprise an otherwise naturally occurring nucleotide sequence having one or more point mutations, insertions or deletions relative to the naturally occurring nucleotide sequence.
  • nonnaturally occurring nucleic acids of the present invention comprise a naturally occurring nucleotide sequence and one or more heterologous nucleotide sequences (e.g., one or more heterologous promoter sequences, intron sequences and/or termination sequences).
  • nonnaturally occurring proteins of the present invention may comprise an otherwise naturally occurring protein that comprises one or more mutations, insertions, additions or deletions relative to the naturally occurring protein (e.g., one or more epitope tags).
  • nonnaturally occurring plants, plant parts, bacteria, viruses and algae of the present invention may comprise one more exogenous nucleotide sequences and/or one or more nonnaturally occurring copies of a naturally occurring nucleotide sequence (i.e., extraneous copies of a gene that naturally occurs in that species).
  • Nonnaturally occurring plants and plant parts may be produced by any suitable method, including, but not limited to, transforming/transfecting/transducing a plant or plant part with an exogenous nucleic acid and crossing a naturally occurring plant or plant part with a nonnaturally occurring plant or plant part. It is to be understood that all nucleic acids, proteins, plants, plant parts, bacteria, viruses and algae claimed herein are nonnaturally occurring.
  • nucleic acid can be used interchangeably and encompass both RNA and DNA, including cDNA, genomic DNA, mRNA, synthetic (e.g., chemically synthesized) DNA or RNA and chimeras of RNA and DNA.
  • polynucleotide, nucleotide sequence, or nucleic acid refers to a chain of nucleotides without regard to length of the chain.
  • the nucleic acid can be double-stranded or single-stranded.
  • nucleic acid unless otherwise limited, encompasses analogues having the essential nature of natural nucleotide sequences in that they hybridize to single-stranded nucleic acids in a manner similar to naturally occurring nucleotides (e.g., peptide nucleic acids).
  • the nucleic acid can be a sense strand or an antisense strand.
  • the nucleic acid can be synthesized using oligonucleotide analogs or derivatives (e.g., inosine or phosphorothioate nucleotides). Such oligonucleotides can be used, for example, to prepare nucleic acids that have altered base-pairing abilities or increased resistance to nucleases.
  • the present invention further provides a nucleic acid that is the complement (which can be either a full complement or a partial complement) of a nucleic acid, nucleotide sequence, or
  • Nucleic acid molecules and/or nucleotide sequences provided herein are presented herein in the 5' to 3 ' direction, from left to right and are represented using the standard code for representing the nucleotide characters as set forth in the U.S. sequence rules, 37 CFR ⁇ 1.821 - 1.825 and the World Intellectual Property Organization (WIPO) Standard ST.25.
  • homologues includes homologous sequences from the same and other species and orthologous sequences from the same and other species.
  • nucleotide refers to a monomeric unit from which DNA or
  • RNA polymers are constructed and which consists of a purine or pyrimidine base, a pentose, and a phosphoric acid group. Nucleotides (usually found in their 5 '-monophosphate form) are referred to by their single letter designation as follows: “A” for adenylate or deoxyadenylate (for RNA or DNA, respectively), “C” for cytidylate or deoxycytidylate, "G” for guanylate or deoxyguanylate, "U” for uridylate, “T” for deoxythymidylate, "R” for purines (A or G), “Y” for pyrimidines (C or T), "K” for G or T, “H” for A or C or T, "I” for inosine, and “N” for any nucleotide.
  • A for adenylate or deoxyadenylate (for RNA or DNA, respectively)
  • C for cytid
  • homologous in the context of the invention refers to the level of similarity between nucleic acid or amino acid sequences in terms of nucleotide or amino acid identity or similarity, respectively, i.e., sequence similarity or identity.
  • homologue, and homologous also refers to the concept of similar functional properties among different nucleic acids or proteins.
  • Homologues include genes that are orthologous and paralogous. Homologues can be determined by using the coding sequence for a gene, disclosed herein or found in appropriate database (such as that at NCBI or others) in one or more of the following ways. For an amino acid sequence, the sequences should be compared using algorithms (for instance see section on "identity” and "substantial identity”).
  • the sequence of one DNA molecule can be compared to the sequence of a known or putative homologue in much the same way.
  • Homologues are at least 20% identical, or at least 30% identical, or at least 40% identical, or at least 50% identical, or at least 60% identical, or at least 70% identical, or at least 80%) identical, or at least 88%> identical, or at least 90%> identical, or at least 92%> identical, or at least 95% identical, across any substantial region of the molecule (DNA, RNA, or protein molecule).
  • a homologue of this invention can have a substantial sequence similarity or identity (e.g., 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and/or 100%) to the nucleotide or polypeptide sequences of the invention.
  • a substantial sequence similarity or identity e.g., 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and/or 100%
  • Identity refers to the degree of similarity between two nucleic acid or amino acid sequences.
  • sequence comparison typically one sequence acts as a reference sequence to which test sequences are compared.
  • sequence comparison algorithm test and reference sequences are input into a computer, subsequence coordinates are designated if necessary, and sequence algorithm program parameters are designated.
  • comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.
  • percent sequence identity refers to the percentage of identical nucleotides in a linear polynucleotide sequence of a reference (“query”) polynucleotide molecule (or its complementary strand) as compared to a test ("subject") polynucleotide molecule (or its complementary strand) when the two sequences are optimally aligned.
  • percent identity can refer to the percentage of identical amino acids in an amino acid sequence.
  • Sequence comparison between two or more polynucleotides is generally performed by comparing portions of the two sequences over a comparison window to identify and compare local regions of sequence similarity.
  • the "percentage of sequence identity" for polynucleotides such as about 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 98, 99 or 100 percent sequence identity, can be determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window can include additions or deletions (i.e., gaps) as compared to the reference sequence for optimal alignment of the two sequences.
  • the percentage is calculated by: (a) determining the number of positions at which the identical nucleic acid base occurs in both sequences; (b) dividing the number of matched positions by the total number of positions in the window of comparison; and (c) multiplying the result by 100.
  • Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2: 482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48: 443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Natl Acad. Sci. USA 85: 2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr.,
  • HSPs high scoring sequence pairs
  • initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them.
  • the word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always ⁇ 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when the cumulative alignment score falls off by the quantity X from its maximum achieved value, the cumulative score goes to zero or below due to the accumulation of one or more negative-scoring residue alignments, or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
  • the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89: 10915 (1989)).
  • the BLAST algorithm In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 90: 5873-5787 (1993)).
  • One measure of similarity provided by the
  • BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.
  • P(N) the smallest sum probability
  • a test nucleic acid sequence is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid sequence to the reference nucleic acid sequence is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.
  • CLUSTALW vl .6 Another widely used and accepted computer program for performing sequence alignments is CLUSTALW vl .6 (Thompson, et al. Nuc. Acids Res., 22: 4673-4680, 1994).
  • the number of matching bases or amino acids is divided by the total number of bases or amino acids, and multiplied by 100 to obtain a percent identity. For example, if two 580 base pair sequences had 145 matched bases, they would be 25 percent identical.
  • the number of matches is divided by the shorter of the two lengths. For example, if there were 100 matched amino acids between a 200 and a 400 amino acid proteins, they are 50 percent identical with respect to the shorter sequence. If the shorter sequence is less than 150 bases or 50 amino acids in length, the number of matches are divided by 150 (for nucleic acid bases) or 50 (for amino acids), and multiplied by 100 to obtain a percent identity.
  • substantially identical in the context of two nucleic acids or two amino acid sequences, refers to two or more sequences or subsequences that have at least 25% nucleotide or amino acid residue identity when compared and aligned for maximum correspondence as measured using one of the following sequence comparison algorithms or by visual inspection.
  • substantially identical sequences have at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least about 98%), or at least about 99% nucleotide or amino acid residue identity.
  • substantial identity exists over a region of the sequences that is at least 20 residues in length, at least 30 residues in length, at least 40 residues in length, at least 50 residues in length, or over a region of at least about 100 residues, or the sequences are substantially identical over at least about 150 residues.
  • the sequences are substantially identical when they are identical over the entire length of the coding regions.
  • the substantial identity exists over a region of the sequences that is at least about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120, about 130, about 140, about 150, or more residues in length.
  • the sequences are substantially identical over at least about 150 residues.
  • substantially identical nucleotide or protein sequences perform substantially the same function (e.g., conferring increased cold tolerance).
  • sequence comparison typically one sequence acts as a reference sequence to which test sequences are compared.
  • test and reference sequences are entered into a computer, subsequence coordinates are designated if necessary, and sequence algorithm program parameters are designated.
  • sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.
  • an “identity fraction" for aligned segments of a test sequence and a reference sequence is the number of identical components which are shared by the two aligned sequences divided by the total number of components in the reference sequence segment, i.e., the entire reference sequence or a smaller defined part of the reference sequence.
  • Two nucleotide sequences can also be considered to be substantially complementary when the two sequences hybridize to each other under stringent conditions.
  • PB percent barren
  • PYREC percent yield recovery
  • yield reduction refers to the degree to which yield is reduced in plants grown under stress conditions. YD is calculated as: yield under non-stress conditions - yield under stress conditions
  • phenotype refers to one or more traits of an organism.
  • the phenotype can be observable to the naked eye, or by any other means of evaluation known in the art, e.g., microscopy, biochemical analysis, and/or an
  • a phenotype is directly controlled by a single gene or genetic locus, i.e., a "single gene trait. " In other cases, a phenotype is the result of several genes.
  • plant cell refers to a cell existing in, taken from and/or derived from a plant (e.g., a cell derived from a plant cell/tissue culture).
  • plant cell may refer to an isolated plant cell, a plant cell in a culture, a plant cell in an isolated tissue/organ and/or a plant cell in a whole plant.
  • plant part refers to at least a fragment of a whole plant or to a cell culture or tissue culture derived from a plant.
  • plant part may refer to a plant cell, a plant tissue and/or a plant organ, as well as to a cell/tissue culture derived from a plant cell, plant tissue or plant culture.
  • Embodiments of the present invention may comprise and/or make use of any suitable plant part, including, but not limited to, anthers, branches, buds, calli, clumps, cobs, cotyledons, ears, embryos, filaments, flowers, fruits, husks, kernels, leaves, lodicules, ovaries, palea, panicles, pedicels, pods, pollen, protoplasts, roots, root tips, seeds, silks, stalks, stems, stigma, styles, and tassels.
  • the plant part is a plant germplasm.
  • polynucleotide refers to a deoxyribopolynucleotide, ribopolynucleotide or analogs thereof that have the essential nature of a natural
  • deoxyribopolynucleotide/ribonucleotide in that they hybridize, under stringent hybridization conditions, to substantially the same nucleotide sequence as naturally occurring nucleotides and/or allow translation into the same amino acid(s) as the naturally occurring nucleotide(s).
  • a polynucleotide can be full-length or a subsequence of a native or heterologous structural or regulatory gene. Unless otherwise indicated, the term includes reference to the specified sequence as well as the complementary sequence thereof. Thus, DNAs or RNAs with backbones modified for stability or for other reasons are "polynucleotides" as that term is intended herein.
  • DNAs or RNAs comprising unusual bases are polynucleotides as the term is used herein. It will be appreciated that a great variety of modifications have been made to DNA and RNA that serve many useful purposes known to those of skill in the art.
  • polynucleotide as it is employed herein embraces such chemically, enzymatically or metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including inter alia, simple and complex cells.
  • polypeptide “peptide” and “protein” refer to a polymer of amino acid residues. The terms encompass amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.
  • progeny and “progeny plant” refer to a plant generated from a vegetative or sexual reproduction from one or more parent plants.
  • a progeny plant may be obtained by cloning or selfing a single parent plant, or by crossing two parental plants.
  • promoter refers to nucleic acid sequences involved in the regulation of transcription initiation.
  • a "plant promoter” is a promoter capable of initiating transcription in plant cells. Exemplary plant promoters include, but are not limited to, those that are obtained from plants, from plant viruses and from bacteria that comprise genes expressed in plant cells such Agrobacterium or Rhizobium.
  • a "tissue-specific promoter” is a promoter that preferentially initiates transcription in a certain tissue (or combination of tissues).
  • stress-inducible promoter is a promoter that preferentially initiates transcription under certain environmental conditions (or combination of environmental conditions).
  • developmental stage-specific promoter is a promoter that preferentially initiates transcription during certain developmental stages (or combination of developmental stages).
  • regulatory sequences refers to nucleotide sequences located upstream (5' non-coding sequences), within or downstream (3' non-coding sequences) of a coding sequence, which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences include, but are not limited to, promoters, enhancers, exons, introns, translation leader sequences, termination signals, and polyadenylation signal sequences. Regulatory sequences include natural and synthetic sequences as well as sequences that can be a combination of synthetic and natural sequences.
  • An “enhancer” is a nucleotide sequence that can stimulate promoter activity and can be an innate element of the promoter or a heterologous element inserted to enhance the activity level or tissue specificity of a promoter.
  • the coding sequence can be present on either strand of a double-stranded DNA molecule, and is capable of functioning even when placed either upstream or downstream from the promoter.
  • stringent conditions include reference to conditions under which a nucleic acid molecule will selectively hybridize to a target sequence to a detectably greater degree than other sequences (e.g., at least 2-fold over a non-target sequence), and optionally may substantially exclude binding to non-target sequences.
  • Stringent conditions are sequence-dependent and will vary under different circumstances. By controlling the stringency of the hybridization and/or washing conditions, target sequences can be identified that can be up to 100% complementary to the reference nucleotide sequence. Alternatively, conditions of moderate or even low stringency can be used to allow some mismatching in sequences so that lower degrees of sequence similarity are detected.
  • primers or probes can be used under conditions of high, moderate or even low stringency.
  • conditions of low or moderate stringency can be advantageous to detect homolog, ortholog and/or paralog sequences having lower degrees of sequence identity than would be identified under highly stringent conditions.
  • T m 81.5°C+16.6 (log M)+0.41 (% GQ-0.61 (% formamide)-500/L; where M is the molarity of monovalent cations, % GC is the percentage of guanosine and cytosine nucleotides in the DNA, % formamide is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs.
  • the T m is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridizes to a perfectly matched probe.
  • T m is reduced by about 1°C for each 1% of mismatching; thus, T m , hybridization and/or wash conditions can be adjusted to hybridize to sequences of the desired degree of identity. For example, if sequences with >90% identity are sought, the T m can be decreased 10°C. Generally, stringent conditions are selected to be about 5°C lower than the thermal melting point (T m ) for the specific sequence and its complement at a defined ionic strength and pH.
  • highly stringent conditions can utilize a hybridization and/or wash at the thermal melting point (T m ) or 1, 2, 3 or 4°C lower than the thermal melting point (T m ); moderately stringent conditions can utilize a hybridization and/or wash at 6, 7, 8, 9 or 10°C lower than the thermal melting point (T m ); low stringency conditions can utilize a hybridization and/or wash at 11, 12, 13, 14, 15 or 20°C lower than the thermal melting point (T m ). If the desired degree of mismatching results in a T m of less than 45 °C (aqueous solution) or 32°C (formamide solution), optionally the SSC concentration can be increased so that a higher temperature can be used.
  • stringent conditions are those in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at about pH 7.0 to pH 8.3 and the temperature is at least about 30°C for short probes (e.g., 10 to 50 nucleotides) and at least about 60°C for longer probes (e.g., greater than 50 nucleotides).
  • Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide or Denhardt's (5 g Ficoll, 5 g polyvinylpyrrolidone, 5 g bovine serum albumin in 500 ml of water).
  • Exemplary low stringency conditions include hybridization with a buffer solution of 30% to 35%
  • high stringency conditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37°C and a wash in 0. IX SSC at 60°C to 65 °C.
  • a further non-limiting example of high stringency conditions include
  • Another illustration of high stringency hybridization conditions includes hybridization in 7% SDS, 0.5 M NaP0 4 , 1 mM EDTA at 50°C with washing in 2X SSC, 0.1% SDS at 50°C, alternatively with washing in IX SSC, 0.1% SDS at 50°C, alternatively with washing in 0.5X SSC, 0.1% SDS at 50°C, or alternatively with washing in 0.1X SSC, 0.1% SDS at 50°C, or even with washing in 0.1X SSC, 0.1%) SDS at 65°C.
  • specificity is typically a function of post-hybridization washes, the relevant factors being the ionic strength and temperature of the final wash solution.
  • Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the proteins that they encode are substantially identical (e.g., due to the degeneracy of the genetic code).
  • a nucleic acid sequence is "isocoding with" a reference nucleic acid sequence when the nucleic acid sequence encodes a polypeptide having the same amino acid sequence as the polypeptide encoded by the reference nucleic acid sequence.
  • the term “substantially complementary” means that two nucleic acid sequences are at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%), 99% or more complementary.
  • the term “substantially complementary” can mean that two nucleic acid sequences can hybridize together under high stringency conditions (as described herein).
  • substantially complementary means about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% complementary, or any value or range therein, to a target nucleic acid sequence.
  • hybridizing specifically to refers to the binding, duplexing, or hybridizing of a molecule to a particular nucleic acid target sequence under stringent conditions when that sequence is present in a complex mixture (e.g., total cellular DNA or RNA) to the substantial exclusion of non-target nucleic acids, or even with no detectable binding, duplexing or hybridizing to non-target sequences.
  • a complex mixture e.g., total cellular DNA or RNA
  • Selectively hybridizing sequences typically are at least about 40% complementary and are optionally substantially complementary or even completely complementary (i.e., 100% identical) to a nucleic acid sequence.
  • nucleic acid molecule complementary hybridization between a nucleic acid molecule and a target nucleic acid and embraces minor mismatches that can be accommodated by reducing the stringency of the hybridization media to achieve the desired detection of the target nucleic acid sequence.
  • transformation refers to the introduction of an exogenous/heterologous nucleic acid (RNA and/or DNA) into a host cell.
  • a cell has been “transformed,” “transfected” or “transduced” with an exogenous/heterologous nucleic acid when such nucleic acid has been introduced or delivered into the cell.
  • transgenic and “recombinant” refer to an organism (e.g., a bacterium or plant) that comprises one or more exogenous nucleic acids.
  • the exogenous nucleic acid is stably integrated within the genome such that at least a portion of the exogenous nucleic acid is passed on to successive generations.
  • the exogenous nucleic acid may be integrated into the genome alone or as part of a recombinant expression cassette.
  • Transgenic may be used to designate any organism the genotype of which has been altered by the presence of an exogenous nucleic acid, including those transgenics initially so altered and those created by sexual crosses or asexual propagation from the initial transgenic.
  • transgenic does not encompass the alteration of the genome (chromosomal or extra- chromosomal) by conventional breeding methods or by naturally occurring events such as random cross-fertilization, non-recombinant viral infection, non-recombinant bacterial transformation, non-recombinant transposition or spontaneous mutation.
  • vector refers to a nucleic acid molecule for the cloning of and/or transfer of a nucleic acid into a cell.
  • a vector may be a replicon to which another nucleotide sequence may be attached to allow for replication of the attached nucleotide sequence.
  • a "replicon” can be any genetic element (e.g., plasmid, phage, cosmid, chromosome, viral genome) that functions as an autonomous unit of nucleic acid replication in vivo (i.e., is capable of replication under its own control).
  • vector includes both viral and nonviral (e.g., plasmid) nucleic acid molecules for introducing a nucleic acid into a cell in vitro, ex vivo, and/or in vivo.
  • viral and nonviral (e.g., plasmid) nucleic acid molecules for introducing a nucleic acid into a cell in vitro, ex vivo, and/or in vivo.
  • a large number of vectors known in the art may be used to manipulate nucleic acids, incorporate response elements and promoters into genes, etc.
  • the insertion of nucleic acid fragments corresponding to response elements and promoters into a suitable vector can be accomplished by ligating the appropriate nucleic acid fragments into a chosen vector that has complementary cohesive termini.
  • the ends of the nucleic acid molecules may be enzymatically modified or any site may be produced by ligating nucleotide sequences (linkers) to the nucleic acid termini.
  • Such vectors may be engineered to contain sequences encoding selectable markers that provide for the selection of cells that contain the vector and/or have incorporated the nucleic acid of the vector into the cellular genome. Such markers allow identification and/or selection of host cells that incorporate and express the proteins encoded by the marker. Examples of such markers are disclosed in Messing & Vierra., GENE 19: 259-268 (1982); Bevan et al., NATURE 304: 184-187 (1983); White et al., NUCL. ACIDS RES.
  • a "recombinant" vector refers to a viral or non-viral vector that comprises one or more heterologous nucleotide sequences (i.e., transgenes). Vectors may be introduced into cells by any suitable method known in the art, including, but not limited to, transfection,
  • ThST03 (also referred to herein as TsST03) encodes a calcium-dependent lipid-binding domain-containing protein from Thellungiella halophila (which is now referred to as T salsuginea). ThST03 was identified by a salt tolerance screen with T. halophila, and it has been shown that overexpression of this gene improved salt tolerance in Arabidopsis (Xie et al, Chinese Patent No. 101,747,419, which is hereby incorporated by reference herein in its entirety).
  • ThST03 is a member of the C2 superfamily.
  • the C2 domain is well-characterized and known to have binding affinity for calcium (Ca 2+ ) and lipids. This domain was originally identified as one of the two conserved domains (C1-C2) in the ⁇ , ⁇ , and ⁇ isoforms of
  • C2 domains are unique among membrane targeting domains in that they show a wide range of lipid selectivity for the major components of cell membranes, including phosphatidyl serine and phosphatidylcholine (Karlin & Altschul, Proc. Natl. Acad. Sci. USA 90: 5873-5787 (1993)).
  • the 3D structure of the C2 domain of synaptotagmin has been reported (Sutton, Cell 80
  • the domain forms an eight-stranded beta sandwich constructed around a conserved 4-stranded motif, designated a C2 key .
  • Calcium binds in a cup-shaped depression formed by the N- and C-terminal loops of the C2-key motif.
  • Structural analyses of several C2 domains have shown them to include similar ternary structures in which three Ca 2+ -binding loops are located at the end of an 8 stranded antiparallel beta sandwich (Farah and Sossin, Adv. Exp. Med. Biol. 740: 663-683 (2012).
  • the C2 superfamily is well conserved among different organisms and found in many proteins. In yeast and animals, more than 150 C2 domain- containing proteins have been identified as various signaling molecules with various biological functions, including phospholipid binding (for example, Davletov, J Biol Chem.
  • ThST03 is a relatively small protein. Unlike other well-known C2 domain containing proteins, a class of small C2-domain proteins, which have only been found in plants, have a single C2 domain and lack the additional conserved motifs present in multi-domain proteins such as PKC. Plant small C2-domain proteins are newly characterized in the art, and it has been indicated that these proteins are involved in a diversity of functions, including mRNA longdistance transport, plant defense, heavy metal stress response, leaf senescence, stress tolerance, and membrane targeting (Meijer and Munnik, Annu. Rev. Plant Biol. 54: 265-306 (2003);
  • ThST03 gene was introduced into a transgenic Arabidopsis plant
  • Thellungiella halophila is known to be a close relative of Arabidopsis thaliana, and was chosen as a model system because it can be directly compared with Arabidopsis (Amtmann, Molecular Plant, 2: 3-12 (2009)).
  • an alignment of ThST03 with its ortholog from Arabidopsis shows a very high level of conservation and identity.
  • the ability of ThST03 to confer abiotic stress tolerance when introduced into a more distantly related dicot plant, or in a monocot plant, which is even more distantly related is unpredictable.
  • ThST03 can confer cold tolerance, heat tolerance, and/or drought tolerance. Furthermore, ThST03 expressed transgenically in maize is shown to confer cold tolerance and drought tolerance in the transgenic maize plants.
  • the functionality of ThST03 in multiple abiotic stress responses and in highly divergent plant species is unexpected and points to an important role for ThST03 and its homologs in plants.
  • the present invention discloses an Arabidopsis thaliana ortholog of ThST03, AtST03
  • the present invention provides orthologs of ThST03 in all plant species examined, including corn, rice, soybean, tomato, tobacco, pepper, melon, and orange.
  • An alignment of these protein orthologs shows that there are conserved amino acid residues within the C2 domain and C-terminally to the C2 domain.
  • groups, or clades of homologs with high levels of similarity to each other can be found (for example, Fig. 1).
  • consensus sequences are useful for identifying ST03 polypeptides in plant species. Examples of such consensus sequences include, but are not limited to, the following:
  • Solanoideae consensus sequence (SEQ ID NO: 12)
  • variable positions within the above consensus sequences can be selected based on what amino acids occur at their corresponding positions in specific ST03 polypeptides or alternatively can be conservative substitutions thereof.
  • ST03 polypeptides of the present invention are substantially identical to (e.g., at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical) any of SEQ ID NOs: 4, 5, 19, 20, 21, 22, and/or 23.
  • the ST03 polypeptides of the present invention comprise one or more of the above-described consensus sequences or conservative variants thereof.
  • the polypeptide sequences of the ST03 homologs disclosed herein or parts thereof may be used to isolate nucleic acid sequences that encode said polypeptide sequences. These nucleic acid sequences may be used to confer at least two abiotic stress tolerances and/or cold stress tolerance in a transgenic plant, when compared to a control plant under similar conditions.
  • compositions and methods useful for enhancing abiotic stress tolerance to at least two abiotic stresses in a plant and/or plant part and/or for enhancing cold stress tolerance in a plant and/or plant part may include nucleic acids of the present invention, proteins of the present invention, and/or plants and/or plant parts of the present invention.
  • a composition and method of the present invention may increase yield and/or increase seed germination of a plant and/or plant part grown under abiotic stress conditions, where these stress conditions may comprise one or more abiotic stress conditions.
  • a nucleic acid of the present invention may comprise, consist essentially of, or consist of: (a) a nucleotide sequence of any one of SEQ ID NOs: 1, 2, 3, 6, 7, or 14 to 18;
  • amino acid sequence of the polypeptide comprises any one of SEQ ID NOs: 4, 5, 8, 9, 10, 11, 12, 13, 19, 20, 21, 22, or 23;
  • amino acid sequence of the polypeptide is at least 70% identical, at least 75% identical, at least 80% identical, at least 83%) identical, at least 85% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 95% identical, at least 96% identical, at least 97%) identical, at least 98% identical, or at least 99% identical to the amino acid sequence of any one of SEQ ID NOs: 4, 5, 19, 20, 21, 22, or 23;
  • a nucleic acid of the present invention may encode a polypeptide that comprises a C2 domain and/or binds calcium.
  • a nucleic acid of the present invention may encode a polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more identical to the amino acid sequence from amino acid 5 to 106 of SEQ ID NO:4 and/or a fragment thereof.
  • a nucleic acid of the present invention may be an abscisic acid (ABA) independent gene, optionally from Thellungiella halophila.
  • ABA abscisic acid
  • a nucleic acid of the present invention encodes a polypeptide comprising an amino acid sequence that is SEQ ID NO:4.
  • a nucleic acid of the present invention may comprise any suitable promoter sequence(s), including, but not limited to, constitutive promoters, tissue-specific promoters, chemically inducible promoters, wound-inducible promoters, stress-inducible promoters and developmental stage-specific promoters.
  • a nucleic acid of the present invention may be operably linked to a heat-inducible promoter.
  • a nucleic acid of the present invention may be operably linked to a cold-inducible promoter.
  • a nucleic acid of the present invention may be operably linked to a salt-inducible promoter, where the presence of a certain amount of salt detected by the plant induces transcription from the salt- inducible promoter.
  • a nucleic acid of the present invention may be operably linked to a hybrid promoter.
  • a hybrid promoter may comprise at least two different promoters (e.g., at least two different constitutive promoters and/or heat inducible promoters).
  • a hybrid promoter may drive the expression of a nucleic acid of the present invention more than each of the single promoters making up the hybrid promoter.
  • a hybrid promoter when operably linked to a nucleic acid of the present invention, an increased level of expression may be achieved for the nucleic acid compared to the level of expression of the nucleic acid with either of the promoters making up the hybrid promoter.
  • a hybrid promoter may comprise a Hsp70 promoter and a RbcS2 promoter.
  • a nucleic acid of the present invention may comprise one or more constitutive promoter sequences.
  • the nucleic acid may comprise one or more CaMV 19S, CaMV 35S, Arabidopsis At6669, maize H3 histone, rice actin 1, actin 2, rice cyclophilin, nos, Adh, sucrose synthase, pEMU, GOS2, constitutive root tip CT2, and/or ubiquitin ⁇ e.g., maize Obi) promoter sequences.
  • suitable promoters are disclosed in U.S. Patent Nos.
  • the nucleic acid comprises one or more of the nucleotide sequences described in (a) to (g) above operably linked to one or more constitutive promoters.
  • a nucleic acid of the present invention may comprise one or more tissue-specific promoter sequences.
  • the nucleic acid may comprise one or more flower-, leaf-, ligule-, node-, internode-, panicle-, root-, seed-, sheath-, stem-, and/or vascular bundle-specific promoter sequences.
  • the nucleic acid may comprise one or more reproductive tissue-specific promoter sequences. Examples of suitable promoters are disclosed in U.S. Patent Nos. 5,459,252, 5,604,121, 5,625,136, 6,040,504 and 7,579,516; EP 0452269; WO 93/07278; Czako et al, MOL. GEN.
  • the nucleic acid comprises one or more of the nucleotide sequences described in (a) to (g) above operably linked to one or more tissue-specific promoters.
  • a nucleic acid of the present invention may comprise one or more chemically inducible promoter sequences.
  • suitable promoters are disclosed in U.S. Patent Nos. 5,614,395, 5,789, 156 and 5,814,618; EP 0332104; WO 97/06269; WO 97/06268; Aoyama et al, PLANT J. 11 :605-612 (1997); De Cosa et al. NAT. BIOTECHNOL. 19:71-74 (2001); Daniell et al. BMC BIOTECHNOL. 9:33 (2009); Gatz et al. MOL. GEN. GENET.
  • the nucleic acid comprises one or more of the nucleotide sequences described in (a) to (g) above operably linked to one or more chemically inducible promoters.
  • a nucleic acid of the present invention may comprise one or more wound-inducible promoter sequences.
  • suitable promoters are disclosed in Stanford et al, MOL. GEN. GENET. 215:200-208 (1989); Xu et al., PLANT MOLEC. BIOL. 22:573-588 (1993); Logemann et al, PLANT CELL 1 : 151-158 (1989); Rohrmeier & Lehle, PLANT MOLEC. BIOL. 22:783-792 (1993); Firek et al., PLANT MOLEC. BIOL. 22: 129-142 (1993); and Warner et al, PLANT J. 3 : 191-201 (1993).
  • the nucleic acid comprises one or more of the nucleotide sequences described in (a) to (g) above operably linked to one or more wound-inducible promoters.
  • a nucleic acid of the present invention may comprise one or more stress-inducible promoter sequences.
  • the nucleic acid may comprise one or more drought stress-inducible, salt stress-inducible, heat stress-inducible, light stress-inducible and/or osmotic stress-inducible promoter sequences.
  • the nucleic acid comprises one or more of the nucleotide sequences described in (a) to (g) above operably linked to one or more stress-inducible promoters.
  • the nucleic acid comprises a cold stress-inducible promoter sequence.
  • a nucleic acid of the present invention may comprise one or more developmental stage-specific promoter sequences.
  • the nucleic acid may comprise a promoter sequence that drives expression prior to and/or during the seedling, tillering, panicle initiation, panicle differentiation, reproductive (e.g., flowering, pollination, fertilization), and/or grain filling stage(s) of development.
  • the nucleic acid comprises one or more of the nucleotide sequences described in (a) to (g) above operably linked to one or more developmental-stage specific promoters.
  • the nucleic acid comprises a promoter sequence that drives expression prior to and/or during the seedling and/or
  • a nucleic acid of the present invention may comprise any suitable termination sequence(s).
  • the nucleic acid may comprise a termination sequence comprising a stop signal for RNA polymerase and a polyadenylation signal for polyadenylase.
  • the nucleic acid comprises one or more of the nucleotide sequences described in (a) to (g) above operably linked to one or more termination sequences.
  • a nucleic acid of the present invention may comprise any suitable expression-enhancing sequence(s).
  • the nucleic acid may comprise one or more intron sequences (e.g., Adhl and/or bronzel) and/or viral leader sequences (from tobacco mosaic virus (TMV), tobacco etch virus (TEV), maize chlorotic mottle virus (MCMV), maize dwarf mottle virus (MDMV) or alfalfa mosaic virus (AMV), for example) that enhance expression of associated nucleotide sequences.
  • intron sequences e.g., Adhl and/or bronzel
  • viral leader sequences from tobacco mosaic virus (TMV), tobacco etch virus (TEV), maize chlorotic mottle virus (MCMV), maize dwarf mottle virus (MDMV) or alfalfa mosaic virus (AMV), for example
  • TMV tobacco mosaic virus
  • TMV tobacco etch virus
  • MCMV maize chlorotic mottle virus
  • MDMV maize dwarf mottle virus
  • AMV alfalfa mosaic virus
  • the nucleic acid comprises one or more of the nucleotide sequences described in (a) to (g) above operably linked to one or more expression-enhancing sequences.
  • a nucleic acid of the present invention may comprise any suitable transgene(s), including, but not limited to, transgenes that encode gene products that provide enhanced abiotic stress tolerance (e.g., enhanced drought stress tolerance, enhanced osmotic stress tolerance, enhanced salt stress tolerance and/or enhanced temperature stress tolerance), herbicide-resistance (e.g., enhanced glyphosate-, Sulfonylurea-, imidazolinione-, dicamba-, glufisinate-, phenoxy proprionic acid-, cycloshexome-, traizine-, benzonitrile-, and/or broxynil-resi stance), pest- resistance and/or disease-resistance.
  • abiotic stress tolerance e.g., enhanced drought stress tolerance, enhanced osmotic stress tolerance, enhanced salt stress tolerance and/or enhanced temperature stress tolerance
  • herbicide-resistance e.g., enhanced glyphosate-, Sulfonylurea-, imid
  • a nucleic acid of the present invention may comprise any suitable number of nucleotides.
  • the nucleic acid is 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1 100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000 or more nucleotides in length.
  • the nucleic acid is less than about 2000, 1900, 1800, 1700, 1600, 1500, 1400, 1300, 1200, 1 100, 1000, 900, 800, 700, 600, 500, 400, 300, 200, or 100 nucleotides in length.
  • the nucleic acid is about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1 100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000 nucleotides in length.
  • a nucleic acid of the present invention may be codon optimized.
  • a nucleic acid of the present invention may be codon optimized for expression in bacteria, viruses, algae and/or plants. Codon optimization is well known in the art and involves modification of a nucleotide sequence for codon usage bias using species-specific codon usage tables. The codon usage tables are generated based on a sequence analysis of the most highly expressed genes for the species of interest. When the nucleotide sequences are to be expressed in the nucleus, the codon usage tables are generated based on a sequence analysis of highly expressed nuclear genes for the species of interest.
  • nucleic acid molecule may be codon optimized for expression in a particular species of interest (e.g., a plant such
  • nucleic acids of the present invention may also be GC-optimized. That is, the nucleotide sequences of nucleic acids of the present invention may be selectively altered to optimize their GC content for increased expression in the desired organism. For example, because microbial nucleotide sequences that have low GC contents may express poorly in plants due to the existence of ATTTA motifs that may destabilize messages and/or AATAAA motifs that may cause inappropriate
  • polyadenylation, expression in plants may be enhanced by increasing GC content to at least about 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or more.
  • a nucleic acid of the present invention is an isolated nucleic acid.
  • a nucleic acid of the present invention may comprise, consist essentially of, or consist of:
  • a monocot codon-optimized nucleotide sequence that is at least 70% identical, at least 75% identical, at least 80% identical, at least 83% identical, at least 85% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical or at least 99% identical to the nucleotide sequence of SEQ ID NO: 7;
  • a monocot codon-optimized nucleotide sequence that encodes a polypeptide comprising an amino acid sequence that is at least 70% identical, at least 75% identical, at least 80% identical, at least 83% identical, at least 85% identical, at least 88%) identical, at least 90% identical, at least 92% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:4;
  • a nucleic acid of the present invention comprises a monocot codon-optimized nucleotide sequence that encodes a polypeptide that comprises an eight stranded anti-parallel ⁇ -sandwich, that comprises a C2 domain, and/or that binds calcium.
  • a nucleic acid of the present invention comprises a monocot codon- optimized nucleotide sequence that may encode a polypeptide that comprises amino acids 5 to 106 of SEQ ID NO : 4 and/or a fragment thereof.
  • a nucleic acid of the present invention comprises a monocot codon-optimized nucleotide sequence. In some embodiments, a nucleic acid of the present invention comprises a nucleotide sequence that is codon-optimized for expression in maize (i.e., the nucleotide sequence is a maize codon-optimized nucleotide sequence). In some embodiments, a nucleic acid of the present invention comprises a monocot codon-optimized nucleotide sequence encoding a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:4.
  • a nucleic acid of the present invention comprises a monocot codon-optimized nucleotide sequence that is at least 70% identical to the nucleotide sequence of SEQ ID NO:7. In some embodiments, the nonnaturally occurring nucleic acid is isolated.
  • the present invention also encompasses expression cassettes comprising one or more nucleic acid(s) of the present invention.
  • the expression cassette comprises a nucleic acid that confers at least one property (e.g., resistance to a selection agent) that can be used to detect, identify or select transformed plant cells and tissues.
  • An expression cassette of the present invention may also include nucleotide sequences that encode other desired traits.
  • desired traits can be other nucleotide sequences which confer other agriculturally desirable traits.
  • nucleotide sequences can be stacked with any combination of nucleotide sequences to create plants, plant parts or plant cells having the desired phenotype. Stacked combinations can be created by any method including, but not limited to, cross breeding plants by any conventional methodology, or by genetic transformation. If stacked by genetically transforming the plants, nucleotide sequences encoding additional desired traits can be combined at any time and in any order. For example, a transgenic plant comprising one or more desired traits can be used as the target to introduce further traits by subsequent transformation.
  • the additional nucleotide sequences can be introduced simultaneously in a co- transformation protocol with a nucleotide sequence, nucleic acid molecule, nucleic acid construct, and/or composition of the invention, provided by any combination of expression cassettes.
  • a nucleotide sequence, nucleic acid molecule, nucleic acid construct, and/or composition of the invention provided by any combination of expression cassettes.
  • two nucleotide sequences will be introduced, they can be incorporated in separate cassettes (trans) or can be incorporated on the same cassette (cis).
  • Expression of the nucleotide sequences can be driven by the same promoter or by different promoters. It is further recognized that nucleotide sequences can be stacked at a desired genomic location using a site- specific recombination system. See, e.g., Int'l Patent Application Publication Nos. WO
  • a nucleic acid molecule, expression cassette or vector of the invention can comprise a transgene that confers resistance to one or more herbicides, optionally glyphosate-, sulfonylurea-, imidazolinione-, dicamba-, glufosinate-, phenoxy proprionic acid-, cycloshexome- , traizine-, benzonitrile-, UPPD inhibitor- and/or broxynil-resi stance; a transgene that confers resistance to one or more pests, optionally bacterial-, fungal, gastropod-, insect-, nematode-, oomycete-, phytoplasma-, protozoa-, and/or viral-resistance, and/or a transgene that confers resistance to one or more diseases.
  • herbicides optionally glyphosate-, sulfonylurea-, imidazolinione-, dicamba-, glufosinate-
  • a nucleic acid, expression cassette and/or vector of the present invention may comprise one or more transgenes that confer tolerance to one or more additional abiotic stresses.
  • transgenes that confer an additional abiotic stress tolerance may confer tolerance to an abiotic stress including, but not limited to, cold temperatures (e.g., freezing and/or chilling temperatures), heat or high
  • the present invention also encompasses vectors comprising one or more nucleic acid(s) and/or expression cassette(s) of the present invention.
  • the vector is a pSTK, pROKI, pBin438, pCAMBIA (e.g., pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1391-Xa, pCAMBIA1391-Xb) (CAMBIA Co., Brisbane, Australia) or pBI121 vector.
  • an expression cassette and/or vector of the present invention may comprise a promoter operably linked to an exogenous nucleotide sequence that comprises:
  • nucleotide sequence that is at least 70% identical, at least 75% identical, at least 80%) identical, at least 83%> identical, at least 85%> identical, at least 88%> identical, at least 90%> identical, at least 92%> identical, at least 95%> identical, at least 96% identical, at least 97%> identical, at least 98%> identical or at least 99%> identical to the nucleotide sequence of any one of SEQ ID NOs: 1, 2, 3, 6, 7, or 14 to 18;
  • amino acid sequence of the polypeptide comprises any one of SEQ ID NOs: 4, 5, 8, 9, 10, 1 1, 12, 13, 19, 20, 21, 22, or 23;
  • amino acid sequence of the polypeptide is at least 70%> identical, at least 75%> identical, at least 80%> identical, at least 83%o identical, at least 85%> identical, at least 88%> identical, at least 90%> identical, at least 92%> identical, at least 95%> identical, at least 96%> identical, at least 97%o identical, at least 98%> identical or at least 99%> identical to the amino acid sequence of any one of SEQ ID NOs: 4, 5, 19, 20, 21, 22, or 23;
  • nucleotide sequence that hybridizes to the nucleotide sequence of any one of (a) to (e) above under stringent hybridization conditions;
  • a functional fragment of any one of (a) to (f) above e.g., a functional fragment that binds calcium; and any combination thereof.
  • the present invention also encompasses transgenic cells/organisms comprising one or more expression cassettes, vectors, and/or nucleic acids of the present invention.
  • the transgenic organism is a bacteria, virus, algae, plant, or plant part.
  • the transgenic cell is a propagating plant cell, such as an egg cell or sperm cell.
  • the transgenic cell is a non-propagating plant cell.
  • the transgenic organism is a plant or plant part.
  • the transgenic plant or plant part comprising the expression cassette has enhanced abiotic stress tolerance to at least one abiotic stress, such as, e.g., cold stress, or, in some embodiments, to at least two abiotic stresses, such as, e.g., drought stress and salt stress, as compared to a plant lacking the expression cassette when grown in similar conditions.
  • abiotic stress such as, e.g., cold stress
  • at least two abiotic stresses such as, e.g., drought stress and salt stress
  • the present invention encompasses a transgenic plant comprising an exogenous nucleic acid sequence which confers enhanced abiotic stress tolerance to at least one abiotic stress, such as cold stress, or, in some embodiments, to at least two abiotic stresses such as, e.g., drought stress and salt stress, as compared to a control plant or plant part, wherein the exogenous sequence comprises, consists essentially of, or consists of:
  • nucleotide sequence that is at least 70% identical to the nucleotide sequence of any one of SEQ ID NOs: 1, 2, 3, 6, 7, or 14 to 18;
  • nucleotide sequence that encodes a polypeptide comprising a C2 domain capable of binding calcium, wherein the amino acid sequence of the polypeptide comprises any one of SEQ ID NOs: 4, 5, 8, 9, 10, 11, 12, 13, 19, 20, 21, 22, or 23
  • nucleotide sequence that encodes a polypeptide comprising a C2 domain capable of binding calcium, wherein the amino acid sequence of the polypeptide is at least 70% identical to the amino acid sequence of any one of SEQ ID NOs: 4, 5, 19, 20, 21, 22, or 23;
  • the exogenous nucleic acid comprises a promoter sequence selected from the group comprising a constitutive promoter sequence, a tissue-specific promoter sequence, a chemically-inducible promoter sequence, a wound-inducible promoter sequence, a stress-inducible promoter sequence, and a developmental stage-specific promoter sequence.
  • the exogenous nucleic acid comprises nucleotide sequences that encode for at least one additional desired trait, wherein the desired trait is selected from the group comprising male sterility, herbicide resistance, bacterial disease resistance, fungal disease resistance, viral disease resistance, insect resistance, nematode resistance, modified fatty acid metabolism, modified carbohydrate metabolism, and enhanced abiotic stress tolerance.
  • the transgenic plant comprises an exogenous nucleic acid sequence which comprises SEQ ID NO: 1, 6, or 7.
  • the transgenic plant is a monocotyledonous plant. In some embodiments, the transgenic plant is a dicotyledonous plant. In some embodiments, the transgenic plant is selected from the group consisting of Brassica ssp, millet, switchgrass, maize, sorghum, wheat, oat, turf grass, pasture grass, papaya, flax, peppers, potato, sunflower, tomato, crucifers, soybean, common bean, lotus, grape, peach, cacao, cotton, rice, soybean, sugarcane, sugar beet, tobacco, barley, cassava, cucumber, watermelon, melon, orange, Clementine, castor bean, and grapevine.
  • the present invention also encompasses nonnaturally occurring proteins useful for enhancing abiotic stress tolerance to at least two abiotic stresses and/or cold stress tolerance in a plant or plant part.
  • Proteins of the present invention may comprise an amino acid sequence the expression of which enhances abiotic stress tolerance (e.g., cold stress tolerance) in a plant or plant part, such as, for example, by increasing yield and/or increasing seed germination under abiotic stress conditions.
  • the protein is an isolated protein.
  • a protein of the present invention may comprise any suitable number of amino acids.
  • the protein is 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 450, 500 or more amino acids in length.
  • the protein is less than about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 450, or 500 amino acids in length.
  • the protein is about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 450, or 500 amino acids in length.
  • a protein of the present invention may be produced using any suitable means, including, but not limited to, expression of nucleic acids of the present invention in a transgenic organism.
  • a protein of the present invention may be produced using a transgenic bacterium or algae expressing one or more nucleic acids of the present invention under the control of one or more heterologous regulatory elements (e.g., the nucleotide sequence of SEQ ID NO: 1 under the control of a constitutive promoter suitable for use in Bt).
  • Nucleic acids and proteins of the present invention may be expressed in any suitable cell/organism, including, but not limited to, plants, bacteria, viruses and algae.
  • the nucleic acid/protein is expressed in a monocot plant or plant part ⁇ e.g., in rice or maize).
  • the nucleic acid/protein is expressed in a dicot plant or plant part ⁇ e.g., in soybean).
  • nucleotide sequence Once a nucleotide sequence has been introduced into a particular cell/organism, it may be propagated in that species using traditional methods ⁇ e.g., traditional breeding). Furthermore, once the nucleotide sequence has been introduced into a particular plant variety, it may be moved into other varieties (including commercial varieties) of the same species.
  • the cold stress tolerance and/or abiotic stress tolerance to at least two abiotic stresses of a plant or plant part expressing a nucleic acid/protein of the present invention may be increased by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 175%, 200%, 250%, 300%) or more as compared to a control plant and/or plant part.
  • a "control plant and/or plant part" as used herein, including grammatical variations thereof, can include a plant and/or plant part of the same species ⁇ e.g., a parent plant) optionally grown under the same or substantially the same environmental conditions.
  • Plants and plant parts expressing a nucleic acid/protein of the present invention may exhibit a variety of abiotic stress tolerant phenotypes, including, but not limited to, increased seed germination, increased yield, increased seedling growth, decreased chlorosis, increased leaf expansion, decreased wilting, decreased necrosis, increased reproductive development, and/or decreased cell and/or organelle membrane damage when grown under abiotic stress conditions.
  • one or more abiotic stress tolerant phenotypes is increased in a plant and/or plant part expressing a nucleic acid/protein of the present invention by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 175%, 200%, 250%, 300%, or more as compared to a control plant and/or plant part grown under the same or substantially the same abiotic stress conditions.
  • one or more abiotic stress tolerant phenotypes is decreased in a plant and/or plant part expressing a nucleic acid/protein of the present invention by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 75%, 80%, 85%, 90%, 95%, or more as compared to a control plant and/or plant part grown under the same or substantially the same abiotic stress conditions.
  • the yield (e.g., seed yield, biomass, harvest index, GWTPN, PYREC and/or YGSMN) of a plant and/or plant part expressing a nucleic acid/protein of the present invention and grown under abiotic stress conditions may be increased by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 175%, 200%, 250%, 300% or more as compared to a control plant and/or plant part grown under the same or substantially the same abiotic stress conditions.
  • the seed germination of a plant and/or plant part expressing a nucleic acid/protein of the present invention and grown under abiotic stress conditions may be increased by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 175%, 200%, 250%, 300% or more as compared to a control plant and/or plant part grown under the same or substantially the same abiotic stress conditions.
  • abiotic stress tolerance to at least one abiotic stress such as cold stress, or in some embodiments to at least two abiotic stresses, such as, e.g., cold stress and drought stress, as compared to a control plant or plant part, may be enhanced by introducing and/or expressing an exogenous nucleic acid comprising, consisting essentially of or consisting of:
  • nucleotide sequence that is at least 70% identical, at least 75% identical, at least 80%) identical, at least 83% identical, at least 85% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical or at least 99% identical to the nucleotide sequence of any one of SEQ ID NOs: 1, 2, 3, 6, 7, or
  • amino acid sequence of the polypeptide comprises any one of SEQ ID NOs: 4, 5, 8, 9, 10, 1 1, 12, 13, 19, 20, 21, 22, or 23;
  • amino acid sequence of the polypeptide is at least 70% identical, at least 75% identical, at least 80% identical, at least 83%) identical, at least 85% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 95% identical, at least 96% identical, at least 97%) identical, at least 98% identical or at least 99% identical to the amino acid sequence of any one of SEQ ID NOs: 4, 5, 19, 20, 21, 22, or 23; (e) a nucleotide sequence that is complementary to the nucleotide sequence of any one of (a) to (d) above;
  • the introduction and/or expression of the described exogenous nucleic acid may result in enhanced cold stress tolerance and/or in abiotic stress tolerance to at least two abiotic stresses in the plant or plant part, as compared to a control plant or plant part.
  • the introduction and/or expression of the exogenous nucleic acid may result in enhanced cold stress tolerance in the plant or plant part, as compared to a control plant or plant part.
  • the introduction and/or expression of the exogenous nucleic acid may result in enhanced abiotic stress tolerance to at least one abiotic stress, such as, e.g., cold stress.
  • the introduction and/or expression of the exogenous nucleic acid may result in enhanced abiotic stress tolerance to at least two abiotic stresses, such as, e.g., cold stress and salt stress, cold stress and drought stress, drought stress and heat stress, or drought stress and salt stress.
  • the introduced and/or expressed exogenous nucleic acid is introduced into a plant part, and a plant is produced from the plant part.
  • the introduced and/or expressed exogenous nucleic acid is first introduced into a plant or plant part and then the nucleic acid is expressed in the plant or plant part.
  • the exogenous nucleic acid encodes a polypeptide comprising an amino acid sequence that is at least 70% identical, at least 75% identical, at least 80%> identical, at least 83%> identical, at least 85%> identical, at least 88%> identical, at least 90% identical, at least 92% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to the nucleotide sequence of SEQ ID NO:4.
  • the exogenous nucleic acid encodes a polypeptide comprising an amino acid sequence that is SEQ ID NO:4.
  • the present invention also encompasses methods of identifying, selecting and/or producing a plant and/or plant part having enhanced abiotic stress tolerance to at least one abiotic stress, such as, e.g., cold stress, or in some embodiments to at least two abiotic stresses, such as, e.g., cold stress and salt stress, as compared to a control plant or plant part.
  • Methods of identifying a plant and/or plant part having said enhanced abiotic stress tolerance may involve detecting, in the plant and/or plant part, an exogenous nucleic acid comprising, consisting essentially of or consisting of:
  • nucleotide sequence of any one of SEQ ID NOs: 1, 2, 3, 6, 7, or 14 to 18 (b) a nucleotide sequence that is at least 70% identical, at least 75% identical, at least 80%) identical, at least 83%> identical, at least 85%> identical, at least 88%> identical, at least 90% identical, at least 92% identical, at least 95% identical, at least 96%) identical, at least 97% identical, at least 98% identical or at least 99% identical to the nucleotide sequence of any one of SEQ ID NOs: 1, 2, 3, 6, 7, or 14 to 18;
  • amino acid sequence of the polypeptide comprises any one of SEQ ID NOs: 4, 5, 8, 9, 10, 11, 12, 13, 19, 20, 21, 22, or 23;
  • amino acid sequence of the polypeptide is at least 70% identical, at least 75% identical, at least 80% identical, at least 83%) identical, at least 85% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 95% identical, at least 96% identical, at least 97%) identical, at least 98% identical or at least 99% identical to the amino acid sequence of any one of SEQ ID NOs: 4, 5, 19, 20, 21, 22, or 23;
  • a plant may be produced from a plant part comprising the exogenous nucleic acid, thereby producing a plant having enhanced abiotic stress tolerance to at least one abiotic stress, such as, e.g., enhanced cold stress tolerance, as compared to a control plant or plant part.
  • the plant has enhanced abiotic stress tolerance to at least two abiotic stresses, such as, e.g., enhanced cold stress tolerance and enhanced salt stress tolerance, as compared to a control plant or plant part.
  • the detection of the exogenous nucleic acid described above may result in the identification of a plant or plant part having enhanced abiotic stress tolerance to at least one abiotic stress, such as, e.g., enhanced cold stress tolerance, or in some embodiments having enhanced abiotic stress tolerance to at least two abiotic stresses, such as, e.g., enhanced cold stress tolerance and enhanced salt stress tolerance, as compared to a control plant or plant part.
  • the exogenous nucleic acid may be detected in an amplification product from a nucleic acid sample from or derived from the plant or plant part.
  • the exogenous nucleic acid may be detected using a probe via a Southern blot hybridization analysis.
  • Methods of producing a plant and/or plant part having enhanced abiotic stress tolerance to at least one abiotic stress may comprise, consist essentially of, or consist of:
  • amino acid sequence of the polypeptide comprises any one of SEQ ID NOs: 4, 5, 8, 9, 10, 11, 12, 13, 19, 20, 21, 22, or 23, or that encodes a polypeptide that is at least 70% identical, at least 75% identical, at least 80% identical, at least 83% identical, at least 85%) identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98%) identical or at least 99% identical to the amino acid sequence of any one of SEQ ID NOs: 1, 2, 3, 6, 7, or 14 to 18, or that encodes a polypeptide comprising a C2 domain capable of binding calcium, wherein the amino acid sequence of the polypeptide comprises any one of SEQ ID NOs: 4, 5, 8, 9, 10, 11, 12, 13, 19, 20, 21, 22, or 23, or that encodes a polypeptide that is at least 70% identical, at least 75% identical, at least 80% identical, at least 83% identical, at least 85%) identical, at least 88% identical, at least 90% identical, at least
  • Each of these methods may thereby produce a progeny generation.
  • plants of the progeny generation may be backcrossed to a parent. In some embodiments, plants of the progeny generation may be crossed with each other to produce a further progeny generation.
  • the progeny generation may comprise at least one plant that possesses the exogenous nucleic acid in its genome and may exhibit enhanced abiotic stress tolerance to at least one abiotic stress, such as, e.g., cold stress, or, in some embodiments, to at least two abiotic stresses, such as, e.g., cold stress and salt stress, as compared to a control plant or plant part.
  • a method of producing a plant or plant part having enhanced abiotic stress tolerance to at least one abiotic stress may comprise detecting, in a plant and/or plant part, the presence of an exogenous nucleic acid comprising, consisting essentially of or consisting of:
  • nucleotide sequence that is at least 70% identical, at least 75% identical, at least 80%) identical, at least 83%> identical, at least 85%> identical, at least 88%> identical, at least 90% identical, at least 92% identical, at least 95% identical, at least 96%) identical, at least 97% identical, at least 98% identical, or at least 99% identical to the nucleotide sequence of any one of SEQ ID NOs: 1, 2, 3, 6, 7, or 14 to 18;
  • amino acid sequence of the polypeptide comprises any one of SEQ ID NOs: 4, 5, 8, 9, 10, 11, 12, 13, 19, 20, 21, 22, or 23;
  • amino acid sequence of the polypeptide is at least 70% identical, at least 75% identical, at least 80% identical, at least 83%) identical, at least 85% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 95% identical, at least 96% identical, at least 97%) identical, at least 98% identical, or at least 99% identical to the amino acid sequence of any one of SEQ ID NOs: 4, 5, 19, 20, 21, 22, or 23;
  • a plant from the plant and/or plant part thereby producing a plant that exhibits enhanced abiotic stress tolerance to at least one abiotic stress, such as, e.g., cold stress, or in some embodiments, to at least two abiotic stresses, such as, e.g., cold stress and salt stress, as compared to a control plant or plant part.
  • abiotic stress such as, e.g., cold stress
  • abiotic stress and salt stress such as, e.g., cold stress and salt stress
  • nucleotide sequence that is at least 70% identical, at least 75% identical, at least 80%) identical, at least 83%> identical, at least 85%> identical, at least 88%> identical, at least 90% identical, at least 92% identical, at least 95% identical, at least 96%) identical, at least 97% identical, at least 98% identical, or at least 99% identical to the nucleotide sequence of any one of SEQ ID NOs: 1, 2, 3, 6, 7, or 14 to 18;
  • amino acid sequence of the polypeptide comprises any one of SEQ ID NOs: 4, 5, 8, 9, 10, 11, 12, 13, 19, 20, 21, 22, or 23;
  • amino acid sequence of the polypeptide is at least 70% identical, at least 75% identical, at least 80% identical, at least 83%) identical, at least 85% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 95% identical, at least 96% identical, at least 97%) identical, at least 98% identical, or at least 99% identical to the amino acid sequence of any one of SEQ ID NOs: 4, 5, 19, 20, 21, 22, or 23;
  • a plant from the plant and/or plant part thereby producing a plant that exhibits enhanced abiotic stress tolerance to at least one abiotic stress, such as, e.g., cold stress, or in some embodiments, to at least two abiotic stresses as compared to a control plant.
  • abiotic stress such as, e.g., cold stress, or in some embodiments, to at least two abiotic stresses as compared to a control plant.
  • nucleotide sequence that is at least 70% identical, at least 75% identical, at least 80%) identical, at least 83%> identical, at least 85%> identical, at least 88%> identical, at least 90% identical, at least 92% identical, at least 95% identical, at least 96%) identical, at least 97% identical, at least 98% identical, or at least 99% identical to the nucleotide sequence of any one of SEQ ID NOs: 1, 2, 3, 6, 7, or 14 to 18;
  • amino acid sequence of the polypeptide comprises any one of SEQ ID NOs: 4, 5, 8, 9, 10, 11, 12, 13, 19, 20, 21, 22, or 23;
  • amino acid sequence of the polypeptide is at least 70% identical, at least 75% identical, at least 80% identical, at least 83%) identical, at least 85% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 95% identical, at least 96% identical, at least 97%) identical, at least 98% identical or at least 99% identical to the amino acid sequence of any one of SEQ ID NOs: 4, 5, 19, 20, 21, 22, or 23;
  • a progeny generation that comprises at least one plant that comprises the nucleic acid or a functional fragment thereof and that exhibits enhanced abiotic stress tolerance to at least one abiotic stress, such as, e.g., cold stress, or, in some embodiments, to at least two abiotic stresses, such as, e.g., cold stress and salt stress, as compared to a control plant.
  • abiotic stress such as, e.g., cold stress
  • at least two abiotic stresses such as, e.g., cold stress and salt stress
  • Such methods may further comprise selecting a progeny plant and/or plant part that comprises a nucleic acid of the present invention (or a functional fragment thereof) within its genome and that exhibits enhanced abiotic stress tolerance to at least one abiotic stress, such as, e.g., cold stress, or, in some embodiments, to at least two abiotic stresses, such as, e.g., cold stress and salt stress, as compared to a control plant.
  • a progeny plant and/or plant part that comprises a nucleic acid of the present invention (or a functional fragment thereof) within its genome and that exhibits enhanced abiotic stress tolerance to at least one abiotic stress, such as, e.g., cold stress, or, in some embodiments, to at least two abiotic stresses, such as, e.g., cold stress and salt stress, as compared to a control plant.
  • a method of producing a plant having enhanced abiotic stress tolerance to at least one abiotic stress such as, e.g., cold stress, or, in some embodiments to at least two abiotic stresses, such as, e.g., cold stress and salt stress, comprises, consists essentially of or consists of crossing a first plant that comprises an exogenous nucleic acid that is at least 70% identical, at least 75% identical, at least 80% identical, at least 83% identical, at least 85% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to the nucleotide sequence of any one of SEQ ID NOs: 1, 2, 3, 6, 7, or 14 to 18, or that encodes a polypeptide comprising a C2 domain capable of binding calcium, wherein the amino acid sequence of the polypeptide comprises any one of SEQ ID NOs: 4, 5, 8, 9, 10, 11, 12, 13, 19, 20, 21,
  • the method further comprises selecting an introgressed plant or plant part based upon the presence of a nucleic acid of the present invention (or a functional fragment thereof) and its enhanced abiotic stress tolerance(s). In some embodiments, the method further comprises selecting the introgressed plant or plant part (for inclusion in a breeding program, for example).
  • a method of producing a plant and/or plant part having enhanced abiotic stress tolerance to at least one abiotic stress, such as, e.g., cold stress, or, in some embodiments, to at least two abiotic stresses, such as, e.g., cold stress and salt stress, as compared to a control plant comprises, consists essentially of or consists of crossing a first plant that comprises an exogenous nucleic acid with a second plant that lacks the nucleic acid and repeatedly backcrossing progeny plants comprising a nucleic acid of the present invention (or a functional fragment thereof) with the second plant to produce an introgressed plant or plant part that comprises the nucleic acid (or a functional fragment thereof) and that exhibits enhanced abiotic stress tolerance to at least two abiotic stresses as compared to a control plant, wherein the exogenous nucleic acid comprises, consists essentially of or consists of:
  • nucleotide sequence that is at least 70% identical, at least 75% identical, at least 80%) identical, at least 83%> identical, at least 85%> identical, at least 88%> identical, at least 90% identical, at least 92% identical, at least 95% identical, at least 96%) identical, at least 97% identical, at least 98% identical, or at least 99% identical to the nucleotide sequence of any one of SEQ ID NOs: 1, 2, 3, 6, 7, or 14 to 18;
  • amino acid sequence of the polypeptide comprises any one of SEQ ID NOs: 4, 5, 8, 9, 10, 11, 12, 13, 19, 20, 21, 22, or 23;
  • amino acid sequence of the polypeptide is at least 70% identical, at least 75% identical, at least 80% identical, at least 83%) identical, at least 85% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 95% identical, at least 96% identical, at least 97%) identical, at least 98% identical, or at least 99% identical to the amino acid sequence of any one of SEQ ID NOs: 4, 5, 19, 20, 21, 22, or 23;
  • the method further comprises selecting an introgressed plant or plant part based upon the presence of the nucleic acid (or a functional fragment thereof) and its enhanced abiotic stress tolerance to at least one abiotic stress, such as, e.g., cold stress, or, in some embodiments, to at least two abiotic stresses, such as, e.g., cold stress and salt stress, as compared to a control plant.
  • the method further comprises selecting the introgressed plant or plant part (for inclusion in a breeding program, for example).
  • a nucleic acid of the present invention may be detected in or introduced into a plant and/or plant part.
  • the nucleic acid detected in or introduced into the plant or plant part is a nucleic acid comprising: (a) a nucleotide sequence of any one of SEQ ID NOs: 1, 2, 3, 6, 7, or 14 to 18;
  • nucleotide sequence that is at least 70% identical, at least 75% identical, at least 80%) identical, at least 83%> identical, at least 85%> identical, at least 88%> identical, at least 90% identical, at least 92% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical or at least 99% identical to the nucleotide sequence of any one of SEQ ID NOs: 1, 2, 3, 6, 7, or 14 to 18;
  • amino acid sequence of the polypeptide comprises any one of SEQ ID NOs: 4, 5, 8, 9, 10, 1 1, 12, 13, 19, 20, 21, 22, or 23;
  • amino acid sequence of the polypeptide is at least 70% identical, at least 75% identical, at least 80% identical, at least 83%) identical, at least 85% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical or at least 99% identical to the amino acid sequence of any one of SEQ ID NOs: 4, 5, 19, 20, 21, 22, or 23;
  • Exogenous nucleic acids may be introduced into a plant and/or plant part via any suitable method, including, but not limited to, microparticle bombardment, liposome-mediated transfection, receptor-mediated delivery, bacteria-mediated delivery (e.g., Agrobacterium- mediated transformation and/or whiskers-mediated transformation).
  • the exogenous nucleic acid is introduced into a plant part by crossing a first plant or plant part comprising the exogenous nucleic acid with a second plant or plant part that lacks the exogenous nucleic acid.
  • "Introducing,” in the context of a nucleotide sequence of interest means presenting the nucleotide sequence of interest to the plant, plant part, and/or plant cell in such a manner that the nucleotide sequence gains access to the interior of a cell.
  • these nucleotide sequences can be assembled as part of a single polynucleotide or nucleic acid construct, or as separate polynucleotide or nucleic acid constructs, and can be located on the same or different transformation vectors.
  • these polynucleotides can be introduced into plant cells in a single transformation event, in separate transformation events, or, e.g., as part of a breeding protocol.
  • "introducing” can encompass transformation of an ancestor plant with a nucleotide sequence of interest followed by conventional breeding process to produce progeny comprising said nucleotide sequence of interest.
  • Transformation of a cell may be stable or transient.
  • a plant cell of the invention is stably transformed with a nucleotide sequence encoding a synthetic miRNA precursor molecule of the invention.
  • a plant of the invention is transiently transformed with a nucleotide sequence encoding a synthetic miRNA precursor molecule of the invention.
  • Transient transformation in the context of a polynucleotide means that a polynucleotide is introduced into the cell and does not integrate into the genome of the cell.
  • “Stable transformation” or “stably transformed,” “stably introducing,” or “stably introduced” as used herein means that a nucleic acid is introduced into a cell and integrates into the genome of the cell. As such, the integrated nucleic acid is capable of being inherited by the progeny thereof, more particularly, by the progeny of multiple successive generations.
  • Gene as used herein also includes the nuclear and the plastid genome, and therefore includes integration of the nucleic acid into, for example, the chloroplast genome. Stable transformation as used herein can also refer to a transgene that is maintained
  • extrachromasomally for example, as a minichromosome.
  • Transient transformation may be detected by, for example, an enzyme-linked
  • ELISA immunosorbent assay
  • Western blot which can detect the presence of a peptide or polypeptide encoded by one or more transgene introduced into an organism.
  • transformation of a cell can be detected by, for example, a Southern blot hybridization assay of genomic DNA of the cell with nucleic acid sequences which specifically hybridize with a nucleotide sequence of a transgene introduced into an organism (e.g., a plant).
  • an organism e.g., a plant.
  • transformation of a cell can be detected by, for example, a Northern blot hybridization assay of RNA of the cell with nucleic acid sequences which specifically hybridize with a nucleotide sequence of a transgene introduced into a plant or other organism.
  • Stable transformation of a cell can also be detected by, e.g., a polymerase chain reaction (PCR) or other amplification reactions as are well known in the art, employing specific primer sequences that hybridize with target sequence(s) of a transgene, resulting in amplification of the transgene sequence, which can be detected according to standard methods Transformation can also be detected by direct sequencing and/or hybridization protocols well known in the art.
  • PCR polymerase chain reaction
  • Methods of introducing a nucleic acid into a plant can also comprise in vivo modification of nucleic acids, methods for which are known in the art.
  • in vivo modification can be used to insert a nucleic acid comprising , e.g., a promoter sequence into the plant genome.
  • in vivo modification can be used to modify the endogenous nucleic acid itself and/or a endogenous transcription and/or translation factor associated with the endogenous nucleic acid, such that the transcription and/or translation of said endogenous nucleic acid is altered, thereby altering the expression said endogenous nucleic acid and/or in the case of nucleic acids encoding polypeptides, the production of said polypeptide.
  • Exemplary methods of in vivo modification include zinc finger nuclease, CRISPR-Cas, TALEN, TILLING (Targeted Induced Local Lesions IN Genomes) and/or engineered meganuclease technology.
  • suitable methods for in vivo modification include the techniques described in Urnov et al . Nature Reviews 11 :636-646 (2010); Gao et. al. , Plant J. 61 , 176 (2010); Li et al. , Nucleic Acids Res. 39, 359 (2011); Miller et al 29, 143-148 (201 1 ); Christian et al. Genetics 186, 757-761 (2010); Jiang et al. Nat. Biotechnol. 31 , 233-239 (2013); U.S. Patent Nos.
  • the method comprises cleaving the plant genome at a target site with a TALEN and/or a meganuclease and providing a nucleic acid that is homologous to at least a portion of the target site and further comprises a promoter sequence of the invention (optionally in operable association with a heterologous nucleotide sequence of interest), such that homologous recombination occurs and results in the insertion of the promoter sequence of the invention into the genome.
  • a CRISPR-Cas system can be used to specifically edit the plant genome so as to alter the expression of endogenous nucleic acids described herein.
  • a genetic modification may also be introduced using the technique of TILLING, which combines high-density mutagenesis with high-throughput screening methods. Methods for TILLING are well known in the art (McCallum, Nature Biotechnol. 18, 455-457, 2000, Stemple, Nature Rev. Genet. 5, 145-150, 2004).
  • polynucleotides of the invention can be modified in vivo using the above described methods as well as any other method of in vivo modification known or later developed.
  • a nucleic acid of the present invention may be detected using any suitable method, including, but not limited to, DNA sequencing, mass spectrometry and capillary electrophoresis.
  • the nucleic acid (or an informative fragment thereof) is detected in one or more amplification products from a nucleic acid sample from the plant or plant part.
  • the amplification product(s) comprise(s) the nucleotide sequence of any one of SEQ ID NOs: 1 to 3 or 6 to 7, the reverse complement thereof, an informative fragment thereof, or an informative fragment of the reverse complement thereof.
  • a nucleic acid of the present invention may be detected using any suitable probe.
  • the nucleic acid (or an informative fragment thereof) is detected using a probe comprising the nucleotide sequence of any one of SEQ ID NOs: 1 to 3 or 6 to 7, the reverse complement thereof, an informative fragment thereof, or an informative fragment of the reverse complement thereof.
  • the probe comprises one or more detectable moieties, such as digoxigenin, fluorescein, acridine-ester, biotin, alkaline phosphatase, horseradish peroxidase, ⁇ -glucuronidase, ⁇ -galactosidase, luciferase, ferritin or a radioactive isotope.
  • the present invention extends to uses of nucleic acids, expression cassettes, vectors, bacteria, viruses, algae, proteins, and/or amplification primers of the present invention, including, but not limited to, uses for enhancing abiotic stress tolerance to at least two abiotic stresses and/or cold stress tolerance in a plant and/or plant part, and/or uses for identifying, selecting and/or producing such a plant and/or plant part.
  • the use comprises introducing a nucleic acid of the present invention into a plant cell, growing the transgenic plant cell into a transgenic plant and/or plant part, and, optionally, selecting the transgenic plant and/or plant part based upon enhanced abiotic stress tolerance.
  • Such uses may comprise transforming the plant cell with a transgenic bacterium/virus of the present invention.
  • the use comprises culturing a transgenic bacterium or algae comprising a nucleic acid of the present invention in/on a culture medium; isolating, from the culture medium, a protein encoded by the nucleic acid; and applying the protein to a plant and/or plant part.
  • the use comprises infecting a plant and/or plant part with a transgenic virus comprising a nucleic acid of the present invention.
  • the use comprises applying a protein of the present invention to a plant and/or plant part.
  • a plant and/or plant part suitable for use with the present invention may be of any plant type, including, but not limited to, plants belonging to the superfamily Viridiplantae and thus includes spermatophytes (e.g., angiosperms and gymnosperms) and embryophytes (e.g., bryophytes, ferns and fern allies).
  • a plant or plant part useful with this invention includes any monocot and/or any dicot plant or plant part.
  • the plant or plant part is a fodder crop, a food crop, an ornamental plant, a tree or a shrub.
  • the plant or plant part is a variety of Acer spp., Actinidia spp., Abelmoschus spp., Agropyron spp., Allium spp., Amaranthus spp., Ananas comosus, Annona spp., Apium graveolens, Arachis spp, Artocarpus spp., Asparagus officinalis, Avena spp. (e.g. Avena sativa, Avena fatua, Avena byzantina, Avena fatua var.
  • Avena spp. e.g. Avena sativa, Avena fatua, Avena byzantina, Avena fatua var.
  • Brassica spp. e.g. Brassica napus, Brassica rapa ssp.
  • Eriobotrya japonica Eugenia uniflora, Fagopyrum spp., Fagus spp., Ficus carica, Fortune lla spp., Fragaria spp., Ginkgo biloba, Glycine spp. (e.g. Glycine max, Soja hispida or Soja max ), Gossypium hirsutum, Helianthus spp. (e.g. Helianthus annuus ), Hemerocallis fulva, Hibiscus spp., Hordeum spp. (e.g.
  • Hordeum vulgare Ipomoea batatas, Juglans spp., Lactuca sativa, Lathyrus spp., Lens culinaris, Linum usitatissimum, Litchi chinensis, Lotus spp., Luff a acutangula, Lupinus spp., Luzula sylvatica, Lycopersicon spp. (e.g.
  • Solanum spp. e.g. Solanum tuberosum, Solanum integrifolium or Solanum lycopersicum
  • Sorghum bicolor Spinacia spp., Syzygium spp., Tagetes spp., Tamarindus indica, Theobroma cacao, Trifolium spp., Triticosecale rimpaui, Triticum spp. (e.g. Solanum tuberosum, Solanum integrifolium or Solanum lycopersicum ), Sorghum bicolor, Spinacia spp., Syzygium spp., Tagetes spp., Tamarindus indica, Theobroma cacao, Trifolium spp., Triticosecale rimpaui, Triticum spp. (e.g.
  • the plant and/or plant part is a rice, maize, wheat, barley, sorghum, millet, oat, triticale, rye, buckwheat, fonio, quinoa, sugar cane, bamboo, banana, ginger, onion, lily, daffodil, iris, amaryllis, orchid, canna, bluebell, tulip, garlic, secale, einkorn, spelt, emmer, durum, kamut, grass ⁇ e.g., gramma grass), teff, milo, flax, Tripsacum sp., or teosinte plant or plant part.
  • the plant or plant part is a blackberry, raspberry, strawberry, barberry, bearberry, blueberry, coffee berry, cranberry, crowberry, currant, elderberry, gooseberry, goji berry, honeyberry, lemon, lime, lingonberry, mangosteen, orange, pepper, persimmon, pomegranate, prune, cotton, clover, acai, plum, peach, nectarin, cherry, guava, almond, pecan, walnut, apple, amaranth, sweet pea, pear, potato, soybean, sugar beet, sunflower, sweet potato, tamarind, tea, tobacco or tomato plant or plant part.
  • the plant and/or plant part is rice, maize, or soybean.
  • the plant and/or plant part is not Thellungiella halophila. (now referred to as Thellungiella salsuginea) and/or the transgenic plant is not Arabidopsis thaliana.
  • a harvested product can be a whole plant or any plant part, as described herein, wherein said harvested product comprises a recombinant nucleic acid molecule/nucleotide sequence of the invention.
  • non-limiting examples of a harvested product include a seed, a fruit, a flower or part thereof (e.g., an anther, a stigma, and the like), a leaf, a stem, and the like.
  • a post-harvest product includes, but is not limited to, a flour, meal, oil, starch, cereal, and the like produced from a harvested seed of the invention, wherein said seed comprises in its genome a recombinant nucleic acid molecule/nucleotide sequence of the invention.
  • the exogenous nucleic acid described in the above methods further comprises a promoter sequence selected from the group comprising a constitutive promoter sequence, a tissue-specific promoter sequence, a chemically-inducible promoter sequence, a wound-inducible promoter sequence, a stress-inducible promoter sequence, and a developmental stage-specific promoter sequence.
  • a promoter sequence selected from the group comprising a constitutive promoter sequence, a tissue-specific promoter sequence, a chemically-inducible promoter sequence, a wound-inducible promoter sequence, a stress-inducible promoter sequence, and a developmental stage-specific promoter sequence.
  • the plant or plant part having enhanced abiotic stress tolerance to at least one abiotic stress such as, e.g., cold stress, or, in some embodiments, to at least two abiotic stresses, such as, e.g., cold stress and drought stress, produced by the methods described above has increased yield and/or increased seed germination under at least one abiotic stress condition compared to a control plant or plant part grown under the same abiotic stress conditions.
  • the abiotic stresses described above comprise at least one abiotic stress selected from the group comprising salt stress, drought stress, cold stress, heat stress, osmotic stress, light stress, flooding stress, an edaphic stress, and any combination thereof. In some embodiments, the abiotic stresses described above comprise at least one abiotic stress selected from the group comprising drought stress, cold stress, heat stress, and any combination thereof.
  • the plant or plant part having enhanced abiotic stress tolerance to at least one abiotic stress such as, e.g., cold stress, or, in some embodiments, to at least two abiotic stresses, such as, e.g., cold stress and drought stress, produced by the methods described above is or is derived from a monocotyledonous plant.
  • the plant or plant part is or is from maize, rice, wheat, and sugarcane.
  • the plant or plant part having enhanced abiotic stress tolerance to at least one abiotic stress such as, e.g., cold stress, or, in some embodiments, to at least two abiotic stresses, such as, e.g., cold stress and drought stress, produced by the methods described above is or is derived from a dicotyledonous plant.
  • the plant or plant part is or is from soybean, cotton, and tomato.
  • the harvested product is a plant part capable of producing a plant and/or plant part that expresses one or more nonnaturally occurring proteins of the present invention.
  • the harvested product is a plant part capable of producing a plant and/or plant part that exhibits enhanced abiotic stress tolerance.
  • the harvested product is a plant part capable of producing a plant and/or plant part that exhibits increased yield and/or increased seed germination under abiotic stress conditions.
  • the present invention also extends to products harvested from plants produced according to methods of the present invention, including, but not limited to, dry pellets and powders, oils, fats, fatty acids, starches and proteins.
  • the invention further provides a plant crop comprising a plurality of transgenic plants of the invention planted together in, for example, an agricultural field, a golf course, a residential lawn, a road side, an athletic field, and/or a recreational field.
  • a method of increasing the yield and/or seed germination of a plant crop under abiotic stress conditions comprising cultivating a plurality of plants of the invention as the plant crop, wherein the plurality of plants of said plant crop have enhanced abiotic stress tolerance, thereby increasing the yield and/or seed germination under abiotic stress conditions of said plant crop as compared to a control plant crop grown under the same environmental conditions, wherein the control plant crop is produced from a plurality of plants lacking an exogenous nucleic acid of the present invention.
  • the plant crop may be a maize crop, a rice crop, or a soybean crop.
  • Chlamydomonas reinhardtii the unicellular green alga Chlamydomonas, is a useful model system to study many eukaryotic processes at molecular level (Harris 1989; Gutman and Niyogi 2004). Chlamydomonas has been shown to be a suitable system for overexpression studies (Siripornadulsil et al. 2002; Kumar et al. 2005)
  • Chlamydomonas reinhardtii mutant type CC-251 (cr6 mt+) was used for exogenous nucleic acid transformation and cold stress treatment.
  • CC-251 is a cold sensitive strain which cr6 mutant is deficient in 70S chloroplast ribosomes and accumulates some 41 S and some 54S subunit particles and/or poorly defined mixed subunit material.
  • the Chlamydomonas reinhardtii wild type CC-4414 (mt+ DN2) is a positive control, which is a cold tolerance strain isolated from an environmental sample taken at 13,000 feet in Breckenri and can grow at low temperatures. These C. reinhardtii strain were purchased from Chlamydomonas center (Duke University, Durham, NC, USA). 1.2 Chlamydomonas growth and culture conditions
  • TAP Tris-acetate-phosphate
  • the cultures were grown in liquid Tris-acetate-phosphate (TAP) medium (Harris 1989) in an incubator shaker with 100-120 rpm at 28°C under continuous illumination at light intensity of 2300-3000 lux.
  • the solid cultures were maintained on TAP-agar medium with the same light and temperature conditions.
  • the culture medium containing Hygromycin (10 ⁇ g/mL) was used for transformant cell growth.
  • Expression vectors pChlamy-1 is designed to facilitate cloning of gene of interest (GOI) for expression in C. reinhardtii (Life technology). ThST03 synthesized with optimized
  • Chlamydomonas reinhardtii codon usage by GeneWiz (SEQ ID NO: 6) and downstream with Chlamydomonas reinhardtii RbcS2 3 ' untranslated region (UTR, 234 bp) was cloned into vector through restriction enzymes Notl and Kpnl.
  • the expression of ThST03 nucleic acid was driven by Hybrid constitutive promoter consisting of Hsp70 and RbcS2 and selection marker Aph7 (Hygromycin resistance) was driven by B2-tublin promoter.
  • Nuclear transformations were done by electroporation with linearized DNA as described in Kosuke et al. (1998).
  • the cells used for transformation were incubated for overnight with agitation on a gyratory shaker till the optical density (OD) of 750 nm was 0.5 of the cultures.
  • 15 mL of the cells were harvested by centrifugation at 2,500 rpm for 10 minutes at 25°C. The supernatant was discarded by decanting.
  • the pellet cells were suspended in 5 mL of TAP-40 mM sucrose solution by gently pipetting up and down. 2 ⁇ g plasmid DNA linearized via Seal was added into the 250 ul suspended cells.
  • the cells were electroporated in 0.4 cm cup with the 600V for 8 times (BTX ECM399).
  • the transformation mixture was split into two aliquots of 125iL each and each aliquot was transferred into one well of the 6-well plate containing 5 mL/well of TAP-40 mM sucrose solution at room temperature.
  • the 6-well plate was placed in the plant growth chamber set to 28°C.
  • the cells were incubated for 24 hours with 100-120 rpm agitation to let them recover.
  • the cells were centrifuged at 2,500 rpm for 10 minutes at room temperature and the supernatant was discarded by decanting.
  • transformation was plated on one TAP-agar-Hygromycin plate in plant growth chamber at 28°C 2300-3000 lux for 5-6 days.
  • the cells were grown in TAP medium (for Wild type) or TAP medium containing 10 ⁇ g/mL Hygromycin for transformed cells until cell density reached an OD750 of 1.0 at 28 °C (about 5 days).
  • the cells were spun down and readjusted to an OD750 of 0.05 in 4 ml TAP medium.
  • This culture was used for cold stress following the procedure of 40°C for 0.5h, 28°C for 2h and 8°C for 14 days. The OD750 was measured at 7 days and 14 days separately.
  • OD750 100 ⁇ ⁇ of the cell cultures were placed into 96 well plates, and the OD750 was read in an MD-2 (Molecular Devices)instrument. The initial reading was OD750 (0) , the 7 day reading was OD750(7), and the 14 day reading was OD750 ( i 4 ) . The biomass per unit was calculated to reflect the increased algae biomass at OD750 (7 or i4) which was normalizd via divided by OD750 (0 ):
  • ThST03 A maize codon-optimized coding sequence of ThST03 (SEQ ID NO: 7) was cloned into a binary expression vector. Constitutive expression of ThST03 was selected to target the appropriate expression level and cell type.
  • the expression cassette is composed of promoter (prUbil-10) and terminator (tUbil-01) sequences. The promoter and terminator selected were based on U.S. Patent Nos. 6,054,574 and 6, 147,282.
  • the resulting binary vector, comprising the ThST03 expression cassette described above, is referred to as construct 19692.
  • Construct 19692 was used for Agrobacterium-mediated maize transformation.
  • Construct 19692 carries both phosphomannose isomerase (PMI) and phosphinothricin acetyltransferase (PAT) as plant selection markers.
  • PMI phosphomannose isomerase
  • PAT phosphinothricin acetyltransferase
  • Agrobacterium strain LBA4404 (pSB l) containing the plant transformation plasmid was grown on YEP (yeast extract (5 g/L), peptone (10 g/L), NaCI (5 g/L), 15 g/I agar, pH 6.8) solid medium for 2-4 days at 28°C. Approximately 0.8xl0 9 Agrobacterium were suspended in LS-inf media supplemented with 100 M As (Negrotto et al., supra). Bacteria were preinduced in this medium for 30-60 minutes.
  • Immature embryos from AX5707 or other suitable genotype were excised from 8-12 day old ears into liquid LS-inf+100 M As. Embryos were rinsed once with fresh infection medium. Agrobacterium solution was then added and embryos were vortexed for 30 seconds and allowed to settle with the bacteria for 5 minutes. The embryos were then transferred scutellum side up to LSAs medium and cultured in the dark for two to three days. Subsequently, between 20 and 25 embryos per petri plate were transferred to LSDc medium supplemented with cefotaxime (250 mg/1) and silver nitrate (1.6 mg/1) and cultured in the dark for 28°C for 10 days.
  • Immature embryos, producing embryogenic callus were transferred to LSD1M05S medium. The cultures were selected on this medium for about 6 weeks with a subculture step at about 3 weeks. Surviving calli were transferred to Regl medium supplemented with mannose. Following culturing in the light (16hour light/8 hour dark regiment), green tissues were then transferred to Reg2 medium without growth regulators and incubated for about 1-2 weeks.
  • Plantlets were transferred to Magenta GA-7 boxes (Magenta Corp, Chicago 111.) containing Reg3 medium and grown in the light.
  • Plants tested positive for PMI and the candidate gene coding sequence were verified by Taqman. Expression for trait expression cassette was assayed by qRT-PCR. Fertile, single copy events were identified and transferred to the greenhouse.
  • Transgenic maize events were produced using construct 19692. A total of 32 single-copy 19692 TO events were identified. T1/T2 seeds were generated via backcrossing with AX5707 as female under ideal growth conditions. Messenger RNA produced from transgene was measured in seedling leaf tissue by qRT-PCR. The qRT-PCR data are reported as the ratio of the gene- specific (specifically, the 3 '-terminus plus the tUbil junction region) signal to that of an endogenous control signal multiplied by 1000. Leaf tissues of Tl seedlings from 10 different events of 19692 were sampled for qRT-PCR analysis at VI 0 stage. Each event was assayed in triplicate. The data in Table 2 (mean ⁇ standard deviation) show that the trait expression cassette functions to produce trait transcript in leaf as expected.
  • a modified assay was used to determine if the 19692 trait increases cold tolerance (Journal of Experimental Botany, Vol. 64, No. 12, pp. 3657-3667, 2013). This assay used 12°C instead of 15°C as the cold treatment. Seeds representing Events 4 and 5 were sown directly in soil in 10 x 10 cm pots. Six similarly staged transgenic and null plants were selected for each treatment. The V4 siblings were subject to cold and or normal growth treatments for two weeks.
  • the cold treatment was 12 C (50% humidity, 16h day/8h night).
  • the normal treatment was 26: 18°C (day/night) (50% humidity, 16h day/8h night).
  • Seed germination under cold conditions is usually related to seedling vigor.
  • a method reported in Theor Appl Genet. 2013 Mar; 126(3):733-46 will be used. Pots containing the seeds will be placed in a growth chamber at 10°C and without light for 7 days. The status of germination at days 4 and 7 will be recorded after transferring the pots into an incubator at normal conditions.
  • B2 plants for 10 events were evaluated by comparing null (RFP-) and transgenic (heterozygous, RFP+) siblings representing the same event ("pairs"). The pairs were selected at the V2 stage and watered normally until the V3 stage. Plants were then subject to drought by withholding water until 90% of the null plants reached leaf rolling score of 3 (v shape) to 5 (o shape). The drought was relieved by full irrigation for 2 days before shoots were harvested for dry biomass analysis. A paired T-test was used to determine if the transgenic siblings were significantly different from null siblings in biomass accumulation under drought conditions.
  • Results of the Paired T-test analysis indicate that a number of events, including events 3, 4, 5, 9, and 10, showed a statistically significant increase in biomass accumulation when plants are exposed to drought conditions.
  • ThST03 (SEQ ID NO: 7) was cloned into vector 18083, a binary expression vector.
  • One expression cassette comprises promoter sequence prZmABP3-01 (U.S. Patent No. 8,344,209) operably linked to SEQ ID NO:7, which is operably linked to terminator sequence tZmABP3-01 (U.S. Patent No. 8,344,209).
  • This binary vector (18083) also contained the PAT selectable marker gene.
  • binary vector 18083 was introduced into rice via
  • T2 seed was generated by selfing the TO and Tl transgenic plants.
  • the genotype of T2 plants were verified via glufosinate herbicide application.
  • homozygous transgenic plants and corresponding null plants for each event were grown in one pot and subject to drought treatment.
  • the paired drought assay was performed with five T2 events. At V3, for each event, 25 homozygous plants were paired with 25 null plants based on plant size, and were transferred from germination plates to 31 x28cm pots. Plants were fully watered until the V5 stage, and then subject to water deficit by withholding water until 90% of null plant leaves reached rolling score of 7, corresponding to having a U shape. All plants were then fully re-watered for 2 days. After that, shoots were harvested and dry shoot biomass was analyzed.
  • Figures 26 and 27 show further results demonstrating that transgenic rice expressing TsST03 exhibit increased drought tolerance.
  • ThST03 (SEQ ID NO: 7) was cloned into vector 18704, a binary expression vector.
  • the expression cassette comprises a promoter sequence (prCMP-04; U.S. Patent No. 7, 166,770) operably linked to SEQ ID NO:7, which is operably linked to a terminator sequence (tNOS-03-01; NCBI accession number V00087.1, Bevan et al, 1983, Nucleic Acids Res. 11 : 369-385).
  • Binary vector 18704 also contained the PAT selectable marker gene.
  • variety Williams 82 was transformed with Agrobacterium harboring binary vector 18704 following methods known in the art, for example Zhang et al., 1999 ⁇ Plant Cell, Tissue and Organ Culture, 56: 37-46). Tl seeds were generated by selfing the TO plants. At the Tl generation, 10 events were selected for the paired drought assay.
  • Paired drought experiment with transgenic soybean plants For the paired drought assay with transgenic soybean, zygosity analysis on Tl transgenic soybean plants is performed to identify null, heterozygous and homozygous plants. At the V2 stage for 10 independent events, 8 homozygous plants are paired with 8 null plants based on similar plant size. Then paired plants are transferred into large pots with similar amount of mixed soybean soil substrate (2500-3000g). Plants are watered in the first 2 days after transplanting and then the drought treatment is applied by withholding water until 90% null plants exhibit a wilting phenotype. Subsequently, all the plants are fully watered for 1 day followed by a second round of drought treatment until 90% null plants show a wilting phenotype. Then, shoots from transgenic plants and nulls are harvested for dry biomass analysis.
  • Example 6 Heat tolerance experiments with transgenic maize plants The purpose of this study is to assess the effect of heat-induced stress on the growth and development of transgenic maize plants compared to a null or non-transgenic control. These transgenic maize plants are generated using the binary vector 19692 as described in Example 2. B2 transgenic and null plants are identified as described in Example 3. The pairs are selected at the V2 stage and watered normally until the V3 stage. At the V3 stage, 20 transgenic/null pairs per event are transferred to growth chambers with optimal (30/22°C) and moderate (43/35°C) heat treatments (day/night) for five days. The photoperiod for all chambers is a 16 hour day/8 hour night. All plants are watered as needed for the duration of the experiment. Plants are evaluated for plant height, growth stage, chlorophyll content, vigor and necrosis prior to heat treatment and at 3 and 5 days after treatment (DAT). Fresh and dry weights of aboveground biomass are measured at the conclusion of the experiment.
  • DAT 3 and 5 days
  • Example 7 Heat tolerance of germination in transgenic maize plants.
  • transgenic and null maize seeds are obtained from transgenic maize plants generated using the binary vector 19692 as described in Example 2. B2 transgenic and null plants are identified as described in Example 3. Seed is collected from transgenic and null plants for the heat treatment assay.
  • the heat treatment chamber comprises an inner chamber and an outer chamber.
  • the inner chamber comprises a plastic container with a lid, into which is placed a tray with a wire mesh screen.
  • the outer chamber comprises a water-jacketed incubator capable of maintaining a constant temperature range from 45°C ⁇ 0.3°C. Seed moisture tins comprise metal tins or similar heat resistant containers with lids.
  • At least 300 seed for each of 10 transgenic events and the corresponding null were weighed and the initial seed moisture was determined.
  • 40 mL of distilled water was added to the inner chamber and the tray with the screen was placed on top, being certain not to splash water onto the screen surface.
  • the seeds were placed in seed moisture tins on the screen tray, above the water.
  • the lid was placed on the inner chamber, but not sealed.
  • the inner chamber was then placed inside the outer chamber.
  • the temperature of the outer chamber was maintained at 45° ⁇ 0.3°C and the relative humidity (RH) was maintained at 99% during the aging period.
  • RH relative humidity
  • transgenic and null maize plants are generated using the binary vector 19692 as described in Example 2.
  • B2 transgenic and null plants are identified as described in Example 3.
  • Fourteen days after planting, transgenic and null pairs are selected based on size similarity. Beginning 17 days after planting and continuing for 12 days, pots in each salt treatment are irrigated with either reverse osmosis water or varying
  • Plants are evaluated for plant height, growth stage, chlorophyll content, and vigor three times during the experiment: prior to treatment and at 9 and 12 days after treatment begins. Fresh and dry weights of above-ground biomass are measured at the conclusion of the experiment.
  • a random cDNA library was first constructed from salt-treated seedlings, including rosette leaves and roots.
  • a double CaMV 35S promoter was used to express this library in Arabidopsis and some 1,000 kanamycin resistant Tl lines were screened.
  • T2 seeds of each plant line and control plants (expressing a pGreen-GFP vector) were germinated and grown on soil.
  • seedlings were watered with 200 mM NaCl solution and seeds from individual putative salt-tolerant plants were harvested for a secondary screen.
  • This screen identified some 20 lines as being highly salt tolerant, with line 0003 displaying an enhanced salt tolerance phenotype.
  • the salt cress gene conferring this tolerance to an imposed salinity condition was identified by PCR amplification and sequencing and named TsST03 (also referred to in this example as ST03 in all instances were referenced, such as, e.g., in reference to fusion proteins, transgenic lines, etc.); the cDNA was 767 bp in length and encodes a protein of 155 amino acids. At the amino acid level, ST03 has 90% identity and 96% similarity to At3g55470.
  • Fig. 24 shows further results illustrating that grafting of TsST03 transgenic Arabidopsis to wild type Arabidopsis can also enhance salt tolerance; thereby demonstrating that the protective effect can be obtained across the whole plant.
  • the C2 Domain of ST03 displays phospholipid binding activity
  • ST03 protein and its plant orthologs contain a single C2 domain (Fig. 9), which appears to be plant specific.
  • the Ca 2+ -dependent phospholipid binding activity is a characteristic feature of many C2 domain proteins, and five conserved aspartic acids in this domain are considered to play a crucial role in binding Ca 2+ ions.
  • the second aspartic acid is glutamic acid instead of aspartic acid, but the remaining four of the five aspartic acids are conserved in ST03 (Fig. 9). Nevertheless, analyses of a small protein with a single C2 domain in rice, OsSMCPl, indicated that Ca 2+ and phospholipid binding characteristics cannot be reliably prediced solely from sequence analysis.
  • Proteins containing C2 domains can interact with phospholipids in a Ca 2+ -dependent or
  • Arabidopsis transgenic P 3 ss-'ST03(mCBS) plants were generated and germinated on half-strength MS medium ⁇ 150 mM NaCl; ST03 and wild-type plants served as controls. Similar to wild-type plants, these P35S-'ST03(mCBS) plants were sensitive to NaCl during germination and post-germination growth periods (Figs. 12 and 13).
  • Fig 25 also confirms that plants expressing wild type TsST03 show improved heat stress tolerance, while plants expressing the mutant mCBS form do not. ST03 is involved in maintenance of plasma membrane integrity
  • the fluorescent dye FM4-64 a well-established endocytic marker, was used to stain the Arabidopsis roots, which allows for easy monitoring of cell membranes by using confocal microscopy. FM4-64 can insert into the lipid bilayer and, therefore, it can be used to label the plasma membrane and, subsequently, the endosomal network via endocytosis.
  • 5-day-old seedlings were used with a single root for reproducibility. The effect of 1 h NaCl treatment on membrane integrity in wild-type, P 3 5S-'ST03 and P ⁇ :ST03 ⁇ mCBS) roots was investigated.
  • GFP-ST03 the subcellular localization of GFP-ST03 and GFP-ST03(mCBS) was checked.
  • GFP-ST03, GFP- ST03(mCBS) and free GFP were all expressed in Arabidopsis driven by the ST03 promoter.
  • Confocal imaging of 5-day-old seedling roots revealed that free GFP, GFP-ST03 and GFP- ST03(mCBS) were similarly localized within the cells under control conditions (1/2 MS treated). Plasmolysis treatment indicated that GFP-ST03 is not present within the cell wall, but rather is localized to intracellular components.
  • the GFP-ST03 signal was localized almost entirely to the cell periphery. This change in cellular distribution may reflect GFP-ST03 turnover in the cytosol, or elevated targeting to the plasma membrane.
  • a time course experiment was conducted in which cycloheximide was applied at the beginning of the NaCl treatment to block de novo synthesis of GFP-ST03. As the level of GFP-ST03 did not change during the time-course, these studies indicated that the change in GFP-ST03 signal, under salt stress, likely reflects elevated targeting of ST03 to the plasma membrane.
  • transgenic Oryza sativa (rice) plants were generated.
  • the ST03 CDS was fused with a Myc-tag and the construct was placed under the control of the Zea mays ubiquitin 1 promoter.
  • Thirteen independent transgenic lines were tested and found to display phenotypes equivalent to wild-type plants when grown under standard conditions.
  • RNA gel blot assays and western blot analysis were performed to determine the expression levels of the ST03 in transgenic rice. Under control conditions, ST03 transcripts and translations were detected at different levels and homozygous T2 lines were selected for further analysis.
  • transgenic and wild-type rice lines were grown hydroponically for 2 weeks prior to commencing a 100 mM NaCl treatment. Ten days later, plants were transferred back to control medium to test for recovery; 7 days later, the suvival rate for the control was only 6%, whereas for the two transgenic lines the values were 60% and 91% (Fig. 16).
  • transgenic and wild-type rice lines were germinated and grown for 21 days in MS medium containing 200 mM NaCl. Analysis of the aerial and root parts of these plants indicated better growth overall for the transgenic rice lines compared to wild-type. These studies indicated that overexpression of ST03 can enhance salt tolerance for both the shoot and root systems in rice.
  • ST03 transgenic plant lines Five days after re-watering, ST03 transgenic plant lines exhibited a high survival rate (80%-90%), whereas the corresponding survival rate for the wild-type control was very low (15%). Taken together, these studies indicate that over-expression of ST03 can improve drought tolerance in both the monocotyledon rice and dicotyledon Arabidopsis plants.
  • salinity-tolerance gene ST03 (T. salsuginea (previously (previously referred to as T. halophihf . _ a t Tolerance 03), imparts improved salt tolerance when overexpressed in Arabidopsis.
  • the salt-tolerance of ST03 is demonstrated to be Ca 2+ -dependent by in vivo and in vitro experiments.
  • Transgenic rice plants expressing the ST03 gene were highly salt and drought tolerant, indicating the likely utility of this gene as an important genetic resource for the further development of salinity-tolerant crops. Analyses of salt tolerance.
  • the vector transgenic line, 35S-ST03 20 and 35S-ST03 16 were grown vertically on MS medium without or with 150 mM NaCl and 0.6% agar under continuous light for 4, 6, 12 days at 23°C. At the indicated times of vertical growth, seedlings were photographed and statistically analyzed.
  • the agar contained half- strength growth solution with full-strength micronutrients.
  • the plants were grown in a random arrangement in aerated solution on a 16/8-h light/dark cycle at 21°C with an irradiance of 75 ⁇ m "2 s "1 .
  • solid NaCl was added to the growth solution to make a final concentration of 120 mM.
  • Ca(N0 3 ) 2 was added to make a final concentration of 1, 5, 10 mM Ca 2+ in the different growth solutions.
  • the treatment was conducted in an environment with 70% RH.
  • For drought tolerance tests of soil-grown plants one-week-old Arabidopsis seedlings and two-week-old rice seedlings were transplanted to the soil and grown under standard growth conditions, and then the plants were subjected to progressive drought conditions by withholding water for the specified time. To minimize experimental variation, the same numbers of plants were grown in the same pot. The entire test was repeated a minimum of three times.
  • a 468-bp fragment containing the ST03 full-length ORF was amplified by RT-PCR using 5'- CGGAATTCGATGGCTGTGGGAATCCTC-3 ' (SEQ ID NO:24) and 5'- CGGAATTCCTAATCAAATTGGCTATGCTTCC-3' 3' (SEQ ID NO:25) primers.
  • the PCR products were cloned into the vector pVIP-myc (Xie et al., 2002, Nature 419: 167-170) in which transgene expression is under the control of the CaMV 35S promoter.
  • ST03(mCBS) CDS with five single amino acid mutations (Asp-20, 77, 120, 126 to Asn and Glu-26 to Gin) was amplified by PCR using five pairs of dot mutated primers (1 : 5'-
  • Ngtcga(G/C)(A/T)Gana(A/T)Gaa-3' SEQ ID NO:43.
  • the PCR products were cloned and inserted into vector pCambial300-221-GFP in the front of the ST03-GFP fusion gene.
  • ST03-GFP and ST03(mCBS) -GFP fusion constructs two 468-bp fragments of ST03 and
  • the two fragments were cloned into the vector pCambial300-221-GFP containing the ST03 promoter, respectively.
  • GST-ST03 and GST-ST03(mCBS) constructs two 468-bp fragments of ST03 and ST03(mCBS) CDS were amplified by PCR using 5'-
  • the ST03 CDS was cloned into the pCAMBIA based vector under the control of the maize Ubil promoter.
  • the N terminal of ST03 was fused with Myc-tag. Transformation vectors and construction of transgenic plants.
  • Transformation of Arabidopsis was performed by the vacuum infiltration method using Agrobacterium tumefaciens strains ABI or EHA105.
  • Agrobacterium tumefaciens strains ABI or EHA105 For transformation of rice, the plasmids were introduced into Agrobacterium tumefaciens AGLl and embryogenic calli from mature rice Oryza sativa L. ssp. Nipponbare seeds.
  • T3 or T4 homozygous lines were used.
  • phosphatidylethanolamine (1 :3, w/w) were prepared in 50 mM HEPES-NaOH, pH 7.4, and 100 mM NaCl by soni cation, collected by centrifugation, and equilibrated with 50 mM HEPES- NaOH, pH 7.4, with or without ImM Ca 2+ .
  • GST fusion proteins (2-5 ⁇ g) were incubated with liposomes corresponding to 160 ⁇ g of phospholipid for 15 min at room temperature (Kang et al., 2011. Biochimica Biophysica Acta. 1810: 1317-22.)).
  • the phospholipid pellets were washed in 500 ⁇ of the above equilibration buffer and then extracted with 300 ⁇ of acetone at -20 °C for 30 min to remove excess lipid.
  • the pellets obtained by centrifugation at 12,000 ⁇ g for 15 min at 4 °C were dissolved in SDS sample buffer.
  • the proteins in the supernatants were precipitated by adding an equal volume of 20% trichloroacetic acid. After incubation for 15 min on ice, the samples were centrifuged at 12,000 x g for 15 min at 4°C, and the precipitates were mixed with SDS sample buffer. Equal portions of the supernatants and pellets were analyzed by 10% SDS-PAGE, followed by Coomassie Brilliant Blue R-250 staining. Microscopy.
  • Seedlings of transgenic Arabidopsis were grown on vertically oriented half-strength MS Petri dishes. Five-day-old seedlings were mounted in liquid medium using a spacer of one layer of Parafilm between slide and cover slip or in similar slide growth chambers. For the subcellular localization analyses, Arabidopsis seeds were germinated in half-strength MS Vertical Petri plates. Seven-day-old seedlings were transferred to half MS supplemented with NaCl at the described concentrations. After the corresponding treatment, roots of the seedlings were visualized using a spacer of a single cover slip between slide and cover slip for confocal microscopy. For plasma membrane integrity analyses, Arabidopsis seeds were germinated in half-strength MS Vertical Petri plates.
  • the red fluorescent dye FM4-64 was excited by the 488-nm laser line, and emission was filtered between 620 and 710 nm. Projections of serial confocal sections and contrast enhancement were done using image processing software (Adobe Systems; Leica Application Suite Advanced Fluorescence; Leica Microsystems).
  • Tobacco (Nicotiana benthamiand) infiltration assay was performed using methods known in the art. Local and systemic leaves were harvested 3 days after infiltration and ground into powder with liquid nitrogen for protein gel blot assay.
  • Plant materials were ground in liquid nitrogen and extracted with 2> ⁇ SDS buffer. Crude extracts were separated by SDS-PAGE and transferred to nitrocellulose membranes. The membranes were stained with 0.2% Ponceau S. Antibodies to GFP and c-Myc were purchased from Santa Cruz Biotechnology. Anti-ubiquitin was also produced.
  • the purpose of this study is to identify orthologs of ThST03 and to introduce them into Arabidopsis plants to assess the effects of salt, cold, and stresses on transgenic Arabidopsis plants into which ST03 orthologs have been introduced.
  • Orthologs were identified using publicly available databases. Orthologs were filtered based on BLAST results using ThST03 as a query sequence, with a cut-off e- value of 10. The identified orthologs are represented by the following GenBank accession numbers :
  • XP_002316283.1 C.cajan_31251; NP_001235151.1; XP_007141871.1; XP_004490896.1;
  • Expression vectors suitable for Arabidopsis transformation are produced.
  • a cDNA nucleic acid sequence for each ortholog (SEQ ID NOS: 14 to 18), and for ThST03 (SEQ ID NO: 1), are introduced into the vector operably linked at the 5' end to a 35S promoter derived from Cauliflower mosaic virus (CaMV) (Odell et al. 1985, Nature 313 : 810-812) and are operably linked at the 3' end to a nopaline synthase (NOS) terminator derived from Cauliflower mosaic virus (CaMV) (Odell et al. 1985, Nature 313 : 810-812) and are operably linked at the 3' end to a nopaline synthase (NOS) terminator derived from Cauliflower mosaic virus (CaMV) (Odell et al. 1985, Nature 313 : 810-812) and are operably linked at the 3' end to a nopaline synthase (NOS)
  • Expression vectors also comprise an expression cassette encoding the PAT gene, a selectable marker known in the art to be useful for selecting transformants after transformation.
  • C. reinhardtii strains the construction of vectors, and transformation of the vectors into C. reinhardtii, and selection of transformants are performed as described in Example 1, using the orthologs of ST03 described in Example 10 as the genes of interest.
  • Cold stress treatments and cold stress responses of the C. reinhardtii transformants are performed as described in Example 1.
  • the CmST03, GmST03, and ZmST03 orthologs were found to confer salt resistance in transgenic A. thaliana. This indicates that these orthologs can confer salt tolerance when expressed in transgenic plants.
  • A. thaliana seeds were sown in MS-0 plates, three repeats in each treatment. The plates were placed in the dark at 4°C for 3 days and then transferred to 22°C (normal condition) or 12°C (cold treatment) under long day conditions (16h : 8h/day : night, 50% humidity). Nine days after treatment root length was checked. The T-test was used to compare performance between the transformed seeds (GM) and the WT seeds. Constructs were considered to be functional for conferring cold tolerance when more than two events with significantly higher root length were observed for the transgenic seeds (GM) at a P ⁇ 0.1 when compared to seeds that were not transformed with the same construct (WT) (Table 11; Table 12).
  • the CmST03, GmST03, CcST03, and ZmST03 orthologs were found to confer cold tolerance in transgenic A. thaliana. This indicates that these orthologs can confer cold tolerance when expressed in transgenic plants. 12.3 Drought tolerance
  • A. thaliana seeds were sown directly into soil and maintained in 8h light/16h dark, 22°C, 50% humidity for 47 days. Forty-seven days after sowing, above-ground shoot/rosette leaves were collected. Three plants mixed as one repeat and there were five repeats per event. Shoot weight was checked hourly after removing from soil. The T test was used to compare performance differences between the transformed seeds and the WT seeds. Constructs were considered to be functional for conferring drought tolerance when more than two events with significantly reduced water loss were observed for the transgenic seeds (GM) at a P ⁇ 0.1 when compared to seeds that were not transformed with the same construct (WT) (Table 13, Table 14).
  • the CmST03, S1ST03, and ZmST03 orthologs were all found to confer reduced leaf water loss in transgenic Arabidopsis, which is an indicator for drought tolerance. This indicates that these orthologs can confer drought tolerance when expressed in transgenic plants.
  • Table 17 summarizes the results of salt tolerance, cold tolerance, and drought tolerance of the ST03 orthologs when expressed transgenically in Arabidopsis. These examples show that ST03 orthologs can be expressed transgenically to produce plants that have enhanced abiotic stress tolerance to at least two abiotic stresses and/or enhanced cold stress tolerance.
  • SEQ ID NO:5 (ATST03 aa)
  • SEQ ID NO:8 (Brassicaeae consensus sequence)
  • SEQ ID NO: 10 (Benincaseae consensus sequence)
  • SEQ ID NO: 11 (Citrus consensus sequence)
  • SEQ ID NO: 12 Solanoideae consensus sequence
  • SEQ ID NO: 13 (Monocot consensus sequence)
  • SEQ ID NO: 14 (Zea mays ST03 cDNA)
  • SEQ ID NO: 17 (Cucumis melo ST03 cDNA)
  • SEQ ID NO: 18 (Citrus Clementina ST03 cDNA)
  • SEQ ID NO: 19 (Zea mays ST03 amino acid sequence)
  • SEQ ID NO:20 (Glycine max ST03 amino acid sequence)
  • SEQ ID NO:21 Solanum lycopersicum ST03 amino acid sequence
  • SEQ ID NO:22 (Cucumis melo ST03 amino acid sequence)

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Abstract

La présente invention concerne des compositions et des procédés pour augmenter la tolérance au stress abiotique dans des plantes. Des plantes et/ou parties de plantes identifiées, sélectionnées et/ou produites à l'aide des compositions et/ou des procédés selon l'invention sont également décrites.
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"GENBANK", Database accession no. XP_004970539.1
"GENBANK", Database accession no. XP_006365698.1
"GENBANK", Database accession no. XP_006403454.1
"GENBANK", Database accession no. XP_006435544.1
"GENBANK", Database accession no. XP_006486477.1
"GENBANK", Database accession no. XP_007141871.1
"GENBANK", Database accession no. XP_007218541.1
"GENBANK", Database accession no. XP_008458869.1
"GENBANK", Database accession no. XP_009116242.1
"GENBANK", Database accession no. XP_009139161.1
"GENBANK", Database accession no. XP_009420100.1
"GENBANK", Database accession no. XP_010030954.1
"GENBANK", Database accession no. XP_010232545.1
"GENBANK", Database accession no. XP_010690675.1
"NCBI", Database accession no. V00087.1
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