WO2018076335A1

WO2018076335A1 - Compositions and methods for enhancing abiotic stress tolerance

Info

Publication number: WO2018076335A1
Application number: PCT/CN2016/104005
Authority: WO
Inventors: Qi Xie; Jian LV; Gang Li; Ran XIA; Yongshen SHANG; Jiang Li; Pingsha HU; Michael Nuccio
Original assignee: Institute Of Genetics And Developmental Biology, Chinese Academy Of Sciences
Priority date: 2016-10-31
Filing date: 2016-10-31
Publication date: 2018-05-03
Also published as: WO2018078390A1

Abstract

Compositions and methods for enhancing abiotic stress tolerance in plants are provided. Plants and/or plant parts identified, selected and/or produced using compositions and/or methods of the present invention are also provided.

Description

COMPOSITIONS AND METHODS FOR ENHANCING ABIOTIC STRESS TOLERANCE

FIELD OF THE INVENTION

The present invention relates to compositions and methods for enhancing abiotic stress tolerance in plants.

BACKGROUND OF THE INVENTION

Drought, salinity, and temperature extremes (heat as well as chilling and freezing) 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.

SUMMARY OF THE INVENTION

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. In some embodiments, 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. In some embodiments, plants and/or plant parts having increased yield and/or increased seed germination under abiotic stress conditions, such as, e.g., cold stress conditions, are provided, as well as 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.

In some embodiments, 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, 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.

In some embodiments, 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, 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 any one of the aforementioned nucleotide sequences, one or more nucleotide sequences that specifically hybridize to one of the aforementioned nucleotide sequences under stringent hybridization conditions, and/or a functional fragment of one of the aforementioned nucleotide sequences.

In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

In some embodiments, 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 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； in some embodiments comprises a nucleotide sequence that is complementary to the nucleotide sequence of any one of the sequences described above； and in some embodiments comprises 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 expression of 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. In some embodiments, 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. In some embodiments, 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.

In some embodiments, 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 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； in some embodiments comprises a nucleotide sequence that is complementary to the nucleotide sequence of any one of the sequences described above； and in some embodiments comprises 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 detection of the above-described 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. In some embodiments, 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.

In some embodiments, 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.

In some embodiments, 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 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； in some embodiments comprises a nucleotide sequence that is complementary to the nucleotide sequence of any one of the sequences described above； and in some embodiments comprises 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 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.

In some embodiments, 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 β-sandwich, that comprises a C2 domain, and/or that binds calcium. In some embodiments, 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. In some embodiments, 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.

In some embodiments, 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.

In some embodiments, 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, 22, or 23； in some embodiments comprises a nucleotide sequence that is complementary to the nucleotide sequence of any one of the sequences described above； and in some embodiments comprises 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. In some embodiments, 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.

In some embodiments, 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. In some embodiments, 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 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； in some embodiments comprises a nucleotide sequence that is complementary to the nucleotide sequence of any one of the sequences described above； and in some embodiments comprises 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. In some embodiments, the exogenous nucleic acid sequence comprises SEQ ID NO: 1. In some embodiments, 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. In some embodiments, the transgenic plant is not Thellungiella salsuginea (previously referred to as T. halophila) and/or the transgenic plant is not Arabidopsis thaliana.

The foregoing and other objects and aspects of the present invention are explained in detail in the drawings and specification set forth below.

BRIEF DESCRIPTION OF THE FIGURES

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. 2 is a graph showing the survival rate percentages for transgenic Arabidopsis lines after salt stress treatment, with WT (Col-0) and empty vector (pVIP-Myc) as the control, in which the number 1 to 20 indicates the name of 17 transgenic lines. Plants were subjected to gradient salt solution treatment (0, 50 100, 150, 200 mM NaCl) every 3 days. Statistical analysis was carried out two weeks after treatment. Data presented as mean ± s. d. (n＝40 seedlings for each line tested, with 5 replicates) .

Fig. 3 is a graph showing the seed germination rate percentage in Murashige-Skoog (MS) medium supplemented with 150 mM NaCl. Data presented as mean ± s. d. (n＝100 seeds for each plant line tested, with five biological replicates) . As described in Chinese Patent No. CN101747419B, incorporated by reference herein.

Fig. 4 is a graph showing the seed germination percentage in MS medium supplemented without 150 mM NaCl. Data presented as mean ± s. d. (n＝100 seeds for each plant line tested, with five biological replicates) . As described in Chinese Patent No. CN101747419B, incorporated by reference herein.

Fig. 5 is a graph comparing the percentage of seedlings with green cotyledons 6 days after seed germination in MS medium supplemented with 150 mM NaCl. Data presented as mean ± s. d. (n＝100 seeds for each plant line tested, with five biological replicates) .

Fig. 6 is a graph comparing the percentage of seedlings with true leaves 12 days after seed germination in MS medium supplemented with 150 mM NaCl. Data presented as mean ± s. d. (n＝100 seeds for each plant line tested, with five biological replicates) . As described in Chinese Patent No. CN101747419B, incorporated by reference herein.

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) . As described in Chinese Patent No. CN101747419B, incorporated by reference herein.

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 (Cmpp16-1, SEQ ID NO: 46, and Cmpp16-2, SEQ ID NO: 47； Xoconostle-Cázares et al., 1999. Science 288: 94-98) . Amino acid residues that form the Ca²⁺ binding sites of the C2 domains in Cmpp16-1 and 16-2 are indicated by pentagrams.

Fig. 10 illustrates the results obtained from Ca²⁺-dependent phospholipid binding assays. Fig. 10, 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％PS/75％PC) in the presence of the indicated concentrations of free Ca²⁺. Liposomes were precipitated by centrifugation, and bound proteins were analyzed by SDS-PAGE. No binding was obtained when the liposomes were incubated in the presence of GST alone. 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 Ca²⁺-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 35S-ST03 (ΔCBS) transgenic lines under NaCl treatments. Data are means of three independent assays.

Fig. 13 is a graph showing post-germination phenotype percentages for wild-type, 35S-ST03 and 35S-ST03 (ΔCBS) 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 Ca²⁺ concentrations under salt stress. Wild-type, P_35S: ST03 and P_35S: 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 Ca²⁺ concentrations indicated and 100mM NaCl. Data are means of three independent assays.

Fig. 15 is a graph showing ion leakage percentages for wild-type, P_35S: ST03 and P_35S: 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 Ca²⁺ 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_35S: ST03 transgenic line.

Fig. 16 is a graph showing survival rate percentages for P_Ubi: 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) (SEQ ID NO: 51) , Glycine max (soybean) (SEQ ID NO: 20) , Phaseolus vulgaris (common bean) (SEQ ID NO: 52) , Cicer arietinum (chickpea) (SEQ ID NO: 53) , and Medicago truncatula (SEQ ID NO: 54) . 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 (tomato) (SEQ ID NO: 21) , Solanum pimpinellifolium (currant tomato) (SEQ ID NO: 58) , and Solanum tuberosum (potato) (SEQ ID NO: 59) . Sequences are at least 91％identical to the consensus sequence (SEQ ID NO: 12) .

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) .

DETAILED DESCRIPTION

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. In some embodiments, 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) . Thus, a plant and/or plant part may be tolerant to at least three abiotic stresses (e.g., salt stress, cold stress, and drought stress) .

Although the following terms are believed to be well understood by one of ordinary skill in the art, the following definitions are set forth to facilitate understanding of the presently disclosed subject matter.

All technical and scientific terms used herein, unless otherwise defined below, are intended to have the same meaning as commonly understood by one of ordinary skill in the art. References to techniques employed herein are intended to refer to the techniques as commonly understood in the art, including variations on those techniques or substitutions of equivalent techniques that would be apparent to one of skill in the art.

All patents, patent publications, non-patent publications referenced herein are incorporated by reference in their entireties for the teachings relevant to the sentence or paragraph in which the reference is presented. In case of a conflict in terminology, the present specification is controlling.

As used herein, the terms "a" or "an" or "the" may refer to one or more than one, unless the context clearly and unequivocally indicates otherwise. For example, "an" endogenous nucleic acid can mean one endogenous nucleic acid or a plurality of endogenous nucleic acids.

As used herein, the term "and/or" refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative ( "or" ) .

As used herein, the term "about, " when used in reference to a measurable value such as an amount of mass, dose, time, temperature, and the like, refers to a variation of ± 0.1％, 0.25％, 0.5％, 0.75％, 1％, 2％, 3％, 4％, 5％, 6％, 7％, 8％, 9％, 10％, 15％or even 20％of the specified value as well as the specified value. Thus, if 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％) .

As used herein, the terms "backcross" and "backcrossing" refer to the process whereby a progeny plant is crossed back to one of its parents. In a backcrossing scheme, 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 F1 generation. The term "BC1" refers to the second use of the recurrent parent, "BC2" refers to the third use of the recurrent parent, and so on.

As used herein, the transitional phrase "consisting essentially of" is to be interpreted as encompassing the recited materials or steps and those that do not materially affect the basic and novel characteristic (s) of the claimed invention.

As used herein, the terms "cross, " "crossing" and "crossed" refer to the fusion of gametes to produce 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) .

As used herein, 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 cultivars/varieties within the same species.

As used herein, 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. In some embodiments, the reduction results in no or essentially no activity (i.e., an insignificant or undetectable amount of activity) .

As used herein, the terms "abiotic stress" and "abiotic stress conditions" refer to non-living 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. ) ) .

As used herein, the terms "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) . When used in reference to a plant part, the terms refer to the ability of a plant that arises from that plant part to endure and/or thrive under abiotic stress conditions.

The elucidation of the response pathways to a given abiotic stress and the cross-talk between pathways for different abiotic stresses is still in its infancy. Recent studies have revealed that the response of plants to a combination of two different abiotic stresses is unique and cannot be directly extrapolated from the response of plants to each of the stresses individually (Mittler, 2006, Trends in Plant Science 11: 15-19) . Therefore, the identification of a gene involved in a number of abiotic stresses is unexpected, and the ability of a gene to confer tolerance to more than one abiotic stress is unpredictable. The inventors of the present application surprisingly discovered that 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) . In some embodiments, 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. In some embodiments, a plant or plant part may experience an abiotic stress or stresses that is/are perceived by the plant or plant part. For example, 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. In some embodiments, 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. In some embodiments, 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. As those of skill in the art will recognize 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) may or may not be stressful for another. This is because 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. Thus, for example, while one species may experience cold stress at a temperature of 15℃, another species may not be impacted until at least 10℃, and the like. In some embodiments, a plant or plant part may experience cold stress at a temperature of about 15℃ or less, such as, for example, at a temperature of about 15℃, 14℃, 13℃, 12℃, 11℃, 10℃, 9℃, 8℃, 7℃, 6℃, 5℃, 4℃, 3℃, 2℃, 1℃, 0℃, -1℃, -2℃, -3℃, -4℃, -5℃, -6℃, -7℃, -8℃, -9℃, -10℃, or less.

It is to be understood that 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.

As used herein, 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) . Typically, 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, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 days) depending on, for example, the plant species.

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.

As used herein, “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 “heat tolerance” or “heat stress tolerance” , including any of their variations, refers to the relative ability of a plant to survive in conditions of heat stress.

As used herein, 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. In some embodiments, 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 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 grain sugar content (GSC) , increased grain yield at standard moisture percentage (YGSMN) , increased grain moisture at harvest (GMSTP) , increased grain weight per plot (GWTPN) , increased percent yield recovery (PYREC) , decreased yield reduction (YRED) , and/or decreased percent barren (PB) ) when grown under abiotic stress conditions. 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. "

In some embodiments, the improvement in an abiotic stress tolerance trait (e.g., improvement in a cold stress tolerance trait and/or improvement in two or more different abiotic stress tolerance traits) 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. " In some embodiments, the improvement in an abiotic stress tolerance trait (e.g., improvement in a cold 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) . 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. " For example, 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) . In some embodiments, 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. ) . In some embodiments, 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, 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, osmotic stress, and an edaphic stress, drought stress, heat stress, cold stress, salt stress, light stress, osmotic stress, an edaphic stress, and oxidative stress. In some embodiments, when a plant and/or plant part has enhanced stress tolerance to at least one or at least two abiotic stresses, 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.

As used herein, 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. Typically, 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) .

As used herein, with respect to nucleic acids, the term "exogenous" refers to a nucleic acid that is not in the natural genetic background of the cell/organism in which it resides. Thus, an exogenous nucleic acid may also be referred to as a nonnaturally occurring nucleic acid. In some embodiments, the exogenous nucleic acid comprises one or more nucleic acid sequences that are not found in the natural genetic background of the cell/organism. In some embodiments, 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.

As used herein, the term "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.

As used herein with respect to nucleotide sequences, the terms "express" and "expression" refer to transcription and/or translation of the sequences.

As used herein with respect to nucleic acids, the term "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. Such a nucleic acid fragment may be, where appropriate, included in a larger polynucleotide of which it is a constituent. In some embodiments, 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. In some embodiments, 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.

As used herein with respect to polypeptides, the term "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. Such a polypeptide fragment may be, where appropriate, included in a larger polypeptide of which it is a constituent. In some embodiments, 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. In some embodiments, 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.

As used herein with respect to nucleic acids, the term "functional fragment" refers to nucleic acid that encodes a functional fragment of a polypeptide.

As used herein with respect to polypeptides, 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. This can be useful in those situations in which the peptide termini tend to be degraded by proteases prior to cellular uptake. 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. For example, one or more non-naturally occurring amino acids, such as D-alanine, can be added to the termini. Alternatively, 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. Additionally, the peptide terminus can be modified, e.g., by acetylation of the N-terminus and/or amidation of the C-terminus. Likewise, the peptides can be covalently or noncovalently coupled to pharmaceutically acceptable "carrier" proteins prior to administration.

As used herein, 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.

As used herein, 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.

As used herein, the term "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. For example, 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: 1.

As used herein with respect to nucleic acids, polynucleotides and polypeptides, the term "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. In some embodiments, 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. In certain embodiments, 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.

As used herein with respect to cells, the term "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. In some embodiments, the cell is separated from other components with which it is normally associated in its natural state. For example, an isolated plant cell may be a plant cell in culture medium and/or a plant cell in a suitable carrier. "Isolated" does not mean that the preparation is technically pure (homogeneous) , but rather that it is sufficiently pure to provide the cell in a form in which it can be used for the intended purpose. In certain embodiments, 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.

As used herein with respect to nucleic acids, the term "nonfunctional fragment" refers to nucleic acid that encodes a nonfunctional fragment of a polypeptide.

As used herein with respect to polypeptides, the term "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.

As used herein with respect to nucleic acids, proteins, plants, plant parts, bacteria, viruses and algae, the term "nonnaturally occurring" refers to nucleic acids, proteins, plants, plant parts, bacteria, viruses or algae that do not naturally exist in nature. In some embodiments, 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. Thus, 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. In some embodiments, 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. For example, 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. In some embodiments, 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) . Likewise, 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) . Similarly, 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.

Also as used herein, the terms "nucleic acid, " "nucleic acid molecule, " "nucleotide sequence" and "polynucleotide" 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. The term 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. The term "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) .

Where single-stranded, 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 polynucleotide of this invention.

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.

Different nucleic acids or proteins having homology are referred to herein as "homologues. " The term homologue includes homologous sequences from the same and other species and orthologous sequences from the same and other species.

As used herein, the term "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 (Aor 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.

The term "homology" 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. Homology, 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" ) . For nucleotide sequences 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) .

In some embodiments, 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.

"Identity" or "percent identity" refers to the degree of similarity between two nucleic acid or amino acid sequences. For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. When using a 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. The 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.

"Identity" can be readily calculated by known methods including, but not limited to, those described in: Computational Molecular Biology (Lesk, A. M., ed. ) Oxford University Press, New York (1988) ； Biocomputing: Informatics and Genome Projects (Smith, D. W., ed. ) Academic Press, New York (1993) ； Computer Analysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H. G., eds. ) Humana Press, New Jersey (1994) ； Sequence Analysis in Molecular Biology (von Heinje, G., ed. ) Academic Press (1987) ； and Sequence Analysis Primer (Gribskov, M. and Devereux, J., eds. ) Stockton Press, New York (1991) .

As used herein, the term "percent sequence identity" or "percent 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. In some embodiments, "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. Nat'l. 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., Madison, WI) , or by visual inspection (see generally, Ausubel et al., infra) .

One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al., J. Mol. Biol. 215: 403-410 (1990) . Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http: //www. ncbi. nlm. nih. gov/) . This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., 1990) . These 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 BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, a cutoff of 100, M＝5, N＝-4, and a comparison of both strands. For amino acid sequences, 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) ) .

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. For example, 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.

Another widely used and accepted computer program for performing sequence alignments is CLUSTALW v1.6 (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. If the two compared sequences are of different lengths, 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.

The phrase "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. In certain embodiments, 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. In certain embodiments, 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. In further embodiments, the sequences are substantially identical when they are identical over the entire length of the coding regions.

Thus, in some embodiments of the invention, 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. In some particular embodiments, the sequences are substantially identical over at least about 150 residues. In representative embodiments, substantially identical nucleotide or protein sequences perform substantially the same function (e.g., conferring increased cold tolerance) .

For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated if necessary, and sequence algorithm program parameters are designated. The 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. In some representative embodiments, two nucleotide sequences considered to be substantially complementary hybridize to each other under highly stringent conditions.

As used herein with respect to nucleic acids, the term "operably linked" refers to a functional linkage between two or more nucleic acids. For example, a promoter sequence may be described as being "operably linked" to a heterologous nucleic acid sequence because the promoter sequences initiates and/or mediates transcription of the heterologous nucleic acid sequence.

As used herein, the term "percent barren" (PB) refers to the percentage of plants in a given area (e.g., plot) with no grain. It is typically expressed in terms of the percentage of plants per plot and can be calculated as:

As used herein, the term "percent yield recovery" (PYREC) refers to the effect a nucleotide sequence and/or combination of nucleotide sequences has on the yield of a plant grown under stress conditions (e.g., cold stress conditions) as compared to that of a control plant that is genetically identical except insofar as it lacks the nucleotide sequence and/or combination of nucleotide sequences. PYREC is calculated as:

By way of example and not limitation, if a control hybrid plant yields 200 bushels under optimal temperature conditions, but yields only 100 bushels under cold stress conditions, then its percentage yield loss would be calculated at 50％. If an otherwise genetically identical hybrid that contains the nucleotide sequence (s) of interest yields 125 bushels under cold stress conditions and 200 bushels under optimal temperature conditions, then the percentage yield loss would be calculated as 37.5％and the PYREC would be calculated as 25％ [1.00- (200-125) / (200-100) x100) ] .

As used herein, the term "yield reduction" (YD) refers to the degree to which yield is reduced in plants grown under stress conditions. YD is calculated as:

As used herein, the terms "phenotype, " "phenotypic trait"or "trait" refer 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 electromechanical assay. In some cases, 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.

As used herein, the term "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) . Thus, the term "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.

As used herein, the term "plant part" refers to at least a fragment of a whole plant or to a cell culture or tissue culture derived from a plant. Thus, the term "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. In some embodiments, the plant part is a plant germplasm.

As used herein, the term "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. Moreover, DNAs or RNAs comprising unusual bases, such as inosine or modified bases, such as tritylated bases, to name just two examples, 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. The term 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.

As used herein, the terms "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.

As used herein, the terms "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.

As used herein, the terms "promoter" and "promoter sequence" refer 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) . A "stress-inducible promoter" is a promoter that preferentially initiates transcription under certain environmental conditions (or combination of environmental conditions) . A "developmental stage-specific promoter" is a promoter that preferentially initiates transcription during certain developmental stages (or combination of developmental stages) .

As used herein, the term "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.

The terms "stringent conditions" or "stringent hybridization 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. For example, those skilled in the art will appreciate that to function as a primer or probe, a nucleotide sequence only needs to be sufficiently complementary to the target sequence to substantially bind thereto so as to form a stable double-stranded structure under the conditions employed. Thus, primers or probes can be used under conditions of high, moderate or even low stringency. Likewise, 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.

For DNA-DNA hybrids, the T_m can be approximated from the equation of Meinkoth and Wahl, Anal. Biochem., 138: 267-84 (1984) : T_m ＝ 81.5℃+16.6 (log M) +0.41 (％GC) -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℃ 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℃. Generally, stringent conditions are selected to be about 5℃ lower than the thermal melting point (T_m) for the specific sequence and its complement at a defined ionic strength and pH. However, highly stringent conditions can utilize a hybridization and/or wash at the thermal melting point (T_m) or 1, 2, 3 or 4℃ 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℃ 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℃ lower than the thermal melting point (T_m) . If the desired degree of mismatching results in a T_m of less than 45℃ (aqueous solution) or 32℃(formamide solution) , optionally the SSC concentration can be increased so that a higher temperature can be used. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes, part I, chapter 2, "Overview of principles of hybridization and the strategy of nucleic acid probe assays, " Elsevier, New York (1993) ； Current Protocols in Molecular Biology, chapter 2, Ausubel, et al., eds, Greene Publishing and Wiley-Interscience, New York (1995) ； and Green &Sambrook, In: Molecular Cloning, A Laboratory Manual, 4th Edition, Cold Spring Harbor Press, Cold Spring Harbor, N. Y. (2012) .

Typically, 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℃ for short probes (e.g., 10 to 50 nucleotides) and at least about 60℃ 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％formamide, 1 M NaCl, 1％SDS (sodium dodecyl sulfate) at 37℃ and a wash in 1X to 2X SSC (20X SSC ＝ 3.0 M NaCl/0.3 M trisodium citrate) at 50℃ to 55℃. Exemplary moderate stringency conditions include hybridization in 40％to 45％formamide, 1 M NaCl, 1％SDS at 37° C and a wash in 0.5X to 1X SSC at 55℃ to 60℃. Exemplary high stringency conditions include hybridization in 50％formamide, 1 M NaCl, 1％SDS at 37℃ and a wash in 0.1X SSC at 60℃ to 65℃. A further non-limiting example of high stringency conditions include hybridization in 4X SSC, 5X Denhardt's , 0.1 mg/ml boiled salmon sperm DNA, and 25 mM Na phosphate at 65℃ and a wash in 0.1X SSC, 0.1％SDS at 65℃. Another illustration of high stringency hybridization conditions includes hybridization in 7％SDS, 0.5 M NaPO₄, 1 mM EDTA at 50℃with washing in 2X SSC, 0.1％SDS at 50℃, alternatively with washing in 1X SSC, 0.1％SDS at 50℃, alternatively with washing in 0.5X SSC, 0.1％SDS at 50℃, or alternatively with washing in 0.1X SSC, 0.1％SDS at 50℃, or even with washing in 0.1X SSC, 0.1％SDS at 65℃. Those skilled in the art will appreciate that 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.

As used herein, the term "substantially complementary" (and similar terms) 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. Alternatively, the term "substantially complementary" (and similar terms) can mean that two nucleic acid sequences can hybridize together under high stringency conditions (as described herein) .

In representative embodiments, "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.

The phrase "hybridizing specifically to" (and similar terms) 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. 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.

The term "bind (s) substantially" (and similar terms) as used herein refers to 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.

As used herein, the terms "transformation" , "transfection" and "transduction" refer 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.

As used herein, the terms "transgenic" and "recombinant" refer to an organism (e.g., a bacterium or plant) that comprises one or more exogenous nucleic acids. Generally, 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. As used herein, the term "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.

As used herein, the term "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) . The term "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. A large number of vectors known in the art may be used to manipulate nucleic acids, incorporate response elements and promoters into genes, etc. For example, 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. Alternatively, 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. 18: 1062 (1990) ； Spencer et al., THEOR. APPL. GENET. 79: 625-631 (1990) ； Blochinger &Diggelmann, MOL. CELL BIOL. 4: 2929-2931 (1984) ； Bourouis et al., EMBO J. 2 (7) : 1099-1104 (1983) ； U.S. Patent No. 4,940,935； U.S. Patent No. 5,188,642； U.S. Pat. No. 5,767,378； and U.S. Patent No. 5,994,629. 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, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, lipofection (lysosome fusion) , and use of a gene gun or nucleic acid vector transporter.

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 (Ca²⁺) and lipids. This domain was originally identified as one of the two conserved domains (C1-C2) in the α, β, and γ isoforms of mammalian Ca²⁺-dependent protein kinase C (PKC) . 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 phosphatidylserine and phosphatidylcholine (Karlin &Altschul, Proc. Nat'l. Acad. Sci. USA 90: 5873-5787 (1993) ) .

The 3D structure of the C2 domain of synaptotagmin has been reported (Sutton, Cell 80 (6) : 929-38 (1995) ) . 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 Ca²⁺-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. 15； 268 (35) : 26386-90 (1993) ) , protein-protein interaction, membrane and vesicular trafficking, and signal transduction (Brose, J Biol Chem. 20； 270 (42) : 25273-80 (1995) ) .

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 long-distance 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) ； Xoconostle-Cazares et al., Science 283: 94-98 (1999) ； Yang et al., Mol. Plant 1: 770-785 (2008) ； Yokotani et al., Plant Mol. Biol. 71: 391-402 (2009) ； Kang et al., Mol. Cells 35: 381-387 (2013) ) .

Although Xie et al. show that they were able to cultivate a salt-tolerant plant when the ThST03 gene was introduced into a transgenic Arabidopsis plant, this finding does not make discovering a salt-tolerant maize or soybean plant when the ThST03 gene is introduced into said plants obvious. 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) ) . Additionally, an alignment of ThST03 with its ortholog from Arabidopsis shows a very high level of conservation and identity. However, 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.

Wong et al. (Plant Mol. Biol., 58: 561-574 (2006) ) compared 6578 ESTs representing 3628 unique genes from cDNA libraries of cold, drought, and salinity stressed T. halophila plants and found very little overlap between gene expression in these different conditions. A similar result was obtained when microarrays spotted with the ESTs were probed with mRNA obtained from abiotically stressed plants (Wong et al., Plant Physiol. 140: 1437-1450, (2006) ) . Therefore, prior to the present invention it would not have been expected that a T. halophila gene with a role in salt tolerance would also play a role in a different abiotic stress tolerance, such as, for example, cold, drought, and/or heat tolerance. Surprisingly, the inventors of the present application have discovered that 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 (AT3G55470) . Additionally, 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. When the alignment is viewed as a phylogenetic tree, groups, or clades, of homologs with high levels of similarity to each other can be found (for example, Fig. 1) . Based on this tree, numerous alignments can be made with orthologous sequences from clades or groups of clades and consensus sequences can be generated. These consensus sequences are useful for identifying ST03 polypeptides in plant species. Examples of such consensus sequences include, but are not limited to, the following:

Those of skill in the art will recognize that the variable positions, denoted as “X” , 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. In some embodiments, 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.

Accordingly, in some embodiments, the ST03 polypeptides of the present invention comprise one or more of the above-described consensus sequences or conservative variants thereof. From the information provided herein and using methods known to a person of ordinary skill in the art, 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.

The present invention provides 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. Compositions useful for enhancing abiotic stress tolerance to at least two abiotic stresses and/or 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. In some embodiments, 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；

(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；

(c) 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；

(d) 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, 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；

(f) a nucleotide sequence that hybridizes to the nucleotide sequence of any one of (a) to (e) above under stringent hybridization conditions；

(g) a functional fragment of any one of (a) to (f) above (e.g., a functional fragment that binds calcium) ； and any combination thereof.

In some embodiments, a nucleic acid of the present invention may encode a polypeptide that comprises a C2 domain and/or binds calcium. In some embodiments, 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. In some embodiments, a nucleic acid of the present invention may be an abscisic acid (ABA) independent gene, optionally from Thellungiella halophila. In some embodiments, 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. In some embodiments, a nucleic acid of the present invention may be operably linked to a heat-inducible promoter. In some embodiments, a nucleic acid of the present invention may be operably linked to a cold-inducible promoter. In some embodiments, 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. In some embodiments, a nucleic acid of the present invention may be operably linked to a hybrid promoter. Some embodiments include that a hybrid promoter may comprise at least two different promoters (e.g., at least two different constitutive promoters and/or heat inducible promoters) . In some embodiments, 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. Thus, when a hybrid promoter is 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. In some embodiments, a hybrid promoter may comprise a Hsp70 promoter and a RbcS2 promoter.

In some embodiments, a nucleic acid of the present invention may comprise one or more constitutive promoter sequences. For example, 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 Ubi) promoter sequences. Examples of suitable promoters are disclosed in U.S. Patent Nos. 5,352,605, 5,641,876, 5,604,121, 6,040,504 and 7,166,770； WO 93/07278； WO 01/73087； EP 0342926； Binet et al., PLANT SCI. 79: 87-94 (1991) ； Christensen et al., PLANT MOLEC. BIOL. 12: 619-632 (1989) ； Ebert et al., PROC. NATL. ACAD. SCI USA 84: 5745-5749 (1987) ； Norris et al., PLANT MOLEC. BIOL. 21: 895-906 (1993) ； Walker et al., PROC. NATL. ACAD. SCI. USA 84: 6624-6629 (1987) ； Wang et al., MOL. CELL. BIOL. 12: 3399-3406 (1992) ； and Yang &Russell, PROC. NATL. ACAD. SCI. USA 87: 4144-4148 (1990) . Thus, in some embodiments, 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.

In some embodiments, a nucleic acid of the present invention may comprise one or more tissue-specific promoter sequences. For example, 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. GENET. 235: 33-40 (1992) ； Hudspeth &Grula, PLANT MOLEC. BIOL. 12: 579-589 (1989) ； de Framond, FEBS 290: 103-106 (1991) ； Jeong et al. PLANT PHYSIOL. 153: 185-197 (2010) ； and KIM ET AL. PLANT CELL 18: 2958-2970 (2006) . Thus, in some embodiments, 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.

In some embodiments, a nucleic acid of the present invention may comprise one or more chemically inducible promoter sequences. Examples of 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. 227, 229-237 (1991) ； Gatz, CURRENT OPINION BIOTECHNOL. 7: 168-172 (1996) ； Gatz, ANN. REV. PLANT PHYSIOL. PLANT MOL. BIOL. 48: 89-108 (1997) ； Li et al., GENE 403: 132-142 (2007) ； Li et al., MOL BIOL. REP. 37: 1143-1154 (2010) ； McNellis et al. PLANT J. 14, 247-257 (1998) ； Muto et al. BMC BIOTECHNOL. 9: 26 (2009) ； Schena et al. PROC. NATL. ACAD. SCI. USA 88, 10421-10425 (1991) ； Surzycki et al. BIOLOGICALS 37: 133-138 (2009) ； and Walker et al. PLANT CELL REP. 23: 727-735 (2005) . Thus, in some embodiments, 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.

In some embodiments, a nucleic acid of the present invention may comprise one or more wound-inducible promoter sequences. Examples of 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) . Thus, in some embodiments, 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.

In some embodiments, a nucleic acid of the present invention may comprise one or more stress-inducible promoter sequences. For example, 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. Thus, in some embodiments, 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. In some embodiments, the nucleic acid comprises a cold stress-inducible promoter sequence.

In some embodiments, a nucleic acid of the present invention may comprise one or more developmental stage-specific promoter sequences. For example, 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. Thus, in some embodiments, 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. In some embodiments, the nucleic acid comprises a promoter sequence that drives expression prior to and/or during the seedling and/or reproductive stage (s) of development.

A nucleic acid of the present invention may comprise any suitable termination sequence (s) . For example, the nucleic acid may comprise a termination sequence comprising a stop signal for RNA polymerase and a polyadenylation signal for polyadenylase. Thus, 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) . For example, 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. Examples of suitable sequences are disclosed in Allison et al. VIROLOGY 154: 9-20 (1986) ； Della-Cioppa et al. PLANT PHYSIOL. 84: 965-968 (1987) ； Elroy-Stein et al. PROC. NATL. ACAD. SCI. USA 86: 6126-6130 (1989) ； Gallie et al., GENE 165: 233-238 (1995) ； Gallie et al. NUCLEIC ACIDS RES. 15: 8693-8711 (1987) ； Gallie et al. NUCLEIC ACIDS RES. 15: 3257-3273 (1987) ； Gallie et al. NUCLEIC ACIDS RES. 16: 883-893 (1988) ； Gallie et al. NUCLEIC ACIDS RES. 20: 4631-4638 (1992) ； Jobling et al. NATURE 325: 622-625 (1987) ； Lommel et al. VIROLOGY 81: 382-385 (1991) ； Skuzeski et al., PLANT MOLEC. BIOL. 15: 65-79 (1990) . Thus, 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-resistance) , pest-resistance and/or disease-resistance.

A nucleic acid of the present invention may comprise any suitable number of nucleotides. In some embodiments, the nucleic acid is 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000 or more nucleotides in length. In some embodiments, the nucleic acid is less than about 2000, 1900, 1800, 1700, 1600, 1500, 1400, 1300, 1200, 1100, 1000, 900, 800, 700, 600, 500, 400, 300, 200, or 100 nucleotides in length. In some embodiments, the nucleic acid is about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000 nucleotides in length.

A nucleic acid of the present invention may be codon optimized. In some embodiments, 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. The modifications of the nucleotide sequences are determined by comparing the species specific codon usage table with the codons present in the native polynucleotide sequences. As is understood in the art, codon optimization of a nucleotide sequence results in a nucleotide sequence having less than 100％identity (e.g., 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％, 99％, and the like) to the native nucleotide sequence but which still encodes a polypeptide having the same function as that encoded by the original, native nucleotide sequence. Thus, in some embodiments of the present invention, the nucleic acid molecule may be codon optimized for expression in a particular species of interest (e.g., a plant such as maize, soybean, sugar cane, sugar beet, rice or wheat) .

Because expression levels may also be dependent on GC content, 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.

In some embodiments, a nucleic acid of the present invention is an isolated nucleic acid.

In some embodiments, a nucleic acid of the present invention may comprise, consist essentially of, or consist of:

(a) the nucleotide sequence set forth in SEQ ID NO: 7；

(b) 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；

(c) a monocot codon-optimized nucleotide sequence that encodes a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 4；

(d) 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；

(g) a functional fragment of any one of (a) to (f) above, wherein the functional fragment encodes a polypeptide that comprises an eight stranded anti-parallel β-sandwich, that comprises a C2 domain, and/or that binds calcium； and any combination thereof.

In some embodiments, 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 β-sandwich, that comprises a C2 domain, and/or that binds calcium. In some embodiments, 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.

In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. Such desired traits can be other nucleotide sequences which confer other agriculturally desirable traits. Such 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. For example, if 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 99/25821； WO 99/25854； WO 99/25840； WO 99/25855 and WO 99/25853. In representative embodiments, 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-, HPPD inhibitor-and/or broxynil-resistance； 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. In some embodiments, 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. Thus, for example, 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 temperatures, drought, flooding, high light intensity, low light intensity, extreme osmotic pressures, extreme salt concentrations, high winds, ozone, poor edaphic conditions (e.g., extreme soil pH, nutrient-deficient soil, compacted soil, etc. ) , and/or combinations thereof.

The present invention also encompasses vectors comprising one or more nucleic acid (s) and/or expression cassette (s) of the present invention. In some embodiments, the vector is a pSTK, pROKI, pBin438, pCAMBIA (e.g., pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1391-Xa, pCAMBIA1391-Xb) (CAMBIA Co., Brisbane, Australia) or pBI121 vector.

In some embodiments, an expression cassette and/or vector of the present invention may comprise a promoter operably linked to an exogenous nucleotide sequence that comprises:

(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；

(d) 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, 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 present invention also encompasses transgenic cells/organisms comprising one or more expression cassettes, vectors, and/or nucleic acids of the present invention. In some embodiments, the transgenic organism is a bacteria, virus, algae, plant, or plant part. In some embodiments, the transgenic cell is a propagating plant cell, such as an egg cell or sperm cell. In some embodiments, the transgenic cell is a non-propagating plant cell. In some embodiments, the transgenic organism is a plant or plant part. In some embodiments, 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.

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:

(b) a 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；

(c) 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

(d) 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, 22, or 23；

In some embodiments, 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. In some embodiments, 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.

In some embodiments, the transgenic plant comprises an exogenous nucleic acid sequence which comprises SEQ ID NO: 1, 6, or 7.

In some embodiments, 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. In some embodiments, the protein is an isolated protein.

A protein of the present invention may comprise any suitable number of amino acids. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, the nucleic acid/protein is expressed in a monocot plant or plant part (e.g., in rice or maize) . In some embodiments, the nucleic acid/protein is expressed in a dicot plant or plant part (e.g., in soybean) .

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.

In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

In some embodiments, 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.

In some embodiments, 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.

According to some embodiments, provided are methods of enhancing abiotic stress tolerance in a plant and/or plant part. In some embodiments, 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:

(d) 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, 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 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, the introduced and/or expressed exogenous nucleic acid is introduced into a plant part, and a plant is produced from the plant part. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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:

Following detection of the exogenous nucleic acid described above, 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. In some embodiments, 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.

In further embodiments, 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. In some embodiments, 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. In some embodiments, 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, 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, may comprise, consist essentially of, or consist of:

(a) detecting, in a plant part, the presence of 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, 22, or 23, or 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: 4, 5, 19, 20, 21, 22, or 23, and producing a plant from the plant part；

(b) introducing, into a plant part, 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, 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: 4, 5, 19, 20, 21, 22, or 23, and growing the plant part into a plant； such methods may further comprise detecting the exogenous nucleic acid in the plant part and/or in the plant produced from the plant part；

(c) crossing a first parent plant with a second parent plant, wherein the first parent plant comprises within its genome 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, 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: 4, 5, 19, 20, 21, 22, or 23； and/or

(d) introgressing 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, 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: 4, 5, 19, 20, 21, 22, or 23 into a plant or plant part lacking the exogenous nucleic acid.

Each of these methods may thereby produce a progeny generation. In some embodiments, 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.

In some embodiments, a method of producing a 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 salt stress, as compared to a control plant or plant part, may comprise detecting, in a plant and/or plant part, the presence of an exogenous nucleic acid comprising, consisting essentially of or consisting of:

(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；

(f) a nucleotide sequence that hybridizes to the nucleotide sequence of any one of (a) to (e) above under stringent hybridization conditions； and/or

(g) a functional fragment of any one of (a) to (f) above (e.g., a functional fragment that binds calcium) ； and

producing 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.

In some embodiments, 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, as compared to a control plant or plant part comprises, consists essentially of, or consists of introducing, into a plant and/or plant part, an exogenous nucleic acid comprising, consisting essentially of or consisting of:

producing 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.

In some embodiments, 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, as compared to a control plant or plant part comprises, consists essentially of or consists of crossing a first parent plant with a second parent plant, wherein the first parent plant comprises within its genome an exogenous nucleic acid comprising, consisting essentially of or consisting of:

(g) a functional fragment of any one of (a) to (f) above (e.g., a functional fragment that binds calcium) ； and any combination thereof；

thereby producing 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. 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. Such selections may be made based upon the presence of the nucleic acid (or a functional fragment thereof) and the observation of enhanced abiotic stress tolerance of the progeny plant or part.

In some embodiments, 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, 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: 4, 5, 19, 20, 21, 22, or 23 with a second plant that lacks the nucleic acid and repeatedly backcrossing progeny plants comprising the nucleic acid (or a functional fragment thereof) with the second plant to produce an introgressed plant that comprises the nucleic acid (or a functional fragment thereof) and that exhibits enhanced abiotic stress tolerance (s) as compared to a control plant. In some embodiments, 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) .

In some embodiments, 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:

In some embodiments, 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. In some embodiments, 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. In some embodiments, the nucleic acid detected in or introduced into the plant or plant part is a nucleic acid comprising:

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) . In some embodiments, 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 (e.g., a nucleotide sequence encoding a synthetic miRNA precursor molecule of the invention) , 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. Where more than one nucleotide sequence is to be introduced 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. Accordingly, 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. Thus, for example, "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. Thus, in some embodiments, a plant cell of the invention is stably transformed with a nucleotide sequence encoding a synthetic miRNA precursor molecule of the invention. In other embodiments, 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. "Genome" 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 immunosorbent assay (ELISA) or Western blot, which can detect the presence of a peptide or polypeptide encoded by one or more transgene introduced into an organism. Stable 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) . Stable 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.

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. For example, in vivo modification can be used to insert a nucleic acid comprising , e.g., a promoter sequence into the plant genome. In a further non-limiting example, 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.

For example, 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 (2011) ； Christian et al. Genetics 186, 757–761 (2010) ； Jiang et al. Nat. Biotechnol. 31, 233–239 (2013) ； U.S. Patent Nos. 7,897,372 and 8,021,867； U.S. Patent Publication No. 2011/0145940 and in International Patent Publication Nos. WO 2009/114321, WO 2009/134714 and WO 2010/079430； U.S. Patent Nos. 8,795,965 and 8, 771, 945 For example, one or more transcription affector-like nucleases (TALEN) and/or one or more meganucleases may be used to incorporate an isolated nucleic acid comprising a promoter sequence of the invention into the plant genome. In representative embodiments, 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. Alternatively, in some embodiments, 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. In some embodiments, 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) .

As would be understood by the skilled artisan, the 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. In some embodiments, 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. In some such embodiments, 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. In some embodiments, 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. In some embodiments, the probe comprises one or more detectable moieties, such as digoxigenin, fluorescein, acridine-ester, biotin, alkaline phosphatase, horseradish peroxidase, β-glucuronidase, β-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.

In some embodiments, 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.

In some embodiments, 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.

In some embodiments, the use comprises infecting a plant and/or plant part with a transgenic virus comprising a nucleic acid of the present invention.

In some embodiments, 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) . In some embodiments, a plant or plant part useful with this invention includes any monocot and/or any dicot plant or plant part. In some embodiments the plant or plant part is a fodder crop, a food crop, an ornamental plant, a tree or a shrub. For example, in some embodiments, 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. sativa, Avena hybrida ) , Averrhoa carambola, Benincasa hispida, Bertholletia excelsea, Beta vulgaris, Brassica spp. (e.g. Brassica napus, Brassica rapa ssp. [canola, oilseed rape, turnip rape] ) , Cadaba farinosa, Camellia sinensis, Canna indica, Capsicum spp., Carex elata, Carica papaya, Carissa macrocarpa, Carya spp., Carthamus tinctorius, Castanea spp., Cichorium endivia, Cinnamomum spp., Citrullus lanatus, Citrus spp., Cocos spp., Coffea spp., Colocasia esculenta, Cola spp., Coriandrum sativum, Corylus spp., Crataegus spp., Crocus sativus, Cucurbita spp., Cucumis spp., Cynara spp., Daucus carota, Desmodium spp., Dimocarpus longan, Dioscorea spp., Diospyros spp., Echinochloa spp., Elaeis (e.g. Elaeis guineensis, Elaeis oleifera ) , Eleusine coracana, Eriobotrya japonica, Eugenia uniflora, Fagopyrum spp., Fagus spp., Ficus carica, Fortunella spp., Fragaria spp., Ginkgo biloba, Glycine spp. (e.g. Glycine max, Soja hispida or Soja max ) , Gossypium hirsutum, Helianthus spp. (e.g. Helianthus annuus ) , Hemerocallis fulva, Hibiscus spp., Hordeum spp. (e.g. Hordeum vulgare ) , Ipomoea batatas, Juglans spp., Lactuca sativa, Lathyrus spp., Lens culinaris, Linum usitatissimum, Litchi chinensis, Lotus spp., Luffa acutangula, Lupinus spp., Luzula sylvatica, Lycopersicon spp. (e.g. Lycopersicon esculentum, Lycopersicon lycopersicum, Lycopersicon pyriforme ) , Macrotyloma spp., Malus spp., Malpighia emarginata, Mammea americana, Mangifera indica, Manihot spp., Manilkara zapota, Medicago sativa, Melilotus spp., Mentha spp., Miscanthus spp., Momordica spp., Morus nigra, Musa spp., Nicotiana spp., Olea spp., Opuntia spp., Ornithopus spp., Oryza spp. (e.g. Oryza sativa, Oryza latifolia) , Panicum miliaceum, Passiflora edulis, Pastinaca sativa, Persea spp., Petroselinum crispum, Phaseolus spp., Phoenix spp., Physalis spp., Pinus spp., Pistacia vera, Pisum spp., Poa spp., Populus spp., Prosopis spp., Prunus spp., Psidium spp., Punica granatum, Pyrus communis, Quercus spp., Raphanus sativus, Rheum rhabarbarum, Ribes spp., Ricinus communis, Rubus spp., Saccharum spp., Sambucus spp., Secale cereale, Sesamum spp., Sinapis sp., Solanum spp. (e.g. Solanum tuberosum, Solanum integrifolium or Solanum lycopersicum ) , Sorghum bicolor, Spinacia spp., Syzygium spp., Tagetes spp., Tamarindus indica, Theobroma cacao, Trifolium spp., Triticosecale rimpaui, Triticum spp. (e.g. Triticum aestivum, Triticum durum, Triticum turgidum, Triticum hybernum, Triticum macha, Triticum sativum or Triticum vulgare ) , Tropaeolum minus, Tropaeolum majus, Vaccinium spp., Vicia spp., Vigna spp., Viola odorata, Vitis spp., Zea mays, Zizania palustris or Ziziphus spp., amongst others.

In some embodiments, 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. In some embodiments, 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.

Some embodiments include that the plant and/or plant part is rice, maize, or soybean. In some embodiments, 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.

The present invention extends to products harvested from plants and/or plant parts produced according to methods of the present invention. 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. Thus, in some embodiments, 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. In other embodiments, 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.

In some embodiments, 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.

In some embodiments, 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.

In some embodiments, 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.

In some embodiments, 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. In some embodiments, the plant or plant part is or is from maize, rice, wheat, and sugarcane.

In some embodiments, 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. In some embodiments, the plant or plant part is or is from soybean, cotton, and tomato.

Some embodiments include that 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. In some embodiments, the harvested product is a plant part capable of producing a plant and/or plant part that exhibits enhanced abiotic stress tolerance. In some embodiments, 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.

In some embodiments, 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.

In some embodiments, a method of increasing the yield and/or seed germination of a plant crop under abiotic stress conditions is provided, the method 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. In some embodiments, the plant crop may be a maize crop, a rice crop, or a soybean crop.

The invention will now be described with reference to the following examples. It should be appreciated that these examples are not intended to limit the scope of the claims to the invention, but are rather intended to be exemplary of certain embodiments. Any variations in the exemplified methods that occur to the skilled artisan are intended to fall within the scope of the invention.

EXAMPLES

The following examples are not intended to be a detailed catalog of all the different ways in which the present invention may be implemented or of all the features that may be added to the present invention. Persons skilled in the art will appreciate that numerous variations and additions to the various embodiments may be made without departing from the present invention. Hence, the following descriptions are intended to illustrate some particular embodiments of the invention, and not to exhaustively specify all permutations, combinations and variations thereof.

Example 1–Cold stress tolerance experiment in Chlamydomonas reinhardtii

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)

1.1 Chlamydomonas strains

The 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 41S 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

The cultures were grown in liquid Tris–acetate–phosphate (TAP) medium (Harris 1989) in an incubator shaker with 100-120 rpm at 28℃ 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 μg/mL) was used for transformant cell growth.

1.3 Vector construction

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 NotI and KpnI. 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.

1.4 Genetic transformation of Chlamydomonas reinhardtii

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℃. 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 μg plasmid DNA linearized via ScaI 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 125 μL 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℃. 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. The entire cell solution from each transformation was plated on one TAP-agar-Hygromycin plate in plant growth chamber at 28℃ 2300-3000 lux for 5-6 days.

1.5 Sensitivity of Chlamydomonas to hygromycin

Chlamydomonas transformant cells of ThST03 showed growth in culture medium containing Hygromycin (10 μg/mL) while wild type showed declined growth. 13 transformed cell lines were selected on 10 μg/mL hygromycin.

1.6 Cold stress treatment

The cells were grown in TAP medium (for Wild type) or TAP medium containing 10 μg/mL Hygromycin for transformed cells until cell density reached an OD750 of 1.0 at 28 ℃ (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℃ for 0.5h, 28℃ for 2h and 8℃ for 14 days. The OD750 was measured at 7 days and 14 days separately.

OD750 measure

100 μL 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 ₍₁₄₎ . The biomass per unit was calculated to reflect the increased algae biomass at OD750 _(7 or 14) which was normalizd via divided by OD750 ₍₀₎ :

1.7 Cold stress responses of Chlamydomonas transformants

Thirteen transformants and wild type CC-251, CC-4414 were subjected to cold stress treatmentwith two biological repeats. The OD750 was measured at 7 days and 14 days and biomass per unit was calculated. Standard deviation (SD) was also calculated. There were 8 colonies (61.5％) that showed significant increased biomass relative to wild type CC-251, and 2 colonies (15.3％) that had a 3 fold increase which is similar to the wild type cold tolerance control CC-4414. The results from the biomass comparison of recombinant ThST03 transformants under cold stress are shown in Table 1.

Table 1: Biomass comparison of ThST03 transformants under cold stress

Colony	7 Days	SD	P Value		14 Days	SD	P Value
1	2.211	0.115	0.058	1.137	0.058	0.104
2	1.099	0.030	0.382	3.217	0.115	0.009
3	2.222	0.066	0.081	3.288	0.299	0.083
4	3.077	0.170	0.018	1.315	0.026	0.326
5	1.477	0.075	0.538	4.613	0.136	0.006
6	2.953	0.068	0.036	3.409	0.135	0.012
7	2.360	0.020	0.096	1.318	0.048	0.322
8	1.430	0.053	0.660	4.559	0.003	0.017
9	2.656	0.038	0.066	4.959	0.033	0.006
10	3.141	0.126	0.016	4.493	0.037	0.006
11	2.691	0.026	0.070	2.173	0.007	0.073
12	1.851	0.011	0.193	1.598	0.026	0.327
13	1.554	0.023	0.403	4.080	0.134	0.007
cc4414	4.710	0.231	0.010	5.259	0.334	0.044
cc251	1.333	0.164		1.456	0.085

Example 2 –Cold stress tolerance experiment in transgenic maize

2.1 Vector construction

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 (prUbi1-10) and terminator (tUbi1-01) 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.

2.2 Maize Transformation

Construct 19692 was used for Agrobacterium-mediated maize transformation. Transformation of immature maize embryos was performed essentially as described in Negrotto et al., 2000, Plant Cell Reports 19: 798-803. For this example, all media constituents were essentially as described in Negrotto et al. However, various media constituents known in the art may be substituted. Construct 19692 carries both phosphomannose isomerase (PMI) and phosphinothricin acetyltransferase (PAT) as plant selection markers. To prepare for plant transformation, Agrobacterium strain LBA4404 (pSB1) 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℃. Approximately 0.8x10⁹ 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/l) and silver nitrate (1.6 mg/l) and cultured in the dark for 28℃ for 10 days.

Immature embryos, producing embryogenic callus were transferred to LSD1MO5S 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 Reg1 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 Ill. ) 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.

2.3 Production of Transgenic Maize

Transgenic maize events were produced using construct 19692. A total of 32 single-copy 19692 T0 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 tUbi1 junction region) signal to that of an endogenous control signal multiplied by 1000. Leaf tissues of T1 seedlings from 10 different events of 19692 were sampled for qRT-PCR analysis at V10 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.

Table 2: Relative expression of ThST03 in trangenic maize events

2.4 Cold treatment at seedling stage

Cold treatment at seedling stage hinders plant growth and may damage the plant. This may be due to damage to the photosynthetic apparatus. For example, higher Fv/Fm indicates improved cold tolerance (Sui, Li et al. 2007； Soltesz, Smedley et al. 2013) . 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℃ instead of 15℃ 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℃ (50％humidity, 16h day/8h night) . The normal treatment was 26: 18℃ (day/night) (50％humidity, 16h day/8h night) . Fv/Fm was measured at 0, 1, 2, 4, 6, 8, 10, 12, 14 days after cold treatment at mid-blade of youngest fully-extended leaf using a Handy PEA instrument (Hasha Scientific Instruments Limited) . Data analysis was done with JMP11 using the model Fv/Fm ＝ Genotype (G) + Event (E) + G*E + Error.

Under normal conditions, there was no significant difference in Fv/Fm (P<0.2) between null and transgenic siblings as shown in Table 3. While at 4 and 10 days after cold treatment, transgenic plants produced a higher Fv/Fm than null siblings, which indicates better cold tolerance in transgenic siblings.

Table 3: Fv/Fm response in null and transgenic maize after cold treatment

2.5 Cold treatment at germination stage

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℃ 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.

Example 3–Drought tolerance experiments with transgenic maize

Paired Drought assays were carried out with transgenic maize plants made with the binary vector 19692 as described in Example 2. T0 plants with low-copy number were selected via TaqMan analysis after transformation and B2 (backcrossed twice) seed were produced by using AX5707 as the female for backcrossing, selecting seed for RFP presence, then backcrossing the selected B1 plants to AX5707 as the female a second time. 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.

Table 4: Paired T-test analysis of shoot dry biomass in transgenic maize

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.

Example 4–Drought tolerance experiments with transgenic rice

4.1 Vector construction

ThST03 (SEQ ID NO: 7) was cloned into vector 18083, a binary expression vector. Constitutive expression of ThST03 was selected to target the appropriate expression level and cell type. 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.

4.2 Rice transformation and production of transgenic rice plants

To evaluate ThST03 in rice, binary vector 18083 was introduced into rice via Agrobacterium inoculation following methods known in the art (for example Toki 1997, Plant Molecular Biology Reporter, 15: 16-21) . T2 seed was generated by selfing the T0 and T1 transgenic plants. The genotype of T2 plants were verified via glufosinate herbicide application. For the paired drought assay, homozygous transgenic plants and corresponding null plants for each event were grown in one pot and subject to drought treatment.

4.3 Paired drought experiment with transgenic rice

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 31×28cm 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.

Table 5: Paired T-Test Analysis of Shoot Dry Biomass in Rice

Results of the paired T-test analysis indicate that event 3 showed a statistically significant increase in biomass accumulation under drought conditions.

Example 5–Drought tolerance experiments with transgenic soybean

5.1 Vector construction

ThST03 (SEQ ID NO: 7) was cloned into vector 18704, a binary expression vector. Constitutive expression of ThST03 was selected to target the appropriate expression level and cell type. 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.

5.2 Soybean transformation and production of transgenic soybean plants

To determine the transgenic effects of ThST03 in soybean, 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) . T1 seeds were generated by selfing the T0 plants. At the T1 generation, 10 events were selected for the paired drought assay.

5.3 Paired drought experiment with transgenic soybean plants

For the paired drought assay with transgenic soybean, zygosity analysis on T1 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℃) and moderate (43/35℃) 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.

Example 7–Heat tolerance of germination in transgenic maize plants.

The purpose of this study is to assess the effect of heat-induced stress on the germination of transgenic and null maize seeds. These 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.

7.1 Heat treatment chamber

The heat treatment chamber comprises an inner chamber and an outer chamber. In this example, 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℃±0.3℃. Seed moisture tins comprise metal tins or similar heat resistant containers with lids.

7-2. Germination assay

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℃ and the relative humidity (RH) was maintained at 99％during the aging period. After three days, the seeds were removed, measured for seed moisture, and placed under standard germination conditions. After 7 days, germination rates were recorded as the percentage of the total seeds that germinated in the assay. Additional seeds that did not undergo the aging assay were also used for germination controls. Results from that germination assay indicated no significant difference in germination between untreated transgenic and null seeds. A T-test was conducted to determine if the transgenic plants were significantly different from null plants in germination rates. Germination rates, the standard deviation, and the T-test P values are shown in Table 6.

Table 6: Germination rates of transgenic and null maize following heat treatment

Three events had increased tolerance to the accelerated aging assay compared to corresponding nulls, namely

events

1, 2, and 4.

Example 8—Salt tolerance assay with transgenic maize

The purpose of this study is to assess the effect of salt treatment on the growth and development of transgenic and null maize plants. 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. 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 concentrations of a NaCl-CaCl₂ solution (up to 600 mM) to achieve different soil electrical conductivity (EC) levels for each salt treatment as follows:

Table 7: Salt Tolerance Experiments

Treatment	Target EC (dS/m)	Total salt per pot (g)
No Salt	< 1	0.0
Mild	approx. 2–4	14.4
Moderate	approx. 5–7	40.2
Severe	approx. 8–10	58.4

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.

Example 9

Overexpression of ST03 enhances salt tolerance in Arabidopsis

As described in Chinese Patent No. CN101747419B, incorporated by reference herein, to identify salt tolerance-related genes from salt cress, 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 T1 lines were screened. T2 seeds of each plant line and control plants (expressing a pGreen-GFP vector) were germinated and grown on soil. Three weeks post germination, 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.

To confirm that the salt tolerance phenotype was conferred by ST03, its cDNA was re-cloned from salt cress and placed under the control of the CaMV 35S promoter for generating transgenic Arabidopsis plants. More than 20 independent transgenic plant lines were obtained and the T2 generation was tested for growth in soil treated with an increasing NaCl concentration series from 50 to 200 mM. These lines segregated in the T2 generation and all kanamycin-resistant plants showed enhanced resistant to salinity compared with wild-type plants (Fig. 2) . Two homozygous T3 transgenic lines, P_35S: ST03 16 and P_35S: ST03 20, were selected to assess their tolerance to salt during the different stages of germination and seedling establishment. Equivalent germination rates were obtained when control (wild-type and vector) , ST03 16 and ST03 20 seeds were plated on MS medium (no NaCl) (Fig. 3) . In the presence of 150 mM NaCl, ST03 16 and ST03 20 seeds displayed an initial higher germination rate compared to the seeds of control plants (Fig. 4) .

During the post-germination period, ST03 16 and ST03 20 plants continued to exhibit both a shoot and root growth advantage over the control plants when exposed to a salt stress treatment (Figs. 5, 6, and 7) . These results confirm that the high salt tolerance of transgenic Arabidopsis is conferred by overexpression of ST03.

It is well known that salt accumulation in soils generally occurs after drought stress, especially in arid and semi-arid areas； i.e., drought and salt stress often go together. Drought treatment was next applied to soil-grown 3-week-old ST03 and control plant lines to assess whether the ST03 gene also confers drought tolerance. Both ST03 and control plants became wilted following withholding of water for 14 days. Importantly, within 4 days of re-watering, ST03 plants had recovered, whereas most control plants failed to recover from this stress treatment (Fig. 8) . By contrast, no evident phenotypic differences were observed for plants grown in the presence of an adequate water supply. These findings indicate that overexpression of ST03 can confer an enhanced capacity for drought tolerance in Arabidopsis.

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 Ca²⁺-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 Ca²⁺ ions. In ST03, 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, OsSMCP1, indicated that Ca²⁺ and phospholipid binding characteristics cannot be reliably prediced solely from sequence analysis.

To ascertain whether ST03 has the properties essential for Ca²⁺-dependent phospholipid binding, centrifugation assays that test for the formation of Ca²⁺-dependent complexes between the ST03 C2 domain and negatively charged liposomes (25％PS/75％PC) were performed. Recombinant GST (glutathione S-transferase) -ST03 was first purified using a Glutathione Sepharose 4B column and then incubated with liposomes in the presence of different concentrations of free Ca²⁺. Following centrifugation, isolated liposome-bound proteins were analyzed using SDS-PAGE and Coomassie Blue staining. A marked increase in GST-ST03 was observed in the pelleted liposomes when the Ca²⁺ level was increased from 1 to 5 mM (Fig. 10, panel i) , but a further increase in signal strength was not observed at higher Ca²⁺ levels； this indicated that 5 mM Ca²⁺ may be the saturation condition for the experimental assays. These results are consistent with the hypothesis that the GST-ST03 fusion protein is capable of binding phospholipids in a Ca²⁺-dependent manner.

It was next tested whether the five acidic amino acid residues in the ST03 C2 domain were essential for Ca²⁺/phospholipid binding ability. Amino acid substitutions were engineered at the 5 calcium binding sites (CBS) Asp-20, Glu-26, Asp-77, -120, -126 to give D20N, E26Q, D77N, D120N and D126N and the resultant mutant protein (ST03mCBS) was then fused to the GST C-terminus. Phospholipid binding assays performed with this engineered protein revealed that its Ca²⁺-dependent phospholipid binding ability was abolished (Fig. 10, panel ii) , demonstrating that the five Ca²⁺-binding sites of ST03 are required for this function.

Ca²⁺-binding ability of ST03 is required to confer salt tolerance

Proteins containing C2 domains can interact with phospholipids in a Ca²⁺-dependent or -independent manner to modulate a diverse range of signaling events. High sality stress promotes a transient intracellular increase in Ca²⁺ concentration and, thereby, activates the salt stress-response pathway. To determine whether the ST03 Ca²⁺-binding ability is necessary to impart salt tolerance, 6-day-old ST03 transgenic and control (expressing a pGreen-GFP vector) seedlings were transferred to hydroponic medium to which 120 mM NaCl along with either 0, 1, 5, or 10 mM calcium nitrate was added. As controls for these experiments, seedlings were transferred to hydroponic medium, ±1 mM Ca²⁺. Three days post-transfer, seedlings were photographed and fresh weights were recorded (Fig. 11) . In the absence of NaCl, ST03 transgenic plants did not show any significant differences in growth compared with the control, irrespective of whether 1 mM Ca²⁺ was or was not added to the medium (Fig. 11) . However, in the presence of 120 mM NaCl, the two ST03 transgenic lines exhibited higher growth characteristics relative to control plants, as long as there was Ca²⁺ in the medium. The inhibitory effect of zero Ca²⁺ on growth is well established.

To ascertain whether ST03 (mCBS) was still able to impart salt tolerance, Arabidopsis transgenic P_35S: 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 P_35S: ST03 (mCBS) plants were sensitive to NaCl during germination and post-germination growth periods (Figs. 12 and 13) . Parallel studies conducted with one-tenth MS medium yielded equivalent results, with the exception that growth inhibition of both P_35S: ST03 (mCBS) and wild-type seedlings was more pronounced in the 100 mM NaCl treatment relative to the P_35S: ST03 seedlings (Fig. 14) . Taken together, these studies established that the Ca²⁺ binding ability of ST03 is essential for this protein to confer salt tolerance in Arabidopsis.

ST03 is involved in maintenance of plasma membrane integrity

High salinity causes both hyperionic and hyperosmotic stress, and the latter often leads to altered membrane fluidity and changes in phospholipids. The Arabidopsis SYT1 gene encodes a C2 domain protein whose mutant showed hypersensitvity when grown under elevated NaCl concentrations. Loss of function of SYT1 caused a reduction in plasma membrane integrity resulting in a decrease in cell viability. As overexpression of ST03 results in enhanced salt tolerance, it was hypothesized that ST03 may be involved in maintenance of the plasma membrane. To test this notion, electrolyte leakage was measured as a reporter for the extent to which the plasma menbrane undergoes damage caused by high NaCl in the presence of high or low Ca²⁺ treatments (Fig. 15) . When growing in one-tenth MS medium and high Ca²⁺ (without NaCl) , a lower level of electrolyte loss was detected in P_35S: ST03 seedlings compared with P_35S: ST03 (mCBS) and the wild-type control. These findings indicated that the presence of ST03 contributes to the maintenance of plasma membrane integrity in non-salt stress conditions. Equivalent results were obtained under 150 mM NaCl treatment, with low or high Ca²⁺ concentrations in the medium. These results suggest that ST03 gives a general increase in plasma membrane integrity, and that this capacity is mediated through its Ca²⁺ binding property (Fig. 15) .

To further study plasma membrane integrity in vivo at the cellular level, 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. For these experiments, 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_35S: ST03 and P_35S: ST03 (mCBS) roots was investigated. Under control conditions (without NaCl) , no membrance damage was observed in these three root systems. In the presence of 200 mM NaCl, obvious damage to the plasma membrane in the three roots was observed and, in comparison with the wild-type and P_35S: ST03 (mCBS) , more cells were alive in P_35S: ST03, indicating maintenance of plasma membrane integrity by P_35S: ST03. Taken together, these results established that ST03 confers high salt tolerance to Arabidopsis transgenic plants by contribting to the integrity and stability of plasma membrane.

To futher understand the function of ST03 under salt stress, the subcellular localization of GFP-ST03 and GFP-ST03 (mCBS) was checked. For these studies, 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. Following a 36 h 150 mM NaCl treatment, 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. To test between these possibilities, 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.

Overexpression of ST03 enhances salt and drought tolerance in a crop plant

To determine whether the ST03 gene can confer salt and drought tolerance in a crop plant, transgenic Oryza sativa (rice) plants were generated. Here, 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.

Two representative transgenic lines along with a wild-type control 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) . Next, 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.

To test for the effect of ST03 overexpression on drought tolerance in 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 ST03 transgenic lines recovered.

Application of drought stress at the heading stage (70-day-old plants) , by withholding water for 5 days, caused control plants to undergo severe leaf rolling and accelerated senescence, whereas the ST03 transgenic plants displayed only mild symptoms. 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.

These studies established that salinity-tolerance gene, ST03 (T. salsuginea (previously (previously referred to as T. halophila) . Salt Tolerance 03) , imparts improved salt tolerance when overexpressed in Arabidopsis. In addition, the salt-tolerance of ST03 is demonstrated to be Ca²⁺-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.

Salt tolerance analysis in Arabidopsis.

For salt tolerance assays on plates, Col-0, 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℃. At the indicated times of vertical growth, seedlings were photographed and statistically analyzed.

For salt tolerance assays in soil, Col-0, vector transgenic line, 35S-

ST3

20 and 35S-ST3 16 were grown vertically on MS medium with 0.6％agar under continuous light for 7 days at 23℃. Seedlings were transferred in soil under long-day conditions (16h light/8h dark) for 3 weeks. Subsequently, the soil was irrigated with 0, 50 mM, 100 mM, 150 mM and 200 mM NaCl concentration gradient every 3 days, and plants were grown for an additional 1 week and photographed and statistically analyzed.

For salt tolerance assays by hydroponics, sterilized seeds were sown on 0.8％ (w/v) agar in 1.5-mL centrifuge tubes and vernalized for 48 h at 4℃. The bottom of the tubes was cut off and the tubes were suspended over an aerated growth solution consisting of 1.25mM KNO₃, 0.625 mM KH₂PO₄, 0.5 mM MgSO4, 0.5 mM Ca (NO₃) ₂ and 0.045 mM FeNa-EDTA with the following micronutrients: 0.16 mM CuSO₄, 0.38 mM ZnSO₄, 1.8 mM MnSO₄, 45 mM H₃BO₃, 0.015 mM (NH₄) 6Mo₇O₂₄, and 0.01 mM CoCl₂. 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℃ with an irradiance of 75 μmol m^-2 s^-1. For high NaCl treatments, solid NaCl was added to the growth solution to make a final concentration of 120 mM. Ca (NO₃) ₂ was added to make a final concentration of 1, 5, 10 mM Ca²⁺ in the different growth solutions.

For salt tolerance assays in Vertical Petri Plates, seeds were surface sterilized following standard protocols (Weigel and Glazebrook, 2002) and placed on square Petri dishes with 0.1×Murashige and Skoog medium (M5519) , pH 5.7, with 1％ (w/v) sucrose and 0.3％ (w/v) Phytagel. The seeds were vernalized at 4℃ for 2 d in the dark before the dishes were positioned vertically in a growth chamber under a 16/8-h light/dark cycle at 21℃ with an irradiance of 120 μmol m^-2 s^-1. For high NaCl treatments, solid NaCl was added to the growth solution to make a final concentration of 100 mM. Ca (NO₃) ₂ was added to make a final concentration of 3 mM Ca²⁺ in the growth solutions.

Salt tolerance analysis in rice.

For salt tolerance assays in medium, seeds of Nipponbare and transgenic lines surface sterilized with 70％ethanol for 1 min and 30％bleach for 30mins, and then washed five times with sterile water. The sterilized seeds were germinated and grown on MS medium without or with 200 mM NaCl and 0.6％agar under continuous light for 21 days at 28℃. At the 21^st day, seedlings were photographed and statistically analyzed.

For salt tolerance assays by hydroponics, seeds of Nipponbare and transgenic lines were germinated at 37℃. 3-day-old seedlings were transferred in Hoaglands hydroponic medium without or with 100 mM NaCl. After 7 days, seedlings were photographed and statistically analyzed.

Analyses of drought tolerance.

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.

Cloning.

To create 35S-ST03 construct, 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.

To create 35S-ST03 (mCBS) construct, 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'-GCAAAGGTCTCAAACGCTCTAATTTTTTTGGTAAGATACAACC-3' (SEQ ID NO: 26) , 5'-GGTTGTATCTTACCAAAAAAATTAGAGCGTTTGAGACCTTTGC-3' (SEQ ID NO: 27) , 2: 5'-CTCTGATTTTTTTGGTAAGATACAACCTTATGCTGAGATCCAATAC-3' (SEQ ID NO: 28) , 5'-GTATTGGATCTCAGCAT AAGGTTGTATCTTACCAAAAAAATCAGAG-3' (SEQ ID NO: 29) , 3: 5'-GCTTATCGTCAAAGTCATGAATCATAATACTTTCTCCGCCGACG-3' (SEQ ID NO: 30) , 5'-CGTCGGCGGAGAAAGTATTATGATTCATGACTTTGACGATAAGC-3' (SEQ ID NO: 31) , 4: 5'-CATAATACTTTCTCCGCCAACAATTTCATCGGCGAAGCTAC-3' (SEQ ID NO: 32) , 5'-GTAGCTTCGCCGATGAAATTGTTGGCGGAGAAAGTATTATG-3') (SEQ ID NO: 33) . The ST03 (mCBS) CDS fragment was cloned into the vector pVIP-myc (Xie et al., 2002) in which transgenic expression is under the control of the CaMV 35S promoter.

To create 35S-ST03-GFP and ST03 (mCBS) -GFP constructs, two 468-bp fragments of ST03 and ST03 (mCBS) full-length ORF were amplified by PCR using 5’ -GCTCTAGAATGGCTGTGGGAATCCTC-3’ (SEQ ID NO: 34) and 5’ -GCGGTACCATCAAATTGGCTATGCTTCC-3’ (SEQ ID NO: 35) primers. The two fragments were cloned into the vector pVIP96 in which the insert fragments were operably linked to the CaMV 35S promoter.

To create P_ST03: ST03-GFP and P_ST03: ST03 (mCBS) -GFP constructs, an 1809-bp fragment from -1 to -1089 bp upstream of the translational start site of ST03 was obtained by Tail-PCR using three ST03 genomic specific primer 5’ -GCGGGTTTGTCCTTTGTATTGGATCTCAGCA-3’ (SEQ ID NO: 36) , 5’ -CCTCGAGGATTCCCACAGCCATCTCTGGA-3’ (SEQ ID NO: 37) , 5’ -GATGAAGTAGG TTGTGTCAGG-3’ (SEQ ID NO: 38) and the random primer 5’ -Ngtcga (G/C) (A/T) Gana (A/T) Gaa-3’ (SEQ ID NO: 43) . The PCR products were cloned and inserted into vector pCambia1300-221-GFP in the front of the ST03-GFP fusion gene. To create ST03-GFP and ST03 (mCBS) -GFP fusion constructs, two 468-bp fragments of ST03 and ST03 (mCBS) full-length ORF were amplified by PCR using 5’ -GCTCTAGAATGGCTGTGGGAATCCTC-3’ (SEQ ID NO: 39) and 5’ -GCGGTACCATCAAATTGGCTATGCTTCC-3’ (SEQ ID NO: 40) primers. The two fragments were cloned into the vector pCambia1300-221-GFP containing the ST03 promoter, respectively.

To create GST-ST03 and GST-ST03 (mCBS) constructs, two 468-bp fragments of ST03 and ST03 (mCBS) CDS were amplified by PCR using 5'-GGAATTCCGATGGCTGTGGGAATCCTCGAGG-3' (SEQ ID NO: 41) and 5'-CGCGGCCGCCTAATCAAATTGGCTATGCTT CCATC -3' (SEQ ID NO: 42) primers. The two fragments were cloned into the vector pGEX4T-2 fused with GST.

To create P_Ubi-Myc-ST03 construct, the ST03 CDS was cloned into the pCAMBIA based vector under the control of the maize Ubi1 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. For transformation of rice, the plasmids were introduced into Agrobacterium tumefaciens AGL1 and embryogenic calli from mature rice Oryza sativa L. ssp. Nipponbare seeds. For the phenotypic analysis, T3 or T4 homozygous lines were used.

Phospholipid binding assays.

Phospholipid binding to isolated C2 domains of ST03 was assessed by a centrifugation assay as described previously. Liposomes consisting of phosphatidylserine and phosphatidylethanolamine (1: 3, w/w) were prepared in 50 mM HEPES-NaOH, pH 7.4, and 100 mM NaCl by sonication, collected by centrifugation, and equilibrated with 50 mM HEPES-NaOH, pH 7.4, with or without 1mM Ca²⁺. GST fusion proteins (2-5 μg) were incubated with liposomes corresponding to 160 μg of phospholipid for 15 min at room temperature (Kang et al., 2011. Biochimica Biophysica Acta. 1810: 1317-22. ) ) . After centrifugation at 12, 000 × g for 10 min at room temperature, the phospholipid pellets were washed in 500 μl of the above equilibration buffer and then extracted with 300 μl of acetone at -20 ℃ for 30 min to remove excess lipid. The pellets obtained by centrifugation at 12, 000 × g for 15 min at 4 ℃ 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 × g for 15 min at 4℃, 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. Five-day-old seedlings were transferred to half MS supplemented with NaCl at the described concentrations. After the corresponding treatment, seedlings were incubated with 50 μM FM 4-64 for 3 min and mounted in half MS liquid medium using a spacer of one layer of cover slip between slide and cover slip for confocal microscopy.

The confocal microscopy was performed on a Leica SP5 confocal microscope, equipped with argon and krypton lasers. For excitation of GFP, the 488-nm line of the argon laser was used and emission was detected with the setting of the acousto optical beam splitter set to 505 to 530 nm. 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) .

Electrolyte leakage measurements.

Five-day-old seedlings grown in half-strength MS plates were transferred to one-tenth MS agar plates supplemented with the designated NaCl concentrations and to the same plates plus 3 mM CaCl₂. There were three replicates for each treatment. After the indicated times, seedlings were carefully removed from plates, washed with deionized water, and placed in tubes containing 5 mL of deionized water. The tubes were shaken overnight, and the conductivity of the solution was measured. The tubes with the seedlings were then autoclaved. After cooling down to room temperature, conductivities of the solutions were measured again. The percentage of electrolyte leakage was calculated as the percentage of the conductivity before autoclaving over that after autoclaving.

Tobacco infiltration assays.

Tobacco (Nicotiana benthamiana) 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.

Protein extraction and gel blot analysis.

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.

Example 10–Orthologs of ThST03

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.

10.1 Identification of orthologs

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_004236744.1； XP_004236744.1； XP_006365698.1； MLOC_39313.2； EMT23936.1；

XP_010232545.1； NP_001148526.1； XP_002456619.1； XP_004970539.1； AAC04628.1；

XP_009420100.1； NP_001078296.1； XP_006403454.1； XP_009116242.1； XP_009139161.1；

supercontig 1.414 CARPA； XP_008458869.1； KGN51319.1； XP_010690675.1；

XP_002533151.1； cassava4.1 018256m； |XP_004307500.1； XP_007218541.1；

XP_002316283.1； C. cajan_31251； NP_001235151.1； XP_007141871.1； XP_004490896.1；

XP_003616407.1； AFK38368.1； XP_002283485.1； XP_010030954.1； XP_006435544.1；

XP_006486477.1； KJB73947.1； Tc10_g003090； and EYU33258.1.

Orthologs were aligned and a phylogenic tree was created (Fig. 1) . Based on this tree, alignments with orthologs from other plant species were created (Figs. 17–22) and consensus sequences were generated from these alignments (SEQ ID NOS: 8 to 13) . From this, orthologs were selected for testing their ability to confer enhanced abiotic stress to at least two stresses in a transgenic plant (Table 8) . These orthologs are from the plant species Arabidopsis thaliana (Arabidopsis； SEQ ID NO: 5) , Zea mays (maize； SEQ ID NO: 19) , Glycine max (soybean； SEQ ID NO: 20) , Solanum lycopersicum (tomato； SEQ ID NO: 21) , Cucumis melo (melon； SEQ ID NO: 22) , and Citrus clementina (clementine； SEQ ID NO: 23) .

Table 8: ST03 Orthologs for Arabidopsis Transformation

10.2 Vector construction

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 Agrobacterium (Bevan et al. 1983, Nucleic Acids Res 11: 369-385) . 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.

10.3 Arabidopsis transformation

The expression vectors described above are introduced into Agrobacterium cells following methods well-known to one skilled in the art. Arabidopsis transformation is performed using the floral dip method as described in Clough and Bent, 1998 (Plant J. 16: 735-743) . Transformed plants are selected by germination of seed harvested from the dipped plants on selection media, using methods well-known to one skilled in the art.

Example 11–Cold stress tolerance experiments with ST03 orthologs in Chlamydomonas reinhardtii

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.

Example 12–Abiotic stress tolerance experiments with transgenic Arabidopsis expressing ST03 orthologs

12.1 Salt tolerance assay with transgenic Arabidopsis

A. thaliana seeds were sown in MS plates (+ 0, 50, 75, 100, 125, 150 and 200 mM NaCl) and placed in the dark at 4℃ for 3 days. The MS plates were then transferred to 22℃ (16h : 8h/day : night, 50％humidity) . Twenty hours after transferring the seeds to normal conditions with 125 mM NaCl treatment the seeds having germinated were counted. A seed is considered to have germinated if it has at least 1mm of root showing (extruding from the seed) . Three replicates were done for each NaCl concentration and the Paired T-test was used to compare performance differences between the transformed and the WT seeds.

Constructs were considered to be functional for conferring salt resistance when more than two events with significantly higher germination rates 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 9, Table 10) .

Table 9. Results of the salt germination assay

Table 10. Summary of the results of the salt germination assay

Ortholog	Distance to ThST03	Construct ID	Performance
AtST03	0.089	23286	No difference
CmST03	0.529	23290	resistance
GmST03	0.583	23284	resistance
CcST03	0.583	23288	No difference
SlST03	0.597	23289	No difference
ZmST03	0.765	23287	resistance
ThST03		23283	resistance

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.

12.2 Cold tolerance assay with transgenic Arabidopsis

A. thaliana seeds were sown in MS-0 plates, three repeats in each treatment. The plates were placed in the dark at 4℃ for 3 days and then transferred to 22℃ (normal condition) or 12℃ (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) .

Table 11. Results of cold tolerance root growth assay

Table 12. Summary of cold tolerance root growth assay

Ortholog	Distance to ThST03	Construct ID	Performance
AtST03	0.089	23286	No difference
CmST03	0.529	23290	resistance
GmST03	0.583	23284	resistance
CcST03	0.583	23288	resistance
SlST03	0.597	23289	No difference
ZmST03	0.765	23287	resistance
ThST03		23283	No difference

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℃, 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) .

Table 13. Water loss rate ％for constructs 23283, 23284, and 23286

Table 14. Water loss rate ％for constructs 23288 and 23290

Table 15. Water loss rate ％for constructs 23287 and 23289

Table 16. Summary of the results of the leaf water loss assay

Ortholog	Distance to ThST03	Construct ID	Performance
AtST03	0.089	23286	No difference
CmST03	0.529	23290	reduced loss
GmST03	0.583	23284	No difference
CcST03	0.583	23288	No difference
SlST03	0.597	23289	reduced loss
ZmST03	0.765	23287	reduced loss
ThST03		23283	No difference

The CmST03, SlST03, 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. Summary of the results from the testing of the ST03 orthologues

Ortholog	Distance to ThST03	Construct ID	Salt(a)	Cold(b)	Drought(c)
AtST03	0.089	23286	No difference	No difference	No difference
CmST03	0.529	23290	resistance	resistance	reduced loss
GmST03	0.583	23284	resistance	resistance	No difference
CcST03	0.583	23288	No difference	resistance	No difference
SlST03	0.597	23289	No difference	No difference	reduced loss
ZmST03	0.765	23287	resistance	resistance	reduced loss
ThST03		23283	resistance	No difference	No difference

(a) germination assay with salt treatment； (b) root growth assay under cold treatment； (c) leaf water loss assay √: better performance in more than 2 transgenic events/construct @P<0.1； ×: worse performance in more than 2 transgenic events/construct @P<0.1； No difference: no significant difference

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.

The foregoing is illustrative of the present invention and is not to be construed as limiting thereof. Although a few exemplary embodiments of this invention have been described, those skilled in the art will readily appreciate that many modifications are possible in the exemplary embodiments without materially departing from the novel teachings and advantages of this invention. Accordingly, all such modifications are intended to be included within the scope of the present invention.

Claims

A method of producing a plant having enhanced abiotic stress tolerance to at least two abiotic stresses as compared to a control plant, comprising introducing into a plant part an exogenous nucleic acid comprising:

(a) a 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 to the nucleotide sequence of any one of SEQ ID NOs: 1, 2, 3, 6, 7, or 14 to 18；

(c) a nucleotide sequence that encodes a polypeptide comprising a C2 domain that binds 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；

(d) a nucleotide sequence that encodes a polypeptide comprising a C2 domain that binds 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； and/or

(e) a nucleotide sequence that is complementary to the nucleotide sequence of any one of (a) to (d) above； and

growing the plant part into a plant that expresses the exogenous nucleic acid and that has 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 method of claim 1, wherein the exogenous nucleic acid encodes a polypeptide comprising an amino acid sequence that is at least 70％ identical to SEQ ID NO: 4.
The method of claim 2, wherein the exogenous nucleic acid encodes a polypeptide comprising an amino acid sequence that is SEQ ID NO: 4.
A method of enhancing abiotic stress tolerance to at least two abiotic stresses in a plant, as compared to a control plant or plant part, comprising:

expressing in the plant an exogenous nucleic acid comprising:

(a) a 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 to the nucleotide sequence of any one of SEQ ID NOs: 1, 2, 3, 6, 7, or 14 to 18；

(c) a nucleotide sequence that encodes a polypeptide comprising a C2 domain that binds 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；

(d) a nucleotide sequence that encodes a polypeptide comprising a C2 domain that binds 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； and/or

(e) a nucleotide sequence that is complementary to the nucleotide sequence of any one of (a) to (d) above；

wherein expression of the exogenous nucleic acid results in enhanced abiotic stress tolerance to at least two abiotic stresses in a plant, as compared to a control plant or plant part.
The method of claim 4, further comprising introducing the exogenous nucleic acid into the plant.
The method of claim 4, further comprising introducing the exogenous nucleic acid into a plant part and producing the plant from the plant part.
A method of producing a plant having enhanced abiotic stress tolerance to at least two abiotic stresses as compared to a control plant or plant part, comprising:

detecting, in a plant part, an exogenous nucleic acid comprising:

(a) a 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 to the nucleotide sequence of any one of SEQ ID NOs: 1, 2, 3, 6, 7, or 14 to 18；

(c) a nucleotide sequence that encodes a polypeptide comprising a C2 domain that binds 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

(d) a nucleotide sequence that encodes a polypeptide comprising a C2 domain that binds 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； and/or

(e) a nucleotide sequence that is complementary to the nucleotide sequence of any one of (a) to (d) above； and

producing a plant from the plant part, thereby producing a plant having enhanced abiotic stress tolerance to at least two abiotic stresses as compared to a control plant or plant part.
A method of identifying a plant or plant part having enhanced abiotic stress tolerance to at least two abiotic stresses as compared to a control plant or plant part, comprising:

detecting, in the plant or plant part, an exogenous nucleic acid comprising:

(a) a 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 to the nucleotide sequence of any one of SEQ ID NOs: 1, 2, 3, 6, 7, or 14 to 18；

(c) a nucleotide sequence that encodes a polypeptide comprising a C2 domain that binds 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

(d) a nucleotide sequence that encodes a polypeptide comprising a C2 domain that binds 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； and/or

(e) a nucleotide sequence that is complementary to the nucleotide sequence of any one of (a) to (d) above；

thereby identifying a plant or plant part having enhanced abiotic stress tolerance to at least two abiotic stresses.
The method of claim 8, wherein 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.
A method of producing a plant having enhanced abiotic stress tolerance to at least two abiotic stresses as compared to a control plant or plant part, comprising:

crossing a first parent plant with a second parent plant, wherein the first parent plant comprises within its genome an exogenous nucleic acid that comprises:

(a) a 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 to the nucleotide sequence of any one of SEQ ID NOs: 1, 2, 3, 6, 7, or 14 to 18；

(c) 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

(d) 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, 22, or 23； and/or

(e) a nucleotide sequence that is complementary to the nucleotide sequence of any one of (a) to (d) above；

thereby producing a progeny generation,

wherein the progeny generation comprises at least one plant that possesses the exogenous nucleic acid within its genome and that exhibits enhanced abiotic stress tolerance to at least two abiotic stresses as compared to a control plant or plant part.
The method of any one of claims 1-3, 7, or 10, further comprising selecting a plant having enhanced salt stress tolerance and enhanced cold stress tolerance relative to the control plant.
The method of any one of claims 1-3, 7, or 10, further comprising selecting a plant having elevated expression and/or activity of a protein encoded by the exogenous nucleic acid relative to the control plant.
The method of any one of claims 1-12, wherein the exogenous nucleic acid further comprises a promoter sequence that comprises 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/or a developmental stage-specific promoter sequence.
The method of any one of claims 1-13, wherein the plant or plant part having enhanced abiotic stress tolerance to at least two abiotic stresses 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 method of any one of claims 1-14, wherein the plant or plant part having enhanced abiotic stress tolerance to at least two abiotic stresses has increased yield and/or increased seed germination under at least two abiotic stress conditions compared to a control plant or plant part grown under the same abiotic stress conditions.
The method of any one of claims 1 to 15, wherein at least one of the at least two abiotic stresses is selected from drought stress, cold stress, heat stress, osmotic stress, light stress, flooding stress, and edaphic stress.
The method of any one of claims 1-16, wherein the plant or plant part is a monocotyledonous plant.
The method of claim 17, wherein the plant is selected from the group consisting of maize, rice, wheat, and sugarcane.
The method of any one of claims 1-15, wherein the plant or plant part is a dicotyledonous plant.
The method of claim 19, wherein the plant or plant part is selected from the group consisting of soybean, cotton, and tomato.
An expression cassette or vector comprising a promoter operably linked to an exogenous nucleotide sequence that comprises:

(a) a 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 to the nucleotide sequence of any one of SEQ ID NOs: 1, 2, 3, 6, 7, or 14 to 18；

(c) 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

(d) 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, 22, or 23； and/or

(e) a nucleotide sequence that is complementary to the nucleotide sequence of any one of (a) to (d) above.
A transgenic plant comprising an exogenous nucleic acid sequence that confers enhanced abiotic stress tolerance to at least two abiotic stresses, wherein said exogenous nucleic acid sequence comprises:

(a) a 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 to the nucleotide sequence of any one of SEQ ID NOs: 1, 2, 3, 6, 7, or 14 to 18；

(c) 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

(d) 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, 22, or 23； and/or

(e) a nucleotide sequence that is complementary to the nucleotide sequence of any one of (a) to (d) above.
The transgenic plant of claim 22, wherein said exogenous nucleic acid sequence comprises SEQ ID NO: 1, 6, or 7.
The transgenic plant of claim 22 or 23, wherein said plant is a monocotyledonous plant.
The transgenic plant of claim 22 or 23, wherein said plant is a dicotyledonous plant.
The transgenic plant of claim 22 or 23, wherein said 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.
A transgenic plant comprising the expression cassette of claim 21, wherein the plant has enhanced abiotic stress tolerance to at least two abiotic stresses compared to a plant lacking the expression cassette when grown in similar conditions.
A transgenic plant of any of claims 22 to 27, wherein the exogenous nucleic acid comprises a promoter sequence that comprises 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/or a developmental stage-specific promoter sequence.
The transgenic plant of any of claims 22 to 28, wherein the exogenous nucleic acid comprises nucleotide sequences which encode for at least one additional desired trait, wherein the at least one additional desired trait is selected from 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/or enhanced abiotic stress tolerance.