AU2013204799B2

AU2013204799B2 - Stress responsive expression

Info

Publication number: AU2013204799B2
Application number: AU2013204799A
Authority: AU
Inventors: John Harris; Nataliya Kovalchuk; Peter Langridge; Sergiy Lopato
Original assignee: Australian Centre for Plant Functional Genomics Pty Ltd
Current assignee: Australian Centre for Plant Functional Genomics Pty Ltd
Priority date: 2012-11-05
Filing date: 2013-04-12
Publication date: 2016-02-25
Anticipated expiration: 2033-04-12
Also published as: AU2013204799A1

Abstract

The present invention relates generally to the identification of transcriptional control sequences that are active in plants in response to stress. Accordingly, methods for effecting stress responsive expression of a nucleotide sequence of interest in a plant are provided, the methods including expressing the nucleotide sequence of interest operably connected to the transcriptional control sequence, wherein the nucleotide sequence of interest is heterologous with respect to the transcriptional control sequence. Also provided are nucleic acid constructs including the stress inducible transcriptional control sequences, genetically modified cells including the nucleic acid constructs, and multicellular structures including one or more of the genetically modified cells.

Description

- 1 STRESS RESPONSIVE EXPRESSION This application claims priority from Australian provisional patent application number 2012904869 filed on 5 November 2012, the contents of which is to be taken as incorporated herein by this reference. FIELD OF THE INVENTION [0001] The present invention relates generally to methods and transcriptional control sequences suitable for effecting expression of a nucleotide sequence of interest in a plant. More particularly, the present invention relates to methods and transcriptional control sequences suitable for the stress responsive expression of a nucleotide sequence of interest in one or more cells of a plant. BACKGROUND OF THE INVENTION [0002] Stresses such as temperature, drought, salinity, disease and pathogen attack are common factors that negatively impact on plant growth and development. To overcome these limitations, plants respond and adapt to various stresses at the physiological and biochemical levels. [0003] For example, several families of transcription factors, such as DREB/CBF, ERF, MYK, MYB, AREB/ABF, and NAC, have been shown to be involved in the regulation of stress response in plants. The dehydration-responsive element-binding proteins (DREBs) or C-repeat-binding proteins (CBFs), for instance, are among the first discovered families of transcription factors responsible for gene regulation under conditions of low temperature and water deficiency. [0004] The genetic manipulation of plants has been used to effect transcription of an introduced nucleotide sequence of interest either specifically or preferentially in a plant, plant part, or at a particular developmental stage of the plant. Accordingly, there is substantial interest in identifying transcriptional control sequences, such as promoters or enhancers, which specifically or preferentially direct transcription in plants, particular plant organs, tissues or cell types, or at particular developmental stages of the plant. [0005] Expression of a heterologous nucleotide sequence in a plant is dependent upon the presence of an operably linked transcriptional control sequence which is functional -2 within the plant. The choice of transcriptional control sequence will determine when and where within the organism the heterologous nucleotide sequence is expressed. For example, where continuous expression is desired throughout the cells of a plant, constitutive promoters are utilised. In contrast, where gene expression in response to a stimulus (such as stress) is desired, an inducible promoter may be used. [0006] Since discovery of the role of DREB/CBF factors in the stress response, several attempts have been undertaken to genetically manipulate plants in order to demonstrate the potential of these factors to improve stress tolerance in Arabidopsis, and crop plants such as Brassica junceae, soybean, rice, wheat and other grasses. In the majority of attempts to overexpress DREB/CBF factors in plants, constitutive promoters such as the Cauliflower mosaic virus 35S promoter, rice actin 1 promoter, maize polyubiquitin promoter, and inducible promoters such as AtRd29A, HvDhn8 and maize Rab17 promoters have been used. However, in most cases strong or even moderate constitutive expression (alternatively, high basal level of the inducible promoter activity) led to different degrees of growth retardation (which subsequently led to dwarfism of the transgenic plants), delayed flowering time and smaller spikes. [0007] In light of the above, it would be desirable to be able to increase the expression of nucleotide sequences, including those involved in stress tolerance (such as DREB/CBF factors), in a stress responsive manner. Therefore, isolation and characterization of stress induced transcriptional control sequences, which can serve as regulatory regions for expression of heterologous nucleotide sequences of interest in a plant, would be desirable for use in the genetic manipulation of plants. [0008] Reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in any country. SUMMARY OF THE INVENTION [0009] The present invention is predicated, in part, on the isolation and characterisation of transcriptional control sequences derived from plant genes. Among other things, the present invention has identified that the transcriptional control sequences can effect stress responsive expression of an operably connected heterologous nucleotide sequence in a plant.

-3 [0010] Accordingly, in a first aspect, the present invention provides a method for effecting stress responsive expression of a nucleotide sequence of interest in one or more cells of a plant, the method including expressing in the one or more cells of the plant the nucleotide sequence of interest operably connected to a transcriptional control sequence which is stress inducible in the plant, wherein the nucleotide sequence of interest is heterologous with respect to the transcriptional control sequence, and wherein the transcriptional control sequences includes: (i) the nucleotide sequence set forth in one of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3; or (ii) a functionally active fragment or variant of the nucleotide sequence set forth in one of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, wherein the functionally active fragment or variant is at least 50% identical to the nucleotide sequence set forth in one of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. [0011] In one embodiment, the plant is a monocot plant. In some embodiments, the plant is a cereal crop plant, such as a wheat, rice or barley plant. [0012] In some embodiments, the functionally active fragment or variant includes one or more sequence motifs corresponding to one or more cis-elements selected from the group consisting of MYBR, ABRE, CRT and ABRE (AB15-binding). [0013] Intentionally left blank. [0014] In embodiments where the transcriptional control sequence includes a functionally active fragment or variant of the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2, the fragment or variant includes one or more sequence motifs corresponding to one or more cis-elements selected from the group consisting of a CGCG-box (calmod-binding), ABRE (AB5-binding), auxin-response (AUX-resp), MYBR, ABRE, CRT, and ammonium response (ammonium-resp). [0015] In one embodiment, the functionally active fragment or variant is at least 70% identical to the nucleotide sequence set forth in one of SEQ ID NO: 1 or SEQ ID NO: 2 In some embodiments, the functionally active fragment or variant includes a nucleotide sequence which is at least 94% identical to the nucleotide sequence set forth in SEQ ID NO: 4. In some embodiments, the functionally active fragment or variant includes the -4 nucleotide sequence set forth in SEQ ID NO: 4. [0016] In some embodiments, the nucleotide sequence of interest includes a nucleotide sequence which, when expressed by one or more cells of a plant, improves the stress tolerance of the plant. [0017] In embodiments where the transcriptional control sequence includes the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2, or a functionally active fragment or variant of the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2, the stress is selected from one or more of cold, drought, and salinity, or wherein the stress is induced by ABA. Accordingly, in some embodiments, the nucleotide sequence of interest includes a nucleotide sequence which, when expressed by one or more cells of a plant, improves the cold, drought, and/or salinity tolerance of the plant. [0018] In embodiments where the transcriptional control sequence includes a functionally active fragment or variant of the nucleotide sequence set forth in SEQ ID NO: 3, the fragment or variant includes one or more sequence motifs corresponding to one or more cis-elements selected from the group consisting of ABRE (AB15-binding), ABRE, GCC box, light regulated cis-element, MYBR, ABRE-similar, ABRE-related (Ca dependent), vascular expression cis-element, WRKY specific element, CRT, and carbohydrate metabolite-responsive cis-element. In one embodiment, the nucleotide sequence of interest includes a nucleotide sequence which, when expressed by one or more cells of a plant, improves the stress tolerance of the plant. In one embodiment, expression of the nucleotide sequence of interest does not disturb development of the plant. [0019] In embodiments where the transcriptional control sequence includes the nucleotide sequence set forth in SEQ ID NO: 3, or a functionally active fragment or variant of the nucleotide sequence set forth in SEQ ID NO: 3, the stress is drought. Accordingly, in some embodiments, the nucleotide sequence of interest includes a nucleotide sequence which, when expressed by one or more cells of a plant, improves the drought tolerance of the plant. In one embodiment, expression of the nucleotide sequence of interest does not disturb development of the plant. [0020] In some embodiments of the first aspect of the invention, the nucleotide sequence of interest includes a nucleotide sequence that encodes a DREB polypeptide. In one embodiment, the DREB polypeptide is a TaDREB3-like polypeptide. [0021] In a second aspect, the present invention provides a method for improving the stress tolerance of a plant, the method including expressing a nucleotide sequence of interest in one or more cells of the plant according to the method of the first aspect of the invention. [0022] In a third aspect, the present invention provides a nucleic acid construct including a nucleotide sequence of interest operably connected to transcriptional control sequence which is stress inducible in a plant, wherein the nucleotide sequence of interest is heterologous with respect to the transcriptional control sequence, and wherein the transcriptional control sequences includes: (i) the nucleotide sequence set forth in one of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3; or (ii) a functionally active fragment or variant of the nucleotide sequence set forth in one of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, wherein the functionally active fragment or variant is at least 50% identical to the nucleotide sequence set forth in one of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. [0023] In one embodiment, the transcriptional control sequence is stress inducible in a monocot plant. In some embodiments, the transcriptional control sequence is stress inducible in a cereal crop plant, such as a wheat, rice or barley plant. [0024] In some embodiments, the functionally active fragment or variant includes one or more sequence motifs corresponding to one or more cis-elements selected from the group consisting of MYBR, ABRE, CRT and ABRE (AB15-binding). [0025] Intentionally left blank. [0026] In embodiments where the transcriptional control sequence includes a functionally active fragment or variant of the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2, the fragment or variant includes one or more sequence motifs corresponding to one or more cis-elements selected from the group consisting of a CGCG-box (calmod-binding), ABRE (ABI5-binding), auxin-response (AUX-resp), MYBR, ABRE, CRTF, and ammonium response (ammonium-resp).

-6 [0027] In one embodiment, the functionally active fragment or variant is at least 70% identical to the nucleotide sequence set forth in one of SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the functionally active fragment or variant includes a nucleotide sequence which is at least 94% identical to the nucleotide sequence set forth in SEQ ID NO: 4. In some embodiments, the functionally active fragment or variant includes the nucleotide sequence set forth in SEQ ID NO: 4. [0028] In some embodiments, the nucleotide sequence of interest includes a nucleotide sequence which, when expressed by one or more cells of a plant, improves the stress tolerance of the plant. [0029] In embodiments where the transcriptional control sequence includes the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2, or a functionally active fragment or variant of the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2, the stress is selected from one or more of cold, drought, and salinity, or wherein the stress is induced by ABA. Accordingly, in some embodiments, the nucleotide sequence of interest includes a nucleotide sequence which, when expressed by one or more cells of a plant, improves the cold, drought, and/or salinity tolerance of the plant. [0030] In embodiments where the transcriptional control sequence includes a functionally active fragment or variant of the nucleotide sequence set forth in SEQ ID NO: 3, the fragment or variant includes one or more sequence motifs corresponding to one or more cis-elements selected from the group consisting of ABRE (AB15-binding), ABRE, GCC box, light regulated cis-element, MYBR, ABRE-similar, ABRE-related (Ca dependent), vascular expression cis-element, WRKY specific element, CRT, and carbohydrate metabolite-responsive cis-element. In one embodiment, the nucleotide sequence of interest includes a nucleotide sequence which, when expressed by one or more cells of a plant, improves the stress tolerance of the plant. In one embodiment, expression of the nucleotide sequence of interest does not disturb development of the plant. [0031] In embodiments where the transcriptional control sequence includes the nucleotide sequence set forth in SEQ ID NO: 3, or a functionally active fragment or variant of the nucleotide sequence set forth in SEQ ID NO: 3, the stress is drought. Accordingly, in some embodiments, the nucleotide sequence of interest includes a nucleotide sequence which, when expressed by one or more cells of a plant, improves the drought tolerance of -7 the plant. In one embodiment, expression of the nucleotide sequence of interest does not disturb development of the plant. [0032] In some embodiments of the third aspect of the invention, the nucleotide sequence of interest includes a nucleotide sequence that encodes a DREB polypeptide. In one embodiment, the DREB polypeptide is a TaDREB3-like polypeptide. [0033] In some embodiments of the third aspect of the invention, the nucleic acid construct may further include a nucleotide sequence defining a transcription terminator. In some embodiments, the nucleic acid construct includes an expression cassette including the structure: [N], - TCS - [N]x - Sol - [N]y - TT - [N]z) wherein: [N]w includes one or more nucleotide residues, or is absent; TCS defines the transcriptional control sequence; [N]x includes one or more nucleotide residues, or is absent; Sol includes the nucleotide sequence of interest that is heterologous with respect to the TCS, wherein the nucleotide sequence of interest encodes an mRNA or non translated RNA, and is operably connected to the TCS; [N]y includes one or more nucleotide residues, or is absent; TT includes a nucleotide sequence defining a transcription terminator; and [N]z includes one or more nucleotide residues, or is absent. [0034] In a fourth aspect, the present invention provides a genetically modified cell including a nucleic acid construct of the third aspect of the invention, or a genomically integrated form thereof. [0035] In one embodiment, the cell is a plant cell. In some embodiments, the cell is a monocot plant cell. In some embodiments, the cell is a cereal crop plant cell, such as a wheat, rice or barley plant cell. [0036] In a fifth aspect, the present invention provides a multicellular structure including one or more cells of the fourth aspect of the invention.

-8 [0037] In one embodiment, the multicellular structure includes a plant or a part, organ or tissue thereof. In some embodiments, a nucleotide sequence of interest is expressed in one or more cells of the plant or a part, organ or tissue thereof in response to stress. For example, the stress may be one or more of cold. [0038] In some embodiments, the multicellular structure includes a monocot plant or a part, organ or tissue thereof. In some embodiments, the multicellular structure includes a cereal crop plant or a part, organ or tissue thereof, such as a wheat, rice or barley plant or a part, organ or tissue thereof. [0039] In some embodiments of the fifth aspect of the invention, the plant or a part, organ or tissue thereof has improved stress tolerance relative to a plant or a part, organ or tissue thereof which does not include one or more cells of the fourth aspect of the invention. For example, the stress may be selected from one or more of cold, drought, and salinity, or the stress may be induced by ABA. [0040] In a sixth aspect, the present invention provides an isolated nucleic acid when used to direct expression of an operably connected nucleotide sequence of interest in one or more cells of a plant when the plant is subject to stress, wherein the nucleic acid comprises a nucleotide sequence defining a transcriptional control sequence, wherein said transcriptional control sequence includes: (i) the nucleotide sequence set forth in one of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3; or (ii) a functionally active fragment or variant of the nucleotide sequence set forth in one of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, wherein the functionally active fragment or variant is at least 50% identical to the nucleotide sequence set forth in one of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. [0041] In one embodiment, the plant is a monocot plant. In some embodiments, the plant is a cereal crop plant, such as a wheat, rice or barley plant. [0042] In some embodiments, the functionally active fragment or variant includes one or more sequence motifs corresponding to one or more cis-elements selected from the group consisting of MYBR, ABRE, CRT and ABRE (AB15-binding). [0043] Intentionally left blank.

-9 [0044] In embodiments of the sixth aspect of the invention where the transcriptional control sequence includes a functionally active fragment or variant of the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2, the fragment or variant includes one or more sequence motifs corresponding to one or more cis-elements selected from the group consisting of a CGCG-box (calmod-binding), ABRE (AB15-binding), auxinresponse (AUX resp), MYBR, ABRE, CRT, and ammonium response (ammonium-resp). [0045] In one embodiment, the functionally active fragment or variant is at least 70% identical to the nucleotide sequence set forth in one of SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the functionally active fragment or variant includes a nucleotide sequence which is at least 94% identical to the nucleotide sequence set forth in SEQ ID NO: 4. In some embodiments, the functionally active fragment or variant includes the nucleotide sequence set forth in SEQ ID NO: 4. [0046] In some embodiments, the nucleotide sequence of interest includes a nucleotide sequence which, when expressed by one or more cells of a plant, improves the stress tolerance of the plant. [0047] In embodiments of the sixth aspect of the invention where the transcriptional control sequence includes the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2, or a functionally active fragment or variant of the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2, the stress is selected from one or more of cold, drought, and salinity, or wherein the stress is induced by ABA. Accordingly, in some ernbodiments, the nucleotide sequence of interest includes a nucleotide sequence which, when expressed by one or more cells of a plant, improves the cold, drought, and/or salinity tolerance of the plant. [0048] In embodiments of the sixth aspect of the invention where the transcriptional control sequence includes a functionally active fragment or variant of the nucleotide sequence set forth in SEQ ID NO: 3, the fragment or variant includes one or more sequence motifs corresponding to one or more cis-elements selected frorn the group consisting of ABRE (ABI5-binding), ABRE, GC-box, light regulated cis-element, MYBR, ABRE-similar, ABRE related (Ca dependent), vascular expression cis-element, WRKY -10 specific element, CRT, and carbohydrate metabolite-responsive cis-element. In one embodiment, the nucleotide sequence of interest includes a nucleotide sequence which, when expressed by one or more cells of a plant, improves the stress tolerance of the plant. In one embodiment, expression of the nucleotide sequence of interest does not disturb development of the plant. [0049] In embodiments of the sixth aspect of the invention where the transcriptional control sequence includes the nucleotide sequence set forth in SEQ ID NO: 3, or a functionally active fragment or variant of the nucleotide sequence set forth in SEQ ID NO: 3, the stress is drought. Accordingly, in some embodiments, the nucleotide sequence of interest includes a nucleotide sequence which, when expressed by one or more cells of a plant, improves the drought tolerance of the plant. In one embodiment, expression of the nucleotide sequence of interest does not disturb development of the plant. [0050] In some embodiments of the sixth aspect of the invention, the nucleotide sequence of interest includes a nucleotide sequence that encodes a DREB polypeptide. In one embodiment, the DREB polypeptide is a TaDREB3-like polypeptide. BRIEF DESCRIPTION OF THE FIGURES [0051] Figure 1 represents graphically the results of expression analysis of the TaHDZipl 3 and TaHDZipl-4 genes in selected tissues of wheat. (A) Spatial expression pattern in different tissues of T. aestivum cv Chinese spring. Caryo - Caryopsis 3-5 days after pollination; Coleo - Coleoptile; Emb.Germ - Embryo in germinating seed; Embryo Embryo 22 days after pollination; Endo - Endosperm 22 days after pollination; Imm.Infl Immature inflorescence; Int - Internode; SeedRoot - Seedling root. (B) Spatial expression pattern in developing wheat stem of T. aestivum cv RAC875. Internode length is plotted against the secondary vertical axis. Stem stages are as follows: stem 1 (100 mm); stem 2 (300 mm) awns emerging; stem 3 (400 mm) head emerging; stem 4 (500 mm) anthesis, peduncle emerged. [0052] Figure 2 represents graphically the results of expression analysis of the TaHDZipl 3 and TaHDZipl-4 genes in leaves of wheat cv RAC875 subject to various stress conditions. (A) Induction of expression by treatment with 200 pM ABA. The same amount of DMSO without ABA was used in the control experiment. (B) Induction of expression by cold treatment (constant 40C). Temperature is plotted against the secondary vertical axis.

-11 (C) Induction of expression by drought. Soil water potential is plotted against the secondary vertical axis. [0053] Figure 3 represents graphically the induction of expression of the TaHDZipI-3 and TaHDZipI-4 genes by cyclic drought in wheat cultivars with different tolerance to drought. (A) Schematic representation of drought regime and time points of collecting samples. (B) Gene copy number in drought tolerant wheat cv RAC875 plants subjected to cyclic drought and control (well watered) plants. (C) Gene copy number in drought sensitive wheat cv Kukri plants subjected to cyclic drought and control (well watered) plants. [0054] Figure 4 represents graphically the results of expression analysis of the TaHDZipI 3 and TaHDZipI-4 genes in leaves of wheat plants hydroponically grown from seedlings in the presence of 100 mM NaCl. Control seedlings were grown in the absence of NaCl. [0055] Figure 5 shows the sequence alignment of the first approximately 1000 base pairs (5' from the start codon) of the transcriptional control sequence/promoter sequence of the TdHDZipI-4a (represented as TdB4L (13-22) - SEQ ID NO: 5) and TdHDZipI-4b (represented as TdB4L (16-20) - SEQ ID NO: 6) genes. The predicted TATA-box for each promoter is indicated (empty box). Sequences conserved in both promoters are shaded. Predicted motifs corresponding to various cis-elements are indicated above their respective sequences (which are highlighted in bold). The 5'ends of the TaHDZipI-4a promoter deletions are marked with "Del" and the relevant deletion number. [0056] Figure 6 shows a 1100 base pair fragment of TdHDZipl-3 transcriptional control sequence/promoter sequence (SEQ ID NO: 7). The best candidate sequence for a TATA box (indicated as an empty box) is coupled to a putative enhancer element (GA-repeats, underlined). Other predicted cis-elements are underlined and indicated over respective sequences. The yellow box indicates an ABRE cis-element, which has an overlap with a GCC-box cis-element (underlined). [0057] Figure 7 shows the results of attempts to activate the TdHDZipl-4 and TdHDZipl-3 promoters in a transient expression assay. The activity of each promoter is presented as the number of GUS-stained foci on the y-axis. The influence of ABA on the activity of the TdHDZipl-4 and TdHDZipl-3 promoters is shown in Figure 7A. Two independent bombardment events are shown. The influence of cold treatment on the activity of the -12 TdHDZipl-4 and TdHDZipl-3 promoters is shown in Figure 7B. An average of 3 independent bombardment events is shown. The influence of ethephon and mannitol on activation of the TdHDZipl-3 promoter is shown in Figures 7C and 7D, respectively. [0058] Figure 8 provides pictures of the activity of the TdHDZipl-4 (pBL4) and TdHDZipl-3 (pBL7) promoters in transgenic barley seedlings transformed with promoter-GUS constructs. [0059] Figure 9 is a schematic of transgene (TaDREB3) copy number and expression levels in transgenic barley plants transformed with pBL7-TaDREB3 (G339) and pBL4 TaDREB3 (G345). Null segregants are indicated with arrows. [0060] Figure 10 provides photographs of transgenic barley lines (G339, pBL7-TaDREB3) at flowering (Experiment 1). wt - control plants; arrows indicate null segregants. [0061] Figure 11 provides photographs of transgenic barley lines (G339, pBL7-TaDREB3) at flowering (Experiment 2). WT - control plants; arrows indicate null segregants. [0062] Figure 12 provides photographs of transgenic barley lines (G345, pBL4-TaDREB3) at flowering (Experiment 1). wt - control plants; arrows indicate null segregants. [0063] Figure 13 provides photographs of transgenic barley lines (G345, pBL4-TaDREB3) at flowering (Experiment 2). WT - control plants; arrows indicate null segregants. [0064] Figure 14 shows the results of phenotyping of G339 (pBL7-TaDREB3) and G345 (pBL4-TaDREB3) transgenic barley plants. Measurements of plant size and tiller number were done using 2-month old plants. WT - wild type, Null - null segregant. [0065] Figure 15 shows the results of the flowering time for G339 (pBL7-TaDREB3) and G345 (pBL4-TaDREB3) transgenic barley plants. Flowering time of the first flowering wt plant was taken as a zero day. Wt plants are in green, null segregants are in black, G345 lines are in red, G339 lines are in blue. [0066] Figure 16 shows a graphical representation of the design (A) and results (B) of the frost survival tests for G339 (pBL7-TaDREB3) and G345 (pBL4-TaDREB3) transgenic -13 barley plants. Arrows indicate the time of leaf tissue sampling for the subsequent transgene expression analysis. [0067] Figure 17 is a graph showing the levels of transgene (TaDREB3) expression in selected transgenic G339 (pBL7-TaDREB3) and G345 (pBL4-TaDREB3) barley plants before and during cold treatment. Bars in green - before stress; bars in blue - after acclimation (also represented by a "c" in the transgenic line identifier. The transgene expression is shown on the y-axis and is represented by normalised copy number per pg RNA. [0068] Figure 18 is a schematic representation of the results of the drought survival test for selected transgenic barley lines of Example 6. Speed of soil drying is shown by the change of the weight of each pot that was used in the experiment. The main points for leaf sampling for the analysis of the water potential and transgene expression are indicated with arrows. Survival rate was estimated one week after re-watering. [0069] Figure 19 shows the results of the drought survival test for selected transgenic barley lines of Example 6. (A) shows the experimental design and leaf sampling points; (B) shows survival rates of G339 (pBL7-TaDREB3) plants; (C) shows survival rates of G345 (pBL4-TaDREB3) plants; and (D) is a photo showing the recovery of meristems of transgenic plants 3 days after re-watering. [0070] Figure 20 is a graphic representation of the results of activation of the BL4 promoter by drought in the survived T 1 transgenic barley plants of Figure 19. Arrows indicate wilting points. before - before stress; s.d. - severe drought. [0071] Figure 21 is a graphic representation of the results of activation of the BL7 promoter by drought in the survived T 1 transgenic barley plants of Figure 19. Arrows indicate wilting points. before - before stress; s.d. - severe drought. [0072] Figure 22 shows the various TdHDZipl-4 promoter deletion constructs used in the ABA-response mapping study, and the results of those mapping studies. These studies identify a cis-element responsible for TdHDZip/-4 promoter activation by ABA.

- 14 DESCRIPTION OF THE INVENTION [0073] Nucleotide sequences are referred to herein by a sequence identifier number (SEQ ID NO:). A summary of the sequence identifiers is provided in Table 1. A sequence listing is also provided. TABLE 1 Summary of Sequence Identifiers Sequence Sequence Identified SEQ ID NO: 1 TdHDZipl-4a promoter nucleotide sequence (2,142 bp) SEQ ID NO: 2 TdHDZipl-4b promoter nucleotide sequence (3,167 bp) SEQ ID NO: 3 TdHDZipl-3 promoter nucleotide sequence (1,915 bp) SEQ ID NO: 4 TdHDZipl-4a sequence between deletion 5 and deletion 6 SEQ ID NO: 5 TdHDZipl-4a promoter nucleotide sequence (Figure 5) SEQ ID NO: 6 TdHDZipl-4b promoter nucleotide sequence (Figure 5) SEQ ID NO: 7 TdHDZipl-3 promoter nucleotide sequence (Figure 6) SEQ ID NO: 8 Oligonucleotide primer - HygF SEQ ID NO: 9 Oligonucleotide primer - HygR SEQ ID NO: 10 TaqMan probe - Hyg SEQ ID NO: 11 TdHDZipl-4a promoter nucleotide sequence (5,082 bp) SEQ ID NO: 12 TdHDZipl-3 promoter nucleotide sequence (5,325 bp) SEQ ID NO: 13 TdHDZipl-4a promoter used in GUS construct (2,920 bp) SEQ ID NO: 14 TdHDZipl-3 promoter used in GUS construct (3,040 bp) SEQ ID NO: 15 TdB4L16_R3 primer- cloning SEQ ID NO: 14 into pMDC164 SEQ ID NO: 16 TdB4L16_R4 primer- cloning SEQ ID NO: 14 into pMDC164 SEQ ID NO: 17 C_TdB4L13 primer- cloning SEQ ID NO: 14 into pMDC164 SEQ ID NO: 18 TdB4L13r primer- cloning SEQ ID NO: 14 into pMDC164 SEQ ID NO: 19 C_TdB4L16 primer- cloning SEQ ID NO: 14 into pMDC164 SEQ ID NO: 20 TdB4L16r primer- cloning SEQ ID NO: 14 into pMDC164 SEQ ID NO: 21 C_TdBL7 primer - cloning SEQ ID NO: 15 into pMDC164 SEQ ID NO: 22 TdBL7r primer - cloning SEQ ID NO: 15 into pMDC164 SEQ ID NO: 23 PRTdB4L13H primer - cloning SEQ ID NO: 1 into pMDC32 SEQ ID NO: 24 PRTdB4L1 3Kr primer - cloning SEQ ID NO: 1 into pMDC32 SEQ ID NO: 25 PRTdBL7H primer - cloning SEQ ID NO: 3 into pMDC32 -15 SEQ ID NO: 26 PRTdBL7Kr primer - cloning SEQ ID NO: 3 into pMDC32 SEQ ID NO: 27 Oligonucleotide primer - TxDREB3 SEQ ID NO: 28 Oligonucleotide primer - TxDREB3rnos [0074] As set out above, the present invention is predicated, in part, on the identification of transcriptional control sequences which are active in plants. Accordingly, in a first aspect, the present invention provides a method for effecting stress responsive expression of a nucleotide sequence of interest in one or more cells of a plant, the method including expressing in the one or more cells of the plant the nucleotide sequence of interest operably connected to a transcriptional control sequence which is stress inducible in the plant, wherein the nucleotide sequence of interest is heterologous with respect to the transcriptional control sequence, and wherein the transcriptional control sequences includes: (i) the nucleotide sequence set forth in one of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3; or (ii) a functionally active fragment or variant of the nucleotide sequence set forth in one of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. [0075] Reference herein to a plant may include seed plant species such as monocotyledonous angiosperm plants ("monocots"), dicotyledonous angiosperm plants ("dicots") and/or gymnosperm plants. [0076] In some embodiments, the plant is a monocot plant. In some embodiments, the plant is a cereal crop plant. As used herein, the term "cereal crop plant" may include a member of the Poaceae (grass) family that produces grain. Examples of Poaceae cereal crop plants include wheat, rice, barley, maize, millets, sorghum, rye, triticale, oats, teff, wild rice, spelt and the like. The term cereal crop plant should also be understood to include a number of non-Poaceae plant species that also produce edible grain, which are known as the pseudocereals and include, for example, amaranth, buckwheat and quinoa. [0077] In some embodiments, the plant is a wheat plant. As referred to herein, "wheat" should be understood as a plant of the genus Triticum. Thus, the term "wheat" encompasses diploid wheat, tetraploid wheat and hexaploid wheat. In some embodiments, the wheat plant may be a cultivated species of wheat including, for example, T. aestivum, T. durum, T. monococcum or T. spelta. In some embodiments, the - 16 term "wheat" refers to wheat of the species Triticum durum. [0078] In some embodiments, the plant is a rice plant. As referred to herein, "rice" should be understood to include several members of the genus Oryza, including the species Oryza sativa and Oryza glaberrima. The term "rice" thus encompasses rice cultivars such as japonica or sinica varieties, indica varieties and javonica varieties. In some embodiments, the term "rice" refers to rice of the species Oryza sativa. [0079] In some embodiments, the plant is a barley plant. As referred to herein, "barley" includes several members of the genus Hordeum. The term "barley" encompasses cultivated barley including two-row barley (Hordeum distichum), four-row barley (Hordeum tetrastichum) and six-row barley (Hordeum vulgare). In some embodiments, barley may also refer to wild barley, (Hordeum spontaneum). In some embodiments, the term "barley" refers to barley of the species Hordeum vulgare. [0080] As set out above, the method according to a first aspect of the present invention contemplates effecting stress responsive expression of a nucleotide sequence of interest in one or more cells of a plant. [0081] As referred to herein, "expression" of a nucleotide sequence of interest refers to the transcription of the nucleotide sequence in one or more cells of a plant. However, this definition in no way implies that expression of the nucleotide sequence must occur in all cells of the plant. [0082] "Stress responsive expression", as referred to herein, should be understood to refer to an increase in the transcription of a nucleotide sequence of interest in one or more cells of the plant when the plant experiences stress. In some embodiments, the level of increase in expression of the nucleotide sequence of interest may be at least about 2 times greater as a result of stress compared to the level of expression of the nucleotide sequence of interest in the absence of stress. In some embodiments, the level of increase in expression may be at least about 3, 4 or 5 times greater than in the absence of stress. However, a person skilled in the art would understand that expression level increases greater than this (for example at least about 10 times, 100 times, 1,000 times or even 10,000 times) in the presence of stress are also contemplated by the present invention.

- 17 [0083] "Stress" as referred to herein should be understood to include any environmental condition the plant, or cells of the plant, experiences which is suboptimal for the growth and/or development of the plant or cell thereof. In some embodiments, the stress may be abiotic in nature, for example the stress may include one or more of low temperature (e.g. frost/cold), drought, salinity, high temperature, high irradiance, and nutrient toxicities or deficiencies. In some embodiments, the stress may be biotic in nature, for example the stress may include disease or invasion by a pathogen or pest. Furthermore, the plant hormone abscisic acid (ABA) is responsible for the signal transduction in one of the abiotic stress response pathways. Accordingly, exposure of plants to ABA will mimic conditions of stress, as described in further detail below. In some embodiments, reference herein to "stress" includes environmental conditions of sufficient severity to cause visible symptoms in a plant such as loss of turgor, wilting, rolled leaves, chlorosis, growth retardation and/or death of a plant. [0084] "Cold" as referred to herein should be understood to include any situation where the temperature in which the plant is exposed is less than the optimum temperature of growth for that plant. In some embodiments, cold may include frost which is a result of the formation of ice crystals in cells of the plant due to the temperature of the plant falling below freezing and falling below the dew point of the surrounding air. In some embodiments, cold may include temperatures in the range of less than about 100C, less than about 90C, less than about 80C, less than about 70C, less than about 60C, less than about 50C, less than about 40C, less than about 30C, less than about 20C, less than about 10C, about 00C, or less than about 00C. [0085] "Drought" as referred to herein should be understood to include any situation wherein the amount of water available to a plant, at a physiologically appropriate level of salinity, is less than the optimum level of water for that plant. In some embodiments, drought may include low volumetric water content (VWC) in a soil. In some embodiments, drought may include a soil VWC of less than about 10%, less than about 9%, less than about 8%, less than 7%, less than about 6%, less than 5%, less than about 4%, or less than about 3%. [0086] In some embodiments, drought may also include other forms of osmotic stress such as wherein a relatively high volume of water is available, but the level of salinity in the water is sufficiently high to cause osmotic stress in the plant. As would be understood -18 by a person skilled in the art, "salinity" as used herein generally refers to the level of salt in the growing environment of a plant. A salt in this regard typically includes sodium chloride, magnesium and calcium sulphates, and bicarbonates. However, the most relevant salt for a majority of cropping systems is sodium chloride. [0087] In some embodiments, stress may be mimicked by exposure of the plant to the plant hormone abscisic acid (ABA). ABA is a key regulator of seed development. In addition to promoting seed maturation, ABA inhibits seed germination and seedling growth. ABA is involved in the abscission of plant leaves. In preparation for winter, ABA is produced in terminal buds, which slows plant growth and directs leaf primordia to develop scales to protect the dormant buds during the cold season. ABA also inhibits the division of cells in the vascular cambium, adjusting to cold conditions in the winter by suspending primary and secondary growth. ABA is also produced in the roots in response to decreased soil water potential and other situations in which the plant may be under stress. ABA then translocates to the leaves, where it rapidly alters the osmotic potential of stomatal guard cells, causing them to shrink and stomata to close. The ABA-induced stomatal closure reduces transpiration, thus preventing further water loss from the leaves in times of low water availability [0088] In the method according to a first aspect of the present invention stress responsive expression of the nucleotide sequence of interest is effected by the nucleotide sequence of interest being operably connected to a transcriptional control sequence which is stress inducible in the plant. [0089] As used herein, the term "transcriptional control sequence" should be understood as a nucleotide sequence that modulates at least the transcription of an operably connected nucleotide sequence of interest. As such, the transcriptional control sequence of the present invention may comprise any one or more of, for example, a leader, promoter, 5' or 3' untranslated region (UTR), enhancer or upstream activating sequence. In some embodiments, the transcriptional control sequence may comprise a promoter and/or 5' UTR. A "promoter" as referred to herein, encompasses any nucleic acid that confers, activates or enhances expression of an operably connected nucleotide sequence of interest in a cell. [0090] As used herein, the term "operably connected" refers to the connection of a -19 transcriptional control sequence, such as a promoter, and a nucleotide sequence of interest in such as way as to bring the nucleotide sequence of interest under the transcriptional control of the transcriptional control sequence. For example, promoters are generally positioned 5' (upstream) of a nucleotide sequence to be operably connected to the promoter. In the construction of heterologous transcriptional control sequence/nucleotide sequence of interest combinations, the promoter is generally positioned at a distance from the transcription start site that is approximately the same as the distance between that promoter and the gene it controls in its natural setting, i.e. the gene from which the promoter is derived. As is known in the art, some variation in this distance can be accommodated without loss of promoter function. [0091] As set out above, the transcriptional control sequence contemplated for use in the present invention is "stress inducible". A stress inducible transcriptional control sequence should be understood to include transcriptional control sequences which generate an increased rate and/or increased level of transcription (expression) of an operably connected nucleotide sequence of interest in a plant when the plant is exposed to stress. In some embodiments, the stress inducible transcriptional control sequence may be activated by one or more transcription factors or other polypeptides which are expressed in a plant when the plant is exposed to stress. As referred to above, when the plant is exposed to stress the rate and/or level of transcription (expression) of the nucleotide sequence of interest may be at least about 2 times greater as a result of stress compared to the level of expression of the nucleotide sequence of interest in the absence of stress. [0092] In some embodiments, the transcriptional control sequence may generate an increased rate and/or increased level of transcription of an operably connected nucleotide sequence which is at least about 3, 4 or 5 times greater than the rate and/or level of transcription in the absence of stress. However, expression rate and/or level increases greater than this (for example at least about 10 times, 100 times, 1,000 times or even 10,000 times) in the presence of stress are also contemplated by the present invention. [0093] Methods to determine the rate and/or level of transcription of an operably connected nucleotide sequence of interest would be known in the art. Generally, such methods include Northern blotting and/or quantitative PCR. [0094] Furthermore, the term "stress inducible" is to be assessed in the context of a plant - 20 of interest. For example a particular transcriptional control sequence may exhibit stress inducibility in the presence of stress in a plant of interest, but need not exhibit this characteristic in all plant species to fall within the meaning of the above-referenced term for the purposes of the present invention. In some embodiments, the term "stress inducible" may also be assessed in the context of a particular tissue type. For example, a particular transcriptional control sequence may exhibit stress inducibility in a particular plant tissue of interest in the presence of stress, e.g. the leaves, but need not exhibit this characteristic in all plant tissues to fall within the meaning of the above-referenced term for the purposes of the present invention. [0095] As set out above, in some embodiments the transcriptional control sequence of the present invention includes the nucleotide sequence set forth in one of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, or a functionally active fragment or variant of the nucleotide sequence set forth in one of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. As referred to herein, a "functionally active fragment or variant" refers to a fragment or variant of the nucleotide sequence set forth in one of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 which substantially retains the ability to direct expression of an operably connected nucleotide sequence of interest in one or more cells of a plant in response to stress. [0096] "Functionally active fragments" of the transcriptional control sequence of the present invention may be of any length provided the transcriptional control sequence retains the ability to direct expression of an operably connected nucleotide sequence in response to stress. In some embodiments, a functionally active fragment may be at least about 80 nucleotides (nt), at least about 90 nt, at least about 100 nt, at least about 200 nt, at least about 300 nt, at least about 400 nt, at least about 500 nt, at least about 1000 nt, at least about 1500 nt, or at least about 1900 nt in length. A fragment "at least about 80 nt in length" includes, for example, fragments which include about 80 or more contiguous bases from the nucleotide sequence of any one of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. [0097] "Functionally active variants" of the transcriptional control sequences of the present invention include orthologs, mutants, synthetic variants, analogs and the like. By being "functionally active", the variants retain the capability to direct expression of an operably connected nucleotide sequence of interest. For example, the term "variant" - 21 should be considered to specifically include transcriptional control sequences from other organisms which are orthologous to one of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3; mutants of the transcriptional control sequence of one of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3; variants of one of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 wherein one or more of the nucleotides within the sequence has been substituted, added or deleted; and analogs that contain one or more modified bases or DNA or RNA backbones modified for stability or for other reasons. "Modified" bases include, for example, tritylated bases and unusual bases such as inosine. [0098] As will be appreciated, functionally active fragments or variants of the nucleotide sequence set forth in one of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 may include transcriptional control sequences isolated from other plants and/or synthetic nucleotide sequences. [0099] In some embodiments, a functionally active fragment or variant of a transcriptional control sequence of the present invention may include one or more sequence motifs which are also found in the transcriptional control sequence, and which act as binding sites for cis-acting factors. These sequence motifs (also referred to as cis-elements) typically interact with cis-acting factors such as proteins or other biomolecules to carry out (turn on/off, regulate, modulate, etc.) gene transcription. For example, the transcriptional control sequences set forth in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 all contain the cis-elements MYBR, ABRE, CRT and ABRE (AB15-binding). Accordingly, a functionally active fragment or variant of these transcriptional control sequences will also include one or more of the cis-elements MYBR, ABRE, CRT and ABRE (AB15-binding). [0100] The MYB recognition site (MYBR) has previously been shown to be present in the promoter region of the ABA-inducible gene RD22 of Arabidopsis, and has the consensus sequence (CT)/WAACCA. This cis-acting element is recognised by the AtMYB2 protein. [0101] The ABA-responsive element (ABRE) is a major cis-acting element in the promoter of many ABA-responsive genes. The cis-element was first identified in the Em gene of wheat and has since been found in various other plants including maize, oat, rice, tobacco and Arabidopsis where it functions as an ABRE. Expression of ABA-responsive genes is activated through binding of the AREB/ABF (bZIP-type) transcription factor to the ABRE sequence. Promoter regions of several ABA-inducible genes have since been compared -22 with each other establishing a consensus sequence for the ABRE, namely (T/G/C)ACGT(G/T)GC with ACGT as the core sequence. [0102] CRT is a cis-element involved in ABA-independent expression of drought-inducible genes, and was originally found in the promoter region of the RD29A gene in Arabidopsis. CRT has also been shown to be a cis-element involved in expression of cold-inducible genes in Arabidopsis. This cis-element is recognised by proteins expressed from a family of genes designated either CBF1/DREBib, CBF2/DREB1c and CBF3/DREB1a which are expressed transiently soon after sensing drought and cold thereby up-regulating the target genes involved in drought and cold tolerance. The consensus sequence for CRT elements has been designated (G/a)(T/c)CGAC. [0103] AB15 encodes a transcription factor that regulates gene expression during embryogenesis and in response to ABA. AB15 is induced by stresses such as cold, salinity and drought. As reported by Carles C et al., 2002 ("Regulation of Arabidopsis thaliana Em genes: role of AB15", Plant J. 30: 373-83), the cis-element that binds AB15 (AGACACGTGGCATGT) has the consensus sequences A(C/G)ACACG and ACACNNG. [0104] There are numerous web-based resources for identifying cis-elements in a given nucleotide sequence, and therefore a person skilled in the art could readily determine if a nucleotide sequence qualified as a fragment or variant of the transcriptional control sequences of the present invention. A summary of various resources for identifying cis elements is provided in Rombauts et al., 2003, Plant Physiol. 132: 1162-1176. Furthermore, representative resources include: * PLACE (http://www.dna.affrc.go.jp/PLACE/) * PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) * AGRIS (http://arabidopsis.med.ohio-state.edu/) * MEME (http://meme.ebi.edu.au/meme/intro.html) * AthaMap (http://www.athamap.de/) * AtProbe (http://exon.cshl.org/cgi-bin/atprobe/atprobe.pl) * DoOP (http://doop.abc.hu/) * PlantProm DB (http://linux1.softberry.com/berry.phtml?topic=plantprom&group=data&subgroup= plantprom) * Transfac (http://www.gene-regulation.com/pub/databases.html#transfac) - 23 * RSAT http://rsat.ulb.ac.be/rsat/) * Motiffinder from TAIR (http://www.arabidopsis.org/tools/bulk/motiffinder/) * GeneSprings, TAIR Pattern Match (http://www.arabidopsis.org/cgi bin/patmatch/nph-patmatch.pl) * Genomatix (http://www.genomatix.de) * BioProspector (http://ai.stanford.edu/-xsliu/BioProspector/) * Toucan 2 (http://homes.esat.kuleuven.be/-saerts/software/toucan.php), and * Regulatory Sequence Analysis Tools (http://rsat.ulb.ac.be/rsat/). [0105] In some embodiments, the functionally active fragment or variant has at least about 42%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% nucleotide sequence identity to the nucleotide sequence set forth in any one of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3. [0106] When comparing nucleic acid sequences to calculate a percentage identity, the compared nucleotide sequences should be compared over a comparison window, i.e. a contiguous segment of sequence, of at least about 80 nucleotide residues, at least about 90 nucleotide residues, at least about 100 nucleotide residues, at least about 200 nucleotide residues, at least about 500 nucleotide residues, at least about 1000 nucleotide residues, at least about 1500 nucleotide residues, at least about 1900 nucleotide residues, or over the full length of any one of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3. The comparison window may comprise additions or deletions (i.e. gaps) of about 20% or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Optimal alignment of sequences for aligning a comparison window may be conducted by computerised implementations of algorithms such as the BLAST family of programs as, for example, disclosed by Altschul et al. 1997 (Nucl. Acids Res. 25: 3389-3402). Global alignment programs may also be used to align similar sequences of roughly equal size. Examples of global alignment programs include NEEDLE (available at www.ebi.ac.uk/Tools/psa/embossneedle/) which is part of the EMBOSS package (Rice P et al., 2000, Trends Genet., 16: 276-277), and the GGSEARCH program (available at - 24 fasta.bioch.virginia.edu/fastawww2/fasta-www.cgi?rm=compare&pgm=gnw) which is part of the FASTA package (Pearson W and Lipman D, 1988, Proc. Nat/. Acad. Sci. USA, 85: 2444-2448). Both of these programs are based on the Needleman-Wunsch algorithm which is used to find the optimum alignment (including gaps) of two sequences along their entire length. A detailed discussion of sequence analysis can also be found in Unit 19.3 of Ausubel et al ("Current Protocols in Molecular Biology" John Wiley & Sons Inc, 1994 1998, Chapter 15, 1998). [0107] In some embodiments, the functionally active fragment or variant is at least 42% identical to one of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. In some embodiments, the functionally active fragment or variant is at least 42% identical to one of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 and includes one or more sequence motifs corresponding to one or more cis-elements selected from the group consisting of MYBR, ABRE, CRT and ABRE (AB15-binding). [0108] In some embodiments of the present invention, the transcriptional control sequence includes the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2. These sequences both contain cis-elements selected from a CGCG-box (calmod-binding), ABRE (AB15-binding), auxin-response (AUX-resp), MYBR, ABRE, CRT, and ammonium response (ammonium-resp). Accordingly, a functionally active fragment or variant of these sequences may include one or more sequence motifs corresponding to one or more cis elements selected from the group consisting of a CGCG-box (calmod-binding), ABRE (AB15-binding), auxin-response (AUX-resp), MYBR, ABRE, CRT, and ammonium response (ammonium-resp). [0109] In some embodiments, the functionally active fragment or variant is at least 70% identical to SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the functionally active fragment or variant is at least 70% identical to SEQ ID NO: 1 or SEQ ID NO: 2 and includes one or more sequence motifs corresponding to one or more cis-elements selected from the group consisting of a CGCG-box (calmod-binding), ABRE (AB15 binding), auxin-response (AUX-resp), MYBR, ABRE, CRT, and ammonium response (ammonium-resp). Details regarding the CGCG-box (calmod-binding), auxin-response (AUX-resp), and ammonium response (ammonium-resp) elements are provided in Example 4.

- 25 [0110] Through promoter deletion studies, the inventors have identified a region of a transcriptional control sequence of the present invention which is required for at least an expression response to the presence of ABA. This region consists of 93 nucleotides, the sequence of which is set forth in SEQ ID NO: 4. Accordingly, in some embodiments, the functionally active fragment or variant includes a nucleotide sequence which is at least 94% identical to the nucleotide sequence set forth in SEQ ID NO: 4. In some embodiments, the functionally active fragment or variant includes the nucleotide sequence set forth in SEQ ID NO: 4. [0111] In some embodiments, the functionally active fragment or variant comprises a nucleic acid molecule which hybridises to a nucleic acid molecule defining a transcriptional control sequence of the present invention under stringent conditions. In some embodiments, the functionally active fragment or variant comprises a nucleic acid molecule which hybridises to a nucleic acid molecule comprising the nucleotide sequence set forth in any one of SEQ ID NO: 1, SEQ ID NO: 2 , or SEQ ID NO: 3 under stringent conditions. [0112] As used herein, "stringent" hybridisation conditions will be 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 pH 7.0 to 8.3 and the temperature is at least 300C. Stringent conditions may also be achieved with the addition of destabilising agents such as formamide. In some embodiments, stringent hybridisation conditions may be low stringency conditions, medium stringency conditions or high stringency conditions. [0113] Exemplary, non-limiting, low stringency conditions include hybridisation with a buffer solution of 30 to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 370C, and a wash in 1x to 2x SSC (20x SSC = 3.0 M NaCl/0.3 M trisodium citrate) at 50 to 55C. Alternative low stringency conditions can be utilised as would be known in the art. For example, hybridisation can be performed in 6x SSC at room temperature to 550C for 16 to 20 hours followed by washing at least twice in 2x to 3x SSC at room temperature to 550C for 20 to 30 minutes each. Low stringency conditions typically detect sequences that share as little as 50% sequence identity. [0114] Exemplary, non-limiting, moderate stringency conditions include hybridisation in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at 370C, and a wash in 0.5x to 1x SSC at 55 to - 26 600C. Alternative moderate stringency conditions can be utilised as would be known in the art. For example, hybridisation can be performed in 5x to 6x SSC at 650C to 700C for 16 to 20 hours followed by washing twice in 2x SSC at room temperature for 5 to 20 minutes each, followed by a further two washes in 1x SSC at 550C to 700C for 30 minutes each. Moderate stringency conditions typically detect sequences that share 80% sequence identity or greater. [0115] Exemplary, non-limiting, high stringency conditions include hybridisation in 50% formamide, 1 M NaCl, 1% SDS at 370C, and a wash in 0.1x SSC at 60 to 650C. Optionally, wash buffers may comprise about 0.1% to about 1% SDS. Duration of hybridisation is generally less than about 24 hours, usually about 4 to about 12 hours. Alternative high stringency conditions can be utilised as would be known in the art. For example, hybridisation can be performed in 5x SSC at 650C for 16 hours followed by washing twice in 2x SSC at room temperature for 15 minutes each, followed by a further two washes in 0.5x SSC at 650C for 20 minutes each. High stringency conditions typically detect sequences that share 90% sequence identity or greater. [0116] Specificity of hybridisation is also a function of post-hybridisation washes, with the critical factors being the ionic strength and temperature of the final wash solution. For DNA-DNA hybrids, the Tm can be approximated from the equation of Meinkoth and Wahl (Anal. Biochem. 138: 267-284, 1984), i.e. Tm =81.50C +16.6 (log M)+0.41 (% GC)-0.61 (% form)-500/L; where M is the molarity of monovalent cations, % GC is the percentage of guanosine and cytosine nucleotides in the DNA, % form is the percentage of formamide in the hybridisation solution, and L is the length of the hybrid in base pairs. The Tm is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridises to a perfectly matched probe. Tm is reduced by about 1 C for each 1% of mismatching; thus, Tm, hybridisation, and/or wash conditions can be adjusted to hybridise to sequences of different degrees of complementarity. For example, sequences with 90% identity can be hybridised by decreasing the Tm by about 100C. Generally, stringent conditions are selected to be about 50C lower than the thermal melting point (Tm) for the specific sequence and its complement at a defined ionic strength and pH. However, high stringency conditions can utilise a hybridisation and/or wash at, for example, 1, 2, 3, or 40C lower than the thermal melting point (Tm); medium stringency conditions can utilise a hybridisation and/or wash at, for example, 6, 7, 8, 9, or 10 C lower than the thermal melting point (Tm); low stringency conditions can utilise a hybridisation - 27 and/or wash at, for example, 11, 12, 13, 14, 15, or 200C lower than the thermal melting point (Tm). Using the equation, hybridisation and wash compositions, and desired Tm, those of ordinary skill will understand that variations in the stringency of hybridisation and/or wash solutions are inherently described. If the desired degree of mismatching results in a Tm of less than 450C (aqueous solution) or 320C (formamide solution), the SSC concentration may be increased so that a higher temperature can be used. An extensive guide to the hybridisation of nucleic acids is found in Tijssen (Laboratory Techniques in Biochemistry and Molecular Biology-Hybridisation with Nucleic Acid Probes, Pt I, Chapter 2, Elsevier, New York, 1993), Ausubel et al., eds. (Current Protocols in Molecular Biology, Chapter 2, Greene Publishing and Wiley-Interscience, New York, 1995) and Sambrook et al (Molecular Cloning: A Laboratory Manual, 2 nd ed., Cold Spring Harbor Laboratory Press, Plainview, NY, 1989). [0117] In some embodiments of the present invention where the transcriptional control sequence includes the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2, the nucleotide sequence of interest includes a nucleotide sequence which, when expressed by one or more cells of a plant, improves the stress tolerance of the plant. The term "stress tolerance" as used herein refers to any trait in the plant which allows the plant to survive, recover and/or reproduce during or after experiencing stress. Measures of stress tolerance may include, for example, the ability of a plant to continue to grow, reproduce or yield during or after an episode of stress; the rate or frequency of recovery of plants after an episode of stress; the extent of any yield penalty for a plant after experiencing an episode of stress; the water use efficiency of a plant; and the like. [0118] "Improvement" in the stress tolerance of a plant should be seen as any increase in the ability of a plant to survive, recover or reproduce during or after experiencing stress. For example, "improved" stress tolerance of a plant may include an increased ability of a plant to continue to grow, reproduce or yield during or after an episode of stress; an increased rate or frequency of recovery of plants after an episode of stress; a decrease in or amelioration of any yield penalty associated with an episode of stress; increased water use efficiency of a plant; and the like. [0119] In some embodiments of the present invention where the transcriptional control sequence includes the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2, the stress is is selected from one or more of cold, drought, and salinity, or wherein the - 28 stress is induced by ABA. Accordingly, in some embodiments, the nucleotide sequence of interest comprises a nucleotide sequence which, when expressed by one or more cells of a plant, improves the cold, drought, and/or salinity tolerance of the plant. [0120] In some embodiments of the present invention, the transcriptional control sequence includes the nucleotide sequence set forth in SEQ ID NO: 3. This sequence contains cis-elements which include ABRE (AB15-binding), ABRE, GCC-box, light regulated cis-element, MYBR, ABRE-similar, ABRE-related (Ca dependent), vascular expression cis-element, WRKY specific element, CRT, and carbohydrate metabolite responsive cis-element. Accordingly, a functionally active fragment or variant of this sequence may include one or more sequence motifs corresponding to one or more cis elements selected from the group consisting of ABRE (AB15-binding), ABRE, GCC-box, light regulated cis-element, MYBR, ABRE-similar, ABRE-related (Ca dependent), vascular expression cis-element, WRKY specific element, CRT, and carbohydrate metabolite responsive cis-element. Details regarding the GCC-box, light regulated cis-element, vascular expression cis-element, WRKY specific element, and the carbohydrate metabolite-responsive element are provided in Example 4. [0121] In some embodiments of the present invention where the transcriptional control sequence includes the nucleotide sequence set forth in SEQ ID NO: 3, the nucleotide sequence of interest includes a nucleotide sequence which, when expressed by one or more cells of a plant, improves the stress tolerance of the plant. [0122] As indicated above, constitutive expression of various endogenous genes can lead to an effect on the development of the plant, such as retardation in growth of the plant. Accordingly, in some embodiments of the present invention where the transcriptional control sequence includes the nucleotide sequence set forth in SEQ ID NO: 3, the nucleotide sequence of interest comprises a nucleotide sequence which, when expressed by one or more cells of a plant, may improve the stress tolerance of the plant without disturbing development of the plant. Development of a plant will typically be assessed through a phenotypic analysis of characteristics of the plant. Such developmental characteristics include, but are not limited to, plant height, leaf length, tiller number at flowering, flowering time, spike number, main spike length, grain weight per plant, spikelet number per spike, grain number per spike, and grain weight per 1 or 100 grains.

- 29 [0123] In some embodiments, the stress is drought. Accordingly, in some embodiments, the nucleotide sequence of interest comprises a nucleotide sequence which, when expressed by one or more cells of a plant, improves the drought tolerance of the plant. In one embodiment, expression of the nucleotide sequence does not disturb development of the plant. Development of the plant can be assessed as described above. [0124] As would be understood by one of skill in the art, the nucleotide sequence of interest, which is placed under the regulatory control of a transcriptional control sequence of the present invention, may be any nucleotide sequence which improves the stress tolerance of the plant. Examples include, but are not limited to, genes encoding transcription factors such as DREB/CBF factors, MYC factors, MYB factors, ERF factors, WRKY factors, MADS factors, NAC factors etc.; genes encoding protein kinases, which are activated or transcriptionally up-regulated under stress, such as SAPKs, receptor kinases, MAP kinases, and the like; genes encoding phosphatases related to stress responses such as ZmPP2C, type 1 inositol 5-phosphatase and the like; stress inducible genes which protect cell integrity (e.g. membrane stability, chloroplast/chlorophyll stability, correct protein folding and protein stability, and the like) such as LEA, DHNs, COR, RD, LT and RAB and the like; genes encoding water channels such as aquaporins, PIPs, TIPs and NIPs; genes encoding stomata opening regulators such as AtMRP4, a guard cell plasma membrane ABCC-type ABC transporter; genes responsible for sugar metabolism such as trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphate phosphatase (TPP), ABA2 (or GLUCOSE INSENSITIVE 1 [GIN1]) encoding a short-chain dehydrogenase/reductase; genes delaying stress-induced leaf senescence such as senescence associated receptor protein kinase (SARK), a gene encoding a calcium/calmodulin-regulated receptor protein kinase; calcineurin B-like proteins (CBLs); ice re-crystallisation inhibition (IRI)-like protein coding genes; and genes encoding apoplast antifreeze proteins such as chitinases, glucanases, and thaumatin-like proteins. [0125] In some embodiments, the nucleotide sequence of interest encodes a DREB polypeptide. The dehydration-responsive element-binding proteins (DREBs) or C-repeat binding proteins (CBFs) are among the first discovered families of transcription factors responsible for gene regulation under conditions of water deficiency. [0126] In some embodiments, a "DREB polypeptide" as referred to herein, may comprise an AP2 domain. In some embodiments, the DREB polypeptide may comprise a single - 30 AP2 domain. The AP2 protein domain is described in detail under pfam accession number PF00847. As referred to herein, the term "dehydration-responsive element-binding proteins" or "DREB" may also encompass a C-repeat-binding protein or CBF. [0127] Examples of DREB/CBF polypeptides include polypeptides having the following NCBI protein database accession numbers: from Triticum aestivum - ABC86563; ABC86564; ABK55389; ABK55388; ABK55387; ABK55386; ABK55385; ABK55384; ABK55383; ABK55382; ABK55381; ABK55380; ABK55379; ABK55378; ABK55377; ABK55376; ABK55375; ABK55374; ABK55373; ABK55372; ABW8701 1; ABK55390; ABK55389; ABK55388; ABK55387; ABK55386; ABK55385; ABK55384; ABK55383; ABK55382; ABK55381; ABK55380; ABK55379; ABK55377; ABK55376; ABK55375; ABK55374; ABK55373; ABK55372; ABK55371; ABK55370; ABK55369; ABK55368; ABK55367; ABK55366; ABK55365; ABK55364; ABK55363; ABK55362; ABK55361; ABK55360; ABK55359; ABK55358; ABK55357; ABK55356; ABK55355; ABK55354; AAY32564; AAY32563; AAY32562; AAY32561; AAY32560; AAY32558; AAY32557; AAY32556; AAY32555; AAY32554; AAY32553; AAY32552; AAY32551; AAX28966; AAX28965; AAX28963; AAX28962; AAX28961; ACK99532; ACB69508; ACB69507; ACB69506; ACB69505; ACB69504; ACB69503; BAD66926; BAD66925; ABB90544; ABA08426; ABA08425; ABA08424; AAX13287; AAX13285; AAX13287; AAX13285; AAX13289; AAX13289; AAX13289; AAX13287; AAX13286; AAX13285; AAX13284; AAX13283; AAX13282; AAX13279; AAX13278; AAX13277; AAR05861; ABB84399; AAX28964; ABW87014; AAX13274 from Triticum monococcum - ABW87013; ABW87012; ABW8701 1; ABK55390; AAY32550; AAX28967; from Aegilops speltoides subsp. Speltoides - AC035591; AC035590; AC035589; AC035588; AC035587; AC035586; AC035585; AC035584; AC035583; AAY25517; from Hordeum vulgare subsp. Vulgare - AAG59618; ABA25897; ABA25896; AAZ99830; AAZ99829; ACC63523; ABA25904; ABA01494; ABA01493; ABA01492; ABA01491; AAX28957; AAX28956; AAX28955; AAX28954; AAX28953; AAX28952; AAX28950; AAX28949; AAX28948; AAX23718; AAX23714; AAX23707; AAX23704; AAX23701; AAX23698; AAX23696; AAX23692; AAX23688; AAX23684; AAX23683; ABF18984; ABF18983; ABF18982; AAX28951; AAX23720; AAX23719; AAX23717; AAX23716; AAX23715; AAX23713; AAX23712; AAX23710; AAX23709; AAX23708; AAX23706; AAX23705; AAX23703; AAX23702; AAX23700; AAX23699; AAX23697; AAX23695; AAX23694; AAX23693; AAX23691; AAX23690; AAX23689; AAX23687; - 31 AAX23686; AAX23685; AAX19267; AAX19266 from Arabidopsis thaliana - NP_849340; NP_564496; NP_181551; NP_177844; NP_001031837; NP_567719; NP_563624; NP_196160; NP_201318; NP_191319; NP_181186; NP_181368; NP_172721; NP_176620; NP_565609; NP_172723; NP_001077764; NP_181566; NP_177681; NP_173355; NP_680184; NP_567867; NP_567721; NP_567720; NP_565929; NP_564468; NP_200015; NP_200012; NP_201520; NP_197953; NP_196720; NP_197346; NP_193098; NP_195688; NP_193408; NP_195408; NP_194543; NP_195006; NP_191608; NP_190595; NP_187713; NP_179915; NP_181113; NP_179810; NP_182021; NP_177887; NP_177631; NP_176491; NP_173695; NP_175104; NP_173609; NP_177931; NP_174636; NP_177307; NP_177301; AAP13384; AAS00621; AA039764; AAP92125; AAL40870; AAG59619; AAN85707; NP_181186; AAX57275; Q3T5N4; QOJQF7; Q9LWV3; Q6J1A5; Q64MA1; AAP83325; AAP83323; AAP83324; AAP83322; AAP83321; AAN02487; AAN02488; AAN02486 [0128] In some embodiments, the DREB polypeptide is a TaDREB3-like polypeptide. [0129] A TaDREB3-like polypeptide, as referred to herein, should be understood as any DREB polypeptide which exhibits at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 82%, at least about 84%, at least about 86%, at least about 88%, at least about 90%, at least about 92%, at least about 94%, at least about 96%, at least about 98%, at least about 99%, or 100% sequence identity to NCBI protein accession number ABC86564 and/or CRT/DRE binding factor 5 (AAY32551; Miller et al., 2006, Mol. Genet. Genomics 275(2), 193-203). When comparing amino acid sequences to calculate a percentage identity, the compared sequences should be compared over a comparison window of at least about 50 amino acid residues, at least about 100 amino acid residues, at least about 150 amino acid residues, or over the full length of ABC86564 and/or AAY32551. The comparison window may comprise additions or deletions (i.e. gaps) of about 20% or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Optimal alignment of sequences for aligning a comparison window may be conducted by computerised implementations of algorithms such as the BLAST family of programs as hereinbefore described. [0130] In some embodiments, the TaDREB3-like polypeptide comprises a polypeptide - 32 encoded by an mRNA comprising the nucleotide sequence set forth in NCBI accession number DQ353853. [0131] In light of the above, in a second aspect the present invention provides a method for improving the stress tolerance of a plant, the method including expressing a nucleotide sequence of interest, which when expressed by one or more cells of the plant improves the stress tolerance of the plant, wherein the nucleotide sequence of interest is operably connected to a stress inducible transcriptional control sequence according to a first aspect of the invention, wherein the nucleotide sequence of interest is heterologous with respect to the transcriptional control sequence. [0132] As set out above, the present invention contemplates expression of a nucleotide sequence of interest under the control of a stress inducible transcriptional control sequence. In some embodiments, this is effected by introducing into the plant a nucleic acid molecule such as a vector which comprises a nucleotide sequence of interest operably connected to a stress inducible transcriptional control sequence. [0133] The nucleic acid molecule may be introduced into the plant via any method known in the art. For example, an explant or cultured plant tissue may be transformed with a nucleic acid molecule, wherein the explant or cultured plant tissue is subsequently regenerated into a mature plant including the nucleic acid molecule; a nucleic acid may be directly transformed into a plant seed, either stably or transiently; a nucleic acid may be introduced into a seed via plant breeding using a parent plant that carries the nucleic acid molecule; and the like. [0134] In some embodiments, the nucleic acid molecule is introduced into a plant cell via transformation. Plants may be transformed using any method known in the art that is appropriate for the particular plant species. Common methods include Agrobacterium mediated transformation, microprojectile bombardment based transformation methods and direct DNA uptake based methods. Roa-Rodriguez et al. (Agrobacterium-mediated transformation of plants, 3 'd Ed. CAMBIA Intellectual Property Resource, Canberra, Australia, 2003) review a wide array of suitable Agrobacterium-mediated plant transformation methods for a wide range of plant species. Other bacterial-mediated plant transformation methods may also be utilised, for example, see Broothaerts et al., 2005 (Nature 433: 629-633). Microprojectile bombardment may also be used to transform plant -33 tissue and methods for the transformation of plants, particularly cereal plants, are reviewed by Casas et al., 1995 (Plant Breeding Rev. 13: 235-264). Examples of direct DNA uptake transformation protocols such as protoplast transformation and electroporation are described in detail in Galbraith et al. (eds.), Methods in Cell Biology Vol. 50, Academic Press, San Diego, 1995). In addition to the methods mentioned above, a range of other transformation protocols may also be used. These include infiltration, electroporation of cells and tissues, electroporation of embryos, microinjection, pollen-tube pathway-, silicon carbide- and liposome mediated transformation. Methods such as these are reviewed by Rakoczy-Trojanowska, 2002 (Cell. Mol. Biol. Lett. 7: 849-858). A range of other plant transformation methods may also be evident to those of skill in the art and, accordingly, the present invention should not be considered in any way limited to the particular plant transformation methods exemplified above. [0135] As set out above, a transcriptional control sequence of the present invention is introduced into a plant cell such that the nucleotide sequence of interest is operably connected to the transcriptional control sequence. The present invention contemplates any method to effect this. For example, a nucleotide sequence of interest may be incorporated into the nucleic acid molecule that comprises the transcriptional control sequence, and be operably connected thereto. In this way, the nucleotide sequence of interest and transcriptional control sequence are both introduced into the plant. Alternatively, a transcriptional control sequence of the present invention may be inserted into the plant genome such that it is placed in operable connection with an endogenous nucleic acid sequence. As would be recognised by one of skill in the art, the insertion of the transcriptional control sequence into the plant genome may be either by non-site specific insertion using standard transformation vectors and protocols, or by site-specific insertion, for example, as described in Terada et al., 2002 (Nat. Biotechnol. 20: 1030 1034). [0136] The present invention also contemplates expression of a nucleotide sequence of interest which is "heterologous with respect to the transcriptional control sequence". A nucleotide sequence which is "heterologous with respect to the transcriptional control sequence" should be understood to include any nucleotide sequence other than that which is operably connected to the transcriptional control sequence in its natural state. For example, in embodiments where the transcriptional control sequence includes the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2, in their natural state - 34 SEQ ID NO: 1 and SEQ ID NO: 2 are operably connected to the HDZipl-4 transcription factor gene in wheat. Similarly, in embodiments where the transcriptional control sequence includes the nucleotide sequence set forth in SEQ ID NO: 3, in its natural state SEQ ID NO: 3 is operably connected to the HDZipl-3 transcription factor gene in wheat. [0137] As would be recognised by one of skill in the art a sequence that is "heterologous with respect to the transcriptional control sequence", including a sequence which is "heterologous with respect to SEQ ID NO: 1", which is "heterologous with respect to SEQ ID NO: 2", or which is "heterologous with respect to SEQ ID NO: 3", may be derived from the same organism or a different organism from which the transcriptional control sequence, SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, respectively, is derived. [0138] In a third aspect, the present invention also provides a nucleic acid construct including a nucleotide sequence of interest operably connected to a transcriptional control sequence which is stress inducible in a plant, wherein the transcriptional control sequence is heterologous with respect to the transcriptional control sequence, and wherein the transcriptional control sequences includes: (i) the nucleotide sequence set forth in one of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3; or (ii) a functionally active fragment or variant of the nucleotide sequence set forth in one of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. [0139] The nucleic acid construct of the third aspect of the present invention may comprise any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. For example, the nucleic acid construct may comprise single- and/or double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. In addition, the nucleic acid construct may comprise triple stranded regions comprising RNA or DNA or both RNA and DNA. The nucleic acid construct may also comprise one or more modified bases or DNA or RNA backbones modified for stability or for other reasons. A variety of modifications can be made to DNA and RNA; thus the term "nucleic acid construct" embraces chemically, enzymatically, or metabolically modified forms.

- 35 [0140] In some embodiments, the nucleic acid construct comprises DNA. Accordingly, the nucleic acid construct may comprise, for example, a linear DNA molecule, a plasmid, a transposon, a cosmid, an artificial chromosome and the like. Furthermore, the nucleic acid construct may be a separate nucleic acid molecule or may be a part of a larger nucleic acid molecule. [0141] In some embodiments of the third aspect of the present invention, the stress inducible transcriptional control sequence, and functionally active fragments or variants thereof, may be as hereinbefore described. [0142] In some embodiments, the transcriptional control sequence is stress inducible in a monocot plant as hereinbefore described. For example, in some embodiments the transcriptional control sequence is stress inducible in a cereal crop plant, such as a wheat, rice or barley plant as hereinbefore described. [0143] In some embodiments, the nucleotide sequence of interest includes a nucleotide sequence which, when expressed by one or more cells of a plant, improves the stress tolerance of the plant as hereinbefore described. [0144] In some embodiments, the nucleotide sequence of interest encodes a DREB polypeptide as hereinbefore described. In some embodiments, the DREB polypeptide is a TaDREB3-like polypeptide as hereinbefore described. [0145] In some embodiments, the nucleic acid construct may further include a nucleotide sequence defining a transcription terminator. The term "transcription terminator" or "terminator" refers to a DNA sequence at the end of a transcriptional unit which signals termination of transcription. Terminators are generally 3'-non-translated DNA sequences and may contain a polyadenylation signal, which facilitates the addition of polyadenylate sequences to the 3'-end of a primary transcript. As with promoter sequences, the terminator may be any terminator sequence which is operable in the cells, tissues or organs in which it is intended to be used. Examples of suitable terminator sequences which may be useful in plant cells include: the nopaline synthase (nos) terminator, the CaMV 35S terminator, the octopine synthase (ocs) terminator, potato proteinase inhibitor gene (pin) terminators, such as the pin!! and pin!!! terminators and the like.

- 36 [0146] In some embodiments of the third aspect of the present invention, the nucleic acid construct may include an expression cassette including the structure: [N], - TCS - [N]x - Sol - [N]y - TT - [N]z) wherein: [N]w includes one or more nucleotide residues, or is absent; TCS defines the transcriptional control sequence; [N]x includes one or more nucleotide residues, or is absent; Sol includes the nucleotide sequence of interest that is heterologous with respect to the TCS, wherein the nucleotide sequence of interest encodes an mRNA or non translated RNA, and is operably connected to the TCS; [N]y includes one or more nucleotide residues, or is absent; TT includes a nucleotide sequence defining a transcription terminator; and [N]z includes one or more nucleotide residues, or is absent. [0147] The nucleic acid construct of the third aspect of the present invention may further include other nucleotide sequences as desired. For example, the nucleic acid construct may include an origin of replication for one or more hosts; a selectable marker gene which is active in one or more hosts; or the like. [0148] As used herein, the term "selectable marker gene" includes any gene that confers a phenotype on a cell, in which it is expressed, to facilitate the identification and/or selection of cells which are transfected or transformed with a nucleic acid construct of the invention. A range of nucleotide sequences encoding suitable selectable markers are known in the art. Exemplary nucleotide sequences that encode selectable markers include: antibiotic resistance genes such as ampicillin-resistance genes, tetracycline resistance genes, kanamycin-resistance genes, the AURI-C gene which confers resistance to the antibiotic aureobasidin A, neomycin phosphotransferase genes (e.g. nptl and nptll) and hygromycin phosphotransferase genes (e.g. hpt); herbicide resistance genes including glufosinate, phosphinothricin or bialaphos resistance genes such as phosphinothricin acetyl transferase-encoding genes (e.g. bar), glyphosate resistance genes including 3-enoyl pyruvyl shikimate 5-phosphate synthase-encoding genes (e.g. aroA), bromyxnil resistance genes including bromyxnil nitrilase-encoding genes, sulfonamide resistance genes including dihydropterate synthase-encoding genes (e.g.

- 37 sul) and sulfonylurea resistance genes including acetolactate synthase-encoding genes; enzyme-encoding reporter genes such as GUS and chloramphenicolacetyltransferase (CAT) encoding genes; fluorescent reporter genes such as the green fluorescent protein encoding gene; and luminescence-based reporter genes such as the luciferase gene, amongst others. [0149] The nucleic acid constructs described herein may further include nucleotide sequences intended for the maintenance and/or replication of the construct in prokaryotes or eukaryotes and/or the integration of the construct or a part thereof into the genome of a eukaryotic or prokaryotic cell. [0150] In some embodiments, the nucleic acid construct of the present invention is adapted to be at least partially transferred into a plant cell via Agrobacterium-mediated transformation. Accordingly, in some embodiments, the nucleic acid construct comprises left and/or right T-DNA border sequences. Suitable T-DNA border sequences would be readily ascertained by one of skill in the art. However, the term "T-DNA border sequences" should be understood to include, for example, any substantially homologous and substantially directly repeated nucleotide sequences that delimit a nucleic acid molecule that is transferred from an Agrobacterium sp. cell into a plant cell susceptible to Agrobacterium-mediated transformation. By way of example, reference is made to the paper of Peralta and Ream (Proc. Nat/. A cad. Sci. USA, 82(15): 5112-5116, 1985) and the review of Gelvin (Microbiology and Molecular Biology Reviews, 67(1): 16-37, 2003). [0151] In some embodiments, the present invention also contemplates any suitable modifications to the nucleic acid construct which facilitate bacterial mediated insertion into a plant cell via bacteria other than Agrobacterium sp., for example, as described in Broothaerts et al., 2005 (supra). [0152] Those skilled in the art will be aware of how to produce the constructs described herein, and of the requirements for obtaining the expression thereof, when so desired, in a specific cell or cell-type under the conditions desired. In particular, it will be known to those skilled in the art that the genetic manipulations required to perform the present invention may require the propagation of a nucleic acid construct described herein or a derivative thereof in a prokaryotic cell such as an E. coli cell or a plant cell or an animal cell. Exemplary methods for cloning nucleic acid molecules are described in Sambrook et - 38 al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York, 2000). [0153] In a fourth aspect, the present invention provides a genetically modified cell including a nucleic acid construct of the third aspect of the invention, or a genomically integrated form thereof. [0154] As referred to herein, a "genetically modified cell" includes any cell having a non naturally occurring and/or introduced nucleic acid. Generally, in the case of the cells of the fourth aspect of the present invention, the introduced and/or non-naturally occurring nucleic acid includes a nucleic acid construct of the third aspect of the invention. [0155] Cells of the fourth aspect of the invention may be transformed cells which contain the nucleic acid construct of the third aspect of the invention, or a genomically integrated form thereof, or progeny of such transformed cells which retain the construct or a genomically integrated form thereof. [0156] As set out above, the nucleic acid construct may be maintained in the cell as a nucleic acid molecule, as an autonomously replicating genetic element (e.g. a plasmid, cosmid, artificial chromosome or the like) or it may be integrated into the genomic DNA of the cell. [0157] As used herein, the term "genomic DNA" should be understood in its broadest context to include any and all endogenous DNA that makes up the genetic complement of a cell. As such, the genomic DNA of a cell should be understood to include chromosomes, mitochondrial DNA, plastid DNA, chloroplast DNA, endogenous plasmid DNA and the like. As such, the term "genomically integrated" contemplates chromosomal integration, mitochondrial DNA integration, plastid DNA integration, chloroplast DNA integration, endogenous plasmid integration, and the like. The "genomically integrated form" of the construct may be all or part of the construct. [0158] The cells contemplated by the fourth aspect of the present invention include any prokaryotic or eukaryotic cell. In some embodiments, the cell is a plant cell. In some embodiments the cell is a monocot plant cell. In some embodiments the cell is a cereal crop plant cell, for example a wheat, rice or barley plant cell as hereinbefore described.

- 39 [0159] In some embodiments, the cell may also include a prokaryotic cell. For example, the prokaryotic cell may include an Agrobacterium sp. cell (or other bacterial cell), which carries the nucleic acid construct and which may, for example, be used to transform a plant. In some embodiments, the prokaryotic cell may be a cell used in the construction or cloning of the nucleic acid construct (e.g. an E. coli cell). [0160] In a fifth aspect, the present invention provides a multicellular structure including one or more cells of the fourth aspect of the invention. [0161] In some embodiments, the multicellular structure comprises a plant or a part, organ or tissue thereof. As referred to herein, "a plant or a part, organ or tissue thereof" should be understood to specifically include a whole plant; a plant tissue; a plant organ; a plant part; a plant embryo; and cultured plant tissue such as a callus or suspension culture. [0162] In some embodiments of the fifth aspect of the present invention, a nucleotide sequence of interest is expressed in one or more cells of the plant or a part, organ or tissue thereof in response to stress. [0163] In some embodiments, the multicellular structure includes a monocot plant or a part, organ or tissue thereof. In some embodiments the multicellular structure includes a cereal crop plant or a part, organ or tissue thereof. For example, in some embodiments, the multicellular structure includes a wheat, rice or barley plant or a part, organ or tissue thereof, as hereinbefore described. [0164] In some embodiments, the present invention also provides a plant or a part, organ or tissue thereof having improved stress tolerance, wherein the plant comprises one or more cells of the fourth aspect of the invention. [0165] In some embodiments of the fifth aspect of the present invention, the plant or a part, organ or tissue thereof of the multicellular structure has improved stress tolerance relative to a plant or a part, organ or tissue thereof which does not include one or more cells of the fourth aspect of the invention. [0166] A plant or a part, organ or tissue thereof of the multicellular structure according to - 40 the fifth aspect of the invention may be regenerated from transformed plant material such as transformed callus, cultured embryos, explants or the like using standard techniques of the art. Such plants are typically referred to as To plants. Plants according to this aspect of the invention should also be understood to include progeny of To plants. Such progeny plants may result from self fertilisation of the To plants or crossing of the To plants with one or more other plants of the same species, or of a different species to form hybrids. As will be appreciated, the nucleic acid construct of the second aspect of the invention may segregate in progeny plants, and thus the plants of the multicellular structure according to the fifth aspect of the invention extend only to those progeny plants that include the construct. [0167] The inventors, for the first time, have identified the nucleic acid sequence of the promoter region for the TdHDZipI-4 and TdHDZipI-3 genes. Therefore, in a sixth aspect the present invention provides an isolated nucleic acid comprising a nucleotide sequence defining a transcriptional control sequence which directs expression of an operably connected nucleotide sequence of interest in one or more cells of a plant when the plant is subject to stress, wherein said transcriptional control sequence includes: (i) the nucleotide sequence set forth in one of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3; or (ii) a functionally active fragment or variant of the nucleotide sequence set forth in one of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. [0168] In some embodiments, the transcriptional control sequence directs expression of the nucleotide sequence of interest in one or more cells of a monocot plant as hereinbefore described when the plant is subject to stress (i.e. the transcriptional control sequence is stress inducible). For example, in some embodiments the transcriptional control sequence is stress inducible in a cereal crop plant, such as a wheat, rice or barley plant as hereinbefore described. [0169] In some embodiments of the sixth aspect of the invention, the functionally active fragment or variant is as hereinbefore described. [0170] In some embodiments, the nucleotide sequence of interest includes a nucleotide sequence which, when expressed by one or more cells of a plant, improves the stress tolerance of the plant as hereinbefore described.

- 41 [0171] In some embodiments, the nucleotide sequence of interest encodes a DREB polypeptide as hereinbefore described. In some embodiments, the DREB polypeptide is a TaDREB3-like polypeptide as hereinbefore described. [0172] It is to be noted that where a range of values is expressed, it will be clearly understood that this range encompasses the upper and lower limits of the range, and all values in between these limits. [0173] Furthermore, the term "about" as used in the specification means approximately or nearly and in the context of a numerical value or range set forth herein is meant to encompass variations of +/- 10% or less, +/- 5% or less, +/- 1% or less, or +/- 0.1% or less of and from the numerical value or range recited or claimed. [0174] It will be apparent to the person skilled in the art that while the invention has been described in some detail for the purposes of clarity and understanding, various modifications and alterations to the embodiments and methods described herein may be made without departing from the scope of the inventive concept disclosed in this specification. [0175] Finally, reference is made to standard textbooks of molecular biology that contain methods for carrying out basic techniques encompassed by the present invention, including DNA restriction and ligation for the generation of the various genetic constructs described herein. See, for example, Sambrook and Russell, Molecular Cloning: A Laboratory Manual (3rd edition), Cold Spring Harbor Laboratory Press, 2001. [0176] The invention is further illustrated in the following examples. The examples are for the purpose of describing particular embodiments only and are not intended to be limiting with respect to the above description. EXAMPLE 1 Isolation of HD-ZipI Transcription Factors from Wheat [0177] Wheat homologues of the y-clade HD-Zip transcription factors AtHB7 and AtHB12 (from Arabidopsis) and Oshox6, Oshox22, and Oshox24 (from rice) were isolated using the yeast one-hybrid (Y1 H) procedure. A wheat cDNA library (WHSL) was prepared from the flag leaf and spikes of the Australian drought-tolerant cultivar, Triticum - 42 aestivum L. cv RAC875, that had been subjected to high temperatures under drought stress or well-watered conditions. Plants were grown in well-watered conditions to anthesis in a growth cabinet under the 14 hour light at 22C / 10 hour dark at 10 C cycle until flowering; then day temperature was gradually increased to 400C (10 minutes of each 24, 27, 29, 30, 32, 34, 36, and 400C). As soon as the temperature reached 400C the first samples of flag leaves and spikes (at different stages of development) were collected; the plants were kept at 400C for a further 3.5 hours and a second set of samples was collected. After that, temperature was stepwise lowered to 220C (at 280C 2 hours and at 220C for the remainder of the day and overnight), and this temperature cycle was applied for 7 days. Watering was withheld on the second day. Plants demonstrated signs of water deficiency at day 4. The samples were collected at day 4 and day 7 at the same time points as for plants grown under well-watered conditions. [0178] Tissue samples were collected from five plants. Collected plant material was immediately frozen in liquid nitrogen and stored at -800C until RNA extraction. Total RNA was isolated from wild type and transgenic plant leaf tissue using the TRizol kit (Invitrogen, Victoria, Australia). A mixture of equal amounts of total RNA from each plant was used for cDNA library preparation. The cDNA library produced was designated Wheat Drought and Heat Stress library (WHSL). [0179] The bait sequence used in the Y1H procedure for isolation of homologous wheat genes from the WHSL library was based on a cis-element known to be present in the promoter regions of the Arabidopsis and rice genes. The sequence of the cis-element was CAATNATTG, and this bait sequence had been used previously to isolate clones of TaHDZipl-1 (Genbank Accession No. DQ353855) and TaHDZipl-2 (Genbank Accession No. DQ353856) (Lopato et al., 2006, "Isolation of plant transcription factors using a modified yeast one-hybrid system", Plant Methods 2: 3-17). However, the previous study did not yield clones of the desired y-clade homologues. [0180] A wheat homologue of the AtHB7 gene was identified as a contig of several wheat EST sequences from public databases. Nested PCR was used to isolate a y clade sequence of the wheat homologue of Oshox6, designated herein as TaHDZipl-3, from the WHSL cDNA library. As HD-Zip transcription factors are known to heterodimerise within each class, TaHDZipl-3 was then used as bait in a yeast two hybrid (Y2H) screen to isolate dimerisation partners from the WHSL cDNA library - 43 according to the methods described in Kovalchuk et al., 2009, Plant Biotechnol. J. Through this approach, a cDNA for TaHDZipl-4 was isolated (a putative paralogue that is homologous with Oshox22). Coding sequences of TaHDZipl-3 and TaHDZipl-4 were used as probes to screen a BAC library prepared from genomic DNA of Triticum durum cv. Langdon (Cenci et al. 2003, "Construction and characterization of a half million clone BAC library of durum wheat (Triticum turgidum ssp durum)", Theor. Apple. Genet. 107: 931-939). This screen yielded BAC clones containing T. durum orthologues designated TdHDZipl-3, TdHDZipl-4a and TdHDZipl-4b. The identified BAC clones were then directly sequenced, which obtained the full length sequences of genes, including their transcriptional control regions (e.g. promoters and terminators). Comparison of the cDNA and genomic sequences also enabled identification of intron and exon spanning regions and revealed the presence of the single intron, which was inserted in a part of the coding sequence that encodes a leucine zipper domain. EXAMPLE 2 Transcript Expression of TaHDZipi-3 and TaHDZipi-4 in Wheat Tissues [0181] Expression of the TaHDZipl-3 and TaHDZipl-4 genes was analysed by efficiency adjusted real-time quantitative PCR in different wheat tissues of T. aestivum cv Chinese spring. A modified MACt method adjusted for amplification efficiency was used to determine the number of copies of the transgene per genome in each tissue sample (Yuan et al., 2008, "Statistical methods for efficiency adjusted real-time PCR quantification," Biotechnol. J. 3: 112-123). DNA was extracted from various wheat tissue using a freeze-drying method described by Shavrukov et al., 2010, "HvNax3 - a locus controlling shoot sodium exclusion derived from wild barley (Hordeum vulgare ssp. Spontaneum)", Funct. Integ. Genomics 10: 277-291. Prior to use in real-time quantitative PCR, each DNA sample was diluted with sterile deionised water to be within the copy standard serial dilution range (12.5 to 200 ng/pl). For template loading normalisation, PCRs were performed using primers and probes complimentary to single-copy endogenous reference genes. [0182] For the various transgenes analysed, a portion of the hygromycin resistance gene (Hyg) was used as the target sequence. The complimentary oligonucleotide sequences were: HygF 5'-CGCTCGTCTGGCTAAGATCG-3' (SEQ ID NO: 8); HygR 5' AGGGTGTCACGTTGCAAGAC-3' (SEQ ID NO: 9); and dual-labelled TaqMan probe Hyg 5'-FAM-TGCCTGAAACCGAACTGCCCGCTG-BHQ1-3' (SEQ ID NO: 10). Real-time -44 quantitative PCR was performed on a LightCycler 480 thermal cycler. Each PCR comprised 1x IQ Supermix (Bio-Rad); forward and reverse primers (400 nM each); dual labelled probe (200 nM); DNA (2 pl) and deionised water to a total volume of 10 pl. The thermal cycling parameters were 950C for three minutes followed by 40 cycles of 950C for 15 seconds and 600C for 60 seconds with fluorescence readings acquired at each cycle on the yellow and green channels. [0183] To allow for the calculation of transgene copy number from unknown DNA samples, a copy-standard serial dilution series was set up. Genomic DNA from a plant known to contain a single copy of the hygromycin gene in addition to the single-copy endogenous reference gene was extracted and diluted. Amounts of 400 ng, 200 ng, 100 ng, 50 ng and 25 ng were used. Three replicate PCRs for each unknown sample and each diluted copy-standard sample were performed with each primer/probe set. Ct values were calculated using the supplied software for the LightCycler 480 thermal cycler. The PCR efficiency for each primer/probe set was determined via analysis of the Ct values obtained from the diluted copy-standard series. Subsequently, all Ct values were adjusted using these PCR efficiency values prior to calculating ACtadjusted (ACtadjusted = Hyg Ctadjusted - reference gene Ctadjusted). The transgene copy number for each unknown sample was determined by calculating 2-AACtadjusted. (AACtadjusted is defined as the difference between the average ACtadjusted of the copy standard series and the ACtadjusted of the unknown sample). Calculated transgene copy numbers were rounded to the closest integer. [0184] As shown in Figure 1A, both HDZip/ genes (in the absence of stress) were predominantly expressed in bracts, pistils and endosperm tissues of wheat. The level of expression of the HDZip/ genes in other tissues was relatively low. [0185] The spatial expression pattern of the TaHDZipI-3 and TaHDZipI-4 genes in the developing wheat stem of T. aestivum cv RAC875 was studied in 4 different internodes and peduncle at different stages of stem development. Briefly, RAC875 seeds were surface sterilised and left to germinate for two days at 400C in petri dishes on wet filter paper. Seeds were then left to resume germination at room temperature for 2-3 days. Five seeds each were planted in 16 pots with coco peat, and grown under room conditions of 8/16 hour day/night lighting and 220C/180C day/night temperatures. 10 stems were harvested at each developmental stage of: about 100 mm long (stem 1); about 300 mm - 45 long with emerging awns (stem 2); about 400 mm long with emerging heads (stem 3); and about 500 mm long with spikes at anthesis and emerged peduncles (stem 4). At each developmental point stems with 5 internodes were harvested and the internodes were measured for length and pooled by type. For RNA extraction, internodes were ground in liquid nitrogen using a mortar and pestle and ground powder was processed for RNA extraction using the TRIzol kit (Invitrogen, Victoria, Australia). cDNA was then generated from the extracted total RNA. [0186] The results of the spatial expression studies are shown in Figure 1B. The stem developmental series revealed that wheat y-clade TaHDZipI-3 and TaHDZipI-4 genes are differentially expressed during normal stem development. The pattern of TaHDZipI-3 expression during stem development differs between internodes. Internodes 1, 2, 3, and 5 show a general increase in expression from stem stage 1 to stem stage 3 followed by a decrease at stem stage 4. However, expression of TaHDZipI-3 at internode 4 displays a different expression pattern where a steady increase from stem stage 1 to stem stage 4 is seen. Expression of TaHDZipI-4 shows a similar pattern of expression in all five internodes, where there is an increase from stem stage 1 to reach maximal expression at stem stage 3, followed by a dramatic decrease at stem stage 4. The peak in expression of TaHDZipI-4 at stem stage 3 is not correlated with the rate of stem elongation as it is seen in both internode 1 and 2 which have reached their maximal length by stem stage 1 and are no longer elongating. Also, the peduncle is likely still elongating after anthesis at stem stage 4 yet shows the same decreases in expression as the other internodes. EXAMPLE 3 Abiotic Stress Responses of TaHDZipi-3 and TaHDZipI-4 in Leaf Tissues [0187] Wheat cv RAC875 plants were subjected to ABA, cold, and water deficit treatments using the following methodology. ABA treatment of wheat cv RAC875 using hydroponics [0188] RAC875 seeds were surface sterilised and left to germinate in growth solution (Shavrukov et al., 2006, "Screening for sodium exclusion in wheat and barley", In: Proceedings of the 13 th Australian society of Agronomy conference, Perth, Australia) for two days at 40C in petri dishes on wet filter paper. Seeds were then left to resume germination at room temperature for 2-3 days until they were transplanted to two hydroponic growth units. Growth continued for one week in a glasshouse and the growth - 46 solution was renewed the day before ABA treatment. ABA was dissolved in DMSO to make a 50 mM stock and 2 ml was added to the growth solution to a final concentration of 200 pM. Control plants received 2 ml of DMSO. Aerial portions of three seedlings were harvested at each time point (0, 1, 2 and 4 hours), put into 10 ml tubes, snap frozen in liquid nitrogen and stored at -800C until processing for RNA extraction. Total RNA was isolated from the samples using the TRIzol kit (Invitrogen, Victoria, Australia). [0189] The hydroponics unit consisted of a 500 ml container with an insert that could hold up to 100 0.5 ml bottomless microtubes to support the seedlings. 30 seedlings of similar size were transferred to the microtubes so that the grain sat just above, but the roots were in contact with, the growth solution. The growth solution was identical to that described in Shavrukov, et al., 2012, "The use of hydroponics in abiotic stress tolerance research. In: Hydroponics: A Standard Methodology for Plant Biological Researchers", (Ed.: Toshiki Asao), ISBN: 979-953-307-480-0, InTech International Publisher (in Press) except that Na 2 SiO 3 was excluded. Growth solution was aerated with an aquarium pump using a hose and an air stone. Cold treatment of wheat cv RAC875 [0190] RAC875 seeds were surface sterilised and left to germinate for two days at 40C in petri dishes on wet filter paper. Seeds were then left to resume germination at room temperature for 2-3 days until they were planted, four seeds each, in 12 x 15 cm pots with coco peat. Growth continued for three weeks in a glasshouse until the commencement of cold treatments in a growth cabinet (BINDER, Tuttlingen, Germany). Plants were grown under 14 hour/10 hour day/night lighting in the cabinet. Growth for two days was at a constant 180C. At the beginning of the cold treatment the temperature was rapidly decreased at 09:00 until the required 40C was reached (at around 10:30), and maintained for 47 hours until the temperature was gradually increased to 180C from 08:00 until 22:00. Temperature was restored to 180C and plants recovered for a further 36 hours. The 3 rd and 4 th leaf of randomly selected plants were harvested at -0.25, 1, 2, 4, 8, 12, 24, 36, and 48 hours for cold treatment and 60, 72, 84, and 96 hours for recovery. Leaves were snap frozen in liquid nitrogen and kept at -800C until processing for RNA extraction using the TRIzol kit (Invitrogen, Victoria, Australia).

- 47 Exposure of wheat cv RAC875 to pot based drought [0191] Eight RAC875 plants were grown in 22 cm pots under room conditions of 8/16 hour day/night lighting and 220C/180C day/night temperatures. Water was withheld after 3 weeks of growth and the youngest fully expanded leaf was sampled from a single different tiller at each time point. Re-watering occurred 31 days after initiation of withholding (day 0). Soil volumetric water content was recorded throughout the experiment using a FieldScout TDR 300 Soil Moisture Meter (Spectrum Technologies, Inc., U.S.A.) and three plants with a similar drying pattern were used for the analysis of downstream genes by quantitative PCR. [0192] The results of the abiotic stress responses are shown in Figure 2. TaHDZipI-4 was strongly induced 4 hours after addition of 200 pM ABA, whereas TaHDZipI-3 expression appeared to be unaffected (Figure 2A). [0193] With respect to cold treatment, exposure to 40C led to the sustained induction of TaHDZip/-4 over the 48 hours of treatment (Figure 2B). However, TaHDZipI-3 showed no measureable transcript levels under cold conditions. [0194] Pot grown RAC875 subjected to a prolonged water deficit had increasing levels of expression of both genes from day 8 to day 25 as soil water potential decreased (Figure 2C). The dramatic reduction of expression seen at day 31 in response to rewatering, also confirms a water deficit response in expression. [0195] The transcriptional responses of TaHDZipI-3 and TaHDZipI-4 were also analysed under cyclic drought conditions in two wheat cultivars, namely RAC875 and Kukri. These two cultivars are derived from the same ancestors but differ in their yield responses under different drought conditions. The drought regime and time points for collection of samples from the plants for RNA extraction and subsequent expression analysis are shown in Figure 3A. In the experiment, where plants were subjected to severe cyclic water-limiting conditions, RAC875 (drought-tolerant cultivar) showed significantly higher grain yield under cyclic water availability (Figure 3B) compared to Kukri (drought-susceptible cultivar - Figure 3C), producing 44% more grain compared to Kukri, respectively. In the second experiment, where plants were subjected to a milder drought stress, the differences between cultivars were less pronounced, with only RAC875 showing significantly higher grain yield under the cyclic water treatment. Grain number per spike and the percentage - 48 of aborted tillers were the major components that affected yield under cyclic water stress (Izanloo et al., 2008, "Different mechanisms of adaptation to cyclic water stress in two South Australian bread wheat cultivars", J. Exp. Bot. 59: 3327-3346). Both cultivars show different patterns of y-clade transcript regulation as well as a difference in patterns of regulation of particular genes in the same cultivar. TaHD-ZipI-3 expression is very weakly induced in RAC875 submitted to water deficit when compared with expression in well watered plants. This weak expression is maintained through the first wilting cycle but is much lower at day 23, the second wilt point. Kukri displays much higher TaHD-ZipI-3 basal expression under well-watered conditions than RAC875. Also, a stronger drought response is shown for TaHD-ZipI-3 expression in Kukri than RAC875, the strongest expression is seen at the wilting points, days 14 and 23. By comparison with TaHD-Zip/-3, expression of TaHD-Zip/-4 is dramatically up regulated by day five of the first water withholding cycle in both cultivars. In RAC875 the rapid induction of TaHD-Zip/-4 is followed by a decline throughout the first wilting cycle and is also greatly reduced at the second wilt point. The drought responsiveness of TaHD-ZipI-4 expression in Kukri is markedly different to that of RAC875, where TaHD-ZipI-4 expression is maintained at a relatively consistent level of induction during the first wilting cycle and at the final wilt point of the second cycle. [0196] The transcriptional responses of TaHDZipI-3 and TaHDZipI-4 were also analysed under high salinity conditions according to the methods described in Shavrukov et al., 2012 (supra). As shown in Figure 4, the expression of TaHDZipI-4 is moderately induced in leaves of T. aestivum cv Chinese spring seedlings grown on hydroponics after addition of 100 mM NaCl, whereas TaHDZipI-3 expression appears to be weakly and negatively affected. EXAMPLE 4 Identification and Analysis of Promoter Sequences of HDZipI-3 and HDZipI-4 Genes [0197] The sequence of the BAC clones identified in Example 1 which contained the T. durum orthologues designated TdHDZipl-3, TdHDZipI-4a and TdHDZipI-4b was analysed upstream of the start codon for each gene to identify the transcriptional control sequence/promoter sequence for each gene. The nucleotide sequence of the transcriptional control sequence for TdHDZipI-4a is set forth in SEQ ID NO: 1 (1,915 bp) and SEQ ID NO: 11 (5,082 bp). The nucleotide sequence of the transcriptional control sequence for TdHDZipI-4b is set forth in SEQ ID NO: 2. The nucleotide sequence of the - 49 transcriptional control sequence for TdHDZipI-3 is set forth in SEQ ID NO: 3 (2,142 bp) and SEQ ID NO: 12 (5,325 bp). [0198] When the first approximately 1000 base pairs of each promoter sequence (5' to the start codon for each gene) was aligned, no significant homology between the sequences was observed. In fact, the transcriptional control sequence for TdHDZipI-3 (SEQ ID NO: 3) shares 42% identity to the transcriptional control sequence of TdHDZipI-4a (SEQ ID NO: 1). However, as discussed in detail below, the transcriptional control sequence of the genes did share a number of sequence motifs corresponding to various cis-elements. [0199] As shown in Figure 5, the first approximately 1000 base pairs of the promoter sequences of TdHDZipI-4a and TdHDZipI-4b were significantly identical, particularly in the first 650 base pairs immediately 5' to the start codon, confirming that these genes are homeologues. In fact, the transcriptional control sequence for TdHDZipI-4a (SEQ ID NO: 1) shares 70% identity to the transcriptional control sequence of TdHDZipI-4b (SEQ ID NO: 2) over their full lengths. Figure 6 provides the first approximately 1000 base pairs of the promoter sequences of TdHDZipI-3 and highlights the predicted cis-elements present in this region, as discussed in detail below. Identification of cis-elements [0200] The promoter regions of the TdHDZipI-3, TdHDZipI-4a and TdHDZipI-4b genes shared a number of sequence motifs corresponding to the cis-elements MYBR, ABRE, CRT and ABRE (AB15-binding), as predicted by PLACE (http://www.dna.affrc.go.jp/PLACE/). [0201] ABRE elements function in ABA signalling during plant development and abiotic stresses through the interaction with bZIP transcription factors (Marcotte et al., 1989, "Abscisic acid-responsive sequences from the Em gene of wheat", Plant Cell 1: 969-976; Busk and Pages, 1998, "Regulation of abscisic acid-induced transcription", Plant Mol. Biol. 37: 425-435; Skriver and Mundy, 1990, "Gene expression in response to abscisic acid and osmotic stress", Plant Cell 2: 503-512; Choi et al., 2000, "ABFs, a family of ABA responsive element binding factors", J. Biol. Chem. 275: 1723-1730; Kang et al., 2002, "Arabidopsis basic leucine zipper proteins that mediate stress-responsive abscisic acid signalling", Plant Cell. 14: 343-357; Oh et al., 2005, "Arabidopsis CBF3/DREB1A and ABF3 in transgenic rice increased tolerance to abiotic stress without stunting growth", - 50 Plant Physiol. 138: 341-351; Choi et al., 2005, "Arabidopsis Calcium-Dependent Protein Kinase AtCPK32 Interacts with ABF4, a Transcriptional Regulator of Abscisic Acid Responsive Gene Expression, and Modulates Its Activity", Plant Physiol. 139: 1750-1761; and Nakashima et al., 2006, "Transcriptional regulation of AB13- and ABA-responsive genes including RD29B and RD29A in seeds, germinating embryos, and seedlings of Arabidopsis", Plant Mol. Biol. 60: 51-68). [0202] Another predicted element, which is potentially responsible for activation of the TdHDZipl-3, TdHDZipI-4a and TdHDZipI-4b promoters under stress is a MYB responsive (MYBR) cis-element, (CT)IWAACCA, present in each promoter. MYBR was earlier identified in the dehydration responsive gene rd22 of Arabidopsis thaliana (Abe et al., 1997, "Role of Arabidopsis MYC and MYB homologues in drought- and abscisic acid regulated gene expression", Plant Cell 9: 1859-1868; and Busk and Pages, 1998, "Regulation of abscisic acid-induced transcription", Plant Mol. Biol. 37: 425-435). [0203] A CRT cis-element was shared between the TdHDZipl-3, TdHDZipI-4a and TdHDZipI-4b gene promoters. In the TdHDZipI-4a/b promoter, the CRT sequence motif acts as a cis-element for the AP2 transcriptional activator HvCBF2 and has the consensus sequence (G/a)(T/c)CGAC. Binding of HvCBF2 to this element has been shown to be regulated by temperature (Xue, 2003, "The DNA-binding activity of an AP2 transcriptional activator HvCBF2 involved in regulation of low-temperature responsive genes in barley is modulated by temperature", Plant J. 33: 373-383). [0204] AB15 encodes a transcription factor that regulates gene expression during embryogenesis and in response to ABA. AB15 is induced by stresses such as cold, salinity and drought. As reported by Carles C et al., 2002 (supra), the cis-element that binds AB15 (AGACACGTGGCATGT) has the consensus sequences A(C/G)ACACG and ACACNNG. [0205] The promoter regions of the TdHDZipI-4a and TdHDZipI-4b genes shared a number of sequence motifs corresponding to cis-elements selected from a CGCG-box (calmod-binding), ABRE (AB15-binding), auxin-response (AUX-resp), MYBR, ABRE, CRT, and ammonium response (ammonium-resp). Detail regarding the ABRE (AB15-binding), MYBR, ABRE and CRT elements is provided above.

- 51 [0206] The calmodulin-binding/CGCG-box (VCGCGB) has been shown to be involved in different signal transduction pathways (Yang and Poovaiah, 2002, "A calmodulin binding/CGCG box DNA-binding protein family involved in multiple signaling pathways in plants", J. Biol. Chem. 277: 45049-45058). [0207] The auxin-response (AUX-resp) element (CATATG) has been reported to be responsible for auxin responsiveness of the gene encoding Small Auxin-upregulated RNA (SAUR) 15A (Xu et al., 1997, "Multiple auxin response modules in the soybean SAUR 15A promoter," Plant Sci.126: 193-201). [0208] The ammonium response cis-element is represented by the sequence motif ACCCTACC, and has been shown to be involved in the ammonium-response of the Chlamydomonas Nial gene encoding nitrate reductase (Loppes and Radoux, 2001, "Identification of short promoter regions involved in the transcriptional expression of the nitrate reductase gene in Chlamydomonas reinhardtif', Plant Mol. Biol. 45: 215-227). [0209] In contrast to the TdHDZipl-4a and TdHDZipI-4b promoters, the TdHDZipl-3 promoter has no clear TATA box. A putative short TATA sequence can be found adjacent to a long GAGA sequence stretch, which is an inverted CT-rich motif similar to the 60 nucleotide region downstream of the transcription start site of the CaMV 35S RNA. This CT-rich motif has been shown to work as an enhancer of gene expression (Pauli et al., 2004, "The cauliflower mosaic virus 35S promoter extends into the transcribed region", J. Virol. 78: 12120-12128). [0210] The TdHDZipl-3 promoter contained a number of cis-elements, including ABRE (AB15-binding), ABRE, GCC-box, light regulated cis-element, MYBR, ABRE-similar, ABRE-related (Ca dependent), vascular expression cis-element, WRKY specific element, CRT, and carbohydrate metabolite-responsive cis-element. Detail regarding the ABRE (AB15-binding), MYBR, ABRE and CRT elements is provided above. In contrast to the TdHDZipl-4a and TdHDZipI-4b promoters, which have no GCC-boxes, the TdHDZipl-3 promoter contains two such cis-elements. GCC-boxes have been found in many pathogene- and wounding-responsive genes, and have been shown to function as ethylene-responsive elements and regulators of jasmonate-responsive gene expression (Brown et al., 2003, "A role for the GCC-box in jasmonate-mediated activation of the PDF1.2 gene of Arabidopsis", Plant Physiol. 132: 1020-1032; and Chakravarthy et al., - 52 2003, "The tomato transcription factor Pti4 regulates defence-related gene expression via GCC box and non-GCC box cis elements", Plant Cell 15: 3033-3050). [0211] The TdHDZipl-3 promoter also contained a type of cis-element (TTTGACY) shown to be involved in a response to both biotic an abiotic stresses. The W-box (T)(T)TGAC(C/T) can be recognised by WRKY factors, which recently were demonstrated to be involved in ABA-dependent response to drought (Ishiguro and Nakamura, 1994, "Characterization of a cDNA encoding a novel DNA-binding protein, SPF1, that recognizes SP8 sequences in the 5' upstream regions of genes coding for sporamin and beta-amylase from sweet potato", Mol. Gen. Genet. 244: 563-571; Rushton et al., 1995, "Members of a new family of DNA-binding proteins bind to a conserved cis-element in the promoters of alpha-Amy2 genes", Plant Mol. Biol. 29: 691-702; and Rushton et al., 1996, "Interaction of elicitor-induced DNA-binding proteins with elicitor response elements in the promoters of parsley PR1 genes", EMBO J. 15: 5690-5700). [0212] The TdHDZipl-3 promoter also contains a cis-element (AGCGGG), which was previously identified in the promoter of Eucalyptus gunnii Cinnamoyl-CoA reductase as a sequence responsible for the vascular expression (Lacombe et al., 2000, "Characterization of cis-elements required for vascular expression of the cinnamoyl CoA reductase gene and for protein-DNA complex formation", Plant J. 23: 663-676). [0213] The TdHDZipl-3 promoter also contains a Carbohydrate Metabolite Signal Responsive Element 1 (CMSRE-1, TGGACGG) found to be responsible for the sucrose inducible expression of the sweet potato sporamin A gene (Morikami et al., 2005, "Two cis-acting regulatory elements are involved in the sucrose-inducible expression of the sporamin gene promoter from sweet potato in transgenic tobacco", Mol. Genet. Genomics. 272: 690-699). EXAMPLE 5 Promoter Activity Using a Transient Expression Assay in Wheat Cell Culture [0214] Activity of the TdHDZipl-3 and TdHDZipl-4 transcriptional control/promoter regions was analysed using a transient expression assay in wheat cell culture. Approximately 3 kb of each promoter sequence was cloned upstream of a GUS-encoding gene in the pMDC164 vector. The promoter sequence of the TdHDZipl-4 gene used in one of the GUS-constructs is set forth in SEQ ID NO: 13. The promoter sequence of the TdHDZipl-3 - 53 gene used in one of the GUS-constructs is set forth in SEQ ID NO: 14. The activity of each promoter was then tested in a transient expression assay using biolistic bombardment of a suspension of T. monoccocum L cell culture with each plasmid construct. [0215] The oligonucleotide primers used for cloning the TdHDZipl-4 promoter into the pMDC164 vector were as follows: TdB4L16_R3 GATACATGTGCGTTGGGCTAGAC (SEQ ID NO: 15) TdB4L16_R4 TAACACTAGTCCATCAGTTGGAC (SEQ ID NO: 16) C_TdB4L13 CACCTTATGTTCCGTGATCCAG (SEQ ID NO: 17) TdB4L13r TTCGGAGCCGTCGCGGTACCGAAATC (SEQ ID NO: 18) C_TdB4L16 CACCACGCATCCAGACCATGTGTG (SEQ ID NO: 19) TdB4L16r TTCGGAGCCGTCGCGGTATCAAATC (SEQ ID NO: 20) [0216] The oligonucleotide primers used for cloning the TdHDZipl-3 promoter into the pMDC164 vector were as follows: C_TdBL7 CACCGATGAGTTGAATCCCGATGC (SEQ ID NO: 21) TdBL7r CGCCCCCTACCTCCCTAGCTAG (SEQ ID NO: 22) [0217] Preparation of plasmid constructs was performed as described in Kovalchuk et al., 2012, J. Exp. Bot. 63: 2025-2040. Coating of gold particles with the plasmid constructs was performed as follows. 5pg of each plasmid construct was co-precipitated, washed and dried in ethanol and sodium acetate according to standard methods. The dried plasmid DNA was then resuspended in 20 pl of H 2 0, and centrifuged for 5 minutes at maximum speed. The gold particles were prepared by pipetting and vortexing 50 pl of gold suspension solution (gold at 30 mg/ml) in 50 % glycerol. Whilst vortexing, 10 pl of the plasmid DNA was added to the gold suspension and vortexing was continued for 30 seconds. 10 pl of precipitation solution (proprietary) was then added and vortexed for a further 30 seconds. The preparation was left at room temperature for 20 minutes and then centrifuged at 6,000 rpm for 1 minute. The supernatant was removed and the gold pellet was carefully washed and agitated in 100 pl of 100% ethanol. This was followed by centrifugation at 6,000 rpm for 1 minute, removal of the supernatant, and addition of 50 pl -54 of 100% ethanol to the gold pellet. Prior to bombardment, the gold pellet was resuspended by vigorously running the tube containing the pellet along a metal wire grid. [0218] As indicated above, a liquid cell suspension culture of Triticum monococcum was the target tissue used for the co-bombardment of each promoter-GUS fusion construct with a pUbi-TF construct. Cell suspensions were grown in 100 ml of liquid medium containing % MS (Murashige-Skoog medium) and 2 mg/L of 2,4-dichlorophenoxyacetic acid, in the dark at 250C, and were sub-cultured weekly. The cell suspensions were harvested on the 4th day after subculture by filtering 6 ml of a 5% (v/v) cell suspension onto Whatman filter paper and left overnight on MS growth media containing sucrose at 60g.L-1. The filters were transferred to an osmotic MS growth media (supplemented with sucrose at 150g.L-1) 3 hrs before bombardment. [0219] Microprojectile bombardment was performed using the Biolistic PDS-1000/He Particle Delivery System (Bio-Rad, Hercules, CA, USA). Bombardment conditions were 1100 psi, with a 15 mm distance from the macrocarrier launch point to a stopping screen and a 60 mm distance from the stopping screen to the target plant material. The distance between the rupture disk and the launch point of the macrocarrier was 12 mm. The pre cultured cell suspensions were bombarded on a growth media containing 150 g.L isucrose. If a treatment was to be applied after transformation, cells were left for approximately 5 hrs until treatment. During treatment, filters were transferred to 3 dry filter papers and 1.8 ml of cell suspension media was applied with or without added treatment compounds. GUS staining was performed 24hrs after treatment and 0.7 ml of GUS staining buffer (X-Gluc 1 mg.ml- 1 ) was applied. The stained cells were left at 37 0 C overnight and then moved to 4 0 C until GUS foci were counted by eye using a Leica DC 300F stereomicroscope (Leica Microsystems GmbH, Nussloch, Germany). [0220] The results of these experiments are shown in Figure 7 and indicate that an about 7-fold activation of the BL4 promoter (TdHDZipl-4) was induced by ABA (Figure 7A). ABA did not influence the basal activity of the BL7 (TdHDZipl-3) promoter (Figure 7A). [0221] Induction of the TdHDZipI-4 promoter by cold was also demonstrated (Figure 7B). After bombardment, plates were kept at 4 0 C for different time periods, so that the end of the incubation was the same point for all samples (incubations started at different time points, first one - 24 hours after bombardment). However, in the same conditions the - 55 TdHDZipl-3 promoter was slightly down-regulated (Figure 7B). [0222] A further attempt to activate the TdHDZipl-3 promoter was made using ethephon and mannitol. As shown in Figure 7, TdHDZipl-3 basal expression was suppressed by 1 mM ethephon (Figure 7C) but not affected by mannitol at the concentrations used (Figure 7D). EXAMPLE 6 Analysis of Promoters in Transgenic Plants [0223] Wheat and barley transgenic plants were generated for the analysis of the TdHDZipl-4a (BL4) and TdHDZipl-3 (BL7) transcriptional control/promoter regions. The constructs used for plant transformation are shown in Table 2. TABLE 2 Construct Promoter Reporter gene Transformed species pBL7-TaDREB3 TdHDZipl-3 TaDREB3 Barley cv Golden promise pBL4-TaDREB3 TdHDZipl-4a TaDREB3 Barley cv Golden promise pBL7-TaCBF5 TdHDZipl-3 TaCBF5 Wheat cv Gladius pBL4-TaCBF5 TdHDZipl-4a TaCBF5 Wheat cv Gladius pBL7-GUS TdHDZipl-3 GUS Wheat cv Gladius pBL4-GUS TdHDZipl-4a GUS Wheat cv Gladius [0224] To create the TdHDZipl-4a (BL4) constructs, the promoter sequence represented by SEQ ID NO: 1 was used. To create the TdHDZipl-3 (BL7) constructs, the promoter sequence represented by SEQ ID NO: 3 was used. [0225] The promoter sequences were amplified by PCR using primers with introduced Hindill and Kpnl restriction sites. The primer sequences used for PCR amplification of the TdHDZipl-4a promoter were PRTdB4L13H 5'-GGCAAGCTTGAAGGACCACATTGG-3' (SEQ ID NO: 23) and PRTdB4L13Kr 5'-GGTGGTACCTTCGGAGCCGTCGCGGTAC-3' (SEQ ID NO: 24), respectively. The primer sequences used for PCR amplification of the TdHDZipl-3 promoter were PRTdBL7H 5'-CCCAAAGCTTGGACCTTTGATACACC-3' (SEQ ID NO: 25) and PRTdBL7Kr 5'-GGTGGTACCCGCCCCCTACCTCCCTAGCTAG-3' (SEQ ID NO: 26), respectively.

- 56 [0226] The PCR-amplified promoter fragments were isolated and cloned into the HindIII KpnI restriction sites of the pMDC32 vector, as briefly described below. The pMDC32 vector was linearised by simultaneous restriction with HindIII and KpnI and purified from a 2% agarose gel using a Gel extraction Kit (Scientifix). The PCR amplified fragments were purified with a PCR clean-up Kit (Scientifix), and was then digested with the restriction enzymes HindIII and KpnI. The digested products were purified from an agarose gel and ligated separately into the linearized pMDC32 vector using T4 ligase (Invitrogen). The ligation mix was transformed into competent DB3.1 E. coli cells (Invitrogen) and plated on Kan/Cm agar plates. Plasmid DNA was purified from transformed colonies using the ISOLATE DNA Kit (BIOLINE). The generated binary vectors were verified by sequencing. The binary vectors were used to clone the TaDREB3 coding sequence downstream of each promoter by recombination with EcoRV linearised pENTR-D-TOPO-TaDREB3 plasmid using Gateway LR Clonase II Enzyme Mix (Invitrogen). Selectable marker genes present in the construct conferred hygromycin resistance in plants and kanamycin resistance in bacteria. [0227] Wheat and/or barley plants were transformed with three types of constructs: promoter-GUS, promoter-TaDREB3 and promoter-TaCBF5. The promoter-GUS and promoter-TaCBF5 constructs were linearised using a unique Pmel restriction site and transformed into wheat (Triticum aestivium L cv Gladius), using biolistic bombardment transformation as described by Kovalchuk et al., 2009 ("Characterization of the wheat endosperm transfer cell-specific protein TaPR60. Plant Mol. Biol. 71: 81-98). The promoter-TaDREB3 constructs were transformed into barley (Hordeum vulgare L cv Golden Promise), using Agrobacterium-mediated transformation as described in Tingay et al., 1997, "Agrobacterium tumefaciens-mediated barley transformation", Plant J. 11: 1369 1376. Transgene integration was confirmed by PCR using the forward primer from the 3' end of each promoter and the reverse primer from the 5' end of the nos terminator. [0228] The basal level of BL4 and BL7 promoter activity (the level of expression of the transgene under normal growth conditions) in leaves of T 1 transgenic plants was assessed by Northern blot analysis and quantitative PCR. For Northern analysis, RNA isolated from leaves was electrophoretically separated on a 1.3% agarose gel containing 6% formaldehyde, transferred to nylon membrane and hybridized with 3 2 P-labeled DNA probes corresponding to each transgene according to the protocol described by Church and Gilbert (1984). For quantitative PCR the method used was essentially the same as - 57 set out in Example 2. The transgene copy numbers were estimated by quantitative PCR using T 1 progeny of transgenic lines pre-selected in hydroponics-based experiments (as described below). Oligonucleotide primers used for quantitative PCR were TxDREB3 5' GTATCTCGCATATGGACGGAG-3' (SEQ ID NO: 27) and TxDREB3r_nos 5' TTGCCAAATGTTTGAACGATC-3' (SEQ ID NO: 28). [0229] Four types of hydroponics-based experiments were performed: (1) quick selection of lines for transgene expression in hydroponics using ABA, dehydration, salt and/or cold treatment (hydroponic selection); (2) plant phenotyping in soil in a glasshouse without stress application (phenotyping experiment); (3) assessment of the frost tolerance at the vegetative stage of development in a frost cabinet (frost tolerance test); and (4) assessment of survival under severe drought (drought survival test). These experiments were carried out according to the methods described in Morran et al., 2011, "Improvement of stress tolerance of wheat and barley by modulation of expression of DREB/CBF factors",. Plant Biotechnol. J. 9: 230-249. [0230] Wild type plants and, in some experiments, null segregants were used as control plants. Progeny from six To transgenic barley lines, three for each of the pBL4-TaDREB3 (G345; lines 32, 26, 17) and pBL7-TaDREB3 (G339; lines 3, 7 and 12) constructs were grown in soil in pots under glasshouse conditions and plants were used either for the phenotyping experiment, the drought survival test of for the frost tolerance test. Non transgenic (wild type, wt) plants were used as control plants. T 1 transgenic barley seedlings were analysed for the presence of the transgene by PCR and/or GUS staining according to the methods described in Li et al., 2008, Plant Biotechnol. J., 6: 146-159. Analysis of pBL4-GUS and pBL 7-GUS transgenic barley seedlings - hydroponic selection [0231] T 1 progeny of fifteen independent pBL4-GUS transgenic lines were analysed in the pilot experiment using hydroponics. Conditions of treatments were: 20 mM NaCl for 24 hours, 2 pM ABA, dehydration for 1-7 hours, and incubation at 40C. Weak staining of seedlings shoots was observed in 8 lines (1a, 2, 4, 6, 8, 10, 12, and 13) after cold treatment (Figure 8A). T 1 progeny of the seven independent pBL7-GUS transgenic lines were analysed using the same conditions. Three lines (3, 5 and 7) showed GUS activity in shoots and roots of seedlings under cold stress and one line (3) under dehydration (Figure 8B). However, the GUS activity was too weak to proceed with histochemical analysis of the spatial pattern of the promoter activity.

- 58 Phenotyping of pBL4-TaDREB3 and pBL7-TaDREB3 transgenic barley plants [0232] Expression of the TaDREB3 gene under constitutive promoters has been shown to negatively influence plant development and grain yield (Morran et al., 2011, supra). In the following experiments, we tested if application of moderate drought or abiotic stress inducible HD-ZipI promoters with expected low basal level of activity in barley instead of strong constitutive promoters (e.g. 35S or maize polyubiquitine) or strong inducible promoter with high basal activity in barley (e.g. maize Rab17 promoter) can improve phenotypes, grain yield and stress tolerance of transgenic barley. Because no significant influence on expression levels of TaHDZip/-3 and -4 genes were detected in transgenic wheat plants with constitutive over-expression of TaDREB3 (Lopato et al., unpublished data), we assume that the transgene in our experiments has not directly influenced the activity of the HD-Zip/ promoters. [0233] T 1 progeny of four pBL4-TaDREB3 (17, 26, 28, 32) and five pBL7-TaDREB3 (3, 7, 12, 18) independent transgenic lines were analysed for transgene copy number, levels of transgene expression, phenotypic characteristics and yield using the methods described above. 6 to 7 plants for each line were characterised and compared with control (wild type, wt) plants. The results of genotyping are shown in Figure 9. Null segregants are marked with arrows. Expression of each transgene was studied in the absence of stress. This reflects the basal levels of promoter activity, which as shown in Figure 9 is very different for different lines and not always correlates with the copy number. All transgenic plants at flowering are shown in Figures 10 to 13 with null-segregants indicated by arrows. [0234] As shown in Figures 14 and 15, three pBL7-TaDREB3 lines (3, 12, and 13) had height, tiller number, flowering time and appearance similar to that of wt plants. These lines were selected for further experiments. Two remaining lines (7 and 18) were different from these three lines; however, the level of transgene expression was much lower than in other lines, and hence observed pleiotropic phenotypes were not produced by the transgene. In contrast, all pBL4-TaDREB3 lines were smaller in size than control plants (Figure 14), although plant appearance (Figures 12 and 13) and flowering time (Figure 15) were not very different from wild type plants. Frost tolerance tests for pBL4-TaDREB3 and pBL7-TaDREB3 transgenic barley plants [0235] Frost tolerance tests were performed as described by Morran et al., 2011 (supra). The temperature schedule of the tests is outlined in Figure 16A. Twelve T 1 plants for each - 59 of three transgenic lines were tested for survival rates. As shown in Figure 16B, overall the G339 (pBL7-TaDREB3) transgenic lines demonstrated better survival rates than the G345 (pBL4-TaDREB3) transgenic lines. This experiment will be repeated using the next generation of homozygous T 2 progeny of the best performing lines. [0236] Transgene expression and activation as a result of the frost/cold exposure was demonstrated by RT-PCR. Detected null segregants (plants with no transgene expression) were removed from the survival rates data. Several lines were also analysed by quantitative PCR using the methods described above to compare basal levels of activity of the two promoters and levels of promoter activation during the 13 hours of plant "acclimation" before the treatment with below zero temperatures (according to the temperature schedule in Figure 16A). [0237] As shown in Figure 17, the BL7 promoter in transgenic barley had a higher constitutive level of activity than the BL4 promoter. Surprisingly, a weak activation of the promoter by frost/cold, which did not exceed 40% of the basal promoter activity, was observed for the BL7 promoter, but was not detected in the previous gene expression studies shown in Figure 2B. In contrast, frost/cold inducible levels of the BL4 promoter activity were 2.5-3 fold higher than basal levels of the promoter activity, although overall they were lower than expected. It is possible that at the moment of sampling, the activity of the promoter had not reached its maximum due to a slow activation of the BL4 promoter by cold (as expected from the gene expression data shown in Figure 2B). A higher activity of the BL7 promoter in combination with the TaDREB3 gene might be explained by a higher basal level of activity, which provides higher levels of target stress inducible genes before the commencement of plant "acclimation" to cold. Drought survival test for selected transgenic barley lines [0238] For the drought survival test, 4 plants were grown per pot: 1 control plant and three for each of the pBL4-TaDREB3 - G345 (lines 32, 26 and 17) and pBL7-TaDREB3 - G339 (lines 3, 7 and 12) transgenic lines. Plants were grown in a growth chamber under the conditions described below. Briefly, eight inch pots and clay-containing soil (mix of 1/3 part of Cocopeat soil and 2/3 parts of clay-rich soil from the field) were used in this experiment. Control and transgenic seedlings were grown under well watered conditions for over six weeks and then watering was withheld. The results of the number of seedlings that germinated and survived the drought experiment are shown in Table 3.

- 60 TABLE 3 Number of Number of T 1 Plants Name of Construct To line Germinated T 1 Survived After Drought Plants Treatment BL7-TaDREB3 (G339) 3 10 3 (30%) BL7-TaDREB3 (G339) 13 10 2 (20%) BL7-TaDREB3 (G339) 18 2 0 wt - 6 0 null 18 1 1 BL4-TaDREB3 (G345) 26 9 4 (44.4%) BL4-TaDREB3 (G345) 28 9 5 (55.6%) BL4-TaDREB3 (G345) 32 8 1 (12.5%) wt - 9 2 (22.2%) [0239] Leaf samples were collected between 2 and 19 days of drought. Relative water content in sampled leaves was measured using a pressure chamber every day until there were clear signs of wilting. Water content in the soil was calculated based on pot weight. The result of the water content analysis based on pot weight is shown in Figure 19. [0240] As shown in Figure 19A, leaf samples were collected from transgenic and control plants before stress, before wilting (5-7 bar), at wilting point (9-10 bar), at very strong drought (12-15 bar) and severe drought (short time before re-watering) and stored at 800C until RNA extraction. The survival rates for T 1 transgenic and control plants were calculated and are shown in Figures 19B and 19C. Survival rates demonstrate an improvement of drought tolerance of transgenic barley plants with both promoters. Figure 20D shows the recovery of meristems of representative transgenic plants 3 days after re watering. Survived plants were transferred to the glasshouse for generation of seeds. [0241] Transgene expression was tested by quantitative PCR using the methods described above in some of the survived plants and the results are shown in Figure 20 (BL4 promoter) and Figure 21 (BL4 promoter). Overall, expression of TaDREB3 was stronger when driven by the BL7 promoter than when its expression was driven by the BL4 promoter. However, the ratio between the maximum promoter induction by drought and the basal level of promoter expression in unstressed leaves in most cases was higher for the BL4 promoter. Although both promoters demonstrated "long-lasting" activity and remained activated until at least day 20 after water was withheld, the BL7 promoter -61 appeared to be more strongly activated by mild stress; however, at more severe conditions the activity of the promoter slowly declined. In contrast, activity of the BL4 promoter at conditions of severe drought either remained the same or even increased compared to activity at mild drought. EXAMPLE 7 Promoter Deletion Studies [0242] Mapping of the BL4 TdHDZipI-4a transcriptional control/promoter segment responsible for the activation by ABA was conducted. T. monococcum cells were bombarded with a series of promoter deletion constructs. The nature of the promoter deletions created, and the results of the mapping experiments on these deletions are shown in Figure 22. Constructs of this promoter deletion series ranged from 1600 to 130 nt and were named 1600 and Del 1 to Del 6 (Figure 22A). ABA treatments were applied 24 hrs post-bombardment. These studies showed that a 93 nucleotide segment of the TdHDZipl-4 promoter (SEQ ID NO: 4) was responsible for activation of the promoter in response to ABA (Figure 22B). This segment contains a potential cis-element (underlined) specific for binding an AB15-like bZIP factor, as determined by in silico analysis performed using the PLACE database. The TdHDZipI-4a and TdHDZipI-4b promoter regions share 94% identity over this 93 nucleotide segment. [0243] Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to, or indicated in this specification, individually or collectively, and any and all combinations of any two or more of the steps or features.

Claims

1. A method for effecting stress responsive expression of a nucleotide sequence of interest in one or more cells of a plant, the method including expressing in the one or more cells of the plant the nucleotide sequence of interest operably connected to a transcriptional control sequence which is stress inducible in the plant, wherein the nucleotide sequence of interest is heterologous with respect to the transcriptional control sequence, and wherein the transcriptional control sequences includes: (i) the nucleotide sequence set forth in one of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3; or (ii) a functionally active fragment or variant of the nucleotide sequence set forth in one of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, wherein the functionally active fragment or variant is at least 50% identical to the nucleotide sequence set forth in one of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

2. A nucleic acid construct including a nucleotide sequence of interest operably connected to a transcriptional control sequence which is stress inducible in a plant, wherein the nucleotide sequence of interest is heterologous with respect to the transcriptional control sequence, and wherein the transcriptional control sequences includes: (i) the nucleotide sequence set forth in one of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3; or (ii) a functionally active fragment or variant of the nucleotide sequence set forth in one of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, wherein the functionally active fragment or variant is at least 50% identical to the nucleotide sequence set forth in one of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

3. An isolated nucleic acid when used to direct expression of an operably connected nucleotide sequence of interest in one or more cells of a plant when the plant is subject to stress, wherein the nucleic acid comprises a nucleotide sequence defining a transcriptional control, wherein said transcriptional control sequence includes: (i) the nucleotide sequence set forth in one of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3; or (ii) a functionally active fragment or variant of the nucleotide sequence set forth in one of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, wherein the - 63 functionally active fragment or variant is at least 50% identical to the nucleotide sequence set forth in one of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

4. The method of claim 1, the nucleic acid construct of claim 2, or the isolated nucleic acid of claim 3, wherein the plant is: (i) a monocot plant; (ii) a cereal crop plant; or (iii) a wheat, rice or barley plant.

5. The method of claim 1 or claim 4, the nucleic acid construct of claim 2 or claim 4, or the isolated nucleic acid of claim 3 or claim 4, wherein the functionally active fragment or variant includes one or more sequence motifs corresponding to one or more cis-elements selected from the group consisting of MYBR, ABRE, CRT and ABRE (AB15-binding).

6. The method of any one of claims 1, 4 and 5, the nucleic acid construct of any one of claims 2, 4 and 5, or the isolated nucleic acid of any one of claims 3 to 5, wherein the transcriptional control sequence includes: (i) the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2; or (ii) a functionally active fragment or variant of the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2, wherein the functionally active fragment or variant is at least 50% identical to the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2, and includes one or more sequence motifs corresponding to one or more cis-elements selected from the group consisting of a CGCG-box (calmod binding), ABRE (AB15-binding), auxin-response (AUX-resp), MYBR, ABRE, CRT, and ammonium response (ammonium-resp).

7. The method, nucleic acid construct or isolated nucleic acid of claim 6, wherein the functionally active fragment or variant includes a nucleotide sequence which is at least 94% identical to the nucleotide sequence set forth in SEQ ID NO: 4, or includes the nucleotide sequence set forth in SEQ ID NO: 4.

8. The method, nucleic acid construct or isolated nucleic acid of claim 6 or claim 7, wherein the nucleotide sequence of interest includes a nucleotide sequence which, when expressed by one or more cells of a plant, improves the stress tolerance of the plant. - 64

9. The method, nucleic acid construct or isolated nucleic acid of any one of claims 6 to 8, wherein the stress is selected from one or more of cold, drought, and salinity, or wherein the stress is induced by ABA.

10. The method, nucleic acid construct or isolated nucleic acid of claim 9, wherein the nucleotide sequence of interest includes a nucleotide sequence which, when expressed by one or more cells of a plant, improves the cold, drought, and/or salinity tolerance of the plant.

11. The method of any one of claims 1, 4 and 5, the nucleic acid construct of any one of claims 2, 4 and 5, or the isolated nucleic acid of any one of claims 3 to 5, wherein the transcriptional control sequence includes: (i) the nucleotide sequence set forth in SEQ ID NO: 3; or (ii) a functionally active fragment or variant of the nucleotide sequence set forth in SEQ ID NO: 3, wherein the functionally active fragment or variant is at least 50% identical to the nucleotide sequence set forth in SEQ ID NO: 3, and includes one or more sequence motifs corresponding to one or more cis-elements selected from the group consisting of ABRE (AB15-binding), ABRE, GCC-box, light regulated cis element, MYBR, ABRE-similar, ABRE-related (Ca dependent), vascular expression cis-element, WRKY specific element, CRT, and carbohydrate metabolite-responsive cis-element.

12. The method, nucleic acid construct or isolated nucleic acid of claim 11, wherein the nucleotide sequence of interest includes a nucleotide sequence which, when expressed by one or more cells of a plant, improves the stress tolerance of the plant.

13. The method, nucleic acid construct or isolated nucleic acid of claim 11 or claim 12, wherein the stress is drought.

14. The method, nucleic acid construct or isolated nucleic acid of claim 13, wherein the nucleotide sequence of interest includes a nucleotide sequence which, when expressed by one or more cells of a plant, improves the drought tolerance of the plant.

15. The method, nucleic acid construct or isolated nucleic acid of any one of - 65 claims 12 to 14, wherein expression of the nucleotide sequence of interest does not disturb development of the plant.

16. The method of any one of claims 1 and 4 to 15, the nucleic acid construct of any one of claims 2 and 4 to 15, or the isolated nucleic acid of any one of claims 3 to 15, wherein the nucleotide sequence of interest includes a nucleotide sequence that encodes a DREB polypeptide, including a TaDREB3-like polypeptide.

17. A method for improving the stress tolerance of a plant, the method including expressing a nucleotide sequence of interest in one or more cells of the plant according to the method of any one of claims 1 and 4 to 16.

18. A genetically modified cell including a nucleic acid construct according to any one of claims 2 and 4 to 16, or a genomically integrated form thereof.

19. A multicellular structure including one or more cells of claim 18.

20. A method according to claim 1, a nucleic acid construct according to claim 2, or an isolated nucleic acid according to claim 3, substantially as herein described with reference to any one or more of the Examples and/or Figures.