WO2011020163A1 - Plant gene expression - Google Patents

Abstract

The present invention relates to a nucleic acid molecule for forming a promoter for regulating transcription of a gene in a plant in anoxic conditions including one or more nucleotide sequences as shown in SEQ. ID Nos. 1 - 14 wherein each of the sequences are spaced apart from each other to define regions for forming regulatory elements for interacting with a transcription factor. Also encompassed by the present invention are uses of this nucleic acid molecule for producing an expression product in a plant in anoxic conditions and/or producing a plant that is tolerant in anoxic conditions.

Description

Plant gene expression

Background of the invention

The invention relates to the regulation of gene expression in plants, especially in anoxic conditions. There is a need for plants, particularly commercial crops, to withstand abiotic stresses caused by extreme weather conditions such as flood, drought and temperature. As the demand for additional land for commercial crops increases, there is also a need for these plants to be able to grow in areas which would traditionally have been avoided because they are exposed to extreme weather conditions. Reference to any prior art in the 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 Australia or any other jurisdiction or that this prior art could reasonably be expected to be ascertained, understood and regarded as relevant by a person skilled in the art. Summary of the invention

In one embodiment there is provided a nucleic acid molecule for forming a promoter for regulating transcription of a gene in a plant in anoxic conditions including one or more nucleotide sequences as shown in SEQ. ID Nos. 1 - 14 wherein each of the sequences are spaced apart from each other to define regions for forming regulatory elements for interacting with a transcription factor.

Preferably, the nucleic acid molecule includes all nucleotide sequences as shown in SEQ. ID Nos. 1 - 14.

In further embodiments there is provided a nucleic acid having a sequence defined by general formula 1 : Xi A X₂ B X₃ C X₄ D X₅ E X₆ F X₇ G X₈ H X₉ I Xio J Xii K Xi₂ L Xi₃ M X_u wherein:

X₁ has a sequence shown in SEQ ID No: 1

X₂ has a sequence shown in SEQ ID No: 2

X₃ has a sequence shown in SEQ ID No: 3 X₄ has a sequence shown in SEQ ID No: 4

X₅ has a sequence shown in SEQ ID No: 5

X₆ has a sequence shown in SEQ ID No: 6

X₇ has a sequence shown in SEQ ID No: 7

X₈ has a sequence shown in SEQ ID No: 8 X₉ has a sequence shown in SEQ ID No: 9

X₁₀ has a sequence shown in SEQ ID No: 10

Xn has a sequence shown in SEQ ID No: 11

X₁₂ has a sequence shown in SEQ ID No: 12

X₁₃ has a sequence shown in SEQ ID No: 13 X₁₄ has a sequence shown in SEQ ID No: 14 and wherein

A, B, C, D, E₁ F, G, H, I J, K, L and M are sequences each having a length of about 50 to 1000 nucleotides, preferably about 10 to 700 nucleotides. In further embodiments there is provided a nucleic acid molecule for forming a promoter for regulation of transcription of a gene in a plant in anoxic conditions including the nucleotide sequence as shown in SEQ. ID No. 15.

In another embodiment there is provided a peptide having a sequence shown in SEQ. ID No. 17, or fragments thereof.

In other embodiments there are provided vectors, constructs, cells, plants and plant tissues containing a nucleic acid or peptide described above.

Brief description of the drawings

Fig. 1 Normalized transcript levels of V-PPase genes in a range of rice tissues. Levels of mRNA are presented as the number of copies per microliter total RNA after normalization, (a) OVP1, OVP2 and OVP6 mRNA ievels. (b) OVP3, OVP4 and OVP5 mRNA levels. Note that A and B differ only with respect to the mRNA abundance scale and are shown separately. The data are the mean of at least three independent measurements with an indication of the standard deviation Fig. 2 Normalized transcript levels of V-PPase genes in Calrose coleoptiles. cDNA was synthesized from total RNA extracted from rice coleoptiles flushed either by air or nitrogen and used for Q-PCR analysis. Statistical analysis is the same as for Fig. 1

Fig. 3 Normalized transcript levels of the OVP3 gene in coleoptiles of Calrose, Amaroo and IR22. RNA extraction, anoxic treatment and statistical analysis are the same as for Fig. 2

Fig. 4 Western blot analysis of V-PPase and V-ATPase. Tonoplast membrane preparations were extracted from coleoptiles pretreated with either air or anoxia and separated by SDS PAGE, (a) lmmunobloting with antibodies against the 75-kD catalytic subunit of V-PPase. (b) lmmunobloting with the antibodies against the 70-kD subunit A of V-ATPase. Ten micrograms of total protein was loaded per lane. Numbers in the left lane indicate the molecular marker position. Lane 1 : aerated control; lane 2: anoxia for 24 h; lane 3 anoxia for 48 h Fig. 5 GUS expression patterns driven by the promoter of OVP3. The seedlings of wild- type segregant (A and B) and transgenic plants (C to E) were treated with anoxia for 24 h. The roots were sectioned and stained 4 h for GUS activity, (a) Root tip, scale bar, 200 μm (from A to E). (b) Transverse section of root tip (1 cm from seminal root cap). (c) Seminal root, (d) Root tip. (e) Transverse section of root tip (1 cm from the seminal root cap) C: cortex; E: epidermis; S: stele

Fig. 6 GUS activity of rice coleoptiles from WT₁ T₁ wild type segregant and transgenic seedlings treated with anoxia. Numbers 1 , 2, 3 and 4 represent four independent lines. The data are the mean of at least three independent measurements with an indication of the standard deviation.

Fig. 7 GUS activity of flag leaves and seminal roots from WT and T₁ seedlings of OVP3 promoter::GUS transformants treated with anoxia, salt or cold. Numbers 1 , 2, 3 and 4 represent four independent lines. Statistical analysis is the same as for Fig. 6

Fig. 8A Nucleotide sequence of OVP6; Fig 8B Predicted amino acid sequence of OVP6 Detailed description of the embodiments

Reference will now be made in detail to certain embodiments of the invention. While the invention will be described in conjunction with the embodiments, it will be understood that the intention is not to limit the invention to those embodiments. On the contrary, the invention is intended to cover all alternatives, modifications, and equivalents, which may be included within the scope of the present invention as defined by the claims.

One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. The present invention is in no way limited to the methods and materials described.

It will be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative aspects of the invention. In one embodiment, there is provided a nucleic acid molecule for forming a promoter for regulating transcription of a gene in a plant in anoxic conditions including one or more nucleotide sequences as shown in SEQ. ID Nos. 1 - 14 wherein each of the sequences are spaced apart from each other to define regions- for forming regulatory elements for interacting with a transcription factor.

Preferably, the nucleic acid molecule includes all nucleotide sequences as shown in SEQ. ID Nos. 1 - 14.

In another embodiment there is provided a nucleic acid molecule for forming a promoter for regulating transcription of a gene in a plant in anoxic conditions including the nucleotide sequences as shown in SEQ. ID Nos. 1 - 14 wherein each of the sequences are spaced apart from each other to define regions for forming regulatory elements for interacting with a transcription factor.

Typically the regions are arranged in the following order from 5' to 3' with reference to the nucleotide sequences defining each region: SEQ. ID Nos. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14.

The regions may be spaced apart by between about 50 tolOOO nucleotides.

In one embodiment, the region defined by SEQ ID No: 1 is spaced apart from the region defined by SEQ ID No: 2 by about 25 and 95 nucleotides, typically 45 nucleotides.

In one embodiment, the region defined by SEQ ID No: 2 is spaced apart from the region defined by SEQ ID No: 3 by about 5 and 15 nucleotides, typically 10 nucleotides.

In one embodiment, the region defined by SEQ ID No: 3 is spaced apart from the region defined by SEQ ID No: 4 by about 35 and 145 nucleotides, typically 75 nucleotides.

In one embodiment, the region defined by SEQ ID No: 4 is spaced apart from the region defined by SEQ ID No: 5 by about 45 and 180 nucleotides, typically 90 nucleotides. In one embodiment, the region defined by SEQ ID No: 5 is spaced apart from the region defined by SEQ ID No: 6 by about 90 and 360 nucleotides, typically 180 nucleotides.

In one embodiment, the region defined by SEQ ID No: 6 is spaced apart from the region defined by SEQ ID No: 7 by about 225 and 800 nucleotides, typically 450 nucleotides. In one embodiment, the region defined by SEQ ID No: 7 is spaced apart from the region defined by SEQ ID No: 8 by about 350 and 1400 nucleotides, typically 735 nucleotides.

In one embodiment, the region defined by SEQ ID No: 8 is spaced apart from the region defined by SEQ ID No: 9 by about 35 and 140 nucleotides, typically 75 nucleotides.

In one embodiment, the region defined by SEQ ID No: 9 is spaced apart from the region defined by SEQ ID No: 10 by about 35 and 130 nucleotides, typically 65 nucleotides.

In one embodiment, the region defined by SEQ ID No: 10 is spaced apart from the region defined by SEQ ID No: 11 by about 25 and 100 nucleotides, typically 55 nucleotides.

In one embodiment, the region defined by SEQ ID No: 11 is spaced apart from the region defined by SEQ ID No: 12 by about 0 and 10 nucleotides, typically 1 nucleotides.

In one embodiment, the region defined by SEQ ID No: 12 is spaced apart from the region defined by SEQ ID No: 13 by about 60 and 240 nucleotides, typically 120 nucleotides.

In one embodiment, the region defined by SEQ ID No: 13 is spaced apart from the region defined by SEQ ID No: 14 by about 10 and 30 nucleotides, typically 20 nucleotides.

In one embodiment the nucleic acid molecule further includes a nucleotide sequence defining one or more further regions for forming regulatory elements for interacting with a transcription factor. Typically the sequence defines an anoxia regulatory element, preferably a sequence as shown in any one of SEQ ID No:s 1 to 14. Without being bound by any theory or mode of action the regulatory element may interact with a MYB or MYB-related transcription factor. Accordingly, in one embodiment of the invention the regulatory element interacts with a MYB or MYB- related transcription factor under anoxic conditions. In further embodiments there is provided a nucleic acid having a sequence defined by general formula 1 :

X₁ A X₂ B X₃ C X₄ D X₅ E X₆ F X₇ G X₈ H X₉ I X₁₀ J Xn K X₁₂ L X₁₃ M X₁₄ wherein:

X_I has a sequence shown in SEQ ID No: 1 X₂ has a sequence shown in SEQ ID No: 2

X₃ has a sequence shown in SEQ ID No: 3 X₄ has a sequence shown in SEQ ID No: 4 X₅ has a sequence shown in SEQ ID No: 5 X₆ has a sequence shown in SEQ ID No: 6 X₇ has a sequence shown in SEQ ID No: 7 X₈ has a sequence shown in SEQ ID No: 8 X₉ has a sequence shown in SEQ ID No: 9 X₁₀ has a sequence shown in SEQ ID No: 10

X_{I I} has a sequence shown in SEQ ID No: 11 X₁₂ has a sequence shown in SEQ ID No: 12 X₁₃ has a sequence shown in SEQ ID No: 13 Xi₄ has a sequence shown in SEQ ID No: 14 and wherein

A, B, C, D₁ E, F, G, H, I J, K, L and M are sequences each having a length of about 50 to 1000 nucleotides, preferably about 10 to 700 nucleotides.

In one aspect of this embodiment the regulatory elements Xi to X₁₄ may be arranged in any order. For example, the sequence may have the general formula:

X₁₁ A X₂ B X₃ C X₉ D X₅ E X₁₄F X₇ G X₈ H X₄I X₁₀ J Xi K X₁₂ L X₁₃ M X₆

In further embodiments there is provided a nucleic acid molecule for forming a promoter for regulation of transcription of a gene in a plant in anoxic conditions including the nucleotide sequence as shown in SEQ. ID No. 15.

In certain embodiments, the nucleic acid molecule has a sequence that is homologous or identical to SEQ ID No. 15.

Percent sequence identity is determined by conventional methods, by means of computer programs known in the art such as GAP provided in the GCG program package (Program Manual for the Wisconsin Package, Version 8, August 1994,

Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA 53711) as disclosed in Needleman, S. B. and Wunsch, CD., (1970), Journal of Molecular Biology,

48, 443-453, which is hereby incorporated by reference in its entirety. Sequence identity of polynucleotide molecules is determined using GAP with the following settings for

DNA sequence comparison: GAP creation penalty of 5.0 and GAP extension penalty of

0.3.

Typically, the nucleic acid molecule contains the sequences shown in SEQ ID No.s: 1 to

14 and otherwise has at least 75% identity, preferably 80%, preferably 85%, preferably 90%, preferably 95%, preferably 97%, preferably 98% or 99% identity with the sequence shown in SEQ ID No: 15. In these embodiments, the sequence identity below 100% may occur in the regions of the nucleic acid molecule sequence flanking SEQ ID No. s 1 to 14. For example, where the nucleic acid sequence is defined by general formula 1 above, the sequence identify below 100% occurs at one or more of regions A to M.

The promoter may cause regulation of transcription when a plant containing the same is exposed to an abiotic stress. An abiotic stress includes exposure to an extreme environmental or weather condition, such as extreme temperatures, drought, flood. Such abiotic stresses may result in for example anoxic soil conditions or high salinity. Typically the promoter causes up regulation of transcription when a plant containing same is exposed to anoxic conditions or abiotic stress. Typically the up regulation causes at least a 2 fold increase in RNA transcript production or accumulation as compared with non anoxic conditions, preferably a 5 fold increase, or 10 fold, or 20 fold or 50 fold increase in RNA transcript production or accumulation. Anoxic conditions generally refer to conditions in which there is a substantial absence of oxygen. These conditions may be observed where plants have been submerged in water, for example in flood conditions. Examples of anoxic conditions are discussed in the Examples contained further herein.

In another embodiment there is provided a vector or like construct having a gene for encoding an expression product and a nucleic acid for forming a promoter as described above.

The expression product encoded by the gene may be one that would benefit from production in plants under an abiotic stress, particularly anoxic conditions. Examples include expression products that are readily oxidised in an oxygen containing environment. Examples of genes encoding an expression product include: pyruvate decarboxylase (PDC), sucrose synthase (SuS), pyrophosphate-dependent phosphofructokinase (PFP), uridine diphosphoglucose pyrophosphorylase (UGPase), alcohol dehydrogenase (ADH), glucokinase and frgctokinase, lactate dehydrogenase, vacuolar pyrophosphatase and enzymes involved in fatty acid synthesis. Other expression products more generally may be those involved in fermentation or glycolytic pathways.

Typically the vector or like construct contains regions necessary for the expression of the expression product including start and stop transcription signals, enhancers and origin of replication.

In one embodiment the vector or like construct does not contain an expression product but instead contains a cloning site for insertion of a gene for encoding an expression product. In these embodiments the vector or like construct may not contain one or more of regions necessary for the expression of the expression product including start and stop transcription signals, enhancers and origin of replication.

In another embodiment there is provided a plant or cell or tissue derived therefrom including a nucleic acid, vector or like construct as described above. Typically the plant or cell or tissue derived therefrom is a monocotyledon, such as a grass. The grass may be a grain crop such as Maize, Rice, Wheat, Barley, Sorghum, Millet, Oats, Rye, Tricale, Fonio, Teff, Buckwheat, Quinoa, or a lef and stem crop such as bamboo, marram grass, meadow grass, reed, ryegrass, sugarcane, or a lawn grass such as bahia grass, bent grass, Bermuda grass, centipede grass, fescue, meadow grass, ryegrass, st Augustine grass, zoysia, or an ornamental grass such as calamagrostisis spp, cortaderia spp., deschampsia spp., fetuca spp., Melica spp., Muhlenbergia spp., stipa spp.

The plant or cell or tissue derived therefrom may also be a dicotyledon, including cotton or hemp. Typically, the cotton is a commercial cotton species, such as Gossypium arboretum L., Gossypium barbadense L., Gossypium herbaceum L., Gossypium hirsutum L.

Preferably, the plant or cell or tissue derived therefrom is from rice or other grain. Alternatively, the a plant or cell or tissue derived therefrom is suitable as a fibre producing plant or crop such as cotton, hemp, jute, flax, milkweed, bamboo, sisal. In another embodiment there is provided a method for producing an expression product in a plant in anoxic conditions including the step of culturing a plant, or tissue or cell derived therefrom as described above in anoxic conditions. Generally the plant is a transgenic plant and the transgene takes the form of the vector or like construct. The expression product may be a product that is syngeneic or endogenous to the wild type background on which the transgene has integrated. In these embodiments the vector or like construct may be essentially constituted of the nucleic acid molecule for forming a promoter as described above. The nucleic acid molecule may have randomly integrated into the wild type background so as to regulate the expression of downstream genes contained within that background. This facilitates the generation of new plant phenotypes in anoxic conditions. There is also provided a use of a vector or like construct including the nucleic molecule described above, for producing expression in a plant in anoxia conditions. Alternatively, the method according to this embodiment may be useful for producing an expression product in a plant under other abiotic stress as described above.

In other embodiments the transgene is a vector or like construct including a vacuolar pyrophosphatase or other molecule or enzyme for- conferring resistance in a plant to oxygen starvation or flooding. Thus there is provided a process for producing a plant, or tissue or cell therefrom that is tolerant to anoxia including the step of providing a vector or construct having a gene encoding a vacuolar pyrophosphatase and a nucleic acid for forming a promoter as described above, the nucleic acid for forming the promoter being linked to the gene for encoding the vacuolar pyrophosphatase for regulation of transcription of the gene when the construct is provided in a plant cell in anoxic conditions, and introducing the vector or construct into a plant cell, tissue or whole plant. There is also provided a use of a vector or like construct including the nucleic molecule described above for producing a plant or tissue or cell therefrom that is tolerant to anoxia. Alternatively, the method according to this embodiment may be useful for producing a plant, or tissue or cell therefrom that is tolerant to other abiotic stress as stated above. In other embodiments the transgene is a vector or like construct including an expression product, the latter not being found in the wild type genetic background into which the transgene has been inserted. These embodiments allow plants to be used in anoxic conditions to express high value products.

In other embodiments the cell is a protozoan, algal, yeast, bacteria or archaebacterial cell. In another embodiment there is provided a peptide having a sequence shown in SEQ. ID No. 17, or fragments thereof. This peptide may function as a vacuolar pyrophosphatase (V-PPase), a proton pump that catalyses the hydrolysis of inorganic pyrophosphate (PPj) to energize proton transport across the tonoplast. The V-PPase proteins consist of a single polypeptide of 75-81 kDa with 14-16 membrane-spanning domains: they belong to a category of primary ion translocases distinct from the F-, P-, and vacuolar H⁺-ATPase families. Particularly preferred fragments are those shown in SEQ ID No: 18 (encoding a C1 domain), SEQ ID No: 19 (encoding a C2 domain), SEQ ID No: 20 (encoding a C3 domain).

In embodiment, the peptide has a sequence that has homology or identity with SEQ ID No. 17. Percent sequence identity is determined by conventional methods, by means of computer programs known in the art such as GAP provided in the GCG program package (Program Manual for the Wisconsin Package, Version 8, August 1994,

Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA 53711) as disclosed in Needleman, S. B. and Wunsch, CD., (1970), Journal of Molecular Biology, 48, 443-453, which is hereby incorporated by reference in its entirety. GAP is used with the following settings for polypeptide sequence comparison: GAP creation penalty of 3.0 and GAP extension penalty of 0.1.

In certain embodiments there is provided a nucleic acid molecule that has a sequence that is complementary to any one of the sequences described herein. The examples that follow are intended to illustrate but in no way limit the present invention. EXAMPLE 1 Materials and methods

Analysing partial sequences of V-PPases

After searching the rice genome and protein databases with two known nucleotide sequences of rice (OVP1 and OVP2; accession no. D45383 and D45384), six distinct genes encoding vacuolar pyrophosphatases were identified. In order to amplify partial V-PPase cDNA sequences from Calrose, Amaroo and IR22, six pairs of oligonucleotide primers were initially designed from the six isoforms of the OVP gene found in the rice genome (http://www.qramene.org/). The second pairs of primers were required for amplification of IR22 OVP4 and Calrose OVP6 genes due to sequence polymorphism among the cultivars (Table 1). A total of 18 PCR products were obtained and sequenced (each gene had a cDNA sequence from Calrose, Amaroo and IR22, respectively). These sequences were used for designing Q-PCR primers from conserved regions among the three rice cultivars so that only one pair of Q-PCR primers for each gene was required for Q-PCR analysis of transcript levels in three rice cultivars (Table 1).

Rice tissue series for quantitative PCR

Dehulled seeds of rice (Oryza sativa L. cv. Nipponbare) were surface-sterilized by washing in 70% ethanol for 5 min, 2 x 1 min in distilled water, 3 min in 4% HgCI₂, and 5 x 1 min in distilled water and germinated in Petri dishes at 28^°C/25^°C. After two days germination, developing embryos were isolated and the whole embryo was collected after seven days. Seven-day-old seedlings were transferred to hydroponic nutrient solution (NH₄NO₃ and KNO₃, 5 mM; Ca(NO₃)₂, 2 mM; MgSO₄, 2 mM; NaFe(III)EDTA, 0.05 mM and micronutrients). After 30 d young roots about 9 cm long, a 5 cm section of young leaf sheath and a 12 cm section of the young leaf blade were collected.

Mature rice plants were grown in a greenhouse under a day/night temperature regime of 28^°C/25^°C. A 16 cm mature leaf blade section was excised from the base of fifth leaf when it was 32 cm long. Young developing grain at various stages were collected and frozen whole and for other seeds the developing endosperm was dissected away from the other tissues of the young grain.

Coleoptiles for quantitative PCR

Dehulled seeds of rice (Oryza sativa L cv. Calrose, Amaroo and IR22) were surface- sterilized as above. About 240 seeds were transferred to 2 L 0.8 mM KH₂PO₄ and 0.5 mM CaCI₂ solution (about 4.5 cm deep) in plastic containers (separate containers for each time point). The seeds of Calrose , Amaroo and IR22 were germinated in this unstirred solution at 28^°C in the dark for 1 d (Calrose and Amaroo - japonica subspecies) or 2 d (IR22 - indica subspecies) by which time coleoptiles had begun to emerge. For anoxic treatment, high-purity nitrogen gas was continuously bubbled through the solution at about 1 L h^'1 until coleoptiles were 3-5 mm long. Coleoptiles were cut from the seed, frozen under liquid N₂ and stored at -80^°C. Control seedlings were grown in aerated solution and coleoptile tissues were collected at the same time as anoxic samples. RNA extraction and cDNA synthesis

Total RNA was extracted from at least three individual samples of coleoptiles and other tissues, using a commercially prepared guanidine reagent, TRIzol (Invitrogen, Mt Waverley, Victoria, Australia) according to the manufacturer's instructions. Purified RNA was treated with DNasel using the DNA-free kit (Ambion, Austin, TX, USA) according to the manufacturer's instructions. RNA integrity was checked on a 1.6% (w/v) agarose gel containing ethidium bromide.

Two microgram of each RNA was mixed with 1 μl_ 50 nM oligo-dT (12-20) primer, 1 μl_ 10 mM dNTP mix and sterile water to a volume of 10 μl_. The reaction was heated at 65°C for 5 min and snap cooled on ice. A master mix (10 μL) was added to each reaction, which contained 2 * RT buffer, 0.1 M dithiothreitol, 50 mM MgCI2, 40 units of RNAseOUT (Invitrogen, Australia) and 50 units of Superscript III RT. The reaction was incubated at 42°C for 1 h and for 15 min at 75°C. The cDNA was stored at -20⁰C. Quantitative PCR analysis of transcript levels

Quantitative PCR primers were designed from conserved regions so that each pair of primers could be used for Q-PCR analysis of all three cultivars (Table 1), after aligning partial cDNA sequences for each member of the OVP gene family from three cultivars. Levels of rice V-PPase mRNA were determined by Q-PCR using a PG 2000 Rotor- Gene Real Time Thermal Cycle. Briefly, PCR products for making standard dilutions were obtained from a coleoptile cDNA in a reaction mixture containing 10 μL QuantiTect SYBR Green PCR reagent (Qiagen, Valencia, CA,- USA), 3 μL each of 4 μM forward and reverse primers (Table 1) and 3 μL water under the following cycling parameters: pre-step, 15 min at 95^°C; 45 cycles of 20 s at 95^°C, 30 s at 55^°C, and 30 s at 72^°C; and extension step, 15 s at 80^°C. The amplification products pooled from 4 to 6 independent 20 μL PCR reactions were purified and quantified by HPLC on a Varian HELIX DNA column (2.1 mm x 15 cm, 3.5 micron; Varian, Middelburg, Netherlands). The PCR product was sequenced to confirm its identity and used to make a dilution series covering seven orders of magnitude.

An optimal temperature for data acquisition for each pair of primers was obtained by performing melting curve analysis after heating PCR products at the end of the amplification from 7O C to 99 C and monitoring fluorescence intensity. Actin (accession number X15865.1), alpha tubulin (accession number L19598.1), glyceraldehyde-3- phosphate dehydrogenase (accession number EF125181.1). peptidylprolyl isomerase (accession number AK102337.1) and elongation factor 1 B2 (accession number D83727.1) sequences from rice were used as control genes (Table 1). For measuring transcript levels in tissue samples, 1 μL 1 :10 dilution of a cDNA population was used to prepare a similar PCR reaction mixture as described above but with the addition of 0.6 μL10 x SYBR Green and 2.4 μL water. Quantitative PCR was performed with the cycling parameters given above. The Rotor-Gene v4.6 software (Corbett Research) was used for data acquisition and manipulation.

For comparisons of mRNA levels in different tissues, a normalization factor was calculated from at least three of five control genes run simultaneously with samples. Western blot analysis

Proteins from the tonoplast preparations were separated on SDS-PAGE. Ten percent acrylamide gels were run in the Bio-Rad Mini-protean Il apparatus. Proteins were transferred to nitrocellulose membranes, lmmunoblotting was performed using a rabbit polyclonal antibody raised against the purified 67 kD subunit of mung bean V-PPase and the 70 kD subunit A of Arabidopsis V-ATPase both kindly provided by M. Maeshima (Nagoya University, Japan). Colour was developed after incubation with goat anti-rabbit IgG-conjugated horseradish peroxidase and the addition of peroxidase substrates. Only one band was seen on the membranes blotted with tonoplast membrane preparations. Promoter sequences and preparation of OVP3 promoter: :GUS constructs

DNA fragments containing the promoter region (2.1 kb) and the 5 -untranslated region (UTR, 105 bp) upstream of the translation start codon of the OVP3 gene were amplified by PCR from rice genomic DNA of Calrose, Amaroo and IR22 using the primer pair (CACCCGCGAACGCTGCAATTTGCn-GGAGATTCTTGGAGGTGA). The PCR products were sequenced for comparisons of anaerobic motifs and regulatory elements. To make the OVP3 promoter::GUS construct, DNA from Amaroo was used. The sequence CACC was added at the 5 end of the DNA sequence, to facilitate directional incorporation of the OVP3 promoter DNA into the pENTR/D-TOPO vector (Invitrogen, Mt Waverley, Victoria, Australia), generating a pENTR/D-TOPO-O\/P3 promoter construct. A recombinant reaction using the LR clonase mix (Invitrogen) was carried out with the pENTR/D-TOPO-O\/P3 promoter DNA and the vector pMDC162 to make pQZ1. pMDC162 is a binary vector designed for cereal transformation and incorporates a hygromycin (Hpt) selectable marker gene under the control of the CaMV 35S promoter and Nos terminator . The constructs were transformed into Agrobacterium tumefaciens strain AgIO by heat shock.

Rice transformation

Oryza sativa L. cv. Calrose was transformed. Embryogenic nodular units arising from scutellum-derived callus were inoculated with the super-virulent A. tumefaciens strain Ag10 and 50 mg I^"1 hygromycin-resistant shoots were regenerated after nine weeks. Overall, the inoculated calli yielded regenerated plants with an average transformation efficiency of four primary transformants per callus. Rooted T₀ plantlets were transferred to the greenhouse in Jiffy peat pots (Jiffy Products International AS, Norway), and moved to soil after 15 d. Total genomic DNA was extracted from the leaves of T₀ plants. Southern analysis was performed. Plant DNA was digested with Hind\\\, which cuts once only in the OVP3 promoter sequences. The full-length GUS gene was used as a probe. Ti seed was harvested from fertile lines, dried for 3 d at 37°C, and placed into cold storage (4°C, 30% humidity) until required.

Analysis of T₁ seedlings

PCR was performed on Ti seedlings to differentiate those plants transformed with OVP3 promoter: :GUS construct and wild type segregants using the primer pairs: ATGTTACGTCCTCTAGAAACCCC/AGCTCGGTAGCAATTCCCG. Total genomic DNA was extracted from the leaves of Ti seedlings as described above. This PCR produced a 2068 bp fragment in transgenic seedlings, with no product for wild type segregants. Histochemical location of GUS expression

Histochemical analysis of GUS activity was performed. Excised seminal root tissues from Ti transformants treated under anoxia for 24 h^' were incubated at 37°C for at least

4 h in a 100 mM sodium phosphate buffer (pH 7.2), containing 0.5 mM potassium ferrocyanide, 0.5 mM potassium ferricyanide, 0.1% Triton X-100 and 1 mM 5-bromo-4- chloro-3-indoyl glucuronide. After fixation, roots were embedded in paraffin and sectioned then visualized using a Leica AS LMD Differential Interference Contrast Laser Dissection microscope (Leica Microscopie Systerhes SA, Heerbrugg, Switzerland). Images were collected using a DFC 480 CCD digital camera.

Preparation of coleoptiles for GUS assay under anoxia

Coleoptiles of Ti seeds from four independent lines and WT were grown as the previous methods given for Q-PCR. Three-day-old coleoptiles were transferred to sealed Falcon tubes with 50 ml of 0.8 mM KH₂PO₄ and 0.5 mM CaCI₂ solution. Each tube contained one coleoptile of 7-8 mm in length still attached to their seed. High purity N₂ was continuously flushed through the tubes for 24 h. Coleoptiles were excised and ground. The extracts were used to measure GUS activity.

Preparation of seedlings for GUS assays under different stresses

The T₁ seeds containing a single locus of the 0VP3 promoter: :GUS construct (selected by PCR as described above) from four independent lines and WT were germinated on wet filter paper in Petri dishes at 28^°C for 5 d then transferred to hydroponic nutrient solution (as above) growing aerobically for a further 15 d. For anoxic treatments, 20-d- old seedlings were exposed to a 16 h hypoxic pre-treatment in the dark, after which they were transferred to sealed Falcon tubes containing 50 ml growth solution. High purity N₂ was continuously flushed through the tubes for 24 h. For salt treatment, seedlings were transplanted to a fresh growth solution supplemented with 25 mM NaCI for 5 d and then 50 mM NaCI for a further 5 d. To maintain the calcium activity during salt treatment, the activity of Ca²⁺ was calculated by Visal MINTEQ and 0.5 mM CaCI₂ was added to the medium. For cold treatment, seedlings were transferred to 20^°C for 2 d and then cooled to 15^°C and 10^°C for 2 d, respectively.

Protein extraction and GUS assays

GUS activity was assayed in the 3-d-old coleoptiles, terminal 20 mm of seminal roots and 50 mm from the tip of the flag leaf. Plant tissues were ground with fine sand in sodium phosphate buffer (50 mM, pH 7.0, 10 mM EDTA, 10 mM β-mercaptoethanol,

0.1% Triton X-100). After centrifugation, the soluble protein was assayed and GUS activity was measured.

EXAMPLE 2

Transcript levels of OVP genes in different tissues

Transcript levels of six isoforms of V-PPase (OVP1, 2, 3, 4, 5 and 6) were determined by Q-PCR using gene-specific primers across a range of tissues. The transcript profiles of the OVP genes showed that OVP1 and OVP6 mRNA levels were very abundant in young leaf blades (50- to 600- fold higher than levels of the other OVP mRNAs) whereas OVP2 transcript levels were dominant in particular growing tissues such as roots and leaf sheaths (Fig. 1a). In contrast, OVP5 mRNA levels were extremely low in most tissues (Fig. 1b) and overall transcript levels of OVP3, OVP4 and OVP5 were markedly lower than levels of OVP1 and OVP6 (Fig. 1).

EXAMPLE 3

Transcript levels of OVP genes in response to anoxia in Calrose

Transcript levels of the six isoforms of V-PPase in anaerobically treated rice coleoptiles were also examined by Q-PCR. Calrose was chosen as a traditional flood-tolerant rice cultivar. After exposure to anoxia for 2 h, OVP3 mRNA levels doubled and peaked at a nine-fold increase after 12 h compared to levels in aerated coleoptiles. A small decline followed after 12 h (Fig. 2b). The greatest rate of increase in the highly responsive OVP3 gene thus occurred during the first 6 h of anoxia (Fig. 2b). In contrast, the mRNA levels of OVP1 and OVP2 decreased significantly under anoxia (Fig. 2a), while OVP4 mRNA levels were nearly unchanged under anoxia over 24 h (Fig. 2b). There was a slight increase in OVP6 mRNA levels but only after 24 h under anoxia (Fig. 2c).

EXAMPLE 4

Transcript levels of OVP3 in rice cultivars with contrasting anoxia tolerance

Having established that OVP3 was an anoxia-responsive gene, we tested OVP3 transcript levels in coleoptiles of three rice cultivars with variable anoxia-tolerance: Calrose (moderate), Amaroo (high) and IR22 (intolerant). OVP3 gene expression in Amaroo was dramatically induced with an increase of approximately 17-fold compared to the mRNA level in aerated coleoptiles 2 h after the start of the anoxic treatment (Fig. 3). OVP3 mRNA then decreased slightly but still remained 10-fold higher than the levels in aerated coleoptiles. In Calrose, there was also a significant increase in OVP3 transcript levels (10-fold) after 2 h of anoxia compared to the aerated coleoptiles (Fig. 3) and this level remained high. The levels of OVP3 mRNA in Calrose were about 3 fold lower than the levels in Amaroo. OVP3 gene expression in IR22 was also induced during the first 6 h of anoxia but the mRNA level was only half of that seen in Calrose (Fig. 3).

EXAMPLE 5

Effect of anoxia on V-PPase and V-ATPase protein abundance

The V-PPase protein was hardly detectable in coleoptiles of rice (Calrose) grown in aerated solution (Fig. 4a) using a polyclonal antibody raised against the 67 kD subunit of mung bean V-PPase, which is consistent with the relatively low transcript levels of all OVP genes during aeration (Fig. 2). By contrast, when coleoptiles were subjected to anoxia, a strong protein band was detected, which was corresponding to the size of rice V-PPase (75 kD). The amount of V-PPase protein increased substantially with time (Fig. 4a). The substantial increase in protein was possibly attributable to the increased expression of OVP3 since the mRNA levels of other isoforms were comparatively low. By contrast, western blots showed that V-ATPase protein level decreased with time under anoxia (Fig. 4b). EXAMPLE 6

Transgenic rice plants

Transgenic T₀ rice plants were produced using the super-virulent Agrobacterium tumefaciens strain Ag 10 transformed with vector pQZ1. A total of 14 T₀ plants containing the OVP3 promoter::GUS fusion construct were identified by Southern blot analysis using the full length of the GUS gene as a probe (data not shown). Of these, four independent T₀ plants carried a single locus (data not shown), whose seeds were harvested and sown to produce T₁ seedlings. The Ti plants were tested by PCR to establish a segregation ratio for the locus of OVP3 promoter::GUS fusion construct. A segregation ratio of 3:1 transgenic to wild type segregant was observed for all four lines carrying a single transgenic locus.

EXAMPLE 7 Anoxia-induced tissue-specific GUS expression

Seven-day old T₁ wild type segregant and transgenic seedlings selected by PCR from four independent lines were treated with anoxia. In the wild type segregant seedlings used as controls, there was no GUS expression observed in any tissue after the anaerobic treatment (Fig. 5, a and b). In transgenic seedlings, GUS staining was seen basal to the root apex and especially in the stele (Fig. 5c). There was no GUS expression in the root cap (Fig. 5d). Stele cells in the root elongation and maturation zones showed a high level of GUS expression (Fig. 5d). There was no GUS expression in the epidermis (Fig. 5e). The GUS expression pattern looked the same in all four independent lines but images from only one line are shown in Fig. 5.

EXAMPLE 8

Quantitative assessment of OVP3 promoter activity

Three-day-old coleoptiles of Ti wild type segregant and transgenic seedlings from four independent lines and wild type (WT) were exposed to anoxia for 24 h before GUS activity was assayed enzymatically. The GUS activity in coleoptiles of the OVP3 promoter: :GUS transformants under anoxia was three-fold higher than in those of anoxia-treated wild type segregants and WT (Fig. 6).

Anoxia, salt or cold stress was applied to the OVP3 promoter::GUS Ti transformants from four independent lines and WT for GUS assay in flag leaves and roots. The GUS activity in roots was induced approximately seven-fold by anoxia compared to WT (Fig. 7) and wild type segregants (data not shown). Even though GUS activity in leaves was less than one-third the level in roots, there was nonetheless an induction of about 4-fold in GUS expression in leaves after anaerobic treatment (Fig. 7). Salt or cold did not substantially induce GUS activity in either roots or leaves (Fig. 7). EXAMPLE 9

Analysis of functional motifs in putative promoters of OVPs Since OVP3 was the primary V-PPase whose transcript level increased in the early stages of anoxia, it could be considered a typical anaerobic protein and one might expect its promoter regions to differ from other OVP isoforms. In order to understand the regulation of V-PPase expression, promoter regions of all six OVP genes were analysed for putative anaerobic-responsive motifs. We also analysed the promoter regions of OVP3 from all three cultivars Amaroo, Calrose and IR22. Promoters were sequenced and putative functional motifs identified.

A search of the PLACE database (http://www.dna.affrc.qo.ip/PLACE/) with a 2.1 kb fragment of the genomic DNA upstream from the transcription start site of the six isoforms of V-PPase revealed that six anaerobic motifs reported in the database were present (Table 3). Four of them (AAACAAA, AGCAGC, TCATCAC and GTTHGCAA) are among the 16 motifs found in silico within 13 promoters of anaerobic genes; the other two were an anaerobiosis-specific binding site of tobacco nuclear factor (GCBP-2) found in maize and a TATA-like motif. The OVP3 promoter contained all six of the functional motifs and was the only promoter that had the GTTHGCAA and GTGGGCCCG motifs, the latter being a binding site of GCBP-2. Moreover, the AGCAGC motif occurred twice in the OVP3 promoter, making a total of seven anaerobic motifs. OVP1 and OVP6 contained the AAACAAA, AGCAGC and TCATCAA motifs, while OVP6 also had a TTTATATA motif. 0VP2 did not contain any of the six functional motifs. OVP4 had the AAACAAA and TTTATATA motifs and OVP5 had only the AGCAGC motif.

PlantCare (http://bioinformatics.psb.uqent.be/webtools/plantcare/html/) was also used to search for c/s-acting regulatory elements for anaerobic induction in the putative promoters of all six isoforms of V-PPase (Table 4)._, ARE (TGGTTT), G-Box and TATA box elements were found in the promoters of all six isoforms. The GC motif identified as an enhancer-like element involved in anoxic-specific inducibility was only found in the 0VP3 promoter. Based collectively on the six anaerobic motifs identified in PLACE and these regulatory elements, 0VP3 was predicted to be the most responsive OVP gene to anaerobiosis. EXAMPLE 10 Identification of a new vacuolar pyrophosphatase gene from rice

The rice genome sequence was searched with genpmic and cDNA sequences of OVP1 and OVP2 using the BLAST program available at the Gramene web site (http://www.gramene.org/). In addition to the previously identified V-PPase genes (OVP3, OVP4, OVP5), an additional gene was identified and designated OVP6. The near full-length OVP6 cDNA clone comprised 2310 bp encoding 770 amino acids. The deduced amino acid sequence of OVP6 has 91% identity with OVP2, while the other isoforms have lower percentage identities with OVP6 (Table 2). However, the nucleotide sequence of OVP6 has only 71% identity with OVP2 (Table 2). The deduced protein sequence of OVP6 contained the motif DVGADLVGKVE, which has been identified as a conserved segment corresponding to a putative catalytic site of V-PPase. In view of its distinct expression pattern and predicted promoter sequence, we believe OVP6 to be a previously unidentified gene belonging to the OVP family. The nucleotide sequence of OVP6 is shown in Figure 8.

Table 1 PCR primers for amplifying partial V-PPase sequences and Q-PCR primers

Primers for amplifying partial V-PPase from Calrose, Amaroo, IR22

Gene Forward primer Reverse primer

OVPl CTTGCTGGTGCTCTCGTTTC AGCTTAGCATGATCGACTGAT

0VP2 ACGGTGGTCTGCTGTTCAAGT CCAGACACGCACCAGAGAT

0VP3 AATTTGAGGACGGACGGAGAT GGCTCAGGCAGACAGAAACT

OVP4 CTGGGACAATGCCAAGAAAT ATGATTGTTTACTCCGTGCG

AATGTTGCGTGAATCGAATT (IR22) AATTCGATTCACGCAACATT (IR22)

OVP5 GGACAACGCCAAGAAGTACA CCAGTTGAAACCGATAAGAGT

OVP6 TGAGTTGATGACTCCATTGTCTAC GTGGCACAGTGAACAAGGAG

TGTTGGGGATGAGCAAAATA TTTCCAATGCTCCTTGTTCA

(Calrose) (Calrose)

primers for Q-PCR

OVPl AGGCATCCTCTTCAAGTGG GGACAGACAGTAATAACAAATAGG

OVP2 TCACGAATAACGACTGCCAG ACGCACCAGAGATGGGAG

OVP3 GACACGGGATCATGTGGGCT AAACGACGCAGCAAGTTAA

OVP4 GGTTCGGACGCTCACAA ATACTCCGTGCGATTGTCAC

OVP5 CCATCGCTCAACATCCTCGTCAA TCGTCTTCTTCTTGTTTATCCAT

OVP6 GGGATGAGCAAAATAGAGATGA GGAACCTGGAACTAGAACAAA

Actin GAAGATCACTGCCTTGCTCC CGATAACAGCTCCTCTTGGC

_Tu ^a TACCGTGCCCTTACTGTTCC CGGTGGAATGTCACAGACA

h GGGCTGCTAGCTTCAACATC TTGATTGCAGCCTTGATCTG

CTGGCAAAGAACCTAAACCTCATC TGCTCCTGTGGTGTAACTGCTTT

Pf ATCTGGGAAATCATCGGTTCTG AGATCGTCCACAATGGTCATCA

ELFf

a alpha tubulin

b glyceraldehyde-3-phosphate dehydrogenase

c peptidylprolyl isomerase

d elongation factor 1 B2 Table 2 Comparison of nucleotide and amino acid sequence identities of rice vacuolar pyrophosphatases

Nucleotide sequence Amino acid sequence

~% OVPl OVP2 OVP3 OVP4 OVP5 OVP6 OVPl OVP2 OVP3 OVPi 0VP5 OVPβ

o^vpl 100 100

o^vp2 78 100 88 100

ovP3 _{67 71 1 O}o 93 88 100

o^vp4 53 54 57 100 74 77 75 100

^OVP5 58 58 61 61 100 73 73. 72 75 100

o^vp6 77 71 68 55 58 100 85 91 87 75 73 100

Table 3 Anaerobic motifs obtained by searching PLACE with 2.1 kb fragments of the genomic DNA upstream from the transcription start site of the six isoforms of rice V- PPase. The number indicates the position from the transcription start site. No motif was found in OVP2. (+): direct sequence; (-): complementary sequence

Motifs/ AAACAA AGCAGC TCATCAC GTTTHGAA GTGGGCCCG TTTATATA Isoforms (Binding site of (TATA-like

GCBP-2) motif

OVP1 776(+) 1721 (-) 1175(+)

842(-) 1183(-)

1666(+)

1713(-)

OVP3 359(+) 88(-) 2025(+) 12(-) 91 (-) 1531 (+)

1945(+)

OVP4 155(+) 2124(-)

558(+)

2005(-)

OVP5 1182(-)

1276(+)

OVP6 1257(+) 361 (-) 1445(-) 840(+)

1886(-)

1988(-) Table 4 Regulatory elements obtained by searching PlantCare with 2.1 bp fragments of the genomic DNA upstream from the transcription start site of the six isoforms of rice V- PPase. The number indicates position from the transcription start site

Regulatory TGGTTT G-Box GC motif TATA-Box

elements/ (ARE³)

Isoforms -GT motif •

OVP1 834(-) 655(-) 1495(+)

1266(-) 1079(+)

OVP2 760(-) 617(-) 1376(+)

OVP3 64(-) 218(+) 1207(+) 1408(+)

1826(-) 2073(-)

1815(-)

OVP4 1049(-) 133(+) 1417(-)

1478(+)

OVP5 211(+) 1469(+) 1454(-)

608(-) <

837(+)

1000(+)

1052(-)

1436(+)

1574(-)

1804(-)

OVP6 271 (-) 1524(+) 1410(+)

306(-)

1590(+)

aARE: Anaerobic Response Element Table 5 List of sequences for SEQ ID No. 1 to 14.

SEQ ID No: Sequence

1 CCGGGGC

TAATA

TCATCAC

AGCAGC

GTCGTG

TTTATATA

CCCCCG

TTTCGAAC

AAACAAA

10 CTACGTATTA

11 CGGGCC

12 CACGTG

13 GCTGCT

14 AAACCA

15 OVP3 TTTGCAAGGCGATTCCACACCGGGGCTTTCAGATGTTGGCGCGAGACGGA promoter TTTTTTGACGCCAATCCCACACATAATATGGTTGTTCATCACTAATTCACT

AGTGTCAGCAGTGTGTCGCTCACTCACTAGCCAAATGCAGCAGGCCTCTC

TACACCAGCAAGGCAGCAGCATGAAACAAGTCCGAAGTCTCATTGGCGTT

GAAACTGCTCTTGCCGCGGGAACTGAAAAACAACAGACCACTCGCCGGAT

CATGTATTCATGTCGTGCGTGCGACGTGCCATGCGAGCTCGGCCACGAAA

GTGCCACTTCATTTTACTACTAGTATCCCCTTCAAAAATAATTTACCACTA

GTATACCCGTGACTGAGCCGTCAAGCTATCGTTTGACTTATTCACAAAATA

AGCTAAACGATATATTTATAAATAAAAAATAATTTATAAATAAAACTTTTA

TATACATAATGATCTAAAAGTAAAAGTTGAAAATAAACATTGATAAAATAT

TCTTAAAATAAATTTTAAATTAAAAATCTAACTTATAAATATAAGTGCAAG

CGAAAAAAAATAAGGCTGTGAGTTTTCTGGCTCTTACAGTAGAGGGAGCA

TGTAGCCGTAGCCTAGTGGTTACAGTAACCTAAGTAGTATTCTAAGGTCC

TGAGTTCAAATCTCATATGGAACGAATTTTAGATTGGGTTGTTTGTGGGG

CTCCCTTGTTCAAATCTTCTATAGAGCGAATTTCAGATTAGGTTGTTTGCG

GGGCTAAGTTTCCTTTTAAGACAAGGGGTTGGATGTAGAGGCCGGGTAAA

AAAAAAAACTTCTCTAAAAAAAACAATAGAGGGAGCAAACTGCAAACCCG

GTCGTGCGAGTCGGTCGTATTCGGGGCAAATCCAGCGGCGTCGCCGAGG

AATCCACCCCCGAAAGCAAAAGCTGCGCCGCCGACGAAACGATGCGGAT

AAGGACGGGCACGGCCTAAAATTTCCATTCTGCAGCTGAAACCGCGACTC

TTTTGTCCCACACCGTCAGCAACCGCTGTTTCTCCTCCCATTTTTTACCAG

GCTAATTAAGTTAACCCCCCACTTGCAGCGTTCATGGCCGTCGATCTCAT

CATCATCTCACTGTGATTATCTTCTTTAATCCACTTCGTTTTAGCTTCTAAT

AACTATCAACTTTCGTTTATAAGCTTCTAACCACACCAGACCAGTACACCA

CTGTTTGTCGCAGGCTGCGAGGACCGAGTTAAATTATTTACTCTTTGGAC

GCAATACTAGCCAAGCATAATCAAATATATACGGGGAGAAATCGAAGTTG

TGCTCTGGGAACACAATTAAAGGGGCCTAAAATCCGAGTGTCAAGTTTAG

TTTGACGTTAATTATTCGATCGGCACATATGTGAATATGATTAGCTCGTCC

TACTGCGAGCTAGACACGCTT AATTACAACAACTATCGCATAAAACTGTT

GATGTAGCACACGGGAAAGTCTCATTGGTTATGGTCTGATTCTTTAAGGA

AGCCGAGAATTCCATTGCTTATCAATCGCGCAATTTGCAAATACGTCATCC

TTTCAAGAGGGATTAGGAAATCGAAGAAGAAGAACATCATAGTGCCATTA

GAAGCCAATTACCTTGATGGCTTAAACCGGGTGAATTGTTTGAATTTCGA

ACGAATCACACAAATCAAATTCATTTCTAGTGATACGTACGCCCAATTGAG

CTTGGAACAATCTAACATTCTAAGCAAGAAACAAATCCCGCTGTAAGATT

CGGATTTCGGACAGTGAGATTCTGGATTAGTAGGATCATGCTTATCTTTG

GACTACGTATTATTATCTTCTGCCTACAAAGTCCAGAAAGACACATGTTAG

CCAGTCAGGTGCGTTTCGGGCCCACGTGGCAGTGTGAGCCTGGCAAAAG

GGCCCAGAAATCGCAGCCCACTCGATCCATCAAGAATATTTACAATTACA

CCCTTGGAAAAAACATAGAATTGCAAAACAACCCACGCACTCCCTCTCCG

CTGCTCGTCTTCTACCTCTCGCCCAAACCAGGCAGCTCCACCTCCACCCA

CCCAGCTATATAAGAGACCTCACCTCCAAGAATCT 16 OVP6 ORF ATGGCGGCGGCGGCGATACTGCCGGAGCTGGCGGCGCAGGTGGTGATCC

CGGTGGCAGCGGCGGTGGGGATCGCGTTCGCGGTGCTGCAGTGGGCGCT

CGTCTCCAAGGTGAAGCTCACGGCGGAGCCGCGGCGCGGGGAGGCGGGG

GGCGCCGCCGGGGGGAAGAGCGGGCCGAGCGACTACCTCATCGAGGAGG

AGGAGGGGCTCAACGACCACAACGTCGTCTCCAAGTGCGCCGAGATCCA

GACCGCCATCTCCGAAGGAGCGACATCTTTCCTTTTCACTGAGTACAAGT

ATGTCGGATTATTCATGAGCATCTTCGCAGTTCTTATTTTCCTCTTCCTTG

GGTCTGTTGAGGGCTTCAGCACGAAGAGCCAGCCTTGCCACTACAGCAAG

GACAAGACTTGCAAGCCTGCCCTTGCAAATGCTATCTTTAGCACTATAGC

TTTTGTGCTTGGTGCAGTTACCTCTCTGGTATCTGGTTTTCTTGGAATGAA

GATTGCAACTTATGCAAATGCTCGGACGACTCTGGAGGCCAGGAAGGGTG

TTGGAAAGGCTTTTATCACTGCTTTCCGGTCTGGTGCCGTTATGGGTTTCC

TGCTTGCTGCAAGCGGTCTCTTGGTCCTTTACATTGCAATTAATTTGTTTG

GAATTTATTATGGTGATGACTGGGAAGGCCTTTTTGAAGCTATTACTGGTT

ATGGACTTGGTGGTTCTTCTATGGCTCTTTTCGGCCGTGTAGGTGGTGGT

ATTTATACAAAAGCTGCTGATGTTGGTGCTGACCTTGTTGGGAAGGTGGA

AAGAAATATTCCTGAGGACGATCCCAGAAACCCAGCTGTCATTGCAGACA

ATGTTGGTGACAATGTTGGAGATATTGCTGGAATGGGATCAGATCTTTTT

GGTTCTTATGCTGAATCATCGTGTGCTGCACTTGTTGTGGCATCAATCTCT

TCTTTTGGAATCAACCATGAATTTACTCCCATGGTGTACCCTCTTCTTGTC

AGCTCTGTGGGAATTATAGCGTGTCTCATAACCACCTTGTTTGCAACTGAT

TTCTTTGAGATAAAGGCTGTGAGTGAAATAGAGCCAGCTCTCAAGAAACA

GCTTATAATTTCCACTGCTTTTATGACTGTTGGCATTGCCCTTGTCAGTTG

GTTGGGCCTTCCTTACACCTTCACCATCTTTAACTTTGGTGCCCAGAAGAC

AGTGCAAAGCTGGCAATTATTCTTGTGTGTGGCGGTTGGTCTTTGGGCTG

GTCTAATCATTGGATTTGTTACCGAGTATTACACCAGCAACGCATATAGCC

CTGTACAAGATGTTGCTGATTCTTGCAGAACTGGAGCTGCCACTAATGTC

ATTTTTGGGCTTGCTTTGGGTTACAAATCAGTCATCATCCCTATTTTTGCT

ATTGCTTTCAGCATTTTCCTCAGCTTCAGCCTTGCTGCCATGTATGGTGTT

GCTGTGGCTGCTCTTGGAATGCTGAGCACAATTGCCACAGGTCTTGCTAT

CGACGCCTATGGTCCCATCAGTGACAATGCTGGAGGAATTGCTGAAATGG

CCGGCATGAGCCACAGAATTCGGGAGAGAACTGATGCTCTGGATGCTGCC

GGAAACACAACTGCCGCAATTGGAAAGGGTTTTGCCATCGGCTCTGCAGC

CTTGGTGTCACTTGCACTTTTTGGTGCCTTTGTGAGCCGTGCGGCTATCTC

TACGGTTGATGTTCTGACACCAAAAGTGTTCATTGGGCTTATTGTTGGTG

CTATGCTCCCATACTGGTTCTCAGCAATGACTATGAAGAGTGTAGGCAGC

GCGGCGCTAAAGATGGTGGAGGAAGTCCGCAGGCAGTTCAACACCATCC

CTGGACTCATGGAGGGCACAACTAAGCCTGACTATGCAACTTGTGTCAAG

ATCTCCACTGATGCATCCATCAAGGAGATGATCCCTCCTGGTGCTCTTGTT

ATGCTCACCCCACTTATCGTCGGAATTCTGTTCGGCGTCGAGACCCTCTC

TGGAGTCCTTGCTGGTGCTCTCGTCTCTGGTGTTCAGGATTACAATTGCA

TCCAATGGTCCGTGCTCCTTATCCGTTTCACCTTCTCTCAGATTGCCATCT

CCGCATCAAACACTGGCGGTGCTTGGGACAATGCAAAGAAATACATTGAG

GCTGGAGCTTCAGAGCATGCCAGGACCCTTGGTCCCAAAGGTTCCGATCC

CCACAAGGCGGCCGTCATCGGTGACACCATCGGAGACCCTCTCAAGGACA

CTTCAGGACCCTCTCTCAACATCCTCATCAAGCTCATGGCGGTCGAATCC

CTCGTTTTCGCCCCCTTCTTCGCCACCCACGGTGGTATCCTCTTCAAGTTG

TTCTAA

17 OVP6 MAILSDVATEVLIPIAAIIGIGFSIAQWVLVARVKLAPSQPGASRSKDGYGDSLI predicted aa EEEEGLNDHNVVAKCAEIQNAIAEGATSFLFTEYQYVGVFMSIFAWIFLFLGS

VEGFSTKTHPCTYSKDKECKPALFNALFSTVSFLLGAITSVVSGFLGMKIATY

ANARTTLEARKGVGKAFITAFRSGAVMGFLLASNGLLVLYIAINLFKMYYGD

DWEGLFESITGYGLGGSSMALFGRVGGGIYTKAADVGADLVGKVERNIPEDD

PRNPAVIADNVGDNVGDIAGMGSDLFGSYAESSCAALVVASISSFGINHDFTG

MCYPLLVSSMGHVCLITTLFATDFFEIKA VKEIEPSLKKQLIISTALMTVGIAL

VSWLALPYKFTIFNFGEQKEVTNWGLFLCVSIGLWAGLIIGYVTEYYTSNAYS

PVQDVADACRTGAATNVIFGLALGYKSVIIPIFAIALGIYVSFTIAAMYGIAVAA

LGMLSTIATGLSIDAYGPISDNAGGIAEMAGMSHRIRERTDALDAAGNTTAAI

GKGFAIGSAALVSLALFGAFVSRAGVKVVDVLSPKVIIGLIVGAMLPYWFSAM

TMKSVGSAALKMVEEVRRQFNTIPGLMEGTGKPDYANCVKISTDASIKQMIP

PGALVMLTPLIVGTLFGVQTLSGVLAGALVSGVQVAISASNTGGAWDNAKKY

IEAGASEHARSLGPKGSDCHKAAVIGDTIGDPLKDTSGPSLNILIKLMAVESLV

FAPFFATHGGILFKLF

18 Cl TKAADVGADLVGKVERNIPE

Claims

1. A nucleic acid molecule for forming a promoter for regulating transcription of a gene in a plant in anoxic conditions including one or more nucleotide sequences as shown in SEQ. ID Nos. 1 - 14 wherein each of the sequences are spaced apart from each other to define regions for forming regulatory elements for interacting with a transcription factor.

2. A nucleic acid molecule according to claim 1 including all nucleotide sequences as shown in SEQ. ID Nos. 1 - 14.

3. A vector or like construct having a gene for encoding an expression product and a nucleic acid molecule according to either claim 1 or 2.

4. A plant or cell or tissue derived therefrom including a nucleic acid molecule, vector or like construct according to any one of the preceding claims.

5. A method for producing an expression product in a plant in anoxic conditions including the step of culturing a plant, or tissue or cell derived therefrom according to claim 3 in anoxic conditions.

6. A peptide having a sequence shown in SEQ. ID No. 17, or fragments thereof.

7. A process for producing a plant, or tissue or cell therefrom that is tolerant to anoxia including the steps of:

-providing a vector or like construct according to claim 3 having a gene for encoding a vacuolar pyrophosphatase for regulation of transcription of the gene when the construct is provided in a plant cell in anoxic conditions, and

- introducing the vector or construct into a plant cell, tissue or whole plant.

8. A plant according to claim 2 or process according to claim 5 or 7, wherein the plant is either a monocotyledon or a dicotyledon.

9. A plant or process according to claim 7 wherein the monocotyledon is a grass.

10. A plant or process according to claim 7 wherein the monocotyledon is rice.

11. A plant or process according to claim 7 wherein the dicotyledon is cotton.

12. A plant or process according to claim 7 wherein the dicotyledon is hemp.

13. Use of a vector or like construct according to claim 3 for producing an expression product in a plant in anoxic conditions.

14. Use of a vector or like construct according to claim 3 for producing a plant or tissue or cell therefrom that is tolerant to anoxia.