WO2003083121A1 - A calcium ion channel - Google Patents

Abstract

A method of providing a plant having an altered calcium uptake. The method comprises the step of altering the expression or activity of a protein in a cell of the plant thus altering the transport of calcium ions into the cytoplasm of the cell of the plant by the protein. The protein comprises a sequence at least 25% identical to SEQ ID NO: 1 over an alignment of at least 450 amino acids.

Description

A CALCIUM ION CHANNEL

The present invention relates to a protein calcium ion channel; nucleic acids encoding the protein and cells and organisms transformed with such nucleic acids. The invention also relates to methods of providing a plant having an altered calcium uptake; methods of providing a plant having an altered stress response and methods of conducting phytoremediation.

Background

An important requirement for cells in all organisms is the ability to regulate the transfer of inorganic ions between the exterior of the cell and its cytoplasm and between the cytoplasm and certain organelles. Channel proteins form hydrophilic pores across membranes in the cell, in particular the plasma membrane, to allow inorganic ions to diffuse down their electrochemical gradients and be transported through the membranes. Calcium ion channels are a particular type of channel protein which allow the passage of calcium ions.

In animal cells, different kinds of calcium channels have been described. They can be divided in two categories : VDDC (Voltage Dependent Calcium Channels) and ligand gated calcium channels. The main point of focus will be on VDCC, localised, as AtTPCI, in the plasma membrane. Voltage-gated calcium channels were first identified in crustacean muscle by Fatt and Katz (Fatt, P. & Katz, B. (1953) The electrical properties of crustacean muscle fibres. Journal of Physiology, 120 : 171- 204), but were subsequently found in all types of excitable cells in vertebrates and invertebrates. They are localised in the plasma membrane. A number of different type of channels have been shown to fulfil different functions according to the cell type. They can be involved in excitation, in the regulation of secretion, in muscle contraction or gating of other ion channels (notably potassium and chloride channels). The VDCCs are a diverse group of multi-subunit proteins, composed of a pore- forming subunit («ι) with several auxiliary subunits (α₂δ, β, γ) as will now be described.

The a-, subunit (~ 200kDa) comprises four internal homologous repeats (domains I to IV), with domain I being responsible for the channel activation kinetics. Each domain comprise 6 transmembrane spanning regions (S1 to S6), in which S4 is positively charged and forms part of the voltage sensor. S4 and S5 form the pore region of the channel. The «ι subunit comprises cAMP dependent protein kinase phosphorylation sites. So far, 10 <*ι genes have been described.

The α₂δ, subunit comprises two membrane spanning subunits (~ 160kDa) bound by a disulphide bridge. It associates with the «■-, subunit to increase the current amplitude. Three α₂δ, subunit genes have been described.

The β subunit is an intracellular protein (50kDa) that associates with the <*-, subunit to regulate the activation, the inactivation, the current and voltage dependence of the channel. The protein contains a cAMP dependent protein kinase phosphorylation site. Four β subunits have been described

The Y subunit is a membrane spanning protein that associates with the «-, subunit and shifts activation to more hyperpolarised membrane potentials, causing a small increase in peak Ca²⁺ currents and activation rate.

Within the category of Voltage Dependent Calcium Channels (VDDCs), L~, T- and N- type calcium channels can be distinguished according to their electrophysiological properties. L-type channels have a long lasting current when Ba²⁺ is the current carrier. They also have a high voltage of activation and a slow voltage dependent inactivation. T-type channels carry a transient current with a low voltage of activation and a rapid inactivation. N-type channels have electrophysiological properties which are in between that of the L- and T- type channels. They can be divided into subclasses according to their pharmacological profile. It has been shown that the ^αι subunit on its own can form a functional channel despite abnormal kinetics and voltage dependence. The co-expression of the «■-, subunit with other subunits can change these properties. The main factor which seems to define the calcium currents is the type of «ι subunit integrated in the channel complex.

A putative calcium channel, with a putative structure of two repeats of six transmembrane domains, has been cloned in rat. The calcium channel activity of the protein has not yet been demonstrated.

Ligand gated calcium channels are channels localised in the endomembranes of cells such as the ER (endoplasmic reticulum), and, in plants, the vacuole. The changes in the levels of certain second messengers (such as IP3) as well as the alteration of various proteins and cofactors can mediate the opening of channels. This type of regulation may be slower than that of voltage gating, but it can last longer.

Two main types of intracellular gated calcium channels are the inositol triphosphate receptor calcium channel and the ryanodine receptor calcium channel .

In yeast, only two proteins have been identified as involved in the entry of calcium into the cell, namely Midi and CCH1. Some results suggest that these two proteins could interact as part of the same calcium channel. Deletion of either of the genes leads to a strong decrease in calcium uptake and to an α-mating factor phenotype when yeast cells are grown at low calcium concentrations (Fischer, M. et al (1997) The Saccharomyces cerevisiae CCH1 gene is involved in calcium influx and mating. FEBS Letters.419 : 259-62.).

Midi is a plasma membrane protein and has four putative transmembrane segments (lida, H et al. (1994) MIDI, a novel Saccharomyces cerevisiae gene encoding a plasma membrane protein, is required for Ca2+ influx and mating. Molecular and Cellular Biology 14 : 8259-71). CCH1 is a 235kDa protein localised to the plasma membrane, with a putative structure of twenty four transmembrane domains organised in four repeats of six. The CCH1 protein sequence is 25% identical to that of the ct-i subunit of the animal voltage gated channels. In S. cerevisiae, CCH1 is the only ^i homologue.

In plants, the evidence for the presence of calcium channels has come from electrophysiological and pharmacological experiments (White, P.J. (2000) Calcium channels in higher plants. Biochimica et Biophysica Ada. 1465 : 171-189). It is possible to distinguish between different types of calcium channels in plants according to their localisation and to their mode of opening/regulation.

Two different types of calcium channels are present in the plant plasma membrane: DACC (Depolarisation Activated Calcium Channels) and HACC (Hyperpolarisation Activated Calcium Channels). They are both voltage gated calcium channels.

DAAC have been subdivided in three classes according to their sensitivity to La³⁺, Gd³⁺ and verapamil. They have a relatively high affinity to calcium but are also permeable to other cations such as Ba²⁺, Sr²⁺ or Mg ₂+

HACC are mainly selective for Ca ⁺ and Ba²⁺, and are sensitive to various inhibitors such as La .3+ , Gd and nifepedine.

With regard to the endomembranes of plants, several classes of calcium channel have been shown to exist in the tonoplast as well as in the ER, the chloroplast and the nuclear membrane. Depolarisation activated channels, such as the slow vacuolar channels (SV), hyperpolarisation channels and ligand gated channels can be distinguished. These latter channels can be activated by cADPR or IP₃ and are highly selective for divalent cations over monovalent cations.

The importance of calcium uptake to plants is known in the art, such as in plant nutrition and plant stress responses. It is known, for example, that lack of calcium in citrus fruits can lead to delays in fruit ripening. In fast-growing tissues and organs, well-known calcium deficiency related disorders can occur including blackheart in celery, tipburn in lettuce, bitter pit in apple and blossom end rot in tomato and watermelon. In all plants, calcium is important for root growth and is particularly important for stolon growth in potatoes. Adequate calcium uptake is therefore essential for all plants though there are differences in the amounts required.

Calcium is also known to be a ubiquitous second messenger in plants, the transduction of a wide range of environmental and hormonal stimuli occurring via an increase in cytosolic calcium concentration. Thus the plant stress response to such stimuli as cold and oxidative stresses is mediated by calcium.

Summary of the invention

The present invention relates, in general, to a calcium ion channel, named AtTPCI, that has been identified in Arabidopsis thaliana, variants of this channel and uses of the channel and similar channels.

According to one aspect of the resent invention, there is provided a method of providing a plant having an altered calcium uptake comprising the step of: altering the expression or activity of a protein in a cell of the plant, the protein comprising a sequence at least 25% identical to SEQ ID NO: 1 over an alignment of at least 450 amino acids, thus altering the transport of calcium ions into the cytoplasm of the cell of the plant by the protein.

Conveniently the expression of the protein is increased thus increasing the transport of the calcium ions into the cytoplasm of the cell.

Preferably the step of increasing the expression of the protein comprises transforming the plant with a nucleic acid encoding the protein.

Advantageously the nucleic acid further comprises a promoter, expression of the protein being under control of the promoter. Conveniently the promoter is inducible.

Preferably the promoter is inducible by an external agent, the method further comprising the step of providing the agent.

Advantageously the agent is a steroid, preferably dexamethasone.

Conveniently the promoter is inducible in specific tissues of the plant.

Preferably the plant is a potato and the promoter is inducible specifically in the stolon of the potato.

Advantageously the promoter is a constitutive promoter and the method further comprises the step of increasing the plant's external concentration of calcium ions, preferably by administering calcium nitrate fertiliser to the plant.

Alternatively the expression of the protein is decreased thus decreasing the transport of calcium ions into the cytoplasm of the cell.

Conveniently, the method further comprises the step of transforming the plant with a nucleic acid sequence comprising the antisense sequence of a sequence encoding a protein comprising a sequence at least 25% identical to SEQ ID NO: 1 over an alignment of at least 450 amino acids.

Preferably, the method comprises the step of breeding a plant having an altered expression of the protein comprising a sequence at least 25% identical to SEQ ID NO: 1 over an alignment of at least 450 amino acids.

Advantageously, the method comprises providing an agent to the plant, the agent altering expression of the wild-type gene in the genome of the plant which encodes the protein. Conveniently, the activity of the protein is increased by providing an agent to the plant, preferably hydrogen peroxide.

Preferably, the activity of the protein is decreased, the method further comprising the step of transforming the plant with a nucleic acid sequence encoding a non-functional variant of the protein, which dimerizes with the protein to form a non-functional dimer.

Conveniently the plant is a crop plant.

According to another aspect of the present invention, there is provided method of providing a plant having an altered stress response comprising the steps of any one of the preceding claims.

Conveniently the stress response is the response to: the presence of heavy metals adjacent to the plant; a temperature of between 0°C and 20°C; drought; salt adjacent to the plant; a hypotonic stress; red or blue light; or abscisic acid.

Preferably the stress response is an oxidative response.

Advantageously the oxidative response is a response to hydrogen peroxide or a fungal infection of the plant.

According to a further aspect of the present invention, there is provided a method of conducting phytoremediation on a surface comprising the steps of locating an organism on the surface and increasing the expression or activity in a cell of the organism of a protein comprising a sequence at least 25% identical to SEQ ID NO: 1 over an alignment of at least 450 amino acids, thus increasing transport of metal ions into the cytoplasm of the cell of the organism.

Preferably the organism is a plant.

Conveniently the plant is Festuca rubra. Preferably the method further comprises the step of harvesting the organism.

Advantageously the heavy metal comprises manganese or thallium.

Preferably the step of increasing the expression of the protein comprises transforming the organism with a nucleic acid encoding the protein.

Conveniently the nucleic acid further comprises a promoter, expression of the protein being under the control of the promoter.

Preferably the promoter is inducible.

Advantageously the promoter is inducible by an external agent, the method further comprising the step of providing the agent.

Conveniently the agent is a steroid, preferably dexamethasone.

Preferably the promoter is a constitutive promoter and the method further comprises the step of increasing the plant's external concentration of calcium ions, preferably by administering calcium nitrate fertiliser to the plant.

Advantageously, the activity of the protein is increased by providing an agent to the plant, preferably hydrogen peroxide.

According to another aspect of the present invention, there is provided the use of a nucleic acid encoding a protein comprising a sequence at least 25% identical to SEQ ID NO: 1 over an alignment of at least 450 amino acids to increase the stress response of a plant.

Conveniently the nucleic acid further comprises a promoter, expression of the protein being under the control of the promoter.

Preferably the promoter is inducible, preferably by an external agent. Advantageously, the promoter is inducible by an external agent ,

Advantageously the agent is a steroid, preferably dexamethasone.

Conveniently the promoter is a constitutive promoter.

According to a further aspect of the present invention, there is provided a polypeptide comprising a sequence at least 30% identical to SEQ ID NO: 1 and being capable of transporting calcium ions across the plasma membrane of a cell.

According to another aspect of the present invention, there is provided a polypeptide comprising the sequence of SEQ ID NO: 1.

According to a further aspect of the present invention, there is provided a nucleic acid encoding a polypeptide as described above.

Conveniently the nucleic acid further comprises a promoter, expression of the polypeptide being controllable by the promoter.

Preferably the promoter is inducible by an external agent.

Advantageously the promoter is inducible by a steroid, preferably dexamethasone.

Conveniently the promoter is a constitutive promoter.

According to a further aspect of the present invention, there is provided a vector comprising a nucleic acid as described above.

According to another aspect of the present invention, there is provided a cell transformed by a nucleic acid as described above.

Preferably, the cell is a plant. According to a further aspect of the present invention, there is provided a plant transformed with a nucleic acid as described above.

Preferably the plant is a crop plant.

Conveniently the plant is Arabidopsis.

Advantageously the plant is Festuca rubra.

Throughout this specification the word "comprises" has the meaning of "includes or consists of.

In this specification, the percentage "identity" between two sequences is determined using the BLASTP algorithm version 2.2.2 (Altschul, Stephen F., Thomas L Madden,

Alejandro A. Schaffer, Jinghui Zhang, Zheng Zhang, Webb Miller, and David J.

Lipman (1997), "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Res. 25:3389-3402) using default parameters. In particular, the BLAST algorithm can be accessed on the internet using the URL www.ncbi.nlm.nih.gov/blast. Where reference is made to alignment over a particular number of amino acids, this refers to the length of the alignment of the two sequences as determined using the BLAST algorithm.

In this specification, percentage "overall similarity" between two sequences is determined using the Smith-Waterman sequence alignment algorithm as implemented in the USC sequence alignment package. In particular this can be accessed on the internet using the URL www-hto.usc.edu/software/seqaln/seqaln-query.html. The default parameters are used except that the type of alignment is set to "global".

In some aspects of the present invention, the above described proteins and nucleic acids are provided in isolated form. In this specification, a "wild-type plant" is a naturally occurring plant, which, in particular, has not undergone any genetic manipulation. A "wild-type gene" is that form of the gene (i.e. the sequence, promoter and genomic location) which is present in the wild-type plant. In other words, the "wild-type gene" is the endogenous gene.

Description

In the detailed description of embodiments of the invention, reference is made to the following figures.

Figure 1 is a hydropathy analysis of Arabidopsis thaliana AtTPCI according to TopPred2 (Topology prediction of membrane proteins (von Heijne, 1992)).

Figure 2 is a representation of the putative secondary structure of the AtTPCI protein. Shaded transmembrane domains are predicted as certain (upper cutoff). Clear domains are predicted as putative (lower cutoff).

Figure 3 shows a graph of experimental results confirming that AtTPCI expression enhances calcium uptake in yeast. AMidlAcCHI yeast cells transformed with EV- pFL61 (black circles and squares) or AtTPC1-pF 6l (clear circles and squares) grown in SD100 and assayed for calcium uptake in the growth medium using ⁴⁵CaCI₂. For the o-factor experiments (black and clear squares) cells were incubated for 1 h in 12 μM α-factor prior to the uptake experiment. Aliquots were taken at different time points and radioactivity was measured. Bars represent SD (n=6). Letters indicate significant differences between according to Student's t-test (p<0.05).

Figure 4 shows a graph of experimental results following the protocol explained in relation to Figure 3 but with the yeast cells grown in SD680. Corresponding "-factor experiments are not shown. Figure 5 shows a graph of experimental results confirming that hydrogen peroxide activates AtTPCI -mediated calcium uptake. Measurements of Ca²⁺ uptake were performed on EV-pFL61 (first and second columns) and /4-TPC7-pFL61 (third and fourth columns) transformed yeast in SD100. 1 mM H₂O₂ was added to the incubation medium along with ⁴⁵CaCI₂ and the incorporated radioactivity was measured after 20 min. Bars represent SD (n=6). Letters indicate significant differences according to Student's t-test (p<0.05).

Figure 6 shows a graph of experimental results following the protocol explained in relation to Figure 5 but with the transformed yeast in SD680.

Figure 7 shows the results of Northern blot analysis of AtTPCI transcript in empty vector (EV) and A-TPC7-pFL61 transformed yeast. Total RNA was extracted and separated according to standard protocols and probed using a full length AtTPCI cDNA.

Figure 8 shows a graph of experimental results on the effects of oc-factor on the viability of EV-pFL61 and .4fTPC7-pFL61 transformed yeast cells in SD100 (black columns) or SD680 (clear columns) growth medium. Results are the means of three independent experiments. Bars represent SD. Letters indicate significant differences according to Student's t-test (p<0.05).

Figure 9 shows the results of experiments observing the growth of A MidlAcCHI S. cerevisiae transformed with pFL61 and \-TPC7-pFL61 on plates containing SD680 medium supplemented with various concentrations of (MnSO₄).

Figure 10 is a graph showing changes in cytosolic calcium concentrations following the cooling of plants from 20°C to 0°C in seedlings overexpressing AtTPCI (line 27, grey line) or in control plants (black line). The cytosolic calcium concentration was assessed by aequorin-related luminescence. The data represents the average of 4 seedlings. Figure 11A and 11B is an alignment of the amino acid sequence of AtTPCI with mammalian calcium channels.

The invention will now be described, by way of example, as a series of embodiments.

The present invention relates, in general, to methods of providing a plant having an altered calcium uptake.

In preferred embodiments, there is provided a method of providing a plant having an altered stress response, due to the altered calcium uptake. In particularly preferred embodiments of the present invention, the stress response is altered by increasing the stress response. In these embodiments, the resulting plant, when subjected to the stress, has a higher level of response to the stress than an equivalent plant that has not undergone the method. The advantage of an increased stress response is that the plant has a better tolerance to stresses, notably by triggering an earlier and/or more rapid stress response.

The level of stress response in a plant is modulated by the concentration of calcium ions in the cytoplasm of a cell of the plant. Therefore, by determining the concentration of calcium ions in the cytoplasm of a cell of a plant, the stress response in the plant can be determined (see, for instance, Example 8). Alternatively, the stress response can be measured by observing external attributes of the plant such as root hair length, symptoms, stomatal aperture or gene expression

In one embodiment of the invention, the stress is the subjecting of the plant to a temperature of between 0°C and 20°C and thus the resulting response is a cold stress response. In another embodiment the stress is the subjecting of the plant to drought and thus the stress response is an osmotic response. In a further embodiment, the stress is the subjecting of the plant to a salt or abscisic acid adjacent to the plant, such as in the medium (e.g. soil of other growing media) surrounding the roots of the plant. In a further embodiment, the stress is the presence of heavy metals adjacent to the plant, such as in the medium surrounding the roots of the plant. In particular embodiments, the heavy metal forms divalent cations, for example, a metal such as manganese. In other embodiments, the heavy metal forms cations having other valencies such as thallium. In some embodiments, the stress is a hypotonic stress, while in other embodiments, the stress is the presence of red or blue light on the plant. In some other embodiments of the invention, the stress response is an oxidative response such as a response to hydrogen peroxide, ozone or a fungal infection of the plant, such as Phytophthora. In the case of a stress response to cold or to an oxidative stress such as ozone, the increase in cytosolic calcium concentration induces a downstream response via an increase in the transcript quantities of proteins such as Kin1 or GST (Clayton H. et al. (1999) Dissection of the ozone-induced calcium signature, Plant J. 17 : 575-579; and Knight H. et al. (1996) Cold calcium signalling in Arabidopsis involves two cellular pools and a change in calcium signature after acclimation, Plant Cell. 8: 489-503).

As is explained above, in certain preferred embodiments of the invention, there is provided a method of providing a plant having an altered stress response, due to the altered calcium uptake. However, in certain alternative embodiments, the calcium uptake is altered for a different purpose, such as improved nutrition. Plants contain about 5 to 30 mg Ca/g dry matter, which is more the result of the relatively high Ca²⁺ concentration in a typical soil solution than of the efficiency of the Ca²⁺ uptake mechanism of root cells. The lack of calcium in a plant can lead to delays in fruit ripening and in fast-growing tissues and organs, calcium-deficiency related disorders occur. Ca²⁺ uptake can be competitively depressed by the presence of other cations such as K⁺ and NH₄ ⁺ which are rapidly taken up by the roots of the plant. Thus, in some embodiments of the invention, raising the calcium uptake of plants results in improved nutrition. This can be of particular use in growing plants in soils having poor calcium concentrations.

In one embodiment of the invention, the expression of the AtTPCI protein (whose sequence is set out in SEQ ID NO: 1) is increased. Thus the concentration of the protein within the cells of the plant is higher than in the wild-type plant under the same conditions. Because the AtTPCI protein is a calcium ion channel, the increased concentration of the protein within the cell results in an increased number of calcium ion channels in the plasma membrane of each cell. This in turn increases the level of calcium ions transported into the cytoplasm of the cell and thus increases the concentration of calcium ions within the cytoplasm. Since cytosolic calcium ion concentration mediates the stress response of the plant, this increase in the concentration results in an increased stress response in the plant in some embodiments.

In some alternative embodiments of this invention, the expression of a protein having the exact sequence of AtTPCI recited in SEQ ID NO: 1 is not altered. Instead, a variant protein having a similar sequence to SEQ ID NO: 1 is used. Thus, in some embodiments, a variant protein is used having a polypeptide sequence with at least 25% identity to SEQ ID NO: 1 over an alignment of 450 amino acid residues or, more preferably 480 amino acid residues. In some embodiments, a variant protein having a sequence with 30%, 50% or 80% identity to SEQ ID NO: 1 is used. Furthermore, in some embodiments, a variant protein is used having an overall similarity of at least 30% or more preferably 40% to SEQ ID NO:1. In addition, in certain embodiments a variant protein is used having 40%, 50% or even 100% identity to residues 281 to 296 and/or 656 to 677 of SEQ ID NO: 1. However, in these embodiments, the protein still has the activity of a calcium ion channel and thus increasing the expression of the protein still results in an increase in the level of calcium ions transported into the cytoplasm of the cell. This, in turn, modulates the stress response of the plant in certain embodiments. It can be determined whether or not a particular protein acts as a calcium channel by performing the complementation test in a yeast knockout mutant as described in Example 3.

In some embodiments of the invention, the expression of the AtTPCI protein (or a variant protein as defined above) is increased by transforming plant cells with an exogenous nucleic acid sequence (usually a DNA sequence) encoding the protein. Thus the cells are transformed with the AtTPCI gene or a variant gene. In some embodiments, cells are transformed by transferring the nucleic acid into agrobacterium tumefaciens which is then used to infect and transform the plant cells. However, in alternative embodiments of the invention, other methods of transformation are used such as by bombardment of plant cells with particles coated in the DNA sequences.

In preferred embodiments of the invention, the nucleic acid sequence also comprises, in addition to the sequence encoding the protein, a promoter sequence, upstream of the coding sequence such that expression of the coding sequence is under the control of the promoter. In some embodiments, an inducible promoter is provided, which, in preferred embodiments, is activated by the presence of an agent that can be added externally of the plant. For example, in some embodiments the promoter is a steroid inducible promoter, such as that described in Zuo and Chua, Current Opinion in Biotechnology, 11: 146-151). In one particular embodiment the steroid is dexamathasone, and the steroid is sprayed onto the plant in order to activate the promoter. However, a wide variety of possible promoters and corresponding agents are used in alternative embodiments of the invention. In some embodiments, the promoter is the wild-type AtTPCI promoter which is set out in SEQ ID NO: 2.

By placing AtTPCI or a variant under the control of an inducible promoter, the uptake of calcium can be controlled. For example, if the promoter is a calcium inducible promoter then uptake can be controlled by the application of external calcium. Uptake of calcium can be manipulated to meet the exact growing conditions of the plant and can be restricted according to the properties of the promoter used to drive the expression of the gene. This has benefits in creating conditions whereby the uptake of calcium is tissue specific and can also be tailored to the composition of calcium formulations used (e.g. solutions of calcium nitrate containing a higher percentage of soluble calcium). In certain embodiments of the invention, the promoter is a tissue specific promoter. Uptake of calcium can thus be restricted to certain tissues of the plant such as the foliage alone or the roots alone. In one embodiment, in which the plant is a potato, the AtTPCI gene is placed under the influence of a stolon-specific, calcium-inducible promoter. In this way, one ensures that external calcium applied in the form of a fertiliser is taken up by the stolon roots, resulting in a higher yield and better quality of tubers. This is particularly useful since normal (wild-type) potato cultivars show little uptake of calcium via stolon roots. In further embodiments of the invention, the promoter is a constitutive promoter, instead of an inducible promoter, such as the 35S promoter of the cauliflower mosaic virus. In these embodiments, the stress response of the plant is modulated or altered, by increasing the plant's external concentration of calcium ions as this causes an increase in the transport of calcium ions into the cytoplasm of the cells of the plant and thus modulates the stress response. Alternatively, the cells of the plant are exposed to an increased concentration of hydrogen peroxide, which also increases the activity of the AtTPCI protein.

By placing the AtTPCI gene or a variant under the control of a constitutive promoter plants such as crop plants take up external calcium that is applied, irrespective of the growth stage of the plant in question. By using such a strategy, the uptake of calcium into the plant via the roots and foliage is enhanced upon the application of calcium-containing preparations such as calcium nitrate fertiliser. The increased uptake by plants due to constitutive expression of the AtTPCI or a variant gene results in the beneficial effects of calcium exhibited as increased resistance to fungal diseases (in plants such as brassicas, potatoes and citrus fruits) and ripening of fruits. In embodiments in which the plant is a crop plant then the benefits are less wastage of the crop due to disease and due to delays in ripening at harvest.

In some embodiments of the invention, the cells of the plant are not transformed with a nucleic acid sequence encoding the AtTPCI protein or variant protein. Instead, the expression of this protein is increased by exposing the plant to an external agent which increases the expression of the wild-type, that is to say endogenous, gene encoding the protein. The external agent causes the increased expression of the wild-type gene in the cells of the plant, thus increasing the levels of the protein in the plasma membrane of the cells and thus increasing the cytoplasmic concentration of calcium ions and the stress response of the plant.

In some alternative embodiments, the stress response of the plant is modulated or altered, not by increasing the expression of AtTPCI or variant protein, but by increasing the activity of the calcium ion channel. This is achieved by exposing the plant to an external actuating agent, such as hydrogen peroxide, that increases the level of calcium ions within the cell, thus modulating the stress response of the plant. Thus, in these embodiments the activity of the calcium channel is post-translationally upregulated.

Within certain embodiments of the invention, the expression of the naturally occurring, wild-type gene encoding the AtTPCI or variant protein in the plant can be decreased. This modulates the stress response of the plant by decreasing the concentration of calcium ions within the cells of the plant. Thus, in some embodiments, the plant is provided particularly in order to survive in an environment in which a reduced sensitivity to a stress is advantageous, such as a plant provided to survive in soils having relatively high concentrations of toxic metal. In these embodiments, reducing the expression of the AtTPCI or variant gene modulates the plant's sensitivity to the stress.

A decrease in the expression of the AtTPCI or variant protein is achieved, in some embodiments, by exposing the plant to an agent which inhibits the activity of the endogenous promoter of the wild-type gene encoding the protein. In other embodiments, the plant cells are transformed with a nucleic acid sequence which is an antisense sequence to that of the sequence encoding the AtTPCI protein. Thus in these embodiments, when the antisense sequence is transcribed into RNA in the plant cell, it will hybridise to the RNA transcripts of the gene encoding the AtTPCI or variant protein, thus preventing translation of the transcript into a protein. In other embodiments, the plant is transformed to produce a mutated, non-functional variant AtTPCI protein. Since the protein channel is dimeric, the mutated AtTPCI , when combined with native AtTPCI or a functional variant protein will result in a nonfunctional dimer and thus decreased channel activity through negative dominance.

In preferred embodiments of the present invention, the plant is a crop plant such as tobacco or soybean. However, depending on the use that the plant is required for, other types of plant are used in alternative embodiments, as is described in greater detail below. In further embodiments of the invention, organisms other than plants are used. In particular, in some embodiments of the invention, organisms are used in phytoremediation (i.e. the biological removal of substances). In particular, the removal of toxic heavy metals from surfaces such as soils is the aim of certain methods. In these embodiments, an increase in the expression of the AtTPCI protein (or a variant protein as defined above) is effected in the cell or cells of the organism in order to increase the level of calcium ion channels in the plasma membrane of the cell. This, in turn, increases the transport of metal ions into the cytoplasm of the cell of the organism. Accordingly, the metal ions are removed from the surface, into the organism. The organism can then be removed from the surface (i.e. harvested in the cases where the organism is a plant) in order to remove the metal ions. This occurs because the AtTPCI protein transports not only calcium ions but also other divalent metal ions into the cytoplasm of a cell.

The organism is preferably a plant. The advantage of plants is the ease with which they can be harvested and in order to remove the metal ions. In preferred embodiments, the metal ions are manganese or thallium. In a particularly preferred embodiment, the plant used is Festuca rubra, a grass cultivar, which in its wild-type form, is able to withstand very high concentrations of cadmium (up to 500 ppm) and fairly high concentrations of copper (up to 100 ppm). Thus this plant is able to survive in the presence of heavy metals and is thus suited for use in taking them up from the soil.

In these embodiments, the expression of the gene encoding the AtTPCI or variant protein is increased by transforming the plant as is explained above, or by exposing the plant to an external agent, again, as explained above.

In certain embodiments of the invention, the AtTPCI protein, itself, (or a variant protein as defined above) is provided. Similarly, in some embodiments the nucleic acid encoding the protein or a variant protein, is provided. Furthermore, the nucleic acid also comprises a promoter as described above in some embodiments. Vectors, such as plasmids and cosmids, comprising these nucleic acid sequences are also prepared and used in some embodiments and can be used to prepare cells transformed by these nucleic acids in various embodiments. These cells can then be grown into transformed plants which form further embodiments of the invention. As explained above, the plants are typically crop plants but may be other types of plant in certain embodiments.

In some embodiments of the invention, the promoter of the AtTPCI gene is provided, which is set out in SEQ ID NO: 2.

Examples

The present invention is now further illustrated by way of the following examples

Example 1

A full length AtTPCI cDNA was amplified with the 5' (5' GCGGCCGCATCGGAGA GAGAAAAAAAAAATGGAAGACCCGTTGATTGGTAGAGATAGTCTTGGTGGTGGT GGTAC 3') and 3' (5' GCGGCCGCTGTTTTATGTGTCAGAAGTGGAACACTCTG 3') primers using the EST H4H9 (ace. W43766) as a template. The fragment was amplified using a pfu polymerase, following the manufacturer's instructions (Promega, Madison, Wl, USA), under the following conditions: one cycle at 94°C, 1 min 30 s followed by 20 cycles of 94°C, 30 s, 43°C 2 min, 72°C, 3 min. A final extension cycle at 72°C for 5 min was performed. The resulting fragment was subcloned into a commercial pUC18 Smal BAP vector (Amersham Pharmacia Biotech, Piscataway, NJ, USA). The AtTPCI fragment obtained after digestion by Notl was ligated into the yeast vector pFL61 at the same restriction site under the control of the phosphoglycerate kinase promoter and terminator. This vector is called /tfTPC7-pFL61 in the rest of this Application. The resulting cDNA for the complete AtTPCI was sequenced. The cDNA was subsequently subcloned in various vectors depending on the type of experiment to be performed.

Example 2

Analysis of the AtTPCI sequence revealed the following.

The full-length cDNA, cloned from an EST, comprises an open reading frame of 2202 nucleotides, encoding an 84.9kDa protein of 733 amino acids shown in SEQ. ID NO: 1. Sequence alignments showed that AtTPCI shares homologies with the «(pore- forming) subunits of rat (AB018253), mouse (AF217002) and human (AB03995) voltage-gated calcium channels. A hydropathy analysis of the protein revealed that AtTPCI shares a topology of 12 potential transmembrane spanning (TMS) domains arranged in 2 repeats (I and II) of a basic 6 TMS structure (Figures 1 and 2). The presence of two putative EF-hands in the hydrophilic loop linking the two repeats (sequences MLEKAFGLIDSDKN and RTLPKISKEEFGLIFDELDDTRDFKINKDEF of SEQ. ID NO: 1) indicates that the channel is regulated by cytosolic calcium concentration.

A putative pore forming region is identified, on the basis of sequence alignments, as lying between the transmembrane domains 5 and 6 in each repeat (sequences NNPDVWIPAY and MESYKDL of SEQ ID NO:1).

Example 3

The following experiments were carried out to determine whether the AtTPCI gene was able to complement the phenotype of the JKmc yeast strain, which is defective in Ca²⁺-dependent processes.

The Saccharomyces cerevisiae strains JKmc (MATa leu2-3, 112, his4, trpl, ura3-52, rmel, mid1::KAN, cch1::KAN, (Fischer, M., Schnell, N., Chattaway, J., Davies, P., Dixon., G. & Sanders, D. (1997) FEBS Letters. 419, 259-262)) and G19 (MATa Ieu2- 3, 2-112, trp1-1, ura3-3, ade2-1, his3-11 can1-100, 15 (Ψ) ena1::HIS3::ena4, (Quintero, F.J., Garciadeblas, B. & Rodriguez-Navarro, A. (1996) Plant Cell. 8, 529- 527)) were transformed using the pFL61 vector (empty vector, EV) or the AtTPCI- pFL61 vector and selected on standard minimal growth medium (SD, (Sherman, F., Fink, G. & Hicks, J. (1986) in Methods in yeast genetics. Cold Spring Harbour Laboratory Press, Cold Spring Harbour, NY)) supplemented with the appropriate amino acids, lacking uracil. The yeast strain JKmc used is deleted in the endogenous. calcium transporter genes Midi and CCH1 (Fischer, M., Schnell, N., Chattaway, J., Davies, P., Dixon., G. & Sanders, D. (1997) FEBS Letters. 419, 259-262) and therefore exhibits a phenotype that is defective in Ca²⁺-dependent processes such as (i) mating response i.e. decreased viability after addition of "-factor, and (ii) ⁴⁵Ca uptake.

Depending upon experiments, SD100 or SD680 (100 or 680 μM CaCI₂, (lida, H., Nakamura, H., Ono, T., Okumara, M. & Anraku, Y. (1994) Molec. Cell. Biol. 14, : 8258-8271)) was used. Cell growth was assessed spectrophotometrically by measuring the ODeoo. The presence of AtTPCI transcripts in the ArTPC7-pFL61 yeast strain was checked by Northern blotting on total yeast RNA using standard techniques.

In order to determine calcium uptake, the following assays were used. Uptake assays were performed in parallel for A TPC7-transformed cells and control cells harbouring the empty pFL61 plasmid. Cells were grown in shaking cultures at 30°C in SD100 or SD680 to an OD600 of ^« 0.6. The cells were incubated with 100 kBq of ⁴⁵CaCI₂ (NEN Life Science, Boston, MA, USA) per ml in the growth medium at 30°C. At various time points, 1.5 ml aliquots were taken and filtered under vacuum through 0.45 μm nitrocellulose filters (Whatman, Clifton, NJ, USA) pre-soaked in 10 mM CaCI₂. The filters were washed twice with 15 ml of 10 mM CaCI₂, then dried and the radioactivity measured by scintillation counting. Results were expressed as pmol Ca²⁺ per 10⁶ cells. For the hydrogen peroxide experiments, 1 mM H2O2 was added to the growth medium at the same time as the isotope.

Expression of AtTPCI in yeast induced an increase in ⁴⁵Ca²⁺ uptake when cells were grown in SD medium containing 100 μM Ca ⁺ (Figure 3). After 90 min incubation with the isotope, calcium uptake by /4 TPC -pFL61 transformed cells was twice that measured in EV-pFL61 transformed yeast. The difference between the two strains could be detected as rapidly as 25 min after incubation. When uptake was measured at a higher calcium concentration (medium SD680), no difference in calcium uptake was apparent between EV-pFL61 and /4-TPC7-pFL61 transformed strains (Figure 4).

The plant transporter LCT1 translocates a range of cations including Na⁺ and reduces growth of the salt sensitive yeast strain G19 when grown at moderate NaCl concentrations (20-100 mM, (Amtmann, A., Fischer, M., March, E.L., Stefanovic, A., Sanders, D. & Schachtman, D.P. (2001) Plant Physiol. 126, 1061-1071)). To determine whether AtTPCI might only form a low selectivity cation pathway, AtTPCI was expressed in yeast strain G19. Growth was measured in arginine phosphate medium supplemented with 20-50 mM NaCl, and AtTPCI was expressed in a K⁺ uptake yeast mutant with growth measured at low potassium concentration. No differences were observed between AtTPCI expressing and EV controls.

Calcium currents at the plasma membrane of guard cells are activated by H₂O₂ at 5 mM (Pei, Z.M., Muratar, Y., Benning, G., Thomine, S., Klusener, B., Allen, G., Grill, E. & Schroeder, J.I. (2000) Nature. 406, 731-734). Therefore the effects of H₂O₂ tested on the activity of AtTPCI expressed in JKmc were tested. The effects of hydrogen peroxide on the viability of the cells was tested prior to the uptake experiments. The hydrogen peroxide concentration used (1 mM) did not have any effect on the viability of both EV-pFL61 and AtTPCI -pFL61 transformed yeasts over the course of the experiment (data not shown).

Calcium uptake in /4fTPC7-pFL61 transformed cells was increased by about 50% when 1 mM hydrogen peroxide was added to the uptake medium at both SD100 and SD680 (Figures 5 and 6). On the contrary, hydrogen peroxide had no effect on calcium uptake by the EV-pFL61 transformed yeast (Figures 5 and 6).

Thus the experiments of this example show that AtTPCI is able to complement partially the Ca²⁺ phenotype of JKmc (Figure 3). Furthermore, addition of hydrogen peroxide increased AtTPCI mediated ⁴⁵Ca uptake (Figure 5). Therefore, these experiments show that AtTPCI is a functional Ca²⁺ channel which is activated by H₂O₂.

Example 4

In order to test the complementation of the mating response phenotype of the JKmc strain, the following experiments were conducted. The yeast strain used was JKmc which is deleted in the endogenous calcium transporter genes Midi and CCH1 (Fischer, M., Schnell, N., Chattaway, J., Davies, P., Dixon., G. & Sanders, D. (1997) FEBS Letters. 419, 259-262). This strain is defective in Ca²⁺ uptake and exhibits Ca²⁺-dependent reduced viability when exposed to α-mating factor. After transformation of JKmc with AtTPC1-pFL6l (see Example 3), AtTPCI mRNA was expressed in yeast (Figure 7). Yeast cell viability was assessed after overnight exposure to 12 μM α-factor (Sigma Aldrich. St Louis, Ml, USA) in SD100 or SD680 at 30°C with shaking. Cells were stained with 0.01% methylene blue and viability checked under a differential interference contrast microscope.

AtTPCI expression in S. cerevisiae partially complemented the α-mating factor phenotype of the JKmc yeast mutant. Thus, after exposing the cells overnight to α- factor at 12 M in a growth medium with a calcium concentration of 100 μM, the percentage viability of the EV-pFL61 strain was reduced from 80% to 28% (Figure 8). In the same conditions, the percentage viability of the >4-TPC7-pFL61 transformed strain was reduced from 80% in the absence of α-factor to only 49%, a value similar to that determined when either control or expressing strains were exposed to α-factor at the higher Ca²⁺ concentration of 680 μM (Figure 8).

Furthermore, when repeating the experiments of Example 4 with cells exposed to α- factor, the increase in calcium uptake was higher for AtTPCI expressing yeast compared to the empty vector control at early stages (up to 50 min) of the experiment in SD100 (Figure 3)- No differences in calcium uptake, following exposure to α- mating factor, between pFL61 and AtTPC1-pFL61 transformed yeast was apparent in SD680 (data not shown).

These experiments show that AtTPCI is able to complement partially the α-mating factor phenotype of the JKmc yeast mutant. AtTPCI therefore plays a role in calcium uptake and/or signalling since the mating response is a calcium ion dependent process. Example 5

Experiments into the Na⁺ permeability of AtTCPI were carried out on the salt sensitive Saccharomyces cere visiae strain G19 (MATα leu2-3, 2-112, trp1-1, ura3-3, ade2-1, his3-11 can1-100, 15(φ) ena::HIS::ena4). Yeast was transformed with the pFL61 vector alone; empty vector, EV; or the pFL61 -AtTPCI vector and selected on standard minimal growth medium (SD) supplemented with the appropriate amino acids (see Example 3 for further details). Growth of EV and AtTPCI transformed yeast on plates and in liquid medium was measured in arginine phosphate medium supplemented with various concentrations of NaCl.

In comparison with known Na⁺ transporting plant proteins (e.g. LCT1, Amtmann et al. 2001) AtTPCI did not reduce growth of this yeast strain in moderate salinity (20-100 mM NaCl). These experiments therefore indicate that AtTPCI is not a Na⁺ channel.

Example 6

In order to assess the involvement of AtTPCI in the transport of toxic ions, AtTPCI- pFL61 transformed yeast (Δmldl ΔCCH1 S. cerevisiae) was tested for thallium and manganese resistance by the drop assay method. Several dilutions of a 0.8 OD liquid culture were spotted on agar plates containing SD680 medium supplemented with various concentrations of TINO₃ or MnSO . Colony density of the spots was monitored after 2 days' growth at 30°C.

The results are shown in Figure 9. The results indicate that expression of AtTPCI induced an increased sensitivity of the yeast mutant to manganese.

The sensitivity of /4-TPC7-pFL61 transformed yeast towards thallium was tested in a salt sensitive Saccharomyces cerevisiae strain G19 (MATα leu2-3, 2-112, trp1-1, ura3-3, ade2-1, his3-11 can1-100, 15(<P) ena::HIS::ena4). The results are presented in Table I. These results indicate that AtTPCI transformed yeast is more sensitive towards thallium than wild-type yeast. Table 1: Growth of G19 yeast transformed with EV and AtTPCI on plates containing SD680 medium supplemented with various concentrations of thallium nitrate (TINO₃).

Thus these experiments show that AtTPCI is involved in the transport of toxic metals.

Example 7 0

The following studies were carried out to determine the number of copies of the AtTPCI gene in the wild-type genome of Arabidoposis thaliana.

Total genomic DNA was extracted, digested and separated according to standard 5 protocols. Following detection using an AtTPCI cDNA, it was possible to show that AtTPCI is present as a single copy gene. The completion of the Arabidopsis genome also revealed the presence of only one copy of TPC7 gene in Arabidopsis genome. Therefore it can be concluded that there is only a single copy of the AtTCPI gene in Arabidopsis thaliana. 0 Example 8

The following experiments were carried out to assess the involvement of AtTPCI in the stress response of Arabidopsis thaliana.

Transgenic Arabidopsis lines were produced expressing the full length TPC1 sequence under the control of a steroid-inducible promoter, as follows. Arabidopsis thaliana (ecotype Columbia) were grown in a greenhouse under long-day conditions (16/8 h light/dark cycle) for 4 weeks before cutting the bolts. Seven days after cutting, the plants with newly grown floral buds were transformed by the 'floral dip' method (Clough, S. J. & Bent, A.F. (1998) Plant J 16, 735-743). For this purpose, Agrobacterium stains (GV3101) containing 35S or inducible constructs were grown in LB containing 50μg/ml for 24 hr at 28°C. The cells were pelleted and resuspended in infiltration media (5% (w/v) Sucrose, 0.05% (v/v) triton X-100) to an ODeoo between 0.7 and 1. Plants were dipped in this suspension for 40 s and grown under long-day conditions in greenhouse. Seeds harvested from these plants were screened on selection media (0.5x MS and kanamycin (50 μg/ml)). The putative transformants were rescued from plates and grown under long-day conditions in greenhouse.

It has been demonstrated that after 48 hour exposure to the steroid hormone dexamethasone, expression of the TPC1 transcript increases markedly in these lines. The plants also constitutively expressed the luminescent calcium indicator protein, aequorin.

The levels of blue light emitted by intact whole plants were monitored using a photon counting camera and gave a real-time measure of the levels of cytosolic free calcium ([Ca²⁺]_Cyt) in the plants as they responded to stress stimulation. Such measurements were made on plants responding to low temperature, simulated drought stress, salinity and oxidative stress. All of these forms of stress are known to increase cytosolic [Ca²⁺]cyt in plants.

The resulting data show that in three independent transgenic lines overexpressing TPC1 , the [Ca²⁺]cyt elevation occurring in response to gradual cooling in the range 20 to 0°C was larger in magnitude than that seen in control plants (Figure 10, average of 4 plants form line 27 and control). In particular, in the time between 80 and 200 seconds after cooling began, the transgenic lines had between approximately 1.5 and 1.9 times the response of the control plants as measured in relative response units. This indicates that increasing the expression levels of the TPC1 calcium channel protein facilitates calcium entry into the plant cells.

Similar measurements have been made on plants responding to hydrogen peroxide (oxidative stress), mannitol (simulated drought) or sodium chloride treatment and qualitative differences in the response compared to controls were frequently observed.

These data show that, in planta, AtTPCI is involved in the regulation of cytosolic calcium concentration, particularly in response to stresses.

Example 9

The following experiments were carried out to clone and analyse the promoter of the AtTPCI gene.

Using specific primers AtTPCIPF (5' AAGCTTCTATTGGTATGTGGCGAAGGAA 3') and AtTPCI PR (5' GGATCCCGGAGTACCATGCGTAATAGC 3'), a 931 bp fragment (upstream of ATG) was cloned from genomic DNA and is shown in SEQ ID NO: 2. Analysis of the sequence shows the presence of the putative CAAT and TATA boxes at positions -458 and -430 respectively (nucleotides 474 and 502, respectively, of SEQ ID NO: 2). This indicates that the transcription starts at about 400bp upstream of the ATG, constituting quite a long 5' UTR.

PLACE (a database containing a list of sequence present in 5' regulatory regions of plant genes) analysis of the promoter sequence shows that it is rich in root expression and drought responsive motifs. In particular, drought responsive elements are present from nucleotide 23 to 28 and from 170 to 175 of SEQ ID NO: 2. Furthermore, root expression elements are present from nucleotide 191 to 195, 330 to 334 and from 435 to 439 of SEQ ID NO:2.

Further details of PLACE may be found in K. Higo, Y. Ugawa, M. Iwamoto and T. Korenaga (1999) Plant cis-acting regulatory DNA elements (PLACE) database: 1999, Nucleic Acids Research Vol.27 No. 1 pp. 297-300. In particular, the database may be accessed on the Internet at the URL www.dna.affrc.go.jp/htdocs/PLACE/.

Example 10

Sequence alignments between AtTPCI and the amino acid sequences of calcium channels in mice, humans and rats were carried out and the results are shown in Figure 11A and B.

The alignments were carried out using the Clustal W alignment program (Thompson, J.D., Higgins, D.G. and Gibson, T.J.(1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions- specific gap penalties and weight matrix choice. Nucleic Acids Research, 22, 4673- 468).

In the alignments, "*" denotes identity between the amino acids in the respective sequences; ":" denotes strong similarity between amino acids; and "." denotes weak similarity between amino acids.

In the alignments, amino acid residues 281 to 296 and 656 to 677 (corresponding to amino acid residues 260 to 275 and 618 to 639 of SEQ ID NO: 1) are the putative pore forming region. In this region, the percentage of similarity (i.e. identical or strongly similar amino acids) between AtTPCI and the mammalian channels reaches 40 to 50%. The percentage identity is 56% and 50% respectively. Amino acid residues 347 to 360 and 379 to 409 are EF-binding hands. Thus these alignments show that it is the conserved, pore forming region of the protein that is responsible for its characteristic of being a calcium channel.

Claims

1. A method of providing a plant having an altered calcium uptake comprising the step of: altering the expression or activity of a protein in a cell of the plant, the protein comprising a sequence at least 25% identical to SEQ ID NO: 1 over an alignment of at least 450 amino acids, thus altering the transport of calcium ions into the cytoplasm of the cell of the plant by the protein.

2. A method according to Claim 1, wherein the expression of the protein is increased thus increasing the transport of the calcium ions into the cytoplasm of the cell.

3. A method according to claim 2, wherein the step of increasing the expression of the protein comprises transforming the plant with a nucleic acid encoding the protein.

4. A method according to claim 3 wherein the nucleic acid further comprises a promoter, expression of the protein being under control of the promoter.

5. A method according to claim 4 wherein the promoter is inducible.

6. A method according to Claim 5 wherein the promoter is inducible by an external agent, the method further comprising the step of providing the agent.

7. A method according to claim 5 wherein the agent is a steroid, preferably dexamethasone.

8. A method according to any one of Claims 5 to 7 wherein the promoter is inducible in specific tissues of the plant.

9. A method according to Claim 8 wherein the plant is a potato and the promoter is inducible specifically in the stolon of the potato.

10. A method according to claim 4 wherein the promoter is a constitutive promoter and the method further comprises the step of increasing the plant's external concentration of calcium ions, preferably by administering calcium nitrate fertiliser to the plant.

11. A method according to Claim 1 , wherein the expression of the protein is decreased thus decreasing the transport of calcium ions into the cytoplasm of the cell.

12. A method according to claim 11 further comprising the step of transforming the plant with a nucleic acid sequence comprising the antisense sequence of a sequence encoding a protein comprising a sequence at least 25% identical to SEQ ID NO: 1 over an alignment of at least 450 amino acids.

13. A method according to any one of Claims 1, 2 or 11 comprising the step of breeding a plant having an altered expression of the protein comprising a sequence at least 25% identical to SEQ ID NO: 1 over an alignment of at least 450 amino acids.

14. A method according to any one of Claims 1, 2 or 11 comprising providing an agent to the plant, the agent altering expression of the wild-type gene in the genome of the plant which encodes the protein.

15. A method according to Claim 1 , wherein the activity of the protein is increased by providing an agent to the plant, preferably hydrogen peroxide.

16. A method according to Claim 1, wherein the activity of the protein is decreased, further comprising the step of transforming the plant with a nucleic acid sequence encoding a non-functional variant of the protein, which dimerizes with the protein to form a non-functional dimer.

17. A method according to any one of the preceding claims wherein the plant is a crop plant.

18. A method of providing a plant having an altered stress response comprising the steps of any one of the preceding claims.

19. A method according to Claim 18 wherein the stress response is the response to the presence of heavy metals adjacent to the plant.

20. A method according to Claim 18 wherein the stress response is the response to a temperature of between 0°C and 20°C.

21. A method according to Claim 18 wherein the stress response is response to drought.

22. A method according to Claim 18 wherein the stress response is a response to salt adjacent to the plant.

23. A method according to Claim 18 wherein the stress response is a response to a hypotonic stress.

24. A method according to Claim 18 wherein the stress response is a response to red or blue light.

25. A method according to Claim 18 wherein the stress response is a response to abscisic acid.

26. A method according to Claim 18 wherein the stress response is an oxidative response.

27. A method according to claim 26 wherein the oxidative response is a response to hydrogen peroxide or a fungal infection of the plant.

28. A method of conducting phytoremediation on a surface comprising the steps of locating an organism on the surface and increasing the expression or activity in a cell of the organism of a protein comprising a sequence at least 25% identical to SEQ ID NO: 1 over an alignment of at least 450 amino acids, thus increasing transport of metal ions into the cytoplasm of the cell of the organism.

29. A method according to claim 28 wherein the organism is a plant.

30. A method according to Claim 29 wherein the plant is Festuca rubra.

31. A method according to any one of claims 28 to 30 further comprising the step of harvesting the organism.

32. A method according to any one of claims 28 to 31 wherein the heavy metal comprises manganese or thallium.

33. A method according to any one of Claims 28 to 32, wherein the step of increasing the expression of the protein comprises transforming the organism with a nucleic acid encoding the protein.

34. A method according to Claim 33, wherein the nucleic acid further comprises a promoter, expression of the protein being under the control of the promoter.

35. A method according to Claim 34, wherein the promoter is inducible.

36. A method according to Claim 35 wherein the promoter is inducible by an external agent, the method further comprising the step of providing the agent.

37. A method according to Claim 36, wherein the agent is a steroid, preferably dexamethasone.

38. A method according to Claim 34, wherein the promoter is a constitutive promoter and the method further comprises the step of increasing the plant's external concentration of calcium ions, preferably by administering calcium nitrate fertiliser to the plant.

39. A method according to Claim 28, wherein the activity of the protein is increased by providing an agent to the plant, preferably hydrogen peroxide.

40. Use of a nucleic acid encoding a protein comprising a sequence at least 25% identical to SEQ ID NO: 1 over an alignment of at least 450 amino acids to increase the stress response of a plant.

41. Use according to Claim 40, wherein the nucleic acid further comprises a promoter, expression of the protein being under the control of the promoter.

42. Use according to Claim 41 , wherein the promoter is inducible, preferably by an external agent.

43. Use according to Claim 42, wherein the agent is a steroid, preferably dexamethasone.

44. Use according to Claim 41 , wherein the promoter is a constitutive promoter.

45. A polypeptide comprising a sequence at least 30% identical to SEQ ID NO: 1 and being capable of transporting calcium ions across the plasma membrane of a cell.

46. A polypeptide comprising the sequence of SEQ ID NO: 1.

47. A nucleic acid encoding a polypeptide according to claim 45 or 46.

48. A nucleic acid according to claim 47, further comprising a promoter, expression of the polypeptide being controllable by the promoter.

49. A nucleic acid according to claim 48 wherein the promoter is inducible by an external agent.

50. A nucleic acid according to claim 49 wherein the promoter is inducible by a steroid, preferably dexamethasone.

51. A nucleic acid according to claim 49 wherein the promoter is a constitutive promoter.

52. A vector comprising a nucleic acid according to any one of claims 47 to 51.

53. A cell transformed by a nucleic acid according to any one of claims 47 to

51.

54. A cell according to claim 53 when the cell is a plant cell.

55. A plant transformed with a nucleic acid according to any one of claims 47 to

51.

56. A plant according to claim 55 wherein the plant is a crop plant.

57. A plant according to claim 55 when the plant is Arabidopsis.

58. A plant according to Claim 55 wherein the plant is Festuca rubra.