WO2014094073A1 - Gaba responsive motif - Google Patents

Abstract

The invention relates to methods and agents for modulating the binding or responsiveness of GABA to the GABA-responsive motif of ALMT in plants.

Description

GABA Responsive motif

TECHNICAL FIELD

[0001] The present invention relates to a peptide motif and proteins containing the motif that are required for GABA efficacy in plants. Accordingly, the disclosure also includes methods of regulating the interaction of GABA with proteins containing the motif in plants.

BACKGROUND ART

[0002] The following discussion of the background art is intended to facilitate an understanding of the present invention only. The discussion is not an acknowledgement or admission that any of the material referred to is or was part of the common general knowledge as at the priority date of the application.

[0003] The non-protein amino acid gamma-aminobutyric acid (GABA) was first discovered in plant tissue over 60 years ago. In animals, its role as a major neurotransmitter signal is well understood where it regulates ion flow across nerve cell membranes via two classes of receptor, the GABA_A and GABA_B. The GABA_A receptors are chloride (Cl channels, and the GABA_B are G-protein coupled receptors that ultimately regulate cation channels. In plants, GABA has a central role in Carbon and Nitrogen metabolism through the GABA shunt, the pathway that converts glutamate to succinate. GABA also rapidly accumulates in plant tissues in response to various stresses, and it has been shown to regulate important physiological processes such as pollen tube growth, root and hypocotyl elongation, and pathogen defence. In addition, GABA binding sites have been detected on pollen tube and mesophyll cell membranes. Thus, there is potential for manipulation and regulation of plant processes if a mechanism for GABA signalling can be identified.

[0004] It is against this background that the present invention has been developed. It is an object of the invention to overcome some or all of the shortcomings of the prior art.

[0005] General

[0006] Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. The invention includes all such variation and modifications. The invention also includes all of the steps, features, formulations and compounds referred to or indicated in the specification, individually or collectively and any and all combinations or any two or more of the steps or features.

[0007] Each document, reference, patent application or patent cited in this text is expressly incorporated herein in their entirety by reference, which means that it should be read and considered by the reader as part of this text. That the document, reference, patent application or patent cited in this text is not repeated in this text is merely for reasons of conciseness. None of the cited material or the information contained in that material should, however be understood to be common general knowledge.

[0008] Manufacturer's instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein, are hereby incorporated herein by reference, and may be employed in the practice of the invention.

[0009] The present invention is not to be limited in scope by any of the specific embodiments described herein. These embodiments are intended for the purpose of exemplification only. Functionally equivalent products, formulations and methods are clearly within the scope of the invention as described herein.

[0010] The invention described herein may include one or more range of values (eg size, concentration etc). A range of values will be understood to include all values within the range, including the values defining the range, and values adjacent to the range which lead to the same or substantially the same outcome as the values immediately adjacent to that value which defines the boundary to the range.

[001 1] Throughout this specification, unless the context requires otherwise, the word "comprise" or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

[0012] Other definitions for selected terms used herein may be found within the detailed description of the invention and apply throughout. Unless otherwise defined, all other scientific and technical terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which the invention belongs.

SUMMARY OF INVENTION

[0013] The inventors have revealed a GABA-responsive motif on the ALMT family of proteins, including ALMT1 which has led to the development of the present invention.

[0014] In this regard, in a first aspect the present invention provides a GABA-responsive motif located on ALMT proteins as set out in Figure 9.

[0015] In a further aspect, the present invention provides an isolated polypeptide of the form:

S-X¹⁶-T-X¹³-F-X⁴-W-X⁽ (SEQ ID NO: 1) wherein X is an amino acid, and X^{num er} = that number of amino acids. For example X⁴ = XXXX.

[0016] In one embodiment, the isolated polypeptide that shares a percentage sequence homology to SEQ ID NO: 1 , the percentage homology selected from the group consisting of: 99%; 98%; 97%; 96%; 95%; 90%; 85% 80%; 75%; 70%; 65% and 60%.

[0017] In a further aspect, the invention preferably provides an isolated polypeptide of the form:

S-Z¹-X³-Z²-X¹⁰-Z³-T-Z⁴-X-Z⁵-X¹⁰-F-Z⁶-X-P-X-W-X-G-Z⁷-D-Z⁸-H (SEQ ID NO: 2) wherein, X is an amino acid, and X^{num er} = that number of amino acids; Z¹ is a negative, polar amino acid residue, for example, Asp, Glu; Z² is a polar amino acid residue, for example, Ser, Tyr; Z³ and Z⁴ and Z⁵ and Z⁷ are aliphatic amino acid residues, for example Val, lie; and Z⁶ is a polar amino acid residue, for example, Glu, Gin.

[0018] In one embodiment, the isolated polypeptide that shares a percentage sequence homology to SEQ ID NO: 2, the percentage homology selected from the group consisting of: 99%; 98%; 97%; 96%; 95%; 90%; 85% 80%; 75%; 70%; 65% and 60%.

[0019] In a further aspect, the present invention preferably provides an isolated polypeptide sequence of the form:

SSYRVEELIQLAHQRFYTIAVGVFICLCTTVFLFPVWAGEDVH (SEQ ID NO: 3)

[0020] In one embodiment, the isolated polypeptide that shares a percentage sequence homology to SEQ ID NO: 3, the percentage homology selected from the group consisting of: 99%; 98%; 97%; 96%; 95%; 90%; 85% 80%; 75%; 70%; 65% and 60%.

[0021] In a further aspect, the present invention is a fusion protein comprising a peptide according to SEQ ID NO: 1 , 2 or 3.

[0022] In a further aspect, the present invention involves preventing GABA-block of anion currents through an ALMT protein by the application of endogenous or exogenous GABA in a plant cell, and preferably a plant root cell. Said ALMT protein is a member of the family of ALMT proteins which include, ALMT1 , amongst others.

[0023] In one embodiment, prevention of GABA-block of ALMT mediated currents comprises modification of the GABA-responsive motif on ALMT. Preferably, blocking the GABA-responsive motif on ALMT comprises the interaction of a non-GABA molecule or a modified GABA protein to at least a portion of the GABA responsive motif on ALMT to prevent GABA (unmodified) from interacting with the motif on ALMT. [0024] In another embodiment, prevention of GABA-block of ALMT comprises altering the site on ALMT that is required for the GABA response. Altering the site on ALMT required for GABA - block can comprise physically or chemically altering the site. In one embodiment, the method comprises modification of the DNA sequence of ALMT that encodes the GABA-responsive motif, site directed mutagenesis, cysteine scanning mutagenesis, MTSEA-biotin protection assay, and addition of agents that allosterically inhibit GABA response such as bicuculline.

[0025] Preferably, the peptide motif on ALMT which is required for GABA block of ALMT currents (SEQ ID NO: 1 , 2 and 3) is modified. This modification can comprise changing the amino acids in the motif. More preferably the Phe in SEQ ID NO: 1 and 2 is modified to another amino acid. Even more preferably, the Phe in SEQ ID NO: 1 and 2 is modified to Cys. Replacing the Phe with a Tyr also results in GABA-block of ALMT.

[0026] In a further aspect, the present invention provides a method for GABA-responsiveness using a peptide of SEQ ID NO: 1 , 2 or 3. The present invention provides a method for the GABA response using a peptide of SEQ ID NO: 1 , 2 or 3 in a plant cell, and more preferably in a root cell.

[0027] The present invention provides use of a peptide of SEQ ID NO: 1 , 2 or 3 to bind GABA. The present invention provides use of a peptide of SEQ ID NO: 1 , 2 or 3 to bind GABA in plant cells. The present invention provides use of a peptide of SEQ ID NO: 1 , 2 or 3 to bind GABA in plant root cells.

[0028] In a further aspect, the present invention provides a method of inhibiting the GABA response of an ALMT protein, the method comprising modifying the GABA-responsive motif on ALMT. The present invention provides a method of inhibiting the GABA response of an ALMT protein, the method comprising modifying the GABA-responsive motif on ALMT as defined by the motif in SEQ ID NO: 1 , 2 or 3. The present invention provides a method of inhibiting the GABA response of an ALMT protein, the method comprising blocking the GABA-responsive motif on ALMT. Preferably the GABA-responsive motif on ALMT is blocked by another molecule which can interact with the GABA-responsive motif. Said molecule may be an analogue of GABA, for example, amongst others, muscimol. The blocking of the GABA-responsive motif may result in greater organic ion efflux i.e. malate fluxes out of cells.

[0029] In a further aspect, the present invention provides a method of affecting the downstream signalling of ALMT proteins, for example, ALMT1 , in plant cells, and preferably plant root cells, said method comprising interacting analogues of GABA and/or exogenous GABA with an ALMT protein. Some non-limiting examples of effects in a plant from said downstream signalling pathways includes: pH regulation, plant development and defence, nitrogen storage and an alternate pathway for glutamate utilization; in pollen tube growth, guidance and fertilization of the egg by the sperm in angiosperm reproduction. Other such effects include modfying anion and malate flux; increasing wheat roots in terms of number and density, at a range of soil pH especially above pH 7.5; assist plants to release carbon to the soil; improve tolerance of plants to extremes of soil pH.

[0030] In a further aspect, the invention is a method of modulating a parameter in a plant, said parameter selected from the group consisting of: pH regulation; plant development; plant defence; nitrogen storage; glutamate utilization; in pollen tube growth; fertilization; ion influx; tolerance to salinity; tolerance to draught; anion and malate flux; root number and density; rate of release of carbon into the soil by the plant; and tolerance to extremes of soil pH, wherein said method comprises the steps of modulating the GABA responsive motif of ALMT.

[0031] In one aspect, the method comprises the steps of agonising the interaction of GABA with the GABA-responsive motif of ALMT. In another aspect, the method comprises the steps of antagonising the binding of GABA to the GABA-responsive motif of ALMT.

[0032] In one embodiment, the GABA-responsive motif of ALMT shares a percentage sequence homology to the wild-type GABA-responsive motif of ALMT of the same species, the percentage homology selected from the group consisting of: 99%; 98%; 97%; 96%; 95%; 90%; 85% 80%; 75%; 70%; 65% and 60%.

[0033] In a further aspect, the invention is the use of the GABA-responsive motif on ALMT as defined by the motif in SEQ ID NO: 1 , 2 or 3 as a marker for selection of plants for breeding.

[0034] In a further aspect, the present invention involves preventing the binding of endogenous or exogenous GABA to an ALMT protein in a plant cell, and preferably a plant root cell.

[0035] In one embodiment, prevention of binding of GABA to ALMT comprises blocking the GABA-responsive motif on ALMT. Preferably, blocking the GABA-responsive motif on ALMT comprises binding a non-GABA molecule or a modified GABA protein to at least a portion of the GABA-responsive ALMT to prevent GABA (unmodified) from binding to the motif on ALMT.

[0036] In another embodiment, prevention of binding of GABA to ALMT comprises altering the site on ALMT where GABA binds. Altering the site on ALMT where GABA binds can comprise physically or chemically altering the site. Preferably, the peptide motif on ALMT at which GABA binds to ALMT (SEQ ID NO: 1 , 2 and 3) is modified. This modification can comprise changing the amino acids in the motif. More preferably the Phe in SEQ ID NO: 1 and 2 is modified to another amino acid. Even more preferably, the Phe in SEQ ID NO: 1 and 2 is modified to Cys. [0037] In a further aspect, the present invention provides a method for binding GABA using a peptide of SEQ ID NO: 1 , 2 or 3. The present invention provides a method for binding GABA using a peptide of SEQ ID NO: 1 , 2 or 3 in a plant cell, and more preferably in a root cell.

[0038] In a further aspect, the present invention provides a method of inhibiting binding of GABA to an ALMT protein, the method comprising modifying the GABA-responsive motif on ALMT. The present invention provides a method of inhibiting binding of GABA to an ALMT protein, the method comprising modifying the GABA-responsive motif on ALMT as defined by the motif in SEQ ID NO: 1 , 2 or 3. The present invention provides a method of inhibiting binding of GABA to an ALMT protein, the method comprising blocking the GABA-responsive motif on ALMT. Preferably the GABA-responsive motif on ALMT is blocked by another molecule which can interact with the GABA-responsive motif. Said molecule may be an analogue of GABA, for example, amongst others, muscimol. The blocking of the GABA-responsive motif may result in greater organic ion efflux i.e. malate fluxes out of cells.

[0039] In a further aspect, the invention is a method of modulating a parameter in a plant, said parameter selected from the group consisting of: pH regulation; plant development; plant defence; nitrogen storage; glutamate utilization; in pollen tube growth; fertilization; ion influx; tolerance to salinity; tolerance to draught; anion and malate flux; root number and density; rate of release of carbon into the soil by the plant; and tolerance to extremes of soil pH, wherein said method comprises the steps of modulating the binding of GABA to the GABA-responsive motif of ALMT.

[0040] In one aspect, the method comprises the steps of agonising the binding of GABA to the GABA-responsive motif of ALMT. In another aspect, the method comprises the steps of antagonising the binding of GABA to the GABA-responsive motif of ALMT

BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Further features of the present invention are more fully described in the following description of several non-limiting embodiments thereof. This description is included solely for the purposes of exemplifying the present invention. It should not be understood as a restriction on the broad summary, disclosure or description of the invention as set out above. The description will be made with reference to the accompanying drawings in which:

Figure 1. [¹⁴C] Malate fluxes. (A) Uptake of radioactively labelled malate by Xenopus oocytes. Oocytes injected with TaALMTI cRNA or water as a control were incubated in [¹⁴C] malate at external pH 7.5 or 4.5 for 30 min and then digested in acid to determine the rate of malate uptake. Error bars show the SEM (n=5) columns with different letters are significantly different (P<0.05; n=5). (B) Oocytes injected with either TaALMTI cRNA or water were preloaded with [¹⁴C] malate and the radioactivity of the bathing solution was measured at pH 7.5 or 4.5. Shown is the mean efflux of radioactive malate from the oocytes 2 min after injection. Error bars show the SEM (n=5) and columns with different letters are significantly different (P<0.05;n=5).

Figure 2. Effect of malate on currents in Xenopus oocytes expressing TaALMTI . Oocytes injected with TaALMTI cRNA or water as control were either not loaded or preloaded by incubation with 10mM malate-BTP overnight. The currents across the oocyte membrane were recorded using the two electrode voltage clamp technique, while the oocytes were bathed in either 0 mM malate-BTP or 10 mM malate-BTP at external pH of 7.5. Shown is the current across the membranes of oocytes clamped at 40 mV and -160 mV. Error bars show the SEM (n=5) and columns with different letters are significantly different (P<0.05; n=5).

Figure 3. Effect of external malate on conductance of TaALMTI . Oocytes injected with TaALMTI cRNA or water as control were pre-loaded by incubation with 10 mM malate- BTP overnight. The currents across the oocyte membrane were recorded using the two electrode voltage clamp technique, while the oocytes were bathed in malate as malate- BTP at concentrations ranging from 0 mM - 50 mM at external pH of 7.5. Conductance was calculated from the slope of the l/V curve between 100 mV and 0 mV after subtracting the controls. Error bars show the SEM (n=5).

Figure 4. Effect of external malate on aluminium (Al³⁺) activation by TaALMTI in Xenopus oocytes. Oocytes injected with TaALMTI cRNA or water as control (data not shown) were injected with 0.1 M sodium malate prior to two electrode voltage clamp technique. The currents across the oocyte membrane were recorded while the bathing the oocytes in either ND88 alone; ND88 + 100 μΜ AICI₃; ND88 + 100 μΜ AICI₃ + 100 μΜ malate or ND88 + 100 μΜ AICI₃ + 1 mM malate at an external pH of 4.5. (A, B) Shown is the current across the membrane of oocytes clamped at 60 mV and -140 mV. Error bars represent SEM (n=4) and columns with different letters are significantly different (P<0.05; n=4). (C and D) Shown is the time course of outward and inward currents in TaALMTI expressing oocytes after 6 min at 60 mV and -140 mV.

Figure 5. Selectivity of TaALMTI to extermal ions at pH 7.5. Oocytes injected with TaALMTI cRNA or water as control were pre-loaded by incubation with 10 mM malate- BTP overnight. The current across the oocyte membrane were recorded using the two electrode voltage clamp technique while the oocytes were bathed in bath solution [CI^" = 1.4 mM] with test anions at 10 mM being the major external anions. Shown is the current across the membranes of oocytes clamped at 60 mV abd -140 mV with the controls subtracted from the TaALMTI expressing oocytes. Error bars show the SEM (n=5-7) and columns with different letters are significantly different (P<0.05; n=5-7). Figure 6. Effect of GABA on malate stimulated anion efflux currents at pH 7.5. (A and B)

Oocytes injected with TaALMTI cRNA or water as control were pre-loaded with malate- BTP overnight. The currents across the oocyte membrane were recorded using the two electrode voltage clamp technique while the oocytes were bathed in either 10 mM malate alone or 10 mM malate with GABA added in concentrations ranging from 1 μΜ - 100 μΜ. The error bars represent SEM (n=5). (C and D) Dose response curves for GABA inhibition of malate stimulated efflux were plotted using the dose-response equation for inhibition and the IC50 calculated. The error bars represent SEM (n=5).

Figure 7. Effect of GABA on aluminium activation by TaALMTI in Xenopus oocytes at pH 4.5. Oocytes injected with TaALMTI cRNA or water as control were injected with 0.1 M sodium malate prior to two electrode voltage clamp. The currents across the oocyte membranes were recorded while the oocytes were bathing in either ND 88 alone; ND 88 + 100 μΜ AICI₃ or ND88 + 100 μΜ AICI₃ + 100 μΜ GABA. Shown here are the currents across the membranes of oocytes clamped at 60 mV and -140 mV for TaALMTI expressing oocytes only. There was no effect of Al³⁺ or GABA on control oocytes (data not shown). Error bars represent the SEM (n=4) and columns with different letters are significantly different (PO.05; n=4).

Figure 8. Dose response curves. Data from the l/V curves was used to calculate the IC50 values for GABA inhibition of malate stimulated efflux. The error bars represent SEM (n=5). Shown is the current across the membranes of oocytes clamped at 60 mV and -140 mV. Error bars show the SEM (n=5 -7).

Figure 9. Aligned partial sequences of the rat GABA_A receptor a subunit types. (A) The residues from the rat GABA_A receptor a subunit types span the region from 53-75 (numbered by alignment with subtype) and this region is highly conserved in all species from which a subunits have been cloned in mammalian brains. These residues were aligned with TaALMTI and span the region from 182-224 residues. Residues not identical to CH are boxed and highlighted in grey, a, F64, an identified GABA binding site residue in rat brain and conserved residues are boxed and indicated with an asterisk. Similar residues are indicated with colon and substitutions with a dot. (e) Alignment of rat GABA_A receptor a subunit types with ALMTs identified from wheat, barley, rice, maize and Brassica. Residues not identical to a1 are boxed and highlighted in grey, F64, an identified GABA binding site residue in rat brain and conserved residues are boxed and indicated with an asterisk. Similar residues are indicated with colon and substitutions with a dot.

Figure 10. ALMT proteins contain amino acids essential for GABA-block of anion currents. All assays performed in Xenopus laevis oocytes, a, Representative current traces for TaALMTI injected or control oocytes at -120 mV; all bathed in basal solution (B) plus addition of transactivation solution 10 mM malate (M), 100 μΜ GABA (G), 10 μΜ muscimol (Mus) or 100 μΜ bicuculline (Bic), b, Current-voltage relationship for TaALMTI in symmetrical 10 mM malate (pH₀ = 7.5) ± muscimol. Currents normalized to largest mean outward current at +60 mV ± S.E.M. (n = 5 oocytes), c, Dose response relationship derived from 10b and Extended Data Fig. 1 1 b. d, Amino acid consensus sequence motif between GABA_A a subunits and ALMT proteins (also see Extended Data Fig 12a and 12b); identical residues shaded (black), 80% similar (gray) and <60% similar (unshaded). Also shows EC₅₀ for GABA inhibition of malate stimulated inward currents of ALMT proteins (after 30 sec), or activation of rat GABA_A receptors², e, TaALMTI ^F213C but not TaALMTI ^F215C mutagenesis decreases GABA sensitivity. Residual currents in 100 μΜ GABA are plotted relative to the initial malate-activated current at - 140 mV. f, TaALMTI ^F213C mutagenesis did not affect activation by 100 μΜ Al³⁺, at pH 4.5, but abolishes block of these Al³⁺-activated currents by 10 μΜ muscimol. Different letters indicate significance within a group (P<0.05).

Figure 11. GABA blocks activation of TaALMTI by Al³⁺ or high pH. All experiments carried out in tobacco BY2 cells expressing TaALMTI , for same assay on BY2 cells expressing empty-vector see Extended Fig. 13. a, Al³⁺ stimulated malate efflux (100 μΜ at pH 4.5) ± concurrent treatments, mean ± S.E.M (n = 5). b, High pH stimulated malate efflux, mean ± S.E.M (n = 5). c, High pH dependent efflux ± concurrent treatments, mean ± S.E.M, n = 3. Different letters indicate significance within a group (P<0.05). GABA (100 μΜ), muscimol (10 μΜ), vigabatrin (100 μΜ) or bicuculline (100 μΜ).

Figure 12. GABA abolishes Al³⁺- and high pH tolerance. All assays carried out in wheat near isogenic lines (ET8 = Al tolerant, ES8 = Al sensitive). Root growth and malate efflux: in presence of Al³⁺ at (a) pH 4.5 ± treatments mean ± S.E.M. (n = 5) or (b) pH 9 ± treatments, mean ± S.E.M. (n = 5). Relative root elongation rate versus malate efflux for different treatments (c) 4.5 +AI (d) pH 9). Different letters indicate significant differences (p<0.05).

Figure 13. Muscimol reduces hypocotyl and pollen tube elongation, a, Arabidopsis hypocotyl etiolation after 4 days ± treatments, mean Δ from control mean ± S.E.M. (482.6 ± 33.03 μιη) (n = 79-104 per treatment), b, In vitro grapevine pollen tube elongation after 6 hours ± treatments, mean Δ from control mean ± S.E.M. (12.5 ± 0.16 mm) (n = 157-193 per treatment). Treatments as per Fig 12. Different letters indicate significant differences (p<0.05).

DESCRIPTION OF PREFERRED EMBODIMENTS

[0042] The present invention provides a peptide sequence of the form: -T-X¹³-F-X⁴-W-X⁶ (SEQ ID NO: 1 ) wherein X is an amino acid, and X^number = that number of amino acids. For example X⁴ = XXXX.

[0043] The present invention preferably provides a peptide sequence of the form:

S-Z¹-X³-Z²-X ⁰-Z³-T-Z⁴-X-Z⁵-X ⁰-F-Z⁶-X-P-X-W-X-G-Z⁷-D-Z⁸-H (SEQ ID NO: 2) wherein, X is an amino acid, and X^number = that number of amino acids; Z¹ is a negative, polar amino acid residue, for example, Asp, Glu; Z² is a polar amino acid residue, for example, Ser, Tyr; Z³ and Z⁴ and Z⁵ and Z⁷ are aliphatic amino acid residues, for example Val, lie; and Z⁶ is a polar amino acid residue, for example, Glu, Gin.

[0016] The present invention preferably provides a peptide sequence of the form:

SSYRVEELIQLAHQRFYTIAVGVFICLCTTVFLFPVWAGEDVH (SEQ ID NO: 3)

ALMTs

[0044] Plant species have evolved diverse mechanisms such as exuding organic acids (citrate, malate and oxalate) to cope with aluminium (Al³⁺) toxicity in acidic soils. Organic acids exuded from the root tips chelate the Al³⁺ into non-toxic compounds thus preventing damage to the roots of growing plants. ALMTs are now known to form a large anion channel family that is found in all plant species. Arabidopsis has 14, grapevine at least 13, soybean 33, and there are 9 in rice. Homologous genes in sequence and function to TaALMTI are found in Arabidopsis (AtALMTI) and further species, but most ALMTs are not activated by Al, nor have any role in Al tolerance. Instead, the roles of /ALMTs are considered to be diverse, for example, mineral nutrition (ZmALMTI), vacuolar malate accumulation (AtALMT9,6) and stomatal aperture control (AtALMT12, HvALMTI). Numerous homologs of TaALMTI have since been identified in plants such as Zea mays, Arabidopsis thaliana, Brassica napus, Hordeum vulgare, Seca/e cereale and Oryza sativa. Using heterologous expression, mainly in Xenopus laevis oocytes, activated by Al³⁺ and function similar to TaALMTI while others are not activated by Al³⁺ and function as either non selective anion transporters or organic anion transporters. Localisation of these transporters also varies from plasma membrane, tonoplast membrane to guard cells. TaALMTI enabled Al³⁺ activated currents are inhibited by Niflumate and this protein is permeable to both malate as well as chloride but has approximately 18 fold more affinity for malate over chloride.

[0045] Aluminium-activated anion transporters (ALMTs) are named after a gene that is highly and constitutively expressed in root tips of Al tolerant varieties of wheat. This gene, TaALMTI encodes a protein found on the plasma membrane that catalyses the efflux of malate from root cells when exposed to micromolar concentrations of soil Al³⁺, as can occur in acid soils. Soil and cell wall malate then chelates the toxic Al ion, thus conferring Al tolerance. [0046] Herein is provided evidence that the ALMT family of plants encode the GABA-receptor in plants and propose that the biophysical properties of these ion channels are ideal suited to key signal transduction elements of essential physiological processes.

GABA

[0047] While examining the selectivity of TaALMTI in oocytes to a variety of inorganic and organic anions, the inventors observed an interesting effect of γ amino butyric acid (GABA) on anion efflux. GABA is a four carbon non-protein amino acid, highly soluble in water, zwitterionic at physiological pH values and a significant component of free amino acid pools in both prokaryotes and eukaryotes. In plants tissues, GABA levels range from 0.03 to 2.00 μιηοΙ g-1 fresh weight but increase in response to abiotic stresses such as heat shock, hypoxia and phytohormones. The evidence suggests GABA is involved in pH regulation, plant development and defence, nitrogen storage and an alternate pathway for glutamate utilization. In angiosperm reproduction, GABA plays a major role in pollen tube growth, guidance and fertilization of the egg by the sperm

[0048] The inventors investigated sustained malate efflux, reversibility of Al³⁺ activation and the role and effect of GABA on the activity of TaALMTI in Xenopus oocytes therein gaining a further insight into the structure and function of this transporter.

[0049] GABA strongly inhibits anion channels in plants with high affinity.

[0050] There are conserved GABA responsive motifs present on ALMTs including ALMT1. This presents evidence that GABA signalling occurs in plant cells. Thus, as GABA regulates proteins that release molecules involved in root-soil interactions, the actions of GABA may be manipulated by affecting the interaction of GABA with ALMT proteins resulting in modifications of how a plant responds to, for example, external stress from extremes of soil pH. This may include activation or suppression of the signalling events between GABA and ALMT.

[0051] The AMLT gene may be modified using known molecular biology techniques to modify the GABA-responsive motif (Figure 9). Such modification may affect GABA-block of AMLT such as preventing the GABA response or reducing the degree of response, therein affecting the downstream signalling events from AMLT.

[0052] A peptide which can bind to the AMLT GABA-responsive motif may be used to affect the downstream signalling events from AMLT.

Polypeptides

[0053] Functional variants also include peptides with modified or different amino acids sequences that still retain their ability to respond or bind to GABA. These functional variants include peptides with deletions, insertions, inversions, repeats and/or type substitutions. Preferably, functional variants are at least 70%, 80% or 90% identical to SEQ 10 NO: 1 , 2 or 3, more preferably at least 95% identical to SEQ 10 NO: 1 , 2 or 3.

[0054] Functional variants also include peptides (i) in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue such as synthetic, non-naturally occurring analogues and/or natural amino acid residues; or (ii) in which one or more of the amino acid residues includes a substituent group. Particular conserved substitutions involve the substitution of a charged amino acid with an alternative charged amino acid or a negatively charged or neutral amino acid. Preferably, the changes are minor and do not have a negative impact on the ability of the peptide to respond or bind to GABA. Other conservative substitutions for the purposes of the present invention are exemplified hereunder. However, it will be appreciated that skilled persons may also determine further conservative substitutions not specifically listed.

Aromatic (Phenylalanine, Tryptophan, Tyrosine, Histidine)

Hydrophobic (Leucine, Isoleucine, Valine, norleucine)

Small (Alanine, Serine, Threonine, Methionine, Glycine)

Acidic (Aspartic acid, Glutamic acid)

Basic (Arginine, Lysine, Histidine)

Polar (Glutamine, Asparagine)

[0055] Representative examples of useful peptides include any of the naturally occurring or synthetic di-, tri-, tetra-, pentapeptides or longer peptides derived from any of the above described amino acid sequences. Representative examples of useful polypeptides include both naturally occurring and synthetic polypeptides derived from the above described amino acids and peptides.

Nucleotides

[0056] The present invention also provides polynucleotides encoding the peptides of the invention, for example, the peptides of SEQ 10 NO: 1 , 2 and 3. It will be understood by a skilled person that due to the degeneracy of the amino acid code, numerous different polynucleotides can encode the same peptide as a result of the degeneracy of the genetic code. In addition, it is to be understood that skilled persons may, using routine techniques, make nucleotide substitutions that do not affect the peptide sequence encoded by the polynucleotides of the invention to reflect the codon usage of any particular host organism in which the polypeptides of the invention are to be expressed.

[0057] Polynucleotides of the invention may comprise DNA or RNA. They may be single-stranded or double-stranded. They may also be polynucleotides that include within them synthetic or modified nucleotides. A number of different types of modification to oligonucleotides are known in the art. These include methylphosphonate and phosphorothioate backbones, addition of acridine or polylysine chains at the 3' and/or 5' ends of the molecule. For the purposes of the present invention, it is to be understood that the polynucleotides described herein may be modified by any method available in the art. Such modifications may be carried out in order to enhance the in vivo activity or life span of polynucleotides of the invention.

[0058] Where the polynucleotide of the invention is double-stranded, both strands of the duplex, either individually or in combination, are encompassed by the present invention. Where the polynucleotide is single-stranded, it is to be understood that the complementary sequence of that polynucleotide is also included within the scope of the present invention.

Modified Peptides

[0059] The present peptide or analogues, such as those recited infra may be derivatized by the attachment of one or more chemical moieties to the peptide sequence. Chemical modification of biologically active peptides provides advantages under certain circumstances, such as increasing the stability and circulation time of the peptides, and to enhance specificity.

[0060] Chemical modification of one or more residues may be achieved by chemically derivatizing a functional side group. Such derivatized molecules include for example, those molecules in which free amino groups have been derivatized to form amine hydrochlorides, p-toluene sulfonyl groups, carbobenzoxy groups, t-butyloxycarbonyl groups, chloroacetyl groups or formyl groups. Free carboxyl groups may be derivatized to form salts, methyl and ethyl esters or other types of esters or hydrazides. Free hydroxyl groups may be derivatized to form O-acyl or O-alkyI derivatives. The imidazole nitrogen of histidine may be derivatized to form N-im-benzylhistidine. Also included as chemical derivatives are those peptides which contain one or more naturally occurring amino acid derivatives of the twenty standard amino acids. For examples: 4- hydroxyproline may be substituted for proline; 5-hydroxylysine may be substituted for lysine; 3-methylhistidine may be substituted for histidine; homoserine may be substituted for serine; and ornithine may be substituted for lysine.

[0061] Replacement of naturally occurring amino acids with a variety of uncoded or modified amino acids such as D-amino acids and N-methyl amino acids may also be used to modify peptides. For example, 0 amino acids can be substituted for L amino acids to increase in vivo stability of the peptides, while still retaining biological activity. Likewise, retro-inverso peptides, which contain NH-CO bonds instead of CO-NH peptide bonds, are much more resistant to proteolysis than L-peptides. Moreover, they have been shown to mimic natural L-peptides with respect to poly- and monoclonal antibodies.

[0062] Therefore, peptides having at least one 0 amino acid on the amino terminal and/or carboxy terminal end of the molecule and which retain biological activity are considered part of the invention. In addition, retro-inverso peptides which contain one or more of the amino acid sequences of the invention and which retain biological activity are also considered part of the invention.lt may also be desirable to use derivatives of the peptides of the invention that are conformationally constrained. Conformational constraint refers to the stability and preferred conformation of the three-dimensional shape assumed by a peptide. Conformational constraints include local constraints, involving restricting the conformational mobility of a single residue in a peptide; regional constraints, involving restricting the conformational mobility of a group of residues, which residues may form some secondary structural unit; and global constraints, involving the entire peptide structure.

[0063] Another approach is to include a metal-ion complexing backbone in the peptide structure. Typically, the preferred metal-peptide backbone is based on the requisite number of particular coordinating groups required by the coordination sphere of a given complexing metal ion. In general, most of the metal ions that may prove useful have a coordination number of four to six. The nature of the coordinating groups in the peptide chain includes nitrogen atoms with amine, amide, imidazole, or guanidino functionalities; sulfur atoms of thiols or disulfides; and oxygen atoms of hydroxy, phenolic, carbonyl, or carboxyl functionalities. In addition, the peptide chain or individual amino acids can be chemically altered to include a coordinating group, such as for example oxime, hydrazino, sulfhydryl, phosphate, cyano, pyridino, piperidino, or morpholino. The peptide construct can be either linear or cyclic, however a linear construct is typically preferred. One example of a small linear peptide is Gly-Gly-Gly- Gly that has four nitrogens (an N4 complexation system) in the backbone that can complex to a metal ion with a coordination number of four.

EXAMPLES

Materials and Methods

In Vitro Transcription and Complementary RNA injection

[0064] The plasmid pGEMHE -DEST containing TaALMTI was linearised with Nhe1 and used as the template for capped copy RNA (cRNA) using a Message Machine^® T7 kit (Ambion^®). Oocytes were harvested and maintained using a modification of the method described previously Preuss et al. (2010) Plant Physiology 152:1431-41. The oocytes were injected with 46 nl of cRNA (0.7 g/ l) encoding TaALMTI (HvALMTI , OsALMT5 and OsALMT9) or RNase free water using a micro-injector (Nanoject II automatic nanolitre injector, Drummond Scientific).

Radioactive malate flux experiments

[0065] For influx experiments, the oocytes expressing either TaALMTI cRNA or water (controls) were incubated in Ca-Frog Ringers solution (pH 7.5 or 4.5) containing radioactive malate (L- 1 ,4 (2,3) [¹⁴C] malic acid; Amersham) at a final malate concentration of 2 mM. The oocytes were placed in a 96 well plate with each well containing 100 μΙ of experimental solution at either pH 7.5 or pH 4.5. After 30 min incubation, the oocytes were placed in scintillation vials, digested with 0.1 N HN0₃ before being counted in a scintillation counter (Tri-Carb^® 2100 TR Liquid Scintillation Analyser, Packard).

[0066] For efflux experiments, both TaALMTI expressing oocytes and control oocytes were injected with 46 nl of radioactive malate (Amersham) and placed in 500 μΙ of assay buffer (Ca- Frog Ringer's) at either pH 4.5 or 7.5. The assay buffer was sampled at periodic intervals (2, 5, 10, 20 and 30 min) and replaced with fresh assay buffer. At the end of the experiment, the sampled buffers and the digested oocytes were placed in scintillation vials and counted in scintillation counter. The data from both experiments was subjected to analysis of variance (ANOVA) and treatments means compared by SEM (P<0.05) using GraphPad Prism (GraphPad Software Inc.).

Electrophvsiology

[0067] The injected oocytes were incubated in Ca-Frog Ringers at 18°C and the solution was replaced daily. Experiments were carried out 2 days after injection. Two Electrode Voltage clamp (TEVC) was performed on both TaALMTI expressing and control oocytes that were either pre-treated overnight in 10 mM Malate-BTP or injected with 0.1 M sodium malate prior to clamping. The composition of the basic recording solution was as used by Kovermann et al., (2007) Plant Journal 52:1169-1180, with modifications to reveal malate dependent currents and the permeability or selectivity of TaALMTI to other anions. The solutions consisted of 10 mM malic acid, citric acid, MES, nitrate, sulphate, phosphate, glutamic acid and γ amino butyric acid (GABA) added to a basal solution containing 0.7 mM CaCI₂ (0.5 mM CaCI₂ at pH 4.5), adjusted to a constant osmolarity of 220 mosmol kg-1 with mannitol and buffered with BTP to pH 7.5 or 4.5. To determine the kinetics of TaALMTI , oocytes expressing TaALMTI and controls were subjected to TEVC after pre loading the oocytes with malate. Currents were recorded by constant perfusion with solution containing either no malate or malate (2 μΜ -50 μΜ).

[0068] In the experiments for Dose Response Curves for GABA, TaALMTI , HvALMTI , OsALMT5 and OsALMT9 expressing oocytes (injected with 46 nl of each cRNA) and control oocytes were incubated in 10 mM malate overnight before being subjected to TEVC. Currents across the oocyte membranes were recorded by constant perfusion with bathing solutions containing either 0.7 mM CaCI₂ and 10 mM malate-BTP alone or supplemented with GABA (2 μΜ - 100 μΜ) at pH 7.5.

[0069] For Al³⁺ activation experiments, oocytes injected with either TaALMTI cRNA or water (controls) were incubated in ND88 (pH 7.6) for 48 h. The oocytes were subsequently injected with 46 nl of 0.1 M sodium malate prior to performing TEVC. Bathing solutions for these experiments were adjusted to pH 4.5 and contained 00 μΜ Al³⁺ alone, 00 μΜ Al³⁺ and 00 μΜ malate or 100 μΜ Al³⁺ and 1 mM malate. Oocytes were monitored for 8 min after perfusion by these bathing solutions.

[0070] The whole cell currents were recorded under constant perfusion and temperature (22°C) with a GeneClamp 500 amplifier (Axon instruments) using conventional TEVC. The recording electrodes were filled with 3.0 M KCI and had resistances between 0.5 ΜΩ to 1.2 ΜΩ. The voltage was stepped between 60 mV and -140 mV in 20 mV increments from a holding potential of -40 mV. The duration of each voltage pulse was 0.5 s, with a resting phase of 1.0 s between successive voltage steps. The output was digitized and analyzed using a Digidata 1322A- pClamp 8 data acquisition system (Axon instruments). Data analysis was performed using the Clampfit software (version 8.2; Molecular Devices Corporation) and GraphPad software (GraphPad Software Inc.) to construct the current-voltage curves. Dose response curves for GABA were constructed at 60 mV and -140 mV by subjecting the data to Transform and Nonlinear fit using GraphPad software.

Tobacco BY2 Cells expressing ALMT1 [0071] The methods for generation of the transgenic tobacco BY2 cells containing TaALMTI , and the method for measuring malate fluxes from cells have been previously described by Zhang et al (2009) Plant and Cell Physiology 49:1316-1330. BY2 cells were incubated in fro 20 hours in 10 mM Na₂S0₄ 3 mM CaCI2 and 3 mM sucrose, and held at the appropriate pH using 5 mM BTP/MES with or without the addition of these modifiers of GABA concentration and GABA reception (GABA (100 μΜ), aminooxyacetic acid (AOA, 1 mM) muscimol (10 μΜ), vigabtrin (100 μΜ) and bicuculline (10 μΜ)).

GenBank accession numbers

[0072] The GenBank accession numbers for coding sequences used in this study are:

(i) TaALMTI AB081803

(ii) HvALMTI EF424084

(iii) OsALMT5 NP_001052768.1

(iv) OsALMT9 NP_001065461.1

(v) AtALMTI 3 Q9LS23

(vii) AtALMT14 Q9LS22

(viii) VvALMT9;1 XP_002275995.1

Results

[¹⁴C1 Malate transport by oocytes expressing TaALMTI (Figure 1)

[0073] To study the dependency of currents on external pH, malate uptake by the oocytes was monitored by bathing the TaALMTI expressing oocytes and water injected controls in uptake assay buffer supplemented with 2 mM final concentration of [¹⁴C]malate and measuring the radioactivity taken up after 30 min of incubation at either pH 7.5 or 4.5 (Fig. 1A). Only at pH 7.5 was the rate of malate uptake significantly greater in oocytes expressing TaALMTI

[0074] Malate efflux from oocytes was monitored after they were injected with [¹⁴C]malate, The malate efflux from TaALMTI expressing oocytes over 2 min was 6.8 fold greater than control oocytes at pH 7.5 (Fig. 1 B). This difference was not apparent 5 min after injection or at time points up to 30 min (data not shown).

Electrophysiology of TaALMTI in Xenopus oocytes

Effect of malate on currents in Xenopus oocytes expressing TaALMTI (Figure 2).

[0075] The transport properties of TaALMTI at pH 7.5 were further examined using TEVC in oocytes. The bathing solutions used in the experiments were designed so that observed inward current would be most likely carried by the efflux of anions from the oocyte while outward currents could be mediated by efflux of cations or influx of anions. Oocytes either pre-loaded with malate or not loaded were subjected to TEVC. Currents across the oocyte membranes were recorded by constant perfusion with solution containing either no malate (0 mM) or 10 mM malate. The inward and outward currents were not significantly different in TaALMTI and control oocytes (both preloaded and not loaded with malate) when external malate concentration was 0 mM (Fig. 2 A and B). When external malate concentration was 10 mM, the TaALMTI expressing oocytes showed a significant increase in both inward and outward currents compared to the control oocytes (Fig. 2 C and D), however there was no significant difference in currents between the preloaded with malate or non-loaded TaALMTI expressing oocytes. Current voltage curves showed that the currents were rapidly activated with changes in voltage (data not shown).

Effect of external malate on conductance of TaALMTI (Figure 3).

[0076] To calculate the effect of different external malate concentrations on TaALMTI , malate pre-loaded control oocytes and those expressing TaALMTI were subjected to TEVC with constant perfusion with solutions containing malate ranging from 0 to 50 mM at pH 7.5. Currents were recorded across the oocyte membranes and the slope conductance was calculated between -100 and 0 mV. The slope conductance increased with increasing concentrations of malate added as malate-BTP (Fig. 3). TaALMTI showed both saturating and non-saturating phases with an apparent Km of 1.1 mM at lower concentrations of malate.

[0077] Effect of external malate on aluminium (Al³⁺) activation by TaALMTI in Xenopus (Figure

[0078] Several members of the ALMT family show increases in magnitude of currents on exposure to Al³⁺. It has been shown that in TaALMTI increased inward currents are activated in oocytes by addition of Al³⁺ to the bathing solution at pH 4.5. These anion efflux currents are activated slowly (after 3 min) and increase with time (Fig. 4 D). However little information is available on the effect of added external malate on Al³⁺ activation by TaALMTI . Experiments were carried out to determine the effect of added external malate on the Al³⁺ activation by TaALMTI Oocytes expressing both TaALMTI and controls were injected with 0.1 M sodium malate prior to TEVC. When Al³⁺ was added to the bathing solution, outward current at 60 mV decreased significantly (Fig. 4 A,C) while inward current at -140 mV increased significantly in TaALMTI expressing oocytes (Fig.4 B, D). Addition of 100 μΜ malate to the basal solution with 100 μΜ Al³⁺ resulted in significant reduction in inward currents in TaALMTI expressing oocytes and a spike in outward current for one time point (Fig 4C). Addition of 1 mM malate to the basal solution in the presence of 100 μΜ Al³⁺ resulted in an increase in magnitude of inward currents similar to that seen with Al³⁺ alone (Fig. 4 B and D). Removal of malate from the solution containing Al³⁺ surprisingly resulted in significant decrease in magnitude of inward currents in TaALMTI expressing oocytes to less than basal levels (Fig. 4 B and D) but no change in outward current (Fig. 4 A, C). Controls did not show significant changes in magnitude of inward currents in response to either Al³⁺ alone or Al³⁺ in combination with malate (data not shown).

[0079] Selectivity of TaALMTI to external anions at pH 7.5 (Figure 5).

[0080] The members of the ALMT family studied to date have been shown to be permeable to malate, chloride and citrate. TaALMTI in oocytes has been shown to be permeable to both malate and chloride. However, not much information exists about the permeability of TaALMTI to other organic and inorganic anions or in regards to the transactivation of external anion on the inward currents (anion efflux). Experiments were done to test the permeability of both organic and inorganic anions in oocytes expressing TaALMTI at an external pH of 7.5. Both the control and TaALMTI expressing oocytes were pre-loaded with malate and subjected to TEVC under constant perfusion with bathing solutions containing either 10 mM nitrate, chloride, sulphate, phosphate, MES, malate, citrate, glutamate or GABA.

[0081] Current voltage curves across the oocyte membranes were recorded. The results suggest that TaALMTI expressing oocytes are permeable to a variety of organic and inorganic anions such as succinate, nitrate, chloride, sulphate, phosphate, MES, glutamate, malate and citrate as summarised by both outward (@ 60 mV) (Fig 5 A, C) and inward currents (@-140 mV) (Fig. 5 B, D). Interestingly TaALMTI expressing oocytes gave much smaller outward and inward currents when GABA was present (Fig. 5 C and D).

[0082] Effect of GABA on malate stimulated anion efflux currents at pH 7.5 (Figure 6).

[0083] Since TaALMTI expressing oocytes were observed to be highly permeable to both organic and inorganic anions except GABA, experiments were carried out to study the effect of GABA in more detail. Both control and TaALMTI expressing oocytes pre-loaded with malate were subjected to TEVC. Currents across the oocyte membranes were recorded by constant perfusion with bath solutions containing either 10 mM malate only or 10 mM malate supplemented with different concentrations of GABA (2 μΜ - 100 μΜ). GABA did not affect malate stimulated inward currents in the water injected control oocytes (Fig. 6 A). In TaALMTI expressing oocytes, malate stimulated inward currents reduced in magnitude with increasing concentrations of GABA (Fig. 6 B). These results suggest that GABA inhibits malate stimulated anion efflux, reducing the inward currents to almost basal levels at a GABA concentration of 100 μΜ. Data from l/V curves was analysed to study the GABA inhibition of malate stimulated efflux at 60 mV and-140 mV. Dose response curves were plotted to determine the concentration of inhibitor that provokes a response halfway between the baseline and maximum response (IC50). The calculated IC50 value at 60 mV was 2.8 μΜ (Fig. 6 C) and at -140 mV was 3.6 μΜ (Fig. 6 D) suggesting that GABA is a potent inhibitor of malate stimulated anion efflux by TaALMTI at pH 7.5.

[0084] Effect of GABA on aluminium activation at pH 4.5 (Figure 7).

[0085] Since GABA effectively blocks malate stimulated efflux by TaALMTI at pH 7.5, the effect of GABA on these currents was studied at pH 4.5. Aluminum exposure increases magnitude of anion efflux in TaALMTI expressing oocytes. Both control and TaALMTI expressing oocytes were injected with 0.1 M sodium malate prior to TEVC. Membrane currents across the oocytes were recorded by constant perfusion with the basal solution only, or basal solution supplemented with either 100 μΜ Al³⁺ alone or 100 μΜ Al³⁺ and 100 μΜ GABA (Fig. 7 B). Addition of 100 μΜ Al³⁺ caused a significant increase in the magnitude of inward currents but addition of GABA to bathing solution with Al³⁺ did not cause a significant increase in the inward currents. Also, removal of GABA from the bathing solution with Al³⁺ did not cause a significant increase in the magnitude of inward currents. These results suggest that GABA does not block the inward currents in TaALMTI expressing oocytes exposed to Al³⁺ at pH 4.5. There was no significant change in the magnitude of outward currents in TaALMTI expressing oocytes (Fig. 7 A). There was no effect of Al³⁺ or addition of GABA on the control oocytes (data not shown).

[0086] Effect of GABA on malate stimulated efflux currents on other members of the ALMT family (Figure 8).

[0087] Numerous members of the ALMT1 family have been cloned and characterized from plants such as wheat, barley, rice, maize, Brassica, rye and Arabidopsis. Some of these genes are activated by Al³⁺ while others do not require Al³⁺ to be active in anion efflux. As the malate stimulated anion efflux in TaALMTI from wheat was effectively inhibited by GABA, experiments were done to study the effect of GABA on ALMTs from barley (HvALMTI) and rice (OsALMT5 and OsALMT9). Both control oocytes and oocytes expressing HvALMTI , OsALMT5 and OsALMT9 were pre-loaded with malate overnight before being subjected to TEVC. Currents across the membranes were recorded with constant perfusion of bathing solution containing 10 mM malate alone or 10 mM malate supplemented with GABA (2 μΜ - 100 μΜ). Addition of increasing concentrations of GABA to bath solution containing 10 mM malate significantly inhibited both outward and inward currents from oocytes expressing HvALMTI and the OsALMTs but GABA did not affect the currents in the control oocytes (data not shown). Data from the l/V curves were analysed to plot the IC50 values for GABA inhibition of malate stimulated anion efflux in the ALMTs at 60 mV and -140 mV (Fig. 8). IC50 value at 60 mV was 4.1 μΜ for HvALMTI , 1.45 μΜ for OsALMT9, 2.07 μΜ for OsALMT5, 7.03 μΜ for AtALMT13 and 2.24 for ALMT14. The IC50 values at -140 mV for HvALMTI were 2.08 μΜ for HvALMTI , 1.08 μΜ for OsALMT9, 2.07 μΜ for OsALMT5; 4.6 μΜ for AtALMT13 and 1 .42 for ALMT14. Malate stimulated anion efflux in OsALMT 9 seemed to be highly sensitive to inhibition by GABA. Whereas Vv

[0088] Putative GABA responsive sites in TaALMTs and other ALMT family members (Figure

[0089] It has been well documented that GABA is the major inhibitory neurotransmitter in the mammalian brain and GABA_A receptors are the primary transducers of this action. The cloned GABA_A receptors possess residues which are conserved in all the a subunit types. As malate stimulated anion currents in TaALMTI expressing oocytes are sensitive to inhibition by low concentrations of GABA, it implies that there may be binding sites for GABA on the TaALMTI . Predicted amino acid sequence of TaALMTI was aligned against the known GABA_A a subunit types from rat's brain to identify the putative residues that may bind GABA or line the GABA binding site (Fig. 9 A). Amino acid alignment revealed that residues 182-224 in TaALMTI showed homology to residues 53-75 from the rat GABA_A receptor a subunits. Serine (S182), Glutamic acid (E187), Tyrosine (Y198), Threonine (T199), Valine (V202), Phenylalanine (F213), Tryptophan (W218) and Glutamic acid (E221) are conserved in both TaALMTI and the rat GABAA receptor a subunits. Similar alignment of barley, rice, maize, Arabidopsis and Brassica ALMTs (Fig. 9 B) to the cloned rat GABA_A receptor a subunit types showed that Serine (S), Threonine (T), Phenylalanine (F) and Tryptophan (W) are highly conserved in all the ALMTs. The e F64 in the rat brain has been identified by photoaffinity labelling experiments as forming a part of GABA binding site. Thus it is hypothesized that Phenylalanine (F) conserved in all the ALMTs and rat GABA_A a subunit types, may form a part of the GABA_A receptor binding site in plants.

[0090] Malate flux can be manipulated across plant cells by using compounds that modify GABA concentration or interact with the GABA responsive site (Figure 10).

[0091] At higher extracellular pHs TaALMTI catalyses greater flux of organic acids out of cells. Malate flux through TaAMLTI can be blocked at all pHs by addition of muscimol, which binds to the GABAA GABA binding site, and is blocked by addition of vigabatrin which inhibits the GABA- Transaminase - which converts GABA to succinate - and therefore results in greater GABA concentration in cells. Aminooxyacetic acid, often abbreviated AOA, increases flux of malate through TaALMTI at all pHs. Bicuculline, which binds to the GABA binding site in GABA_A receptors decreases malate flux through TaALMTI at high extracellular pH.

[0092] By surveying all known ALMT proteins and expressing a selection of these in Xenopus laevis oocytes our results suggested that, as is also found for GABA_A._P receptors or GABA_A a subunits, the aromatic amino acid residues phenylalanine (F) or tyrosine (Y) are important for GABA efficacy (Fig. 1 1a). This survey also showed the non-aromatic amino acid cysteine (C) was a common natural substitution for F or Y in the motif. Site directed mutagenesis of the first aromatic residue within this motif in TaALMTI , either by itself (TaALMT1 ^{F2 3C}) or in combination with a second F mutation (TaALMTI ^F213C/F215C), abolished block of malate currents by 100 μΜ GABA and 10 μΜ muscimol (Fig 11 e). Similarly, a Y to C conversion of Vitis vinifera ALMT9 (VvALMT9^Y237C) abolished its sensitivity to 100 μΜ GABA (data not shown). The magnitude of GABA block of TaALMTI was not significantly reduced by mutation of the second residue in isolation (TaALMTI ^F215C). The EC₅₀ of GABA-block of TaALMTI ^{F213C/F2 5C}, as compared to wildtype TaALMTI , increased from 3.2 μΜ to over 1 mM (data not shown). We tested whether these mutations could affect other properties of the channel such as transactivation by external anions or AI³⁺-activation at low pH (processes believed to be dependent upon amino acid residues identified within the C-terminus), but these occurred in TaALMTI ^{F2 3C} as in wildtype (Fig. 1 1 f). In contrast, the Al^3+"induced malate currents of TaALMTI ^{F2 3C} were insensitive to muscimol (Fig. 11f), demonstrating that GABA-block and activation by anions or Al³⁺ are likely to be encoded on separate parts of the protein.

Studies in other plant species

[0093] Wheat TaALMTI was previously expressed in tobacco BY2 cells to study its Al³⁺- activation in a plant-based system. We used this cell line to confirm that at low pH, micromolar concentrations of GABA and muscimol blocked Al³⁺-triggered malate efflux through TaALMTI and that concurrent addition of bicuculline relieved this block (Fig. 12a). Vigabatrin increases GABA concentration in cells by inhibition of GABA transaminase (GABA-T), the enzyme responsible for GABA catabolism, blocked Al³⁺-stimulated malate flux (Fig 12a). In Xenopus oocytes we had observed greater basal activity of TaALMTI at pH7.5 compared to pH4.5. We quantified the effect of pH on TaALMTI activity in both oocytes and BY2 cells and found malate flux through TaALMTI was much greater at alkaline pH (Fig. 12b). GABA, muscimol and vigabatrin reduced malate efflux from BY2 cells to a minimal level at every pH tested, whereas bicuculline stimulated malate efflux at pH4.5 and 7.5 (Fig. 12c).

[0094] To examine the physiological significance of GABA and pH regulation of TaALMTI we used the near isogenic lines of wheat that were initially used to clone and functionally characterize TaALMTI. These lines differ in their tolerance to Al³⁺, as conferred by a greater malate efflux from the roots of the tolerant line (ET8), with the transcript and protein abundance of TaALMTI in the root apex of ET8 being constitutively higher than in the sensitive (ES8) line. At low pH, application of muscimol, vigabatrin and GABA to ET8 roots completely abolished Al³⁺-activated malate efflux and reduced the root growth of ET8 to that of ES8 (Fig. 13a,b). Thus, treatment of the ET8 line with GABA could phenocopy the malate efflux and growth characteristics of ES8 in the presence of Al³⁺. Exposure of ET8 roots to bicuculline abolished the inhibitory effect of muscimol on the efflux of malate stimulated by Al³⁺ and restored root growth to that of ET8 plants in the absence of muscimol (data not presented). This effect of muscimol and bicuculline on Al³⁺ stimulated malate efflux also occurred in barley seedlings constitutively overexpressing TaALMTI (data not presented). Consistent with its pH regulation of TaALMTI in BY2 cells and Xenopus oocytes (Fig 12b) we observed high malate efflux from ET8 roots at pH 9 compared to ES8, and this could be fully inhibited by GABA, muscimol and vigabatrin (Fig 13c,d). Such an inhibition of malate efflux also correlated with a reduction of root growth at high pH (Fig 13d).

Claims

1. An isolated polypeptide comprising SEQ ID NO: 1.

2. An isolated polypeptide that shares a percentage sequence homology to SEQ ID NO: 1 , the percentage homology selected from the group consisting of: 99%; 98%; 97%; 96%; 95%; 90%; 85% 80%; 75%; 70%; 65% and 60%.

3. An isolated polypeptide comprising SEQ ID NO: 2.

4. An isolated polypeptide that shares a percentage sequence homology to SEQ ID NO: 2, the percentage homology selected from the group consisting of: 99%; 98%; 97%; 96%; 95%; 90%; 85% 80%; 75%; 70%; 65% and 60%.

5. An isolated polypeptide comprising SEQ ID NO: 3.

6. An isolated polypeptide that shares a percentage sequence homology to SEQ ID NO: 3, the percentage homology selected from the group consisting of: 99%; 98%; 97%; 96%; 95%; 90%; 85% 80%; 75%; 70%; 65% and 60%.

7. A method for binding GABA comprising contacting with an isolated polypeptide of claims 1 to 6 with GABA.

8. Use of an isolated polypeptide of claims 1 to 6 to bind GABA.

9. A method of inhibiting binding of GABA to an ALMT protein, the method comprising modifying the GABA-responsive motif on ALMT.

10. A method of claim 9, wherein modifying the GABA-responsive motif on ALMT utilizes a method selected from the group consisting of: modification of the DNA sequence of ALMT that encodes the GABA-responsive motif; site-directed mutagenesis; cysteine scanning mutagenesis; MTSEA-biotin protection assay; and addition of agents that allosterically inhibit GABA response.

11. A method of modulating a parameter in a plant, said parameter selected from the group consisting of: pH regulation; plant development; plant defence; nitrogen storage; glutamate utilization; in pollen tube growth; fertilization; ion influx; tolerance to salinity; tolerance to draught; anion and malate flux; root number and density; rate of release of carbon into the soil by the plant; and tolerance to extremes of soil pH, wherein said method comprises the steps of modulating the binding of GABA to the GABA-responsive motif of ALMT.

12. A method of claim 11 , wherein the GABA-responsive motif of ALMT shares a percentage sequence homology to the wild-type GABA-responsive motif of ALMT, the percentage homology selected from the group consisting of: 99%; 98%; 97%; 96%; 95%; 90%; 85% 80%; 75%; 70%; 65% and 60%.

13. A method of modulating a parameter in a plant, said parameter selected from the group consisting of: pH regulation; plant development; plant defence; nitrogen storage; glutamate utilization; in pollen tube growth; fertilization; ion influx; tolerance to salinity; tolerance to draught; anion and malate flux; root number and density; rate of release of carbon into the soil by the plant; and tolerance to extremes of soil pH, wherein said method comprises the steps of modulating the binding of GABA to the GABA-responsive motif of ALMT.

14. A method of claim 13, wherein the GABA-responsive motif of ALMT shares a percentage sequence homology to the wild-type GABA-responsive motif of ALMT, the percentage homology selected from the group consisting of: 99%; 98%; 97%; 96%; 95%; 90%; 85% 80%; 75%; 70%; 65% and 60%.