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Figure 1. GABA regulates the magnitude of Al3+-induced malate flux and the extent of wheat root Al3+ tolerance.Hydroponically grown seedlings of near-isogenic wheat lines ET8 (Al3+ tolerant) and ES8 (Al3+ sensitive)8 were used in all experiments, roots were bathed in basal nutrient solution at pH 4.5 ± 100âμM Al3+ (+Al) ± 1 or 10âmM GABA, or 10âμM muscimol (Mus) for 22âh. (a) The concentration of GABA in ET8 and ES8 wheat roots is decreased in response to Al treatment. (b) Malate efflux from wheat roots is increased by Al and decreased in response to Al and GABA treatment in ET8, not ES8 wheat. (c) Root malate efflux and root relative elongation rate (RER=(loge(length at 22âh)âloge(length at 0âh))/22âh) is positively correlated in ET8 in the presence of Al. Both parameters are negatively regulated by GABA and Mus, which phenocopies the response of ES8 to Al. *, **, *** and **** indicate significant differences between genotypes at P<0.05, 0.01, 0.001 and 0.0001, respectively, using a one-way ANOVA; NS, not significantly different. The significance comparisons between some groups have been omitted for clarity. All data n=5 biological replicates, all error bars are ± s.e.m. All experiments were repeated at least three times.
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Figure 2. GABA regulates Al3+-activated malate efflux through TaALMT1.(a) TaALMT1 expression in barley10 increases malate efflux and root growth of barley in the presence of 100âμm Al3+ at pH 4.5, over 22âh, but this is negatively regulated by 10âμM muscimol (Mus). (b) Representative current traces from TaALMT1-injected X. laevis oocytes voltage-clamped at â120âmV challenged with 100âμm Al3+ ± 100âμm GABA or 10âμM muscimol (Mus) at pH 4.5. (c) Malate efflux from TaALMT1-expressing BY2 cells11 in standard BY2 solution at pH 4.5 ± 100âμm Al3+ ± 10âμm Mus ± 100âμm bicuculline (Bic). For controls for b and c, see Supplementary Fig. 2. *indicates significant differences between genotypes at P<0.05 using a two-tailed t-test (a) or one-way analysis of variance (c). Full-TaALMT1 sequence identifier (DQ072260). All data n=5 biological replicates (except b, which are representative traces from n=5). All error bars are ±s.e.m. Transgenic barley experiments were repeated twice, Xenopus oocyte experiments were repeated with at least three different frogs and BY2 tobacco cell experiments were repeated thrice.
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Figure 3. TaALMT1-mediated fluxes are activated by external anions at alkaline pH and regulated by GABA, muscimol and bicuculline.(a) Malate efflux from TaALMT1-expressing BY2 cells11 in standard BY2 solution +10âmM SO42â increases with increasing pH over 22âh. (b) Malate efflux from TaALMT1-expressing BY2 cells11 in the absence of Al3+ + 10âmM SO42â is negatively regulated by 100âμm GABA or 10âμm muscimol (Mus) at alkaline pH, and increased by 100âμm bicuculline (Bic) at pH 4.5. *, *** and **** indicate significant differences between genotypes at P<0.05, 0.001 and 0.0001, respectively, using a one-way analysis of variance (b). All data n=3 biological replicates, all error bars are ±s.e.m. All experiments were repeated three times.
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Figure 4. TaALMT1 currents are activated by external malate at pH 7.5 and are regulated by GABA, muscimol and bicuculline.All results from X. laevis oocytes injected with TaALMT1 cRNA bathed at pH 7.5 and measured using two-electrode voltage-clamp electrophysiology. (a) Representative current traces at â120âmV; M=10âmM malate; Mus=10âμm muscimol; G=100âμm GABA; Bic=100âμm bicuculline from n=5 biological replicates for top and middle traces, n=3 for the bottom trace. Response of water-injected control oocytes are shown in Supplementary Fig. 6. (b,c) Currentâvoltage relationship of malate-activated current as regulated by muscimol and GABA as indicated applied and recorded 30âs after each solution change. Control-subtracted currents were normalized to the largest mean outward current at +60âmV (n=5 independent oocytes for each treatment). (d) Concentration dependence of GABA- and muscimol-regulated inward current at â140âmV taken from b and c (n=5 for b, and 9 for c). All data are water-control subtracted except in a. All error bars are ±s.e.m. Experiments repeated with oocytes from at least two different frogs.
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Figure 5. Muscimol-regulated anion-stimulated malate efflux at alkaline pH correlates with wheat root growth and modulates membrane potential.All experiments use ET8 and ES8 seedlings. (a) Malate efflux from, or, (b) root growth of, wheat roots after 22âh bathed in basal solution at pH 9 + 10âmM SO42â ± 10âμM muscimol (Mus). (c) Membrane potential difference (PD) across the plasma membrane of wheat root apical cells in response to 10âmM SO42â at pH 8. (d) PD in response to 10âmM SO42â at pH 8 +10âμM muscimol (Mus) treatment. The Black scale bar indicates value prior to treatment and the clear bar is in presence of treatment. * indicates significant differences between genotypes at P<0.05 using a one-way a two-tailed t-test (a,b). Biological replicates for a and b are n=5 and c and d are n=4. All error bars are ±s.e.m. Controls are shown in Supplementary Fig. 8. Experiments in a and b are repeated at least twice.
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Figure 6. GABA regulation of transport activity is a common ALMT property as is the presence of a putative GABA-binding motif.(a) Half-maximal effective concentration (EC50) and efficacy (Emax) of GABA regulation of rat GABAA receptors (average) or selected plant ALMT in cRNA-injected X. laevis oocytes assayed using two-electrode voltage-clamp electrophysiology (Os=rice; Hv=barley) (full data set, Supplementary Fig. 8). (b) Sequence logo of the predicted GABA-binding motif identified using MEME analysis1618 (detailed alignment, Supplementary Fig. 9). (c) Residues corresponding to logo in proteins from a (see Supplementary Figs 10 and 11 for this sequence and residue frequencies at each position within the motif in all other identified ALMT), identical residues shaded (black), 80% similar (grey) and <60% similar (unshaded). Full-sequence identifiers are AtALMT1 (AT1G08430); TaALMT1 (DQ072260); OsALMT5 (Os04g0417000); HvALMT1 (EF424084); AtALMT13 (AT5G46600); AtALMT14 (AT5G46610); OsALMT9 (Os10g0572100); VvALMT9 (XM_002275959). All measurements were carried out at least twice with different frogs.
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Figure 7. GABA regulation of ALMT transport activity is dependent on an aromatic residue within the predicted GABA-binding motif.(a) Sensitivity of wild-type and site-directed ALMT mutants (M, 10âmM malate; GABA, 100âμM), at pH 4.5 (Al, 100âμM; muscimol (Mus), 10âμM) assayed by two-electrode voltage-clamp electrophysiology in cRNA-injected X. laevis oocytes. Currents were normalized to â140âmV value in basal solution (at each pH) for each protein (dotted line). For all treatments, n=3 for TaALMT1, n=4 for TaALMT1F213C, n=5 for TaALMT1F215C, n=3 for TaALMT1F213C,F215C, n=7 for VvALMT9 and n=4 for VvALMT9Y237C. *indicates significant differences from basal currents within each treatment (P<0.05), ·indicates a significance difference between activated currents (P<0.05), using a one-sample t-test on log-transformed data. (b,c) Fluorescence of the plasma membrane of X. laevis oocytes after exposure to the muscimol-BODIPY conjugate, control (water injected) (n=12), TaALMT1- (n=14), TaALMT1F213C-injected (n=8) and oocytes co-incubated with GABA and muscimol-BODIPY, control (water injected) (n=4) and TaALMT1-injected (n=6). (d,e) Fluorescence of wheat roots after exposure to the muscimol-BODIPY conjugate (n=5 for each). **, *** and **** indicates significant differences in fluorescence between control, TaALMT1 and TaALMT1F213C at P<0.01, 0.001, 0.0001, respectively, using one-way analysis of variance and Tukey's post hoc test. All error bars are ±s.e.m., scale bars, 100âμm. Experiments in a were carried out at least twice with two different frogs. (b,c) Measurements were repeated thrice with three different frogs. (d,e) Fluorescence measurements were carried out twice on roots in different experiments.
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Figure 8. Muscimol reduces Arabidopsis thaliana and Vitis vinifera pollen tube elongation in vitro, and bicuculline antagonizes muscimol regulation.(a) In vitro Arabidopsis pollen tube elongation after 3âh±treatments (n=499). Mus (20âμM muscimol and Bic (200âμM bicuculline). (b) In vitro grapevine pollen tube elongation after 6âh ± treatments, mean Î from control mean±s.e.m. (12.5±0.16âmm) (n=157â193 per treatment). **, *** and **** indicate significant differences between genotypes at P<0.01, 0.001 and 0.0001, respectively, using a one-way analysis of variance and Tukey's post hoc test. All error bars are ±s.e.m. (b) The experiments were replicated at least three times.
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