Split personality of Aluminum Activated Malate Transporter family proteins: facilitation of both GABA and malate transport

Plant aluminum activated malate transporters (ALMTs) are currently classified as anion channels; they are also known to be regulated by diverse signals leading to a range of physiological responses. Gamma-aminobutyric acid (GABA) regulation of anion flux through ALMT proteins requires the presence of a specific amino acid motif in ALMTs that shares similarity with a GABA-binding site in mammalian GABAA receptors. Here, we explore why TaALMT1-activation leads to a negative correlation between malate efflux and endogenous GABA concentrations ([GABA]i) in both wheat root tips and in heterologous expression systems. We show that TaALMT1 activation reduces [GABA]i because TaALMT1 facilitates GABA efflux. TaALMT1-expression also leads to GABA transport into cells, demonstrated by a yeast complementation assay and via 14CGABA uptake into TaALMT1-expressing Xenopus laevis oocytes; this was found to be a general feature of all ALMTs we examined. Mutation of the GABA motif (TaALMT1F213C) prevented both GABA influx and efflux, and uncoupled the relationship between malate efflux and [GABA]i. We conclude that ALMTs are likely to act as both GABA and anion transporters in planta. GABA and malate appear to interact with ALMTs in a complex manner regulating each other’s transport, suggestive of a role for ALMTs in communicating metabolic status.


INTRODUCTION
Gamma-aminobutyric acid (GABA) is a four carbon non-proteinogenic amino acid that was first discovered in potato tubers (Steward et al., 1949); however, since this time it has mainly been studied in mammals as an inhibitory neurotransmitter (Sigel and Steinmann, 2012).
While TaALMT1 can be activated by aluminum (Al 3+ ) this is not a general feature of ALMTs; some ALMTs can facilitate anion efflux when trans-activated by external anions such as SO 4 2or malate 2in alkaline solutions (Ramesh et al., 2015). A putative GABA binding motif was discovered in ALMTs with homology to the one found in mammalian GABA A receptors, and treatment with micromolar concentrations of muscimol (an analogue of GABA) resulted in inhibition of anion flux (Ramesh et al., 2015). Addition of bicuculline (a GABA receptor antagonist) attenuated the effect of both muscimol and GABA (Ramesh et al., 2015). These results suggest that there are distinct classes of anion channels from plant and animal cells that have comparable modes of GABA regulation (Žárský, 2015;Gilliham and Tyerman, 2016;Ramesh et al., 2016).
It is well established that in acidic soils TaALMT1 confers Al 3+ tolerance in wheat through exuding malate from the root tips and chelating toxic Al 3+ (Delhaize and Ryan, 1995;Ma et al., 2001;Sasaki T, 2004). Exogenous application of GABA or muscimol to the roots of wheat seedlings with high TaALMT1 expression inhibited malate efflux and impaired root growth in the presence of Al 3+ , which phenocopied a near isogenic line with less expression of TaALMT1 and less Al 3+ tolerance (Ramesh et al., 2015). Interestingly, in these conditions, it was observed that when root efflux of malate was high, endogenous GABA concentrations ([GABA] i ) in the cells were low and vice versa (Ramesh et al., 2015). This reciprocal relationship remained unexplained and may indicate either TaALMT1 activation caused changes in [GABA] i or that [GABA] i is altered in some way that then regulates TaALMT1.
Identification of a putative GABA binding motif in ALMTs provided a possible mechanism by which plant GABA may act as a signal (Ramesh et al., 2015;Žárský, 2015), but we are yet to fully understand the molecular and physiological basis of how this occurs and the relationship between anion flux and GABA regulation in plant cells. A number of pharmacological agents have been used to characterise animal GABA receptors, some of which are plant or fungal derived, either as agonists i.e muscimol, or as regulators of GABA synthesis or catabolism i.e. amino-oxyacetate (AOA) and vigabatrin (Wood and Peesker, 1973;Jackson et al., 1982;Grant and Heel, 1991). Manipulating [GABA] i in plant cells and studying the effect this has on anion efflux mediated by ALMTs will increase our understanding of the role of GABA in plants under different stresses.
It was previously hypothesised that ALMT might sense and signal metabolic status through their regulation by GABA and malate, which alters membrane voltage and transduces the signal into a physiological response (Gilliham and Tyerman, 2016;Xu et al., 2016). Cellular efflux of GABA has been well documented (Bown and Shelp, 1989;Chung et al., 1992).
Micromolar concentrations of GABA are found in root exudates and the apoplast, and among all amino acids exuded from wheat roots, GABA shows the highest efflux (Warren, 2015); it was envisaged that such carbon and nitrogen loss might only be justified energetically if GABA was involved in signaling (Gilliham and Tyerman, 2016). While a high affinity GABA influx transporter (AtGAT1) has been characterised and is expressed in Arabidopsis roots (Meyer et al., 2006), no transporter that can efflux GABA from the cytoplasm into the apoplast has been identified. This raises an interesting question as to how GABA exits the cytoplasm and enters the apoplast.
In this study we used two GABA analogs to manipulate [GABA]

i in cells expressing
TaALMT1: vigabatrin -a GABA transaminase (GABA-T) inhibitor used as an antiepileptic in humans (Livingston et al., 1989;Nanavati and Silverman, 1991), and amino-oxyacetate (AOA) -an inhibitor of both GABA-T and glutamate decarboxylase (GAD) (John and Charteris, 1978;Miller et al., 1991;Snedden et al., 1992). We demonstrate that exogenous application of AOA and vigabatrin has an affect upon anion efflux via TaALMT1. This led to negative correlations between [GABA] i and anion efflux being observed, which are also evident following Al 3+ application. Interestingly, these negative correlations are uncoupled when the site directed mutant TaALMT1 F213C -impaired in its GABA regulation of malate transport -is expressed instead of TaALMT1. Further, we propose that the reduction in [GABA] i upon Al 3+ treatment at low pH is likely to be due, in part, to efflux of GABA via TaALMT1. More broadly, our work reveals that changes in intracellular and apoplasmic concentrations of GABA can be facilitated by an ALMT protein, resulting in exquisite regulation of malate efflux via both a feed-forward (malate) and feed-back (GABA) regulation. Such ALMT activity will strongly affect [GABA] i , resulting in changes in metabolic flux through the GABA shunt. In the case of the wheat root this provides a likely signalling mechanism by which TaALMT1 can regulate root growth in acidic and alkaline conditions.

Validating measurement of intracellular GABA concentration
We routinetly use a GABase enzyme assay to measure GABA concentration, which utilises a GABA-T enzyme. Whilst examining the effects of inhibitors on cell and tissue GABA concentrations ([GABA] i ) we found that this assay was inhibited by AOA when added directly to the enzyme mix. We therefore examined if the GABase assay was compromised when it was used on extracts of tissues that had been treated with AOA, Al 3+ or vigabatrin by performing GABA spike and recovery experiments (Supplemental Figure 1). After 22 h treatment with 1 mM AOA the recovery of spiked GABA from wheat seedling root extract was 97% (Supplemental Figure 1A, B,C); Al 3+ treatment also did not compromise measurement of [GABA] i (Supplemental Figure 1D,E,F). Vigabatrin treated wheat root tips also yielded full recovery of spiked GABA (Supplemental Figure 1G,H,I). Xenopus laevis oocytes treated with these compounds were also examined and similarly full recovery of GABA was obtained (Supplemental Figure 1J,K,L). We also used Ultra High Performance Liquid Chromatography (UPLC) to measure [GABA] i on the same wheat root tip tissue extracts, after treatment with Al 3+ and AOA, validating the results obtained by the enzyme assay (Supplemental Figure 1M). Thus, we expect the GABase assay to faithfully measure [GABA] i following Al 3+ , AOA or vigabatrin treatments.

Wheat roots malate efflux and [GABA] i
External Al 3+ at low pH was previously shown to reduce [GABA] i while stimulating malate efflux from roots of wheat seedlings (Ramesh et al. 2015). The Al 3+ tolerant wheat line ET8 has higher expression of TaALMT1 that is increase by external Al 3+ compared to ES8, its near isogenic line (NIL) (Yamaguchi et al., 2005) (Supplemental Figure 2). We confirmed that ET8 exhibited Al 3+ (100 µM) induced malate efflux from roots of 3-day old wheat seedlings and that this co-incided with a reduction in root tip [GABA] i (Figure 1). Line ES8 had lower [GABA] i than ET8 and ES8 [GABA] i did not respond to Al 3+ treatment (Supplementary Figure 1M). When corresponding values from the same plants were plotted against each other we observed that Al 3+ -stimulated malate efflux from roots had a negative linear relationship with root tip [GABA] i ( Figure 1A). Root tips have previously been shown to be the major site of malate efflux in response to Al 3+ (Delhaize et al., 1993). We confirmed this response ( Figure 1B), which also corresponded to reduced root tip [GABA] i ( Figure 1C). Aminooxyacetate (AOA) treatment (1 mM) at pH 4.5 also stimulated malate efflux ( Figure 1B) and reduced [GABA] i in excised root tips of ET8 seedlings ( Figure 1C) to the same degree as 100 µM Al 3+ .
We treated roots of intact ET8 wheat seedlings with either a basal solution at pH 4.5 or pH 7.5 ± 10 mM Na 2 SO 4 (to add a trans-activating anion) ± 1 mM AOA (at pH 4.5) or ± 100 µM vigabatrin (at pH 7.5) ( Figure 2). Vigabatrin was only used at pH 7.5 since according to its  Figure 2C). At pH 7.5, malate efflux was significantly higher in the presence of SO 4 2but vigabatrin inhibited the SO 4 2stimulated efflux ( Figure 2D). Root tip [GABA] i at pH 7.5 was slightly lower but not significantly so than at pH 4.5 ( Figure 2B Figure 3A, Supplemental Figure 3C); the effect being greatest at pH 4.5 compared to higher pHs (Supplemental Figure 4). At pH 4.5, the stimulated malate efflux corresponded to a lowered [GABA] i at the end of the efflux period ( Figure 3B). There was no effect of SO 4 2by itself on either parameter at pH 4.5 ( Figure 3A In contrast to pH 4.5, SO 4 2at pH 7.5 significantly stimulated malate efflux from BY2 cells expressing TaALMT1 (Supplemental Figure 6A). The addition of vigabatrin inhibited SO 4 2stimulated malate efflux in a dose dependent manner with complete block observed at 100 µM (Supplemental Figure 6A), but had no effect on empty vector expressing controls (Supplemental Figure 6C). In BY2 cells expressing TaALMT1 the reduction of malate efflux correlated with an increase in [GABA] i (Supplemental Figure 6B).
[GABA] i in BY2 cells were elevated by vigabatrin as expected from its proposed action on GABA-T in both TaALMT1 (Supplemental Figure 6B) and empty vector expressing cells (Supplemental Figure   6D). Therefore, following both AOA and vigabatrin treatment the significant relationships between malate efflux and [GABA] i that were observed were dependent upon the expression of TaALMT1.

The F213C mutation in TaALMT1 uncouples the relationship between [GABA] i and malate efflux
A phenylalanine residue (F) has been shown to be important for GABA sensitivity in GABA A receptors and TaALMT1 (Boileau et al., 1999;Ramesh et al., 2015). Mutation of this residue abolishes GABA sensitivity in animals and impairs GABA sensitivity of TaALMT1 but did not abolish activation of the malate efflux by Al 3+ or external anions (Ramesh et al., 2015). Therefore, we tested whether the exogenous application of Al 3+ Figure 3C) but these were uncoupled in cells expressing the TaALMT1 F213C mutant at pH 4.5 ( Figure 3D). Malate efflux increased with exogenous application of Al 3+ or AOA in cells expressing TaALMT1 F213C similarly to cells expressing TaALMT1 (Supplemental Figure 3A,B,C), but unlike for TaALMT1 expressing cells, [GABA] i was not significantly reduced by these treatments (Supplemental Figure 3D, E,F).
At pH 7.5, malate efflux from TaALMT1 F213C and TaALMT1 expressing cells was stimulated by SO 4 2to a similar degree (Supplemental Figure 7A,B,C), but, in contrast to cells expressing TaALMT1, vigabatrin did not inhibit efflux from cells expressing TaALMT1 F213C even though [GABA] i was elevated in both (Supplemental Figure 7D,E,F). Thus, the relationship between malate efflux and [GABA] i was also uncoupled in cells expressing the TaALMT1 F213C mutant at pH 7.5 (Supplemental Figure 7G,H). The empty vector controls also showed increased [GABA] i with vigabatrin treatment (Supplemental Figure 7D,E,F) but did not show a significant correlation between malate efflux and [GABA] i (Supplemental Figure   8).

Activation of TaALMT1 results in GABA efflux
To determine if the reduction in [GABA] i was due to transport of GABA through TaALMT1, we tested if Al 3+ not only activated malate efflux but also GABA efflux from the wheat NILs ET8 and ES8 ( Figure 4). We observed that ET8 showed not only significantly higher malate efflux ( Figure 4A) but also higher GABA efflux when compared to ES8 ( Figure 4B).
Similarly transgenic barley expressing TaALMT1 (OE) (Delhaize et al., 2004;Ramesh et al., 2015) when exposed to Al 3+ at low pH, showed significantly increased malate efflux ( Figure   4C) as well as higher GABA efflux ( Figure 4D) when compared to the Golden Promise background alone. It is also interesting to note that the efflux of GABA was higher than that of malate on a molar basis by 2-fold in ET8 wheat and over 500-fold in TaALMT1 expressing barley roots. GABA efflux was also examined in tobacco BY2 cells expressing TaALMT1  and the TaALMT1 F213C mutant where at low pH we observed very large GABA efflux with Al 3+ treatment only for cells expressing TaALMT1 ( Figure 4E). GABA efflux in response to Al 3+ from BY2 cells expressing TaALMT1 was also higher than malate efflux (compare Figure 4E with Figure 3C or Supplemental Figure 3B).
Given that Al 3+ activates GABA efflux through TaALMT1 in addition to malate, we tested the possibility that GABA may also complex Al 3+ , since there was no data in the literature on possible interactions between GABA and Al 3+ . Using a fluoride competitive ligand method and comparing GABA with the organic anions citrate, oxalate, malate and salicylate, we found that GABA had very low affinity for Al 3+ compared to these other compounds, with the strength of complexation following the order: citrate>oxalate>malate>salicylate>>GABA (Detailed in Supplemental material A). We also found no synergistic or antagonistic interaction between GABA and malate in Al 3+ complexation.

AOA and vigabatrin have direct effects on TaALMT1
Since AOA had similar effects to Al 3+ in activation of malate efflux and on depression [GABA] i , it prompted us to examine if the GABA analogs AOA and vigabatrin may have direct effects on TaALMT1 that may then alter [GABA] I ( Figure 5). Using TEVC on X.
laevis oocytes expressing TaALMT1 and TaALMT1 F213C , we perfused the bath with 1 mM AOA and 100 µM Al 3+ to compare the inward current activation corresponding to activation of malate efflux ( Figure 5A,B). AOA activates TaALMT1 and the TaALMT1 F213C mutant rapidly and with similar kinetics to Al 3+ . Vigabatrin was also examined for its effect on anion-activated currents at pH 7.5 ( Figure 5C). Vigabatrin also acted rapidly on the malateactivated inward current giving 100% inhibition at the applied concentration of 100 µM.

TaALMT1 and other ALMTs also facilitate GABA influx
To test if TaALMT1 also facilitated GABA influx we expressed TaALMT1 and TaALMT1 F213C in X. laevis oocytes and tested their ability to influx [ 14 C] GABA. The GABA transporter AtGAT1 from Arabidopsis was used as a positive control in these uptake experiments ( Figure 6). At pH 4.5, TaALMT1 expressing oocytes showed significantly higher GABA uptake from an external concentration of 1 mM compared to both control and mutant TaALMT1 F213C expressing oocytes and similar to that facilitated by AtGAT1 ( Figure 6A). At pH 7.5 TaALMT1-expressing oocytes (without a transactivation anion) did not show significant GABA influx compared to control oocytes, with the rate being significantly lower than AtGAT1-expressing oocytes ( Figure 6B). AtGAT1 expressing oocytes had significantly reduced influx at pH 7.5 compared to that at pH 4.5, consistent with the hypothesis that this transporter uses the proton motive force to drive transport (Meyer et al., 2006). Activation of TaALMT1 at pH 7.5 with 10 mM Na 2 SO 4 increased GABA uptake significantly into the TaALMT1 expressing oocytes when compared to either the controls or TaALMT1 F213C expressing oocytes ( Figure 6B).
Wheat ALMT1 is the founding member of the ALMT family (Sasaki T, 2004 Figure 6C). In all cases 100 µM Al 3+ reduced this uptake to control levels. The presence of Al 3+ , however, did not affect the uptake of GABA by AtGAT1. These results demonstrate that transport of GABA is a general feature of the ALMT family and that extracellular Al 3+ is a common inhibitor for GABA uptake of this transport across members 1 1

TaALMT1 complements growth of a yeast mutant deficient in the transport of GABA
To further explore GABA transport by TaALMT1 we tested if GABA could be used as a nitrogen source for yeast growth, where TaALMT1 and site directed mutant TaALMT1 F213C was transformed into a yeast triple mutant strain 22754d (MATα ura3-1, gap1-1, put4-1, uga4-1) (Figure 7). This yeast strain carries mutations in general amino acid permease (gap1), proline (put4) and GABA (uga4) and is unable to grow on proline, citrulline or GABA as the sole nitrogen source; it was used to characterise high affinity GABA transport (Meyer et al., 2006). The efflux of malate was observed to be higher in each of the transformants expressing TaALMT1 and the mutant ( Figure 7A), consistent with TaALMT1 and its mutant being located to the plasma membrane and able to efflux malate, as previously shown in X. laevis and tobacco BY2 cells (Ramesh et al., 2015). All the yeast strains were capable of growth on selective drop out medium (SC-ura) supplemented with 2% glucose or galactose as carbon source and ammonium sulphate as nitrogen source (Supplemental Figure   9A,B). When the yeast strains were starved of nitrogen by growth in nitrogen free medium and transferred to medium with no GABA, there were no significant differences in growth of the yeast strains (Supplemental Figure 9C). However in medium supplemented with 1 mM GABA as the sole nitrogen source, yeast cells expressing TaALMT1 showed significantly higher relative growth rate compared to control (empty vector) or mutant TaALMT1 F213C ( Figure 7B, Supplemental Figure 9E). On medium supplemented with GABA at a concentration of 20 or 37.83 mM, which corresponds to SC medium with GABA at the concentration equivalent to that of ammonium sulphate, all the yeast strains showed similar growth (Supplemental Figure 9D) indicating that the stimulation of growth by 1 mM GABA was already saturating. Therefore we examined the GABA dose-response of yeast growth ( Figure 7C). The apparent K d for growth stimulation by GABA was 0.56 mM indicating at least moderate affinity for GABA transport by TaALMT1. This growth stimulation was completely inhibited by the addition of 2 mM malate to the medium for TaALMT1 ( Figure   7D).
This accounts for the negative linear relationship observed between malate efflux and endogenous GABA concentrations in cells expressing TaALMT1. We have shown GABA transport by TaALMT1 using near isogenic lines of wheat that differ in the expression of TaALMT1, transgenic barley expressing TaALMT1, tobacco BY2 cells expressing TaALMT1, X. laevis oocytes expressing TaALMT1, and complementation by TaALMT1 of a yeast mutant deficient in GABA transport. The yeast mutant 22574d that we used to characterise GABA transport and nitrogen utilisation in yeast cells (Grenson et al., 1970;Breitkreuz et al., 1999;Meyer et al., 2006) will likely be a very useful system in which to characterise other ALMTs for interactions with GABA and to further explore the pharmacology of the transporter.

GABA transport may be a general feature of ALMTs
Uptake studies with X. laevis oocytes showed that GABA uptake via ALMTs is not unique to TaALMT1, but ALMTs from barley, rice and Arabidopsis also transported significantly more GABA into the cells than controls. Interestingly, the addition of Al 3+ reduced the uptake in each case. We applied Al 3+ since this activates TaALMT1 to efflux both malate and GABA at low pH. The block of GABA influx by Al 3+ indicates that influx is mutually exclusive of efflux. In contrast the uptake of GABA by AtGAT1 was not reduced in response to Al 3+ . It is interesting to note that there is evidence for interactions of Al 3+ with GABA A receptors (Trombley, 1998) and GABA transporters in animals (Albrecht and Norenberg, 1991;Cordeiro et al., 2003).

GABA the regulator?
Initially we investigated the role of GABA in regulating TaALMT1 based on the negative linear relationship between malate efflux and [GABA] i observed in an earlier study ( Figure 1 in Ramesh et al., 2015) that was hypothesised to be linked to the control of ALMT facilitated anion efflux. It was shown that there is a motif in ALMTs that is likely to be involved in GABA binding. This motif with an important phenylalanine residue (213 in TaALMT1) has similarities to a region in GABA A receptors (Smith and Olsen, 1995;Boileau et al., 1999).
Furthermore, some important pharmacological agents used to probe GABA receptors in animal cells also appear to work on ALMTs (e.g. muscimol and bicuculline). AtGAT1, the high affinity Arabidopsis GABA transporter used as a positive control for GABA transport in the current work, has a region of similarity with this motif (Ramesh et al., 2016).
GABA inhibits malate efflux and inward membrane currents through ALMTs when presented to the external (apoplast) side with high affinity (IC50 1 to 7 µM) (Ramesh et al., 2015). This posed the question as to how GABA is exported from the cell to the apoplast to regulate TaALMT1 if external GABA regulates ALMTs, at least for those ALMTs expressed on the plasma membrane. As far as we are aware no transport system has been identified until now that is able to account for GABA efflux across the plasma membrane into the apoplast (Shelp and Zarei, 2017). This is despite numerous examples of relatively high extracellular concentrations of GABA being measured (Chung I, 1992;Snedden et al., 1992;Crawford et al., 1994) and particularly the case for wheat roots where GABA is by far the most exported amino acid from roots (Warren, 2015).

The location of the F213 that is critical for GABA transport
We note that there is some controversy regarding the membrane topology of ALMT proteins  Figure 8) computed from evolutionary sequence variation over 3688 alignments using the EvFold web portal (Marks et al., 2012). Noteably the GABA motif previously characterised is located towards the end of TMD 6 (or 7) just before the long C terminus, which in most predictions is oriented toward the cytoplasm. It was suggested that this was oriented towards the apoplast to account for the rapid effect of GABA (Ramesh et al., 2015) and corresponding with immunocytochemical evidence for the C terminus to be located on the apoplast side (Motoda et al., 2007). Another 1 4 model formulated from extensive sequence alignments across the family and secondary structure predictions also indicate that the GABA motif may be oriented toward the apoplast, but with a further two TMDs within the C-terminus (Dreyer et al., 2012). If the GABA motif (beginning at F213 in TaALMT1) does orient towards the cytosolic side, the transport of GABA that we demonstrate here may reconcile the rapid action of GABA on malate currents, which presumably also applies to some GABA analogs that block depending on F213, such as muscimol and vigabatrin. Clearly a crystal structure will resolve this issue.

Inhibitors that target GABA metabolism and catabolism directly interact with TaALMT1
Manipulating Apart from the expected associations with the inhibitors described above, we also observed that activation of the TaALMT1 Ramesh et al., 2015). Similarly GABA and muscimol inhibition of the currents was as rapid as could be expected for solution change kinetics (Ramesh et al., 2015). We have shown with X. laevis oocytes that AOA at pH 4.5 will rapidly activate inward current at negative membrane potential consistent with malate anion efflux accounting for the inward current.
The activation occurs with the same velocity (initial current rise) as that induced by Al 3+ .  (Figure 1 in Ramesh et al., 2015) and as was observed here with tobacco BY2 cells expressing TaALMT1 (Supplemental Figure 3) particularly evident at low pH. This is potentially due to increased accumulation of GABA via influx through TaALMT1 when TaALMT1 is not activated for "efflux mode" as was demonstrated by its capacity to influx GABA at low pH when malate efflux was not activated.
It may not be surprising that both AOA and vigabatrin interact with TaALMT1. In the case of AOA it inhibits GAD and GABA-T via interaction with the pyridoxal phosphate cofactor binding site (Wallach, 1961;John and Charteris, 1978;Löscher et al., 1989;Miller et al., 1991). It is interesting that AOA strongly activates TaALMT1, so far the only known external organic compound besides transported anions that has this effect. AOA is a structural analog of GABA that is one atom shorter and is also a zwitterion that has no net change at neutral pH.

Potential roles for GABA transport via ALMTs
Until now the identity of a GABA efflux transporter across the plasma membrane was not known. Our studies show that TaALMT1 in addition to mediating efflux of organic anions also mediates GABA efflux from cells when activated. Its transport capacity for GABA is very high as indicated by its activation being able to significantly reduce [GABA] i in root tips and other cells expressing TaALMT1. In fact, we have shown higher capacity for GABA efflux relative to malate efflux on a molar basis. This is quite novel as changes in [GABA] i are likely to have broad effects on carbon metabolism and signalling. We have shown that GABA does not significantly complex Al 3+ therefore we can exclude the hypothesis that GABA efflux with malate may provide additional protection against Al 3+ . Although it is not clear why high GABA efflux may be an advantage when TaALMT1 is activated by Al 3+ at low pH, or activated at high pH, we may speculate along the following lines as follows.
Firstly GABA may act as an extracellular pH buffer. GABA addition to solutions at both low and high pH tends to bring the pH back towards neutrality. Thus at low pH where GABA synthesis by GAD acts as a cytoplasmic pH stat (Snedden et al., 1992;Crawford et al., 1994;Shelp et al., 1999;Snedden and Fromm, 1999), its efflux to the external medium will also tend to increase the external pH. It has been reported that exogenously supplied GABA (10 µM) significantly improved root growth of barley seedlings at pH 4.5 and when exposed to 20 µM Al 3+ at pH 5 (Song et al., 2010). This was considered to be via alleviation of oxidative damage.
Secondly AtALMT1 has been implicated as the key regulator in the attraction of the beneficial Bacillus subtilis strain FB17 to the Arabidopsis root (Lakshmanan et al., 2013), in addition to its role in Al 3+ tolerance. Pseudomonads are known for their specific GABA receptors and positive chemotactic response to GABA (Reyes-Darias et al., 2015) and Bacillus subtilus has positive chemotaxis to Arabidopsis roots with chemoreceptors that recognise a range of amino acids.
Thirdly GABA transported by TaALMT1 also provides feedback regulation on the cotransport of organic anions. As GABA builds up in the apoplast this will tend to reduce malate efflux. Apoplastic GABA may also signal to adjacent cells via its effect on

The transport mechanism for GABA and its interaction with malate anions
Considering the transport of GABA via TaALMT1, it is necessary to account for the likely ionic charge on GABA. GABA is a zwitterion at neutral pH and is thus uncharged in the cytosol. Only at very high pH (10.43) or at low pH (4.23) does either a negative or positive charge occur respectively. At pH 4.5 we calculate that there would be 35% of GABA in the external media that has a net positive charge due to a proportion of molecules not deprotonated at the carboxyl end while the amino terminal will remain positively charged.

8
Efflux from the cytoplasm (slightly alkaline pH) would not be detectable as an electrical current, but influx at pH 4.5 could be detected as an inward current if the cationic form of GABA is transported, particularly if there is a high affinity of transport as suggested by the IC 50 of block by GABA of the anion current, and from the high influx measured relative to that of AtGAT1 at pH 4.5. It is also possible that GABA fluxes may be coupled to the movement of protons in the same direction similar to GABA transport via AtGAT1 (Meyer et al., 2006), which would be detectable as an electrogenic current. GABA influx into X. laevis oocytes was substantially higher at pH 4.5 than pH 7.5 (10-fold), and the flux at pH 7.5 was similar to that of control water injected oocytes. However, GABA influx was activated at pH 7.5 by the addition of Na 2 SO 4 to the bathing medium, which also stimulates malate efflux.
We have shown that for all the ALMTs that tested positive for GABA influx, external GABA also inhibited the anion efflux current at pH 7.5 at micromolar concentrations (Ramesh et al., 2015). This may indicate some coupling between anion efflux and GABA transport.
GABA on the trans side for anions (normally the external side of the plasma membrane) inhibits anion efflux but results in GABA uptake. Malate on the trans side also inhibits GABA influx. However, GABA on the inside of the cell clearly allows efflux of anions and GABA together, since in the heterologous expression systems used here and in the wheat NIL lines both GABA and malate efflux occur simultaneously. In the case of X. laevis oocyte the [GABA] i is well above the concentrations that blocks malate efflux from the trans side. The F213C mutation in TaALMT1 greatly reduces the external GABA sensitivity of anion efflux currents (Ramesh et al., 2015). We have shown here that this mutation also effectively abolishes GABA influx and GABA efflux via TaALMT1, but still allows activation by external anions and external Al 3+ , further confirming the importance of this site in GABA interactions. The F213C mutation also shows extremely high malate efflux when expressed in X. laevis oocytes compared to the unmutated TaALMT1, suggesting that "co-transport" of malate and GABA has been uncoupled. A summary of the observed interactions between GABA and malate transport via TaALMT1 is presented in Figure 8.
To fully understand the transport mechanism in ALMTs more detailed kinetic studies will be required where GABA and malate concentrations are varied on both cis and trans faces, most probably best achieved via patch clamp studies. Our work reported here indicates that ALMTs are more complicated than a relatively simple ligand gated anion channel, and we suggest that at least some (e.g. TaALMT1) could be considered as GABA transporters with anion channel activity. Why this has not be revealed from the many previous studies is probably related to several factors including the focus upon Al 3+ tolerance resulting from the malate transport through TaALMT1 and the probable lack of or low electrogenic activity associated with GABA transport.
The dual function of TaALMT1 and other ALMTs is analogous to some transporters that display channel activity such as the excitatory amino acid transporters (EAATs) from animals that function as both glutamate transporters and chloride channels (Cater et al., 2016). There is also a precedent for GABA transport in that the mammalian GAT1 transporter displays sodium channel activity (Risso et al., 1996). In the context of the biological link between GABA and anion transport and particularly that of malate, the regulation of efflux of both substrates is titrated finely by apoplastic concentrations of both substrates making them uniquely positioned to provide intercellular and intracellular communication of metabolic status ( Figure 8).

Chemicals
All chemicals were purchased from Sigma. 14

Voltage-clamp electrophysiology
Electrophysiology was performed on X. laevis oocytes 2 days post injection with water/cRNA (Preuss et al., 2010;Ramesh et al., 2015). Oocytes were injected with 46 nl of RNase-free water using a micro-injector (Nanoject II, automatic nanolitre injector, Drummond Scientific) ± 16-32 ng cRNA. Sodium malate (10 mM, pH 7.5) was injected into oocytes 1 h before measurement. Aluminium activation was carried out in ND88 solution at 2 0 pH 4.5 (Sasaki T, 2004;Hoekenga et al., 2006) ± aluminium chloride (AlCl 3 -100 µM) and vigabatrin (100 µM). Basal external solutions for anion activation contained 0.5 mM CaCl 2 (pH 4.5) or 0.7 mM CaCl 2 (pH 7.5) and mannitol to 220 mOsm kg -1 , ± 10 mM sodium sulphate or 10 mM malate and amino oxyacetate (AOA-1 mM) buffered with 5 mM BTP/MES from pH 4.5 to 7.5. In all X. laevis oocyte experiments, solutions were applied to gene-injected oocytes in the same order as controls (water injected). Randomly selected oocytes were alternated between control and gene injected to limit any bias caused by time- For oocyte GABA measurements, Xenopus oocytes injected with TaALMT1 cRNA or water (controls) were imaged in groups of 4-5 using a stereo zoom microscope (SMZ800) with a Nikon (cDSS230) camera 48h post-injection. The oocytes were incubated in treatment solutions indicated in the figure legends for 10 min. After 10 min, treatment solution was removed and oocytes snap frozen in liquid nitrogen and stored at -80 °C until further use.
Oocytes were extracted with same protocol used for root tips and assayed as mentioned above. Samples from root flux assays or tobacco BY2 suspension cell assays were used to measure external GABA using the GABase enzyme as described above.
GABA concentrations were also analysed with UPLC. As described above the same GABA extracts from root tips, oocytes, and tobacco BY2 cells were centrifuged at 16,000 x g in a

Statistics
All graphs and data analysis were performed in GraphPad Prism 7 (version 7.02). All data shown are mean ± SEM.

Accession numbers
Sequence data from this article can be found in the EMBL/GenBank data libraries under  (  M  e  c  h  a  n  i  s  m  s  o  f  i  n  a  c  t  i  v  a  t  i  o  n  o  f  .  g  a  m  m  a  .  -a  m  i  n  o  b  u  t  y  r  i  c   a  c  i  d  a  m  i  n  o  t  r  a  n  s  f  e  r  a  s  e  b  y  t  h  e  a  n  t  i  e  p  i  l  e  p  s  y  d  r  u  g  .  g  a  m  m  a  .  -v  i  n  y  l  G  A  B  A  (  v  i  g  a  b  a  t  r  i  n  )  .  J  o  u  r  n  a  l  o  f   T  h  e  A  m  e  r  i  c  a  n  C  h  e  m  i  c  a  l  S  o  c  i  e  t  y  1  1  3  ,  9  3  4  1  -9  3  4  9  .   N  u  g  e  n  t  ,  T  .  ,  a  n  d  J  o  n  e  s  ,  D  .  T  .  (  2  0  1  2  )  .  D  e  t  e  c  t  i  n  g  p  o  r  e  -l  i  n  i  n  g  r  e  g  i  o  n  s  i  n  t  r  a  n  s  m  e  m  b  r  a  n  e  p  r  o  t  e  i  n   s  e  q  u  e  n  c  e  s  .  B  M  C  B  i  o  i  n  f  o  r  m  a  t  i  c  s  .  2  0  1  2  J  u  l  1  7  ;  1  3  :  1  6  9  .  d  o  i  :  1  0  .  1  1  8  6  /  1  4  7  1  -2  1  0  5  -1  3  -1  6     There was no effect of AOA on malate efflux from BY2 cells expressing empty vector (Supplemental Figure 4A).

(B)
[GABA] i in BY2 cells expressing TaALMT1 at the end of the efflux period. There was no effect of AOA on [GABA] i in BY2 cells expressing empty vector (Supplemental Figure   3D,E,F). All data n=5 replicates; *, **, ***, **** indicate significant differences between treatments at P< 0.05, 0.01, 0.001, 0.0001 respectively, using a one-way ANOVA.      There was no effect of AOA on malate efflux from BY2 cells expressing empty vector (Supplemental Figure 4A).

(B)
[GABA] i in BY2 cells expressing TaALMT1 at the end of the efflux period. There was no effect of AOA on [GABA] i in BY2 cells expressing empty vector (Supplemental Figure  3D,E,F). All data n=5 replicates; *, **, ***, **** indicate significant differences between treatments at P< 0.05, 0.01, 0.001, 0.0001 respectively, using a one-way ANOVA.  Figure 4. A large GABA efflux occurs from roots and BY2 cells expressing TaALMT1 together with malate efflux in response to Al 3+ at pH 4.5. (A) Increased malate efflux is observed from intact seedling roots of NIL ET8 but not ES8 wheat after treatment with 100 µM Al 3+ . (B) Increased GABA efflux is observed from roots of ET8 but not ES8 in response to Al 3+ . (C) Increased malate efflux is observed from intact seedling roots of transgenic barley (Golden Promise) expressing TaALMT1 (OE) compared with Golden Promise (GP) background after treatment with 100 µM Al 3+ . (D) Increased GABA efflux is observed from barley roots TaALMT1 OE but not GP as above. (E) GABA efflux from tobacco BY2 cells over 22 h in response to Al 3+ at pH 4.5 for empty vector (Control), cells expressing TaALMT1 or site directed mutant TaALMT1 F213C . Only TaALMT1 + Al 3+ showed a significant increase in GABA efflux (P<0.0001). For malate efflux see Supplemental Figure 3. All data n=5-12 replicates; *, **, ***, **** indicate significant differences between treatments at P< 0.05, 0.01, 0.001, 0.0001 respectively, using a one-way ANOVA.  Comparison of 14 C GABA uptake into X. laevis oocytes expressing TaALMT1 and AtGAT1 high affinity GABA transporter at pH 4.5 (Exp. 1). Exp. 2: TaALMT1 expressing oocytes showed significantly higher influx of GABA when compared with the mutant TaALMT1 F213C or control (water injected). There was a significant reduction in GABA uptake on addition of 100 µM Al 3+ for oocytes expressing TaALMT1.

(B)
Comparison of 14 C GABA uptake for TaALMT1 and AtGAT1 at pH 7.5 (Exp. 1). Exp. 2: TaALMT1 expressing oocytes showed significantly higher influx of GABA when activated by 10 mM Na 2 SO 4 compared with the TaALMT1 F213C or control (water injected) oocytes. (C) GABA uptake was significantly higher for oocytes expressing ALMTs from wheat (TaALMT1), barley (HvALMT1), Arabidopsis (AtALMT1), rice (OsALMT5 and 9) and Arabidopsis GABA transporter (AtGAT1) at pH 4.5. Fluxes were normalised to the median of AtGAT1.  The majority of prediction algorithms (see text) indicate that TaALMT1 consists of 6 transmembrane helices (red, orange) with F213 oriented towards the cytosol at the end of the 6 th TM (orange, GABA motif=magenta). The N and C termini are predicted also to be on the cytosolic side. The model structure of TaALMT1 was computed from evolutionary sequence variation using the EvFold web portal (http://evfold.org/evfold-web/evfold.d) (Marks et al., 2012;Marks, et al., 2011, Ramesh et al., 2016. (B) Summary scheme of transport interactions and gating modes of TaALMT1. Left hand modes occur when GABA reaches a higher concentration on the outside blocking malate transport both outwards and inwards (bassed on TEVC), but GABA transport can proceed inwards. Right hand side modes occur when TaALMT1 is activated by Al 3+ or AOA at pH 4.5 or the presence of anions (malate 2-, SO 4 2-) on the outside at high pH (>7.5). The F213C mutant is shown on the outer left and right. Malate currents are activated by Al 3+ and AOA, but are unresponsive to GABA and it does not transport GABA inwards.