<figure><img src="image_1.png" /></figure>

HAL
open science

# Heterologous expression reveals that GABA does not directly inhibit the vacuolar anion channel AtALMT9

Justyna Jaślan, Alexis de Angeli

► To cite this version:

Justyna Jaślan, Alexis de Angeli. Heterologous expression reveals that GABA does not directly inhibit the vacuolar anion channel AtALMT9. Plant Physiology, In press, 189 (2), pp.469-472. 10.1093/plphys/kiac132. hal-03619972

HAL Id: hal-03619972

https://hal.science/hal-03619972

Submitted on 25 Apr 2022

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_2.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
1

2

# Heterologous expression reveals that GABA does not directly inhibit the vacuolar anion channel *AtALMT9*

3

Justyna Jaślan 1, Alexis De Angeli 1

1

4

IPSiM, Univ Montpellier, CNRS, INRAE, Institut Agro, Montpellier, France.

5

## One-Sentence Summary:

6

7

GABA, a molecule involved in the regulation of the stomata aperture and drought tolerance, does not modify vacuolar anion fluxes mediated by the anion channel *AtALMT9*.

8

9

10

Dear Editor,

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

GABA (gamma-aminobutyric acid) is a well-known neurotransmitter activating Cl⁻ channels in synapses and having an inhibitory effect on neural activity. In plants, GABA was proposed to have different functions, for example, in the regulation of the carbon/nitrogen balance and resistance/tolerance to different biotic and abiotic stresses (Steward, 1949; Bouché et al., 2003; Bouché and Fromm, 2004; Fait et al., 2008; Renault et al., 2010; Fait et al., 2011; Sigel and Steinmann, 2012). Plants unable to synthesize GABA present a modified leaf transpiration suggesting a role of GABA in the regulation of stomatal movements (Mekonnen et al., 2016). More recently, it was proposed that GABA regulates vacuolar ion transport in guard cells directly targeting the anion channel *AtALMT9* (*Arabidopsis thaliana* Aluminum Activated Malate Transporter 9; Xu et al., 2021a). Indeed, Xu and colleagues proposed that GABA might affect stomata aperture regulation via direct inhibition of *AtALMT9*. However, although crucial, straightforward evidence showing a direct effect of GABA on *AtALMT9* ion transport activity is missing (Xu et al., 2021b). Therefore, we designed patch-clamp experiments to detect possible direct effects of GABA on *AtALMT9* mediated ionic currents. In the following *Letter*, we demonstrate that, in our experimental system, GABA has no direct influence on *AtALMT9* ion transport activity across the vacuolar membrane.

27

28

29

30

*AtALMT9* is a vacuolar anion channel mediating Cl⁻ influx into the vacuole during stomatal opening (De Angeli et al., 2013). Notably, GABA inhibits *TaALMT1* ionic currents in *Xenopus* oocytes from the cytosolic side (Long et al., 2020). Based on these findings, we decided to test whether the application of GABA from the cytosolic side (GABA<sub>cyt</sub>) impacts
AtALMT9 mediated Cl⁻ currents. Since *AtALMT9-GFP* complements *almt9* knock-out phenotypes (Xu et al., 2021a), indicating that GFP tagging does not modify ion channel properties, we transiently expressed *AtALMT9-GFP* into *Nicotiana benthamiana* for electrophysiological experiments. We performed patch-clamp experiments in cytosolic-side-out configuration from isolated vacuoles (De Angeli et al., 2013; Zhang et al., 2013) as this experimental design allows us to directly access the cytosolic face of the vacuolar membrane and to apply different cytosolic conditions to the same patch, *i.e.* to the same population of ion channels (Zhang et al., 2014). In planta GABA concentrations are in the range of 60 µM – 1 mM (Renault et al., 2011; Xu et al., 2021a). Therefore, we spanned a concentration range applying 100 µM, 2 mM and 10 mM GABA at the cytosolic side of *AtALMT9* (Fig 1A). We measured *AtALMT9* mediated Cl⁻ currents in presence of 100 mM Cl⁻ in the cytosol (black triangles; Figure 1B), and subsequently upon addition of different GABA<sub>cyt</sub> concentrations (Figure 1A and B; open triangles). We could observe typical *AtALMT9* mediated inward currents, with no rundown over 20 minutes (Figure 1D). However, we could not measure any significant effect after the application of the three different concentrations of GABA<sub>cyt</sub> on *AtALMT9* mediated Cl⁻ currents (Figure 1A). Notably, even a concentration as high as 10 mM GABA<sub>cyt</sub> was unable to inhibit *AtALMT9* ion transport activity (Figure 1A).

Cytosolic malate is a known activator of *AtALMT9* (De Angeli et al., 2013), thus we tested whether application of GABA<sub>cyt</sub> modifies the activation of *AtALMT9* mediated Cl⁻ currents by 1 mM malate²⁻_cyt. Therefore, we quantified the activation by 1 mM malate²⁻_cyt of *AtALMT9* in the presence and in the absence of 2 mM GABA<sub>cyt</sub> sequentially applied to the same patch (Figure 1A, B; black squares and C). Additionally, we tested the effect of 100 µM or 10 mM GABA<sub>cyt</sub> on the activation by 1mM malate²⁻_cyt. Also under these conditions we did not observe any reduction of the ionic currents over time (*i.e.* no rundown; Figure 1E). Importantly, our data show that the application of GABA<sub>cyt</sub> does not influence the activation of *AtALMT9* mediated ionic currents by malate²⁻_cyt (Figure 1A and B). Since the regulation of the vacuolar ion channels can also occur from the vacuolar lumen side, in a last set of experiments, we tested this possibility for GABA. To check whether GABA<sub>lum</sub> modifies *AtALMT9* mediated Cl⁻ currents and also malate²⁻_cyt activation of *AtALMT9* we used a vacuolar side buffer (*i.e.* pipette solution) containing, in addition to all the components used in the previous experiments, 2 mM GABA (full description in the legend of the Figure 1). We could not detect any effect of GABA<sub>lum</sub> (Figure 2A).
Overall, the results we have obtained answer the question whether GABA directly regulates *AtALMT9* ion transport activity (Xu et al., 2021b). Indeed, the present findings show that GABA, applied from either the cytosolic or the vacuolar side, has no effect on *AtALMT9* mediated Cl⁻ currents. Consequently, GABA does not have a direct influence on the ion transport activity of *AtALMT9* across the vacuolar membrane. Interestingly, by analogy with *TaALMT1*, it was proposed that *AtALMT9* harbors a putative “GABA binding motif” (Ramesh et al., 2015; Xu et al., 2021a). However, the lack of a direct effect of GABA on *AtALMT9* ionic currents shows that direct inhibition by GABA is not a feature of all ALMT’s. Notably, *AtALMT9* and *AtALMT1* belong to different clades of the ALMT family (Kovermann et al., 2007), therefore the different behavior possibly reflects structural differences between the two ion channels.

Overall, evidence suggests that GABA influences stomata aperture (Mekonnen et al., 2016; Xu et al., 2021a) but our data suggest that this does not occur through a direct inhibition of *AtALMT9*. In this context different scenarios are possible. Indeed, we can speculate that *in vivo* GABA could indirectly modify *AtALMT9* transport activity via a so far unknown regulatory factor (e.g. a GABA activated protein, Figure 2B). Alternatively, GABA could regulate other vacuolar ion channels and transporters residing in the vacuolar membrane (Figure 2B). Finally, GABA metabolism modifies organic acid homeostasis in Arabidopsis (*Arabidopsis thaliana*) leaves (Mekonnen et al., 2016) and therefore we cannot exclude that this indirectly modifies guard cell responses. In summary, our data show that more research is needed to identify the molecular mechanism mediating the effects of GABA *in vivo*.

## ACKNOWLEDGMENTS

We acknowledge the Heterologous Expression and Plant Electrophysiology (EHEV) platform from BPMP lab in Montpellier for their advices and assistance. ADA and JJ were funded by CNRS ATIP-AVENIR grant 2018 and ANR PARASOL (2021-2024).

Authors:

Justyna Jaślan and Alexis De Angeli *

Affiliations:
93 Justyna Jaślan: IPSiM, Univ Montpellier, CNRS, INRAE, Institut Agro, Montpellier, France

94 Alexis De Angeli: IPSiM, Univ Montpellier, CNRS, INRAE, Institut Agro, Montpellier, France

95

96 *Corresponding Author: Alexis De Angeli, alexis.deangeli@cnrs.fr

97

98 The author responsible for distribution of materials integral to the findings presented in this
99 article in accordance with the policy described in the Instructions for Authors
100 (https://academic.oup.com/plphys/pages/General-Instructions) is Alexis De Angeli.

101

102 Author contributions:

103 J.J. performed patch-clamp experiments; J.J. and A.DA. designed research; J.J. and A.DA.

104 wrote the manuscript.

105

106 **Figure Legends**

107 **Figure 1. Cytosolic GABA does not directly inhibit AtALMT9-mediated Cl⁻ currents.**

108 (A) AtALMT9 mediated chloride currents without and after activation by 1mM malate at the cytosolic
109 side in presence or absence of GABA (currents are normalized to their initial 100mM cytosolic Cl⁻).
110 Data represent means ± SE (n = 3-5). No statistical differences were found between presence and
111 absence of GABA (paired or un-paired two-sample Student's t-test). (B) Mean current-voltage
112 relationship from vacuolar patches expressing AtALMT9 (black symbols and lines) or mock controls
113 (blue symbols and lines) exposed to different cytosolic conditions. Symbols represent means ± SE (n =
114 3-10). (C) Representative current traces recorded in cytosolic-side-out excised patch from N.
115 benthamiana mesophyll vacuole expressing AtALMT9 under different cytosolic conditions. (D) and
116 (E) Time courses of representative currents upon perfusion with different cytosolic conditions. Each
117 graph displays a representative patch-clamp experiment. Cytosolic side solutions were sequentially
118 exchanged by a perfusion system. Data presented in A, B, C, D and E were recorded from patches of
119 N. benthamiana mesophyll vacuoles from transiently transformed leaves as previously described (De
120 Angeli et al. 2013). For consistency with study performed by Xu et al. (2021a) AtALMT9-GFP fusion
121 was used. Currents were elicited from a holding potential of 0 mV with 3s pulses from +40 mV to -
122 120 mV with -20 mV steps (Figure 1B and C) or from 0 mV to -160 mV (Figure 1A, D and E).
123 Recordings were performed in cytosolic-side-out configuration using as cytosolic side solution: 100
124 mM BTP-Cl⁻, 0.1 mM CaCl₂, pH = 7.5 adjusted with BTP and π = 500 mOsm adjusted with D-sorbitol
125 with or without 1 mM malate²_cyt (as indicated). Pipette solution (i.e. vacuolar side) contained 11.2 mM
126 malate², 100 mM HCl, pH = 6 adjusted with BTP and π = 550 mOsm adjusted with D-sorbitol. To
127 study GABA effect 100 µM, 2 mM or 10 mM GABA was added to the cytosolic side (GABAcyt)
128 solution or 2 mM GABA was added to the pipette solution (GABAlum; Figure 2).

129

130

131

132 **Figure 2. Vacuolar GABA does not directly influence AtALMT9 currents.**

133 (A) Mean current-voltage relationship obtained from vacuolar patches expressing AtALMT9 in
134 absence (grey symbols) or presence (black symbols) of the GABA in the vacuole (GABA<sub>lum</sub>) under
135 different cytosolic conditions. Solutions and applied voltage protocols are described in Figure 1.
136 Symbols represents means ± SE (n = 3-10). (B) Graphical summary showing that GABA has no direct
137 impact on the transport capacities of AtALMT9. Indirect effects of GABA on vacuolar ion transport
138 mediated by AtALMT9 might involve currently unknown regulatory factor (RC). Direct effect of
139 GABA could be mediated by regulation of other vacuolar ion carriers (channel or transporter, IC).

140 REFERENCES

141 Bouché N, Fait A, Bouchez D, Moller SG, Fromm H (2003) Mitochondrial succinic-
142 semialdehyde dehydrogenase of the -aminobutyrate shunt is required to restrict levels
143 of reactive oxygen intermediates in plants. Proceedings of the National Academy of
144 Sciences **100**: 6843–6848

145 Bouché N, Fromm H (2004) GABA in plants: just a metabolite? Trends in Plant Science **9**:
146 110–115

147 De Angeli A, Zhang J, Meyer S, Martinoia E (2013) AtALMT9 is a malate-activated
148 vacuolar chloride channel required for stomatal opening in Arabidopsis. Nat Commun
149 4: 1804

150 Fait A, Fromm H, Walter D, Galili G, Fernie AR (2008) Highway or byway: the metabolic
151 role of the GABA shunt in plants. Trends in Plant Science **13**: 14–19

152 Fait A, Nesi AN, Angelovici R, Lehmann M, Pham PA, Song L, Haslam RP, Napier JA,
153 Galili G, Fernie AR (2011) Targeted enhancement of glutamate-to-γ-aminobutyrate
154 conversion in Arabidopsis seeds affects carbon-nitrogen balance and storage reserves
155 in a development-dependent manner. Plant Physiology **157**: 1026–1042

156 Kovermann P, Meyer S, Hörtensteiner S, Picco C, Scholz-Starke J, Ravera S, Lee Y,
157 Martinoia E (2007) The Arabidopsis vacuolar malate channel is a member of the
158 ALMT family. The Plant Journal **52**: 1169–1180

159 Long Y, Tyerman SD, Gilliham M (2020) Cytosolic GABA inhibits anion transport by
160 wheat ALMT1. New Phytologist **225**: 671–678
161 **Mekonnen DW, Flügge U-I, Ludewig F** (2016) Gamma-aminobutyric acid depletion affects stomata closure and drought tolerance of Arabidopsis thaliana. *Plant Science* **245**: 25–34

164 **Ramesh SA, Tyerman SD, Xu B, Bose J, Kaur S, Conn V, Domingos P, Ullah S, Wege S,**
**Shabala S, et al** (2015) GABA signalling modulates plant growth by directly regulating the activity of plant-specific anion transporters. *Nat Commun* **6**: 7879

167 **Renault H, El Amrani A, Palanivelu R, Updegraff EP, Yu A, Renou J-P, Preuss D,**
**Bouchereau A, Deleu C** (2011) GABA accumulation causes cell elongation defects and a decrease in expression of genes encoding secreted and cell wall-related proteins in Arabidopsis thaliana. *Plant and Cell Physiology* **52**: 894–908

171 **Renault H, Roussel V, El Amrani A, Arzel M, Renault D, Bouchereau A, Deleu C** (2010)
The Arabidopsis pop2-1 mutant reveals the involvement of GABA transaminase in salt stress tolerance. *BMC Plant Biol* **10**: 20

174 **Sigel E, Steinmann ME** (2012) Structure, function, and modulation of GABAA receptors. *Journal of Biological Chemistry* **287**: 40224–40231

176 **Steward FC** (1949) γ-aminobutyric acid: a constituent of the potato tuber? *Science* **110**: 439–440

178 **Xu B, Long Y, Feng X, Zhu X, Sai N, Chirkova L, Betts A, Herrmann J, Edwards EJ,**
**Okamoto M, et al** (2021a) GABA signalling modulates stomatal opening to enhance plant water use efficiency and drought resilience. *Nat Commun* **12**: 1952

181 **Xu B, Sai N, Gilliham M** (2021b) The emerging role of GABA as a transport regulator and physiological signal. *Plant Physiology* **kiab347**

183 **Zhang J, Baetz U, Krügel U, Martinoia E, De Angeli A** (2013) Identification of a probable pore-forming domain in the multimeric vacuolar anion channel *AtALMT9*. *Plant Physiology* **163**: 830–843

186 **Zhang J, Martinoia E, De Angeli A** (2014) Cytosolic nucleotides block and regulate the Arabidopsis vacuolar anion channel *AtALMT9*. *Journal of Biological Chemistry* **289**: 25581–25589

189
A

<figure><img src="image_3.png" /></figure>

B

<figure><img src="image_4.png" /></figure>

C

<figure><img src="image_5.png" /></figure>

D

<figure><img src="image_6.png" /></figure>

E

<figure><img src="image_7.png" /></figure>
A

<figure><img src="image_8.png" /></figure>

B

<figure><img src="image_9.png" /></figure>
## Parsed Citations

Bouché N, Fait A, Bouchez D, Moller SG, Fromm H (2003) Mitochondrial succinic-semialdehyde dehydrogenase of the -aminobutyrate shunt is required to restrict levels of reactive oxygen intermediates in plants. Proceedings of the National Academy of Sciences 100: 6843–6848

Google Scholar: [Author Only Title Only Author and Title](https://scholar.google.com/search?q=Author Only Title Only Author and Title)

Bouché N, Fromm H (2004) GABA in plants: just a metabolite? *Trends in Plant Science* 9: 110–115

De Angeli A, Zhang J, Meyer S, Martinoia E (2013) *AtALMT9* is a malate-activated vacuolar chloride channel required for stomatal opening in *Arabidopsis*. *Nat Commun* 4: 1804

Google Scholar: [Author Only Title Only Author and Title](https://scholar.google.com/search?q=Author Only Title Only Author and Title)

Fait A, Fromm H, Walter D, Galili G, Fernie AR (2008) Highway or byway: the metabolic role of the GABA shunt in plants. *Trends in Plant Science* 13: 14–19

Google Scholar: [Author Only Title Only Author and Title](https://scholar.google.com/search?q=Author Only Title Only Author and Title)

Fait A, Nesi AN, Angelovici R, Lehmann M, Pham PA, Song L, Haslam RP, Napier JA, Galili G, Fernie AR (2011) Targeted enhancement of glutamate-to-γ-aminobutyrate conversion in Arabidopsis seeds affects carbon-nitrogen balance and storage reserves in a development-dependent manner. *Plant Physiology* 157: 1026–1042

Google Scholar: [Author Only Title Only Author and Title](https://scholar.google.com/search?q=Author Only Title Only Author and Title)

Kovermann P, Meyer S, Hörtensteiner S, Picco C, Scholz-Starke J, Ravera S, Lee Y, Martinoia E (2007) The Arabidopsis vacuolar malate channel is a member of the ALMT family. *The Plant Journal* 52: 1169–1180

Google Scholar: [Author Only Title Only Author and Title](https://scholar.google.com/search?q=Author Only Title Only Author and Title)

Long Y, Tyerman SD, Gilliham M (2020) Cytosolic GABA inhibits anion transport by wheat ALMT1. *New Phytologist* 225: 671–678

Google Scholar: [Author Only Title Only Author and Title](https://scholar.google.com/search?q=Author Only Title Only Author and Title)

Mekonnen DW, Flügge U-I, Ludewig F (2016) Gamma-aminobutyric acid depletion affects stomata closure and drought tolerance of Arabidopsis thaliana. *Plant Science* 245: 25–34

Google Scholar: [Author Only Title Only Author and Title](https://scholar.google.com/search?q=Author Only Title Only Author and Title)

Ramesh SA, Tyerman SD, Xu B, Bose J, Kaur S, Conn V, Domingos P, Ullah S, Wege S, Shabala S, et al (2015) GABA signalling modulates plant growth by directly regulating the activity of plant-specific anion transporters. *Nat Commun* 6: 7879

Google Scholar: [Author Only Title Only Author and Title](https://scholar.google.com/search?q=Author Only Title Only Author and Title)

Renault H, El Amrani A, Palanivelu R, Updegraff EP, Yu A, Renou J-P, Preuss D, Bouchereau A, Deleu C (2011) GABA accumulation causes cell elongation defects and a decrease in expression of genes encoding secreted and cell wall-related proteins in Arabidopsis thaliana. *Plant and Cell Physiology* 52: 894–908

Google Scholar: [Author Only Title Only Author and Title](https://scholar.google.com/search?q=Author Only Title Only Author and Title)

Renault H, Roussel V, El Amrani A, Arzel M, Renault D, Bouchereau A, Deleu C (2010) The Arabidopsis pop2-1 mutant reveals the involvement of GABA transaminase in salt stress tolerance. *BMC Plant Biol* 10: 20

Google Scholar: [Author Only Title Only Author and Title](https://scholar.google.com/search?q=Author Only Title Only Author and Title)

Sigel E, Steinmann ME (2012) Structure, function, and modulation of GABAA receptors. *Journal of Biological Chemistry* 287: 40224–40231

Google Scholar: [Author Only Title Only Author and Title](https://scholar.google.com/search?q=Author Only Title Only Author and Title)

Steward FC (1949) γ-aminobutyric acid: a constituent of the potato tuber? *Science* 110: 439–440

Xu B, Long Y, Feng X, Zhu X, Sai N, Chirkova L, Betts A, Herrmann J, Edwards EJ, Okamoto M, et al (2021a) GABA signalling modulates stomatal opening to enhance plant water use efficiency and drought resilience. *Nat Commun* 12: 1952

Google Scholar: [Author Only Title Only Author and Title](https://scholar.google.com/search?q=Author Only Title Only Author and Title)

Xu B, Sai N, Gilliham M (2021b) The emerging role of GABA as a transport regulator and physiological signal. *Plant Physiology* kiab347

Google Scholar: [Author Only Title Only Author and Title](https://scholar.google.com/search?q=Author Only Title Only Author and Title)

Zhang J, Baetz U, Krügel U, Martinoia E, De Angeli A (2013) Identification of a probable pore-forming domain in the multimeric vacuolar anion channel *AtALMT9*. *Plant Physiology* 163: 830–843

Google Scholar: [Author Only Title Only Author and Title](https://scholar.google.com/search?q=Author Only Title Only Author and Title)

Zhang J, Martinoia E, De Angeli A (2014) Cytosolic nucleotides block and regulate the Arabidopsis vacuolar anion channel *AtALMT9*. *Journal of Biological Chemistry* 289: 25581–25589

Google Scholar: [Author Only Title Only Author and Title](https://scholar.google.com/search?q=Author Only Title Only Author and Title)