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

Abstract

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.

γ-Aminobutyric acid may directly or indirectly regulate Arabidopsis ALMT9

“…Recently, Xu et al (2021a) provided multiple lines of genetic and physiological evidence that GABA reduces stomatal opening to improve water use efficiency, and that in Arabidopsis ( Arabidopsis thaliana ) this modulation likely depends upon tonoplast-localized AtALMT9. In a follow-up study, Jaślan and De Angeli (2022) clearly show no impact of GABA on anion currents carried by AtALMT9 transiently expressed in Nicotiana benthamiana mesophyll cells, which was presented as proof, as highlighted by the title, that GABA “does not directly inhibit AtALMT9.” While we welcome the study and do not question the data, such a result allows for such a conclusion to be drawn only in the heterologous system used for testing. Jaślan and De Angeli (2022) transiently expressed the Arabidopsis protein AtALMT9 fused to green fluorescent protein (GFP) using Agrobacterium -based infection of N. benthamiana leaves, followed by enzymatic digestion to remove the cell walls and release protoplasts and osmotic shock to burst protoplasts and isolate vacuoles.…”

“…In a follow-up study, Jaślan and De Angeli (2022) clearly show no impact of GABA on anion currents carried by AtALMT9 transiently expressed in Nicotiana benthamiana mesophyll cells, which was presented as proof, as highlighted by the title, that GABA “does not directly inhibit AtALMT9.” While we welcome the study and do not question the data, such a result allows for such a conclusion to be drawn only in the heterologous system used for testing. Jaślan and De Angeli (2022) transiently expressed the Arabidopsis protein AtALMT9 fused to green fluorescent protein (GFP) using Agrobacterium -based infection of N. benthamiana leaves, followed by enzymatic digestion to remove the cell walls and release protoplasts and osmotic shock to burst protoplasts and isolate vacuoles. These vacuolar membranes were then subjected to patch clamp electrophysiology using highly artificial solutions that diluted vacuolar contents, were devoid of standard cytosolic factors, and were dominated by Bis–Tris propane to reduce native currents ( De Angeli et al, 2013 ).…”

“…(1) Voltage driven by the tonoplast proton (H + ) pump to generate electrical gradients as a driving force for AtALMT9-mediated chloride (Cl − ) and malate uptake ( De Angeli et al, 2013 ); (2) malate stimulated Cl − uptake ( De Angeli et al, 2013 ); (3) cytosolic citrate suppression of AtALMT9-mediated malate transport ( Zhang et al, 2013 ); (4) cytosolic nucleotides inhibition of AtALMT9-mediated Cl − and malate uptake, including adenosine 5′-triphosphate (ATP), guanosine-5′-triphosphate (GTP), and adenosine di-phosphate (ADP) ( Zhang et al, 2014 ); and (5) AtALMT9-dependent GABA regulation of stomatal opening ( Xu et al, 2021a ). As stated by Xu et al (2021a) , AtALMT9 may be indirectly regulated by GABA ( Jaślan and De Angeli, 2022 ), possibly via an unknown intermediate ( x ), or directly (as hypothesized for other ALMTs) (e.g. Long et al, 2019 ).…”

“…These vacuolar membranes were then subjected to patch clamp electrophysiology using highly artificial solutions that diluted vacuolar contents, were devoid of standard cytosolic factors, and were dominated by Bis–Tris propane to reduce native currents ( De Angeli et al, 2013 ). Results from the heterologous system used by Jaślan and De Angeli (2022) have been used previously to suggest regulators when an experimental interaction was observed (e.g. Zhang et al, 2013 ; Figure 1 ), but no definitive statements regarding in planta function can be made, especially with respect to negative results.…”

“… Zhang et al, 2013 ; Figure 1 ), but no definitive statements regarding in planta function can be made, especially with respect to negative results. Therefore, the results of Jaślan and De Angeli (2022) can be used to legitimately cast doubt on whether direct GABA regulation occurs for AtALMT9, but reliance on this single reductionist and heterologous experimental approach without confirmation via other approaches, including in planta assays, precludes irrefutable extrapolation of negative results to the native system. Therefore, we suggest the question of how GABA regulates AtALMT9 remains very much open.…”