The malate-activated ALMT12 anion channel in the grass Brachypodium distachyon is co-activated by Ca2+/calmodulin

Abstract

Edited by Mike Shipston In plants, strict regulation of stomatal pores is critical for modulation of CO 2 fixation and transpiration. Under certain abiotic and biotic stressors, pore closure is initiated through anionic flux, with calcium (Ca 2؉) playing a central role. The aluminum-activated malate transporter 12 (ALMT12) is a malate-activated, voltage-dependent member of the aluminumactivated malate transporter family that has been implicated in anionic flux from guard cells controlling the stomatal aperture. Herein, we report the characterization of the regulatory mechanisms mediating channel activities of an ALMT from the grass Brachypodium distachyon (BdALMT12) that has the highest sequence identity to Arabidopsis thaliana ALMT12. Electrophysiological studies in a heterologous cell system confirmed that this channel is malate-and voltage-dependent. However, this was shown to be true only in the presence of Ca 2؉. Although a general kinase inhibitor increased the current density of BdALMT12, a calmodulin (CaM) inhibitor reduced the Ca 2؉dependent channel activation. We investigated the physiological relevance of the CaM-based regulation in planta, where stomatal closure, induced by exogenous Ca 2؉ ionophore and malate, was shown to be inhibited by exogenous application of a CaM inhibitor. Subsequent analyses revealed that the double substitutions R335A/R338A and R335A/K342A, within a predicted BdALMT12 CaM-binding domain (CBD), also decreased the channels' ability to activate. Using isothermal titration calorimetry and CBD-mimetic peptides, as well as CaM-agarose affinity pulldown of full-length recombinant BdALMT12, we confirmed the physical interaction between the CBD and CaM. Together, these findings support a co-regulatory mechanism of BdALMT12 activation by malate, and Ca 2؉ /CaM, emphasizing that a complex regulatory network modulates BdALMT12 activity.

Structural and Functional Insights into the Role of Guard Cell Ion Channels in Abiotic Stress-Induced Stomatal Closure

“…A Patch-clamp study showed that malate-dependent ALMT12 activity is dependent on cytosolic calcium and the channel current increased with the increasing cytosolic calcium concentration. The study further shows that the channel is co-regulated by calmodulin indicating a complex regulation mechanism for ALMT12 [209].…”

Calmodulin‐like protein CML24 interacts with CAMTA2 and WRKY46 to regulate ALMT1‐dependent Al resistance in Arabidopsis thaliana

“…Many studies have shown that Ca 2+ signalling is involved in the regulation of anion channels (Lewis et al ., 1997; Brault et al ., 2004; Vahisalu et al ., 2008; Siegel et al ., 2009; Chen et al ., 2010; Geiger et al ., 2010; Li et al ., 2016; Gutermuth et al ., 2018; Saito et al ., 2018). In the grass Brachypodium distachyon , the malate‐activated ALMT12 anion channel is activated by Ca 2+ /CaM through the physical interaction between the CaM and BdALMT12 (Luu et al ., 2019). In this study, both ALMT1 expression and malate exudation from roots were lower in the cml24 mutant, but higher in the CML24‐OE transgenic plants compared with the WT plants (Fig.…”

Functional roles of ALMT‐type anion channels in malate‐induced stomatal closure in tomato and Arabidopsis

“…To explore the C-terminal region of ALMT proteins involved in generating the bell-shaped current-voltage patterns, we re-evaluated the amino acid alignments of AtALMT12, SlALMT11 and BdALMT12 orthologs. BdALMT12 is also reported to be an ALMT that is expressed in guard cells of Brachypodium distachyon (Luu et al, 2019).…”