Arabidopsis nanodomain-delimited ABA signaling pathway regulates the anion channel SLAH3

Abstract

The phytohormone abscisic acid (ABA) plays a key role in the plant response to drought stress. Hence, ABA-dependent gene transcription and ion transport is regulated by a variety of protein kinases and phosphatases. However, the nature of the membranedelimited ABA signal transduction steps remains largely unknown. To gain insight into plasma membrane-bound ABA signaling, we identified sterol-dependent proteins associated with detergent resistant membranes from Arabidopsis thaliana mesophyll cells. Among those, we detected the central ABA signaling phosphatase ABI1 (abscisic-acid insensitive 1) and the calcium-dependent protein kinase 21 (CPK21). Using fluorescence microscopy, we found these proteins to localize in membrane nanodomains, as observed by colocalization with the nanodomain marker remorin Arabidopsis thaliana remorin 1.3 (AtRem 1.3). After transient coexpression, CPK21 interacted with SLAH3 [slow anion channel 1 (SLAC1) homolog 3] and activated this anion channel. Upon CPK21 stimulation, SLAH3 exhibited the hallmark properties of S-type anion channels. Coexpression of SLAH3/CPK21 with ABI1, however, prevented proper nanodomain localization of the SLAH3/CPK21 protein complex, and as a result anion channel activation failed. FRET studies revealed enhanced interaction of SLAH3 and CPK21 within the plasma membrane in response to ABA and thus confirmed our initial observations. Interestingly, the ABA-induced SLAH3/CPK21 interaction was modulated by ABI1 and the ABA receptor RCAR1/PYL9 [regulatory components of ABA receptor 1/PYR1 (pyrabactin resistance 1)-like protein 9]. We therefore propose that ABA signaling via inhibition of ABI1 modulates the apparent association of a signaling and transport complex within membrane domains that is necessary for phosphorylation and activation of the S-type anion channel SLAH3 by CPK21.

Modification of Plasma Membrane Organization in Tobacco Cells Elicited by Cryptogein

“…Moreover, immunoelectron microscopy experiments have revealed that lateral segregation of lipids and proteins occurs at the nanoscale level at the tobacco PM, thus correlating detergent insolubility with membrane domain localization of presumptive raft proteins (Raffaele et al, 2009;Furt et al, 2010;Demir et al, 2013). Together, these data point to the existence of specialized lipid domains in plants.…”

“…Similarly, plant-based immunoelectron microscopy experiments have revealed partitioning of both the lipid phosphatidylinositol 4,5-bisphosphate into 25-nm clusters on the PM of tobacco BY2 cells (Furt et al, 2010) and the protein flotillin into clusters of less than 100 nm on the PM of Arabidopsis roots apices (Li et al, 2012). In addition, the protein remorin was observed by immunoelectron microscopy in domains of about 80 nm in the cytosolic leaflet of the membrane of tobacco leaves (Raffaele et al, 2009) and was more recently localized by stimulated emission depletion microscopy within domains of 97 nm in Arabidopsis, whereas the apparent size of these domains was about 250 nm, using classical confocal microscopy (Demir et al, 2013). Similarly, the heterogeneity of RGR values that we observed could correspond to domains of 100 nm or less.…”

“…Recent studies have shown how several abscisic acid signaling components interact within nanodomains in response to this phytohormone (Demir et al, 2013), as well as how the S-acylation of the small G protein Rop6 (which conditions its dynamic association to DIMs) is a critical determinant of its role in the regulation of cell polarity (Sorek et al, 2011). In our system, the PM-localized NADPH oxidase that is responsible for ROS production upon cryptogein treatment was previously revealed to associate exclusively with the DIM fraction; furthermore, its product is localized within 100-nm patches along the membrane within minutes of elicitation .…”

Reconstitution of Abscisic Acid Signaling from the Receptor to DNA via bHLH Transcription Factors

“…ABA modulates both ion channel activity and gene expression in guard cells. Previous studies suggested that ABA-dependent activation of anion channels occurs within a nanodomain in the plasma membranes (Demir et al, 2013). By contrast, it remains unclear where the ABA signal is perceived and how the signal is transmitted to the nucleus in the ABA signaling pathway for gene regulation.…”

A Single-Pore Residue Renders the Arabidopsis Root Anion Channel SLAH2 Highly Nitrate Selective

“…SLAC1, together with SLAC1 HOMOLOGOUS3 (SLAH3), represent the molecular determinants for S-type anion channel activity in guard cells (Geiger et al, 2009b(Geiger et al, , 2010(Geiger et al, , 2011, while in mesophyll cells, S-type anion channel activity appears to be driven by just SLAH3 (Geiger et al, 2009b(Geiger et al, , 2010(Geiger et al, , 2011Demir et al, 2013). Both the SLAC1 and SLAH3 channels interact with distinct kinasephosphatase pairs shown to be associated with water stress signaling (Geiger et al, 2009b(Geiger et al, , 2010(Geiger et al, , 2011Brandt et al, 2012;Scherzer et al, 2012).…”