Abscisic acid regulation of guard-cell K ⁺ and anion channels in Gβ- and RGS-deficient Arabidopsis lines

Abstract

In mammals, basal currents through G protein-coupled inwardly rectifying K ؉ (GIRK) channels are repressed by G␣i/oGDP, and the channels are activated by direct binding of free G␤␥ subunits released upon stimulation of G␣ i/o-coupled receptors. However, essentially all information on G protein regulation of GIRK electrophysiology has been gained on the basis of coexpression studies in heterologous systems. A major advantage of the model organism, Arabidopsis thaliana, is the ease with which knockout mutants can be obtained. We evaluated plants harboring mutations in the sole Arabidopsis G␣ (AtGPA1), G␤ (AGB1), and Regulator of G protein Signaling (AtRGS1) genes for impacts on ion channel regulation. In guard cells, where K ؉ fluxes are integral to cellular regulation of stomatal apertures, inhibition of inward K ؉ (Kin) currents and stomatal opening by the phytohormone abscisic acid (ABA) was equally impaired in Atgpa1 and agb1 single mutants and the Atgpa1 agb1 double mutant. AGB1 overexpressing lines maintained a wild-type phenotype. The Atrgs1 mutation did not affect K in current magnitude or ABA sensitivity, but Kin voltage-activation kinetics were altered. Thus, Arabidopsis cells differ from mammalian cells in that they uniquely use the G␣ subunit or regulation of the heterotrimer to mediate K in channel modulation after ligand perception. In contrast, outwardly rectifying (K out) currents were unaltered in the mutants, and ABA activation of slow anion currents was conditionally disrupted in conjunction with cytosolic pH clamp. Our studies highlight unique aspects of ion channel regulation by heterotrimeric G proteins and relate these aspects to stomatal aperture control, a key determinant of plant biomass acquisition and drought tolerance.G protein-coupled inwardly rectifying potassium or ''GIRK'' channels (also known as Kir3 channels) comprise important targets of heterotrimeric G protein regulation in mammals (1, 2). GIRK channels mediate signals from muscarinic, adrenergic, opioid, dopaminergic, and GABA B receptors (3). Basal activity of GIRK channels is repressed by their direct binding of G␣ i/o GDP (4, 5) within a macromolecular complex that includes G␤␥ (4-7). Upon activation of G i/o -coupled G protein-coupled receptors (GPCRs), formation of the GTP-bound form of G␣ i/o both alleviates G␣-mediated repression and releases ␤␥ dimers that independently interact with the channel (7,8). G␤ 1-4 ␥ binding strengthens GIRK interaction with phosphatidylinositol 4,5-bisphosphate (PtdInsP 2 ), thereby promoting conformational changes that increase channel open time (3, 9-11). Conversely, G␣ q -based activation of phospholipase C opposes GIRK activity via both depletion of PtdInsP 2 and activation of PKC-based phosphorylation events (12).Numerous studies analyzing GIRK activity in Xenopus oocytes and cultured mammalian cells have led to the beautifully intricate model described above. However, studies analyzing GIRK activity in the appropriate native cell context upon genetic depletion of G␤ subunits are lack...

Abscisic Acid-Induced Resistance against the Brown Spot Pathogen Cochliobolus miyabeanus in Rice Involves MAP Kinase-Mediated Repression of Ethylene Signaling

“…This view is consistent with the recent discovery of the Arabidopsis Ga-subunit gene GPA1 as a central element in ABAdependent innate immunity in stomatal guard cells . Moreover, there is ample evidence indicating that ABA signaling processes in both seeds and guard cells involve components of the heterotrimeric G-protein complex, further supporting our hypothesis (Pandey and Assmann, 2004;Fan et al, 2008;Wang et al, 2008). According to the G-protein signaling paradigm, the lack of Ga not only abolishes Ga-mediated signaling but also results in free Gbg, thereby possibly enhancing Gbg signal output (Pandey et al, 2006).…”

ArabidopsisN-MYC DOWNREGULATED-LIKE1, a Positive Regulator of Auxin Transport in a G Protein–Mediated Pathway

“…Current ( Figures 1A and 1B) and previous data from our lab and other labs support the G protein component interactions shown in Figure 9A (e.g., Mason and Botella, 2001;Chen et al, 2003;Kato et al, 2004;Chen et al, 2006c;Fan, et al, 2008).…”

S-type Anion Channels SLAC1 and SLAH3 Function as Essential Negative Regulators of Inward K+ Channels and Stomatal Opening in Arabidopsis

“…SNAREs are involved in regulating K + in currents by affecting the trafficking and subcellular localization of KAT1 (Sutter et al, 2006;Eisenach et al, 2012;Zhang et al, 2015). ABA-mediated inhibition of K + in currents was observed in Arabidopsis guard cells (Fan et al, 2008), and G protein played important roles in this process . K + in currents of guard cell protoplasts were strongly reduced without impairing stomatal opening in the knockout mutants kat1 and kat2 (Szyroki et al, 2001;Lebaudy et al, 2008), whereas both the K + in currents and stomatal opening were inhibited in dominant-negative KAT1 mutant plants in the wild-type background and dominant-negative KAT2 mutant plants in the kat2-1 mutant background (Kwak et al, 2001;Lebaudy et al, 2008).…”