Guard cell anion channel SLAC1 is regulated by CDPK protein kinases with distinct Ca ²⁺ affinities

Abstract

In response to drought stress, the phytohormone abscisic acid (ABA) induces stomatal closure. Thereby the stress hormone activates guard cell anion channels in a calcium-dependent, as well as -independent, manner. Open stomata 1 protein kinase (OST1) and ABI1 protein phosphatase (ABA insensitive 1) represent key components of calcium-independent ABA signaling. Recently, the guard cell anion channel SLAC1 was identified. When expressed heterologously SLAC1 remained electrically silent. Upon coexpression with Ca 2+ -independent OST1, however, SLAC1 anion channels appear activated in an ABI1-dependent manner. Mutants lacking distinct calcium-dependent protein kinases (CPKs) appeared impaired in ABA stimulation of guard cell ion channels, too. To study SLAC1 activation via the calcium-dependent ABA pathway, we studied the SLAC1 response to CPKs in the Xenopus laevis oocyte system. Split YFP-based protein-protein interaction assays, using SLAC1 as the bait, identified guard cell expressed CPK21 and 23 as major interacting partners. Upon coexpression of SLAC1 with CPK21 and 23, anion currents document SLAC1 stimulation by these guard cell protein kinases. Ca 2+ -sensitive activation of SLAC1, however, could be assigned to the CPK21 pathway only because CPK23 turned out to be rather Ca 2+ -insensitive. In line with activation by OST1, CPK activation of the guard cell anion channel was suppressed by ABI1. Thus the CPK and OST1 branch of ABA signal transduction in guard cells seem to converge on the level of SLAC1 under the control of the ABI1/ABA-receptor complex.abscisic acid signaling | drought stress | guard cell | S-type anion channel T he drought hormone abscisic acid (ABA) triggers release of K + and anions from guard cells and thereby causes stomatal closure (1, 2). Recently, SLAC1, a guard cell anion channel, was identified (3-5). In guard cells of these ABA-and CO 2 /O 3 -insensitive mutant plants, anion currents appeared largely suppressed. When SLAC1 was expressed with the open stomata 1 protein kinase (OST1) in Xenopus oocytes, SLAC1-related anion currents, similar to those observed in guard cells, appeared (6). The presence of ABI1, however, prevented SLAC1 activation. This ABA pathway resembles the Ca 2+ -independent activation of SLAC-type anion currents in guard cells. ABA signal transduction, however, has been shown to activate guard cell anion channels in a calcium-independent as well as -dependent manner (7-10). This became evident in abi1-1 mutant plants, where anion channels do not respond to ABA anymore (11) but still activate with calcium (12). Furthermore the described mutant growth controlled by abscisic acid (gca2) (13-14), isolated from the Arabidopsis ecotype Landsberg erecta, was shown to be impaired in ABA-induced stomatal closure in a Ca 2+ -dependent manner. Moreover [Ca 2+ ] cyt elevation was shown to result in activation of S-type anion channels via phosphorylation (12, 15), suggesting a role of phosphorylation events in [Ca 2+ ] cyt signaling.CDPKs resemble Ca 2+ -dependent Ser/Thr pr...

A molecular pathway for CO2 response in Arabidopsis guard cells

“…Further research on the structural basis for the HT1-OST1 interaction and phosphorylation sites on OST1 will provide a more detailed molecular mechanism underlying OST1 inhibition by HT1 kinase. Given SLAC1 is also regulated by other protein kinases including CPKs [30][31][32] and calcium sensor kinases CBL-CIPKs 52 , further work should also be conducted to find out whether HT1, similar to the PP2C A-type phosphatases in ABA signalling, can also interact with and inhibit calcium-dependent kinases.…”

“…A group of kinases such as OST1 (STOMATAL OPEN 1), CPKs (Ca 2 þ -dependent protein kinases) and GHR1 (GUARD CELL HYDROGEN PEROXIDE-RESISTANT 1) can phosphorylate and activate the SLAC1 anion channel 23,24,[29][30][31][32] . Such activation is often considered to be the key step for ABA-induced stomatal closure [21][22][23][24] .…”

Evolution of the Stomatal Regulation of Plant Water Content

“…The phenotype of single gene mutants in the key SnRK2 for stomatal responses to ABA, OST1, are profound, displaying no sensitivity to ABA (Mustilli et al, 2002) or VPD (Merilo et al, 2013;Merilo et al, 2015). While other SnRK2-independent signaling pathways for ABA perception and SLAC activation have been suggested (Geiger et al, 2010;Brandt et al, 2012;Pornsiriwong et al, 2017), these are redundant and many either converge or require cross-talk with functional OST1 to activate anion channels (Brandt et al, 2015). Whether these alternative SnRK2-independent ABA signaling pathways play an adaptively relevant role in stomatal function is yet to be determined.…”

The unique mode of action of a divergent member of the ABA-receptor protein family in ABA and stress signaling

“…Structural analysis revealed that the interaction between SnRK2.6 and HAB1 mimics the interaction between ABA-bound PYL2 and HAB1 [12]. PYLs can inhibit PP2C activity in the presence of ABA, and thereby activating SnRK2.6, which can phosphorylate slow anion channel1 (SLAC1) and activate SLAC1-mediated ion currents [13,14].…”