Reconstitution of abscisic acid activation of SLAC1 anion channel by CPK6 and OST1 kinases and branched ABI1 PP2C phosphatase action

Abstract

The plant hormone abscisic acid (ABA) is produced in response to abiotic stresses and mediates stomatal closure in response to drought via recently identified ABA receptors (pyrabactin resistance/regulatory component of ABA receptor; PYR/RCAR). SLAC1 encodes a central guard cell S-type anion channel that mediates ABA-induced stomatal closure. Coexpression of the calcium-dependent protein kinase 21 (CPK21), CPK23, or the Open Stomata 1 kinase (OST1) activates SLAC1 anion currents. However, reconstitution of ABA activation of any plant ion channel has not yet been attained. Whether the known core ABA signaling components are sufficient for ABA activation of SLAC1 anion channels or whether additional components are required remains unknown. The Ca 2+ -dependent protein kinase CPK6 is known to function in vivo in ABA-induced stomatal closure. Here we show that CPK6 robustly activates SLAC1-mediated currents and phosphorylates the SLAC1 N terminus. A phosphorylation site (S59) in SLAC1, crucial for CPK6 activation, was identified. The group A PP2Cs ABI1, ABI2, and PP2CA down-regulated CPK6-mediated SLAC1 activity in oocytes. Unexpectedly, ABI1 directly dephosphorylated the N terminus of SLAC1, indicating an alternate branched early ABA signaling core in which ABI1 targets SLAC1 directly (downregulation). Furthermore, here we have successfully reconstituted ABA-induced activation of SLAC1 channels in oocytes using the ABA receptor pyrabactin resistant 1 (PYR1) and PP2C phosphatases with two alternate signaling cores including either CPK6 or OST1. Point mutations in ABI1 disrupting PYR1-ABI1 interaction abolished ABA signal transduction. Moreover, by addition of CPK6, a functional ABA signal transduction core from ABA receptors to ion channel activation was reconstituted without a SnRK2 kinase.Arabidopsis | chloride channel T he perception of the phytohormone abscisic acid (ABA) is achieved by the recently discovered 14-member START protein family of ABA receptors named pyrabactin resistance (PYR), or regulatory component of ABA receptor (RCAR) (1, 2). PYR/RCARs have been shown to bind to clade A PP2Cs and inhibit the activity of these PP2Cs in the presence of ABA (1-5). Structural studies show that PYR1, PYL1, and PYL2 function as ABA receptors, with ABA binding in a protein cavity that locks down the ABA molecule (6-10).ABA reduces transpirational water loss of plants by inducing stomatal closure (11). ABA can cause an increase in guard cell intracellular Ca 2+ concentration (12-17), which leads to the down-regulation of inward-rectifying K + channels and activation of both slow-sustained (S-type) and rapid-transient (R-type) anion channels (18)(19)(20). Previous findings have led to the model that S-type anion channels play a key role in controlling stomatal closure (18,21,22). slac1 mutant plants have greatly reduced S-type anion channel activity (23) and display impaired stomatal closure in response to ABA, elevated CO 2 , ozone, reactive oxygen species, calcium, and reduced humidity, underlining that SLAC1 repres...

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].…”

“…In guard cells, the SnRK2s can also phosphorylate and activate the NADPH oxidase catalytic subunit RBOHF, leading to the production of reactive oxygen species (ROS) [17,22,23]. SnRK2s also interact with and phosphorylate plasma membrane anion channels such as SLAC1 to regulate ion transport [13,[24][25][26].…”