Site- and kinase-specific phosphorylation-mediated activation of SLAC1, a guard cell anion channel stimulated by abscisic acid

Abstract

Under drought stress, abscisic acid (ABA) triggers closure of leaf cell pores called stomata, which are formed by two specialized cells called guard cells in plant epidermis. Two pathways downstream of ABA stimulate phosphorylation of the S-type anion channels SLAC1 (slow anion channel associated 1) and SLAH3 (SLAC1 homolog 3), which causes these channels to open, reducing guard cell volume and triggering stomatal closure. One branch involves OST1 (open stomata 1), a calcium-independent SnRK2-type kinase, and the other branch involves calcium-dependent protein kinases of the CPK (calcium-dependent protein kinase) family. We used coexpression analyses in Xenopus oocytes to show that the calcineurin B-like (CBL) calcium sensors CBL1 and CBL9 and their interacting protein kinase CIPK23 also triggered SLAC1 and SLAH3 opening. We analyzed whether regulation of SLAC1 opening by these different families of kinases involved the same or different sites on SLAC1 by measuring channel conductance of SLAC1 with mutations in the putative phosphorylation sites in the amino or carboxyl termini coexpressed with specific kinases in Xenopus oocytes. SLAC1 mutants lacking the OST1-phosphorylated site were still activated by CPK or by CBL/CIPK complexes. Phosphorylation and activation of SLAC1 by any of the kinases were inhibited by the phosphatase ABI1 (ABA insensitive 1), which is inactivated in response to ABA signaling. These findings identified CBL/CIPK complexes as potential regulators of stomatal aperture through S-type anion channels and indicated that phosphorylation at distinct sites enables SLAC1 activation by both calcium-dependent and calcium-independent pathways downstream of ABA.

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

“…Structural analysis revealed that the ion selectivity of SLAC1 may be conferred by its own topological structure (Chen et al, 2010). Recent research showed that the selectivity of SLAC/SLAH is determined by the polarity of pore-lining residues, which are localized in the alpha helix 3 (Maierhofer et al, 2014). Nevertheless, considering the structural difference between the two types of channels, it seems that S-type anion channels may not be able to alter the topological structures of Shaker members and vice versa.…”

“…Structural analysis revealed that SLAC1 forms a trimer, but each subunit of the trimer forms a channel pore independently (Chen et al, 2010). SLAC1 and SLAH3 are activated by protein phosphorylation in a Ca 2+ -independent manner by OST1 (open stomata1) and CPK23 (calcium-dependent protein kinase23), respectively (Geiger et al, 2009bLee et al, 2009), or in a Ca 2+ -dependent manner by GHR1 (guard cell hydrogen peroxideresistant 1), CPK3/6/21, and CBL1 (calcineurin B-like protein 1)/ CIPK23, respectively Brandt et al, 2012;Hua et al, 2012;Scherzer et al, 2012;Demir et al, 2013;Maierhofer et al, 2014). On exposure to drought stress, plants produce ABA, which is perceived by its receptors PYR (pyrabactin resistance)/ PYL (parabactin resistance 1-like)/RCAR (regulatory component of ABA receptor).…”

“…On exposure to drought stress, plants produce ABA, which is perceived by its receptors PYR (pyrabactin resistance)/ PYL (parabactin resistance 1-like)/RCAR (regulatory component of ABA receptor). These ABA receptors interact with a few phosphatases from the PP2C (protein phosphatase 2C) family, mainly including ABI1 (ABA insensitive 1), ABI2, PP2CA, and HAB1 (hypersensitive to ABA1), to release the diverse protein kinases (Ma et al, 2009;Nishimura et al, 2009;Park et al, 2009;Nishimura et al, 2010), which further activate SLAC1 and SLAH3 by phosphorylation (Geiger et al, 2009bLee et al, 2009;Brandt et al, 2012;Hua et al, 2012;Scherzer et al, 2012;Demir et al, 2013;Maierhofer et al, 2014). ALMT12 (aluminum-activated malate transporter 12)/QUAC (quick-activating anion channel 1) is an Rtype anion channel in Arabidopsis guard cells (Meyer et al, 2010;Imes et al, 2013).…”

Reactive Oxygen Species in the Regulation of Stomatal Movements

“…CIPK23 interacted with SLAC1 in planta, and coexpression of CBL1 or CBL9 together with CIPK23 in X. laevis oocytes induced SLAC1 and SLAC1 HOMOLOG3 (SLAH3) currents (Maierhofer et al, 2014). CBL/CIPK complexes are emerging as candidates linking ROS production and subsequent signaling leading to guard cell anion channel activation as the activation of AtRBOHF by CBL1/CBL9-CIPK26 complexes has also been recently described (Drerup et al, 2013).…”

The Arabidopsis AtPP2CA Protein Phosphatase Inhibits the GORK K+ Efflux Channel and Exerts a Dominant Suppressive Effect on Phosphomimetic-activating Mutations

“…This signaling pathway is important for guard cell plasma membrane depolarization, which drives K ϩ efflux and causes stomatal closure. Besides OST1, the calcineurin B-like protein kinase CIPK23 (20) and calcium-dependent protein kinases (CPKs) (21,22) also phosphorylate SLAC1 and its homolog SLAH3. This activation is reversed by the PP2Cs.…”