Guard cell anion channel SLAC1 is regulated by CDPK protein kinases with distinct Ca2+ 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.

Fig. 1.

Interaction studies among proteins involved in the ABA signaling pathway by bimolecular fluorescence complementation (BIFC) in Xenopus oocytes (A–D) and Arabidopsis mesophyll protoplasts (E and F). Pictures of oocytes, taken with a confocal laser scanning microscope, show a quarter of an optical slice of an oocyte. (A) SLAC1::YFP^C coexpressed with CPK23::YFP^N. (B) SLAC1::YFP^C coexpressed with CPK31::YFP^N. (C) ABI1::YFP^C coexpressed with CPK23::YFP^N. (D) HAB1::YFP^C coexpressed with CPK23::YFP^N. (E and F) Interaction of SLAC1 and CPK23 monitored in transiently transformed Arabidopsis mesophyll protoplasts by bimolecular fluorescence complementation. Plasmid combination was as follows: YFP^C::SLAC1 and CPK23::YFP^N. (E) Transmitted light. (F) Overlay of YFP and chlorophyll fluorescence. YFP fluorescence was emitted at the level of the protoplast plasma membrane. (G) CPK23 interacts with the N-terminus of SLAC1 in planta. Arabidopsis protoplasts were transfected with the indicated tagged constructs (+) and total protein extracts (input) were prepared. CPK23-Strep was affinity purified, and proteins were visualized using either anti-Strep detection system (Middle) or anti-GFP antibodies (Upper and Lower). NT-SLAC1-YFP and CT-SLAC1-YFP migrate at approximately 48 and 35 kDa, CPK23-Strep at about 60, respectively.

Fig. 2.

Whole-oocyte current recordings in standard bath solution (30 mM Cl⁻, pH 5.6). (A–D) Whole-oocyte current recordings upon 40-s-lasting voltage pulses ranging from +40 to −180 mV in 20 mV decrements followed by a 3 s voltage pulse to −120 mV. The holding potential was 0 mV. No current responses of ooyctes expressing CPK23 (A) or SLAC1 alone (B) could be recorded. Coexpression of CPK31 with SLAC1 mediated no anion currents either (D). Only coexpression of SLAC1 together with CPK23 (C) resulted in macroscopic anion currents slowly deactivating at negative membrane potentials. Representative cells are shown. (E) Reversal potentials for Cl⁻ and NO₃⁻ were shown as a function of the logarithmic external anion concentration. As expected for an anion-selective channel, the reversal potential shifted to more negative values with increasing anion concentrations (n ≥ 4, mean ± SD). (F) Following a preactivation voltage pulse of 0 mV pronounced instantaneous anion currents (I_T) at −100 mV could be measured with SLAC1/CPK23-injected oocytes. No macroscopic currents were visible in oocytes coexpressing the kinase CPK31 together with SLAC1. SLAC1 activation by CPK21 was weaker than by CPK23. SLAC1 expression alone (indicated as none) as well as SLAC1 coexpression with CPK23 D193A did not result in macroscopic anion currents (n ≥ 4, mean ± SD). (G) Inhibition of SLAC1 activity by coexpression of the protein phosphatases ABI1 and ABI2. Coinjection of the inactive ABI1 G174D, HAB1, and HAB2, however, could not prevent SLAC1/CPK23 mediated anion currents (n ≥ 3, mean ± SD). (H) SLAC1 activation by a constitutive active C-terminal truncated Ca²⁺-independent CPK23 (indicated as deletion) was slightly higher than activation by the wild-type CPK23 (full length). In the case of CPK21, this effect was more pronounced. Instantaneous anion currents (I_T) at −100 mV recorded in standard bath solution are shown (n = 5, mean ± SD).

Fig. 3.

(A) Macroscopic anion current recordings from Arabidopsis thaliana guard cell protoplasts in the presence of 2 μM cytosolic Ca²⁺. Representative current responses from wild type and cpk23 (Salk 007958) to 7.5-s-lasting voltage pulses from +46 mV to −134 mV are shown. (B) Steady-state current densities plotted against the clamped voltages. Data points represent the mean ± SE. The number of at least three independent experiments were n = 7 for cpk23 (open circles) and n = 6 for wild type (closed circles).

Fig. 4.

In vitro kinase assays using recombinant proteins. (A) ABA-dependent phosphorylation of SLAC1 by CPK23 (Upper) and OST1 (Lower) is mediated by the ABA-receptor/PP2C phosphatase complex RCAR1/ABI1. (Lanes 1 and 2) ABA-independent SLAC1 NT phosphorylation by CPK23 or OST1. (Lanes 3 and 4) ABI1 inhibited the phosphorylation of SLAC1 NT by CPK23 or OST1 independent from the presence of ABA. (Lanes 5 and 6) RCAR1 was neither in the presence or absence of ABA capable to prevent SLAC1 phosphorylation by CPK23 or OST1. (Lanes 7 and 8) Combination of SLAC1 NT, CPK23 or OST1, ABI1 and RCAR1 in one reaction mixture: the phosphorylation of SLAC1 was dependent on the availability of ABA. The recombinant proteins SLAC1 NT, CPK23, and OST1 were GST-tagged whereas RCAR1 carried a His-tag. Note that recombinant ABI1 was His-tagged in Upper and GST-tagged in Lower. (B) In vitro phosphorylation activity of CPK21 and 23 as a function of various Ca²⁺ concentrations. The affinity toward Ca²⁺ of CPK21 could be best described by a Hill equation with a Hill factor of four. In contrast CPK23 exhibited a 60% core activity even at very low Ca²⁺ concentrations and was only weak Ca²⁺ dependent.