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Results CPK requirement for ABA activation of anion channels Previous studies have shown that A. thaliana single or double mutants in CPKs cause partial ABA-insensitivities in guard cell signaling (Mori et al., 2006; Zhu et al., 2007; Hubbard et al., 2012). We addressed Erastin in vivo the question whether higher order CPK gene disruption mutant plants display more strongly impaired ABA responses. CPK6 and CPK23 were shown to activate SLAC1 in Xenopus oocytes and disruption of the corresponding genes in plants leads to a partial reduction of S-type anion current activation in guard cells (Mori et al., 2006; Geiger et al., 2010; Brandt et al., 2012). The closest homolog to CPK6, CPK5, is associated with reactive oxygen species signaling (Boudsocq et al., 2010; Dubiella et al., 2013). CPK5 also activates SLAC1 in oocytes (Figure 1��figure supplement 1A,B). Whole-cell patch-clamp analysis showed that mutation of CPK5 alone does not substantially disrupt ABA-activation of S-type anion channels (Figure 1��figure supplement 1C,D), consistent with findings of over-lapping gene functions in this response (Mori et al., 2006; Hubbard Ro3280 et al., 2012). CPK11 is highly expressed in guard cells and involved in ABA responses (Zhu et al., 2007; Geiger et al., 2009). We isolated cpk5/6/11/23 quadruple T-DNA insertion mutant plants and investigated ABA-induced S-type 3-deazaneplanocin A solubility dmso anion channel current regulation. Either ABA treatment (Siegel et al., 2009) or by-passing ABA signaling by exposure of guard cells to a high external Ca2+ shock (Allen et al., 2002) renders wildtype (Col0) guard cells sensitive to physiological [Ca2+]cyt increases. Notably, even when previously exposed to ABA or a high external Ca2+ shock, 2 ��M [Ca2+]cyt did not result in S-type anion current activation in cpk5/6/11/23 quadruple mutant guard cells in contrast to WT plants (Figure 1A�CD). These results show an important role of these calcium sensing protein kinases in ABA-dependent S-type anion channel activation in guard cells. We further investigated ABA-induced stomatal movement responses. Application of 5 ��M ABA to WT leaves significantly decreased stomatal apertures compared to mock-treated control stomatal apertures (Figure 1E; p