Xenbase Image ID: 156825

Figure 3. PAP restores guard cell ion fluxes, ROS burst and global transcriptional response to ABA when accumulated genetically in ost1-2 sal1-8 and when applied exogenously to ost1-2 and wild type.Effects of (A) 500 µM ABA or (B) 500 µM PAP on combined net flux of each of the ion transporters for K+ and Cl− from guard cells in leaf epidermal peels of four-week old Arabidopsis plants. Average net ion fluxes ± SEM (n = 5–7 plants) are shown for control, 10 min and 50 min after ABA or PAP treatment. Asterisk shows statistically significant difference to 0 min (p<0.05, ANOVA). (C) Mean corrected total cell fluorescence of ROS in the presence of 2’,7’-dichlorodihydrofluorescein diacetate (H2DCFDA), a ROS probe that detects primarily H2O2 and to a lesser extent hydroxyl radicals (Wojtala et al., 2014), in guard cells before and after 10 min of 100 µM ABA or 100 µM PAP. Means ± SEM of 45–54 (+ABA) or 73–92 (+PAP) stomata per genotype is shown with significant differences denoted (t-test, p<0.05). (D–E) Hierarchical clustering comparing ABA transcriptional response in wild type (WT) and mutants for (D) transcripts responsive to ABA in WT in this study; and (E) transcripts known to respond to ABA in guard cells (Wang et al., 2011). The blue (down)-red (up) scale is log2 fold change for each genotype +/− ABA, respectively. The scale has been condensed such that the red and blue colours at the end of the scale encompass all fold-changes greater or equal to 2, or less than or equal to 0.5, respectively. Clusters showing co-expression in WT and ost1-2 sal1-8 are marked (I). Also see Supplementary file 1.DOI: http://dx.doi.org/10.7554/eLife.23361.008Figure 3—figure supplement 1. Effect of endogenously accumulated and exogenous PAP on ion fluxes in guard cells and transporter activity in oocytes respectively.(A) Changes in ion fluxes (K+, Cl−) in genotypes treated with CaCl2, a known secondary messenger and inducer of stomatal closure downstream of ABA, transcription, NO and ROS. Bars denote means of at least five plants ± SEM. Asterisk indicates statistically significant difference to 0 min (p<0.05, ANOVA) (B) Effect of 100 µM PAP on the activity of the chloride ion channel SLAC1 and potassium ion channels KAT1 and KAT2 expressed in Xenopus oocytes. Steady-state ion channel currents were measured at −110 mV for SLAC1 and −150 mV for KAT1 and KAT2 (see Materials and methods for voltage clamp protocols and measuring conditions). ‘OST1-activated for SLAC1’ treatments refer to oocytes co-injected by both SLAC1 and OST1 cRNA to allow expression of both proteins and for phosphorylation of SLAC1 by OST1, which activates SLAC1 activity. Means and SE of three to four biological replicates per treatment and ion channel are shown.DOI: http://dx.doi.org/10.7554/eLife.23361.009 Image published in: Pornsiriwong W et al. (2017) © 2017, Pornsiriwong et al. This image is reproduced with permission of the journal and the copyright holder. This is an open-access article distributed under the terms of the Creative Commons Attribution license Larger Image Printer Friendly View

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