Volume 5, Issue 4, Pages 852-864 (July 2012) Extracellular ATP Promotes Stomatal Opening of Arabidopsis thaliana through Heterotrimeric G Protein α Subunit and Reactive Oxygen Species  Li-Hua Hao, Wei-Xia Wang, Chen Chen, Yu-Fang Wang, Ting Liu, Xia Li, Zhong-Lin Shang  Molecular Plant  Volume 5, Issue 4, Pages 852-864 (July 2012) DOI: 10.1093/mp/ssr095 Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 1 ATP-Promoted Stomatal Opening of Arabidopsis thaliana. (A) Time course of stomatal opening in MES buffer containing ATP. (B) Dose-dependence of ATP-promoted stomatal opening. (C) Non- or weakly hydrolyzable ATP analogs promoted stomatal opening. (D) The effect of various purine nucleotides on stomatal movement. (E) Added ATP promoted stomatal opening in two A. thaliana ecotypes. (F) Added ATP or non-hydrolyzable ATP analog (2meATP) promoted stomatal opening in the dark. Stomatal apertures after 60-min treatment were noted. In (A), (C), (D), and (E), the final concentration of added reagents was 0.3 mM. In all figures, data are represented as means ± SE stomatal aperture (n = 6). In all figures, ‘control’ means treatment with MES buffer only. In (B–E), the stomatal aperture was measured after 60-min treatment. In (A–D) and (F), ecotype col-0 was used as the material. In all figures, ATP or ATP analogs promoted stomatal opening predominantly (P < 0.05, Student’s t-test). Molecular Plant 2012 5, 852-864DOI: (10.1093/mp/ssr095) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 2 Added Apyrase Inhibited Stomatal Opening of Arabidopsis thaliana. Col-0 was used as the material, and stomatal movement in light was investigated. (A) Time course of stomatal movement in MES buffer containing apyrase. Data are means ± SE stomatal aperture (n = 6) at different time points. The stomatal aperture was smaller in apyrase-treated epidermis (P < 0.05, Student’s t-test.). Heat-inactivated apyrase (30 units ml−1) did not affect stomatal movement (P > 0.05, Student’s t-test). (B, C) Cell viability measurement after apyrase treatment. FDA was loaded into guard cells, and images were captured with confocal laser scanning microscope. Mean ± SE relative fluorescent intensity in guard cells, which were treated by 10 or 30 units ml−1 apyrase for 4 h, was noted in (B). Images of representative guard cells that were treated with active or denatured apyrase are shown in (C). In all figures, ‘control’ means treatment with MES buffer only. Molecular Plant 2012 5, 852-864DOI: (10.1093/mp/ssr095) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 3 Reactive Oxygen Species (ROS) Participate in Extracellular ATP-Promoted Stomatal Opening in Arabidopsis thaliana (col-0). (A) Added ascorbic acid + CuCl2 mixture had contrasting effects on stomatal movement. Low- and high-concentration hydroxyl promoted stomatal opening and stomatal closing, respectively. (B) Diphenylene iodonium (DPI) inhibited ATP-promoted stomatal opening. (C) Dithiothreitol (DTT) inhibited ATP-promoted stomatal opening. (D) In an NADPH oxidase null mutant (atrbohD/F), ATP-promoted stomatal opening was blocked. In all figures, the data are means ± SE (n = 6) stomatal aperture after 60-min treatment with 0.3 mM ATP. In col-0, which was pretreated by DPI (B) or DTT (C) and in atrbohD/F (D), the stomatal aperture before and after ATP treatment was not significantly different (P > 0.05, Student’s t-test). In all figures, ‘control’ means treatment with MES buffer only. Molecular Plant 2012 5, 852-864DOI: (10.1093/mp/ssr095) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 4 The Heterotrimeric G Protein and NADPH Oxidase Are Involved in ATP-Promoted Cytoplasmic Reactive Oxygen Species (ROS) Generation in Guard Cells. Cytoplasmic ROS in guard cells was stained with H2CDFDA. (A) Fluorescence was captured using a laser confocal scanning microscope and is shown as pseudocolor images. The pseudocolor bar is shown at the bottom of (A). Fluorescent images before (control) and after 0.3 mM ATP treatment for 120 s (ATP), and the corresponding brightfield images, are shown. The scale bar is shown on the left bottom of (A). (B) Means ± SE of relative fluorescent intensities in 30 guard cells. In the two wild-types, ATP markedly stimulated ROS generation (P < 0.05, Student’s t-test). In null mutants of Gα or NADPH oxidase, fluorescent intensity was not remarkably different before and after ATP treatment (P > 0.05, Student’s t-test). In all figures, ‘control’ means treatment with MES buffer only. Molecular Plant 2012 5, 852-864DOI: (10.1093/mp/ssr095) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 5 The Heterotrimeric G Protein Participates in Extracellular ATP-Promoted Stomatal Opening. (A) and (B) show stomatal apertures of wild-type and Gα null mutants before and after 60-min ATP or 2meATP treatment in light (A) and darkness (B), respectively. Data are means ± SE (n = 6) for stomatal aperture. The concentration of ATP or 2meATP was 0.3 mM. In gpa1-1 and gpa1-2, stomatal apertures before and after treatment were not significantly different (P > 0.05, Student’s t-test). In all figures, ‘control’ means treatment with MES buffer only. Molecular Plant 2012 5, 852-864DOI: (10.1093/mp/ssr095) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 6 ATP Stimulates Ca2+ Influx and H+ Efflux in Guard Cells of Arabidopsis thaliana (Ecotype col-0). (A) Gadolinium chloride (50 μM) blocked ATP-promoted stomatal opening. (B) and (C) show time course of Ca2+ flux in MES buffer without (B) or with (C) epidermis before and after 0.6 mM ATP treatment, respectively. The arrow marks time points of ATP treatment. (D) The dose-dependence of ATP-promoted Ca2+ influx; data are the means ± SE (n = 30) of the peak Ca2+ influx value after ATP stimulation. The positive and negative values of ion flux represent ion influx and efflux, respectively. (E) Sodium vanadate (100 μM) blocked ATP-promoted stomatal opening. (F) and (G) show time courses of H+ flux in MES buffer without (F) or with (G) epidermis before and after 0.6 mM ATP treatment, respectively. The arrow marks time points of ATP treatment. In (A) and (E), data are means ± SE (n = 6) for stomatal aperture. In all figures, ‘control’ means treatment with MES buffer only. (H) The dose-dependence of ATP-promoted H+ influx; data are the means ± SE (n = 30) of the peak H+ efflux value after ATP stimulation. Molecular Plant 2012 5, 852-864DOI: (10.1093/mp/ssr095) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 7 The Heterotrimeric G Protein and Reactive Oxygen Species Are Involved in ATP-Stimulated Ca2+ Influx and H+ Efflux. (A) and (C) show the time course of Ca2+ influx before and after 0.6 mM ATP treatment in null mutants of heterotrimeric G protein α subunit (A) and NADPH oxidase D/F subunit (C) and their wild-type epidermis, respectively. The arrow marks the time point of ATP treatment. (B) and (D) show the means ± SE (n = 30) peak Ca2+ influx velocity before and after ATP treatment in the wild-type and null mutants. (E) and (G) show the time course of H+ efflux before and after 0.6 mM ATP treatment in null mutants of heterotrimeric G protein α subunit (E) and NADPH oxidase D/F subunit (G) and their wild-type epidermis, respectively. The arrow marks the time point of ATP treatment. (F) and (H) show the means ± SE (n = 30) peak H+ efflux velocities before and after ATP treatment in the wild-type and null mutants. In all figures, ‘control’ means treatment with MES buffer only. Molecular Plant 2012 5, 852-864DOI: (10.1093/mp/ssr095) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions