Fig. 3.

Induction and localization of intracellular ROS in guard cells after treatment with ABA. (A) Total ROS accumulation in guard cells. Leaf discs of wild-type or antisense plants were treated with ABA as in Fig. 2. ROS were detected by 2′,7′-dichlorodihydrofluorescein diacetate as described in the Materials and methods. Confocal Z-sections of each guard cell (compiled 8-10 sections, each 1 μm thick) were projected to quantify the mean intensity of H₂DCFDA in a whole guard cell. Z section projections and fluorescence measurements were done using the ImagePro Plus program. Different letters within column denote statistical significance after F test (P <0.05, NS, not significant), N=15 ±SE. (B) Intracellular localization of ROS induced by ABA in wild-type and antisense AtVAMP711 guard cells. Simultaneous staining of ROS by H₂DCFDA (green filter) and intracellular membranes by MitoFluor Red 589 (red filter) in guard cells of wild-type and antisense plants (line 2091). Leaf discs were treated with ABA for 2 h as in (A). Loading of dyes and fluorescence detection are described in the Materials and methods. Shown are representative confocal images of a single section in guard cells of control (top panel), or ABA-treated plants (lower panel). For a complete Z stack of each image see Supplementary Fig. S2 at JXB online. Similar results were obtained in the other VAMP711 antisense lines (data not shown). (C, D) Nuclear localization of ABA-induced ROS in wild-type guard cells and epidermal cells. Simultaneous staining of nuclei (DAPI, blue), ROS (H₂DCFDA, green) (D) and intracellular membranes [MitoFluo Red 589 (C)] in guard cells (C) and epidermal pavement cells (D). Leaf discs of wild-type and antisense plants (line 2091) were treated with ABA for 2 h as in (A). Shown are representative confocal images (C, D). Control images are presented in the top panel of (B). Similar results were obtained in the antisense lines (data not shown).