Figure 2.

The atgpx3 Mutants Are More Sensitive to H₂O₂ in Terms of Seedling Growth and Produce More H₂O₂ in Guard Cells.

(A) Sensitivity to H₂O₂ during seedling development. Seeds from the wild type, atgpx3-1, and atgpx3-2 were germinated and allowed to grow on vertical agar medium containing MS nutrients (top panel) or MS nutrients supplemented with 3 mM H₂O₂ (bottom panel). The photographs were taken at 10 d after seed imbibition.

(B) Seedlings with true leaves over total number of seeds planted on MS medium supplemented with H₂O₂ at the indicated concentrations. Data represent means ± sd of four independent experiments (>120 seeds per point).

(C) Exogenous ABA-induced production of ROS in guard cells. A pair of guard cells from the wild type and mutants loaded with H₂DCF-DA before and 5 min after the addition of 10 μM ABA. The micrographs show representative fluorescence images from guard cells of the wild type and atgpx3 mutants in three independent experiments. The pseudocolor key is shown in the bottom right panel and was applied to pixel intensity (0 to 255) for all fluorescence images. Bar = 10 μm for all images.

(D) Effects of ABA on the DCF fluorescence in guard cells of wild-type and atgpx3 plants. The time points represent means ± se from measurements of pixel intensity in whole cells determined before and 5 min after ABA (10 μM) treatment in three independent experiments (for detailed steps for measuring pixel intensity, see Methods). For the wild type, n = 150 cells before ABA treatment, n = 110 cells after ABA treatment; for atgpx3-1, n = 110 cells before ABA treatment, n = 150 cells after ABA treatment; and for atgpx3-2, n = 96 cells before ABA treatment, n = 105 cells after ABA treatment.

(E) Histochemical localization of ABA-induced ROS production in leaves of wild-type and atgpx3 plants visualized using DAB. The detached leaves of plants were treated with 10 μM ABA for 10 min and then stained with DAB. Shown are representative leaves from three independent experiments.

(F) Assays of ATGPX3 peroxidase activity. Line A, complete reaction without thioredoxin; line B, complete reaction without ATGPX3; line C, complete assay in the presence of GSH, without thioredoxin and thioredoxin reductase; line D, complete assay in the presence of ATGPX3, thioredoxin, thioredoxin reductase, NADPH, and H₂O₂.

(G) In vitro reduction of ATGPX3. Lane 1, oxidized ATGPX3; lane 2, reduced ATGPX3; lane 3, oxidized ATGPX3, thioredoxin, thioredoxin reductase, and NADPH; lane 4, reduced ATGPX3, H₂O₂, thioredoxin, thioredoxin reductase, and NADPH; lane 5, oxidized ATGPX3, GSH, glutathione reductase, and NADPH. Shown is a representative gel from three independent experiments.