Article Figures & Data
Figures
- Figure 1.
Characterization of atgpx3 T-DNA Insertion Mutants and Overexpression Transgenic Plants.
(A) The insertion positions of T-DNA in the ATGPX3 gene.
(B) RNA gel blots show ATGPX3 expression in the wild type, atgpx3 mutants, and complemented ATGPX3 (Com) lines. Total RNA was extracted from the wild type, atgpx3-1, atgpx3-2, and complementation lines. Fifteen micrograms of total RNA was loaded in each lane. An Actin gene and ATGPX7 were used as a loading control and a positive control, respectively.
(C) Germination sensitivity to osmotic stress in atgpx3 mutants. Seeds from the wild type, atgpx3-1, and atgpx3-2 were germinated on MS agar medium or MS medium supplemented with different concentrations of mannitol for 7 d. Values are means ± sd of three independent experiments (>120 seeds per point).
(D) Complementation of the phenotype conferred by atgpx3-1 by expression of wild-type ATGPX3. After germinating for 5 d in MS medium, the seedlings were transferred to MS agar medium or MS medium containing 200 mM mannitol and grown for 15 d.
(E) Expression of ATGPX3 in two ATGPX3 overexpression transgenic lines.
(F) Germination insensitivity to osmotic stress in ATGPX3 overexpression transgenic plants. Seeds from the wild type and both overexpression lines were germinated on MS agar medium or MS medium supplemented with different concentrations of mannitol for 7 d. Values are means ± sd of three independent experiments (>120 seeds per point).
(G) RT-PCR analysis of ATGPX3 gene transcripts in response to stress conditions. Arabidopsis seedlings were grown on MS medium plates for 15 d. Wild-type plants were treated with 60 μM ABA, drought stress, or 5 mM H2O2 for 4 h. Total RNA was isolated from treated and untreated wild-type plants. Actin2 primer was used as an internal control.
- 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.
- Figure 3.
atgpx3 Mutants Impair ABA- and H2O2-Induced Stomatal Closure but Not Stomatal Density.
(A) Effects of ABA and H2O2 on stomatal closure in the wild type, atgpx3-1, and atgpx3-2. Stomatal apertures were measured on epidermal peels of the wild type and atgpx3-1. Values are means ± sd of 50 measurements from three independent experiments. Left panel, ABA-induced stomatal closing; middle panel, H2O2-induced stomatal closing; right panel, effects of CAT on ABA- (10 μM) and H2O2- (100 μM) induced stomatal closing.
(B) Stomatal density in the epidermis of the abaxial surface of rosette leaves from the wild type, atgpx3-1, and atgpx3-2. Stomatal density is presented as stomatal numbers per square millimeter ± se, determined from leaves of five individual wild-type and mutant plants. Five independent counts were performed on each leaf.
(C) Expression of ATGPX3-GUS in guard cells and RT-PCR analysis. Left panel, GUS activity in guard cells of wild-type plants expressing the GUS reporter gene under the control of the ATGPX3 promoter; right panel, ATGPX3 transcript detected by RT-PCR analysis of 10 μg of guard cell–enriched total RNA (GC). Ten micrograms of total RNA from whole leaves was run in parallel as a control (Leaf). KAT1 and ATGPX7 were used as positive controls in guard cells.
- Figure 4.
Responses of atgpx3 Mutants and Overexpression Lines to Drought Stress.
(A) Analysis of the drought stress sensitivity of Arabidopsis seedlings. Seven-day-old Arabidopsis seedlings were transferred to soil and grown for 10 d in a growth chamber, after which watering was stopped for the drought stress treatment. The photographs were taken (from top to bottom) 5, 8, and 9 d later and again 1 d after being rewatered. OE, overexpression.
(B) Transpirational water loss in wild-type, atgpx3, and overexpression lines at the indicated time points after detachment. Water loss is expressed as the percentage of initial fresh weight (FW). Values are means ± sd of four samples (each sample had six leaves).
(C) False-color infrared images of drought-stressed plantlets. The thermal images show leaf temperature profiles of 14-d-old wild-type, atgpx3-1, and atgpx3-2 plantlets at the start of drought treatment (middle panel) and 5 d later (bottom panel). The top panel shows the 5-d drought-stressed plants.
(D) Temperature of the leaf surface in ATGPX3 mutants quantified by infrared thermal imaging. Data are means ± sd (n = 20 plants for each condition; data are from ∼4000 measurements of square pixels from multiple leaves of each plant).
(E) False-color infrared images of drought-stressed wild-type and ATGPX3 transgenic plants. The same experimental procedures were used as described for (C). The middle and bottom panels show leaf temperature profiles at the start of drought treatment (middle panel) and 5 d later (bottom panel). The top panel shows the 5-d drought-stressed plants.
(F) Temperature of the leaf surface in ATGPX3 transgenic plants quantified by infrared thermal imaging upon drought for 5 d. Data are means ± sd (n = 20 plants for each condition; values are from ∼4000 measurements of square pixels from multiple leaves of each plant in three independent experiments).
- Figure 5.
ATGPX3 Specifically Interacts with ABI2 and ABI1.
(A) ATGPX3 strongly interacted with ABI2 or ABI1 in the yeast two-hybrid system. Yeast strains containing pAS-ATGPX3 as bait and pACT-ABI2/1 as prey were grown on YEPD medium lacking Trp and Leu for 48 h (left panel) and were assayed for LacZ expression by a filter-lift assay (right panel). pACT-SOS3 and the empty prey vector were used as negative controls. Blue color indicates interaction. β-gal, β-galactosidase activity.
(B) Quantitative analysis of β-galactosidase activity of the yeast strains in liquid culture showing the interaction between ATGPX3 and ABI2 or ABI1 and with the control partner SOS3. Values are means of data from three independent experiments. Error bars indicate sd.
(C) Biotinylated Lys-labeled ABI1/2 protein was pulled down by GST-ATGPX3 but not by GST-SOS3. GST was used as a negative control.
(D) In vivo interaction between ATGPX3 and ABI2 as determined using bimolecular fluorescence complementation. a, control (SYNE-ATGPX3 and SYCE); b, the YFP signal in the cytoplasm indicates a positive interaction between ATGPX3 and ABI2; c, the ATGPX3-GFP protein is localized in the cytoplasm. Left panel, fluorescence images under confocal microscopy; right panel, bright-field images of the cell.
(E) The phenotypes of atgpx3 and abi2 single mutants and double mutants (atgpx3-1 abi2-1 and atgpx3-2 abi2-1). F2 seeds from the crosses between the respective mutants were planted on MS agar medium (top plate) or MS agar medium supplemented 0.5 μM ABA (bottom plate) and allowed to grow for 2 weeks before the photographs were taken. Col, Columbia; Ler, Landsberg erecta.
(F) Comparisons of germination rates of the wild type, atgpx3-1, atgpx3-2, abi2-1, and the double mutants atgpx3-1 abi2-1 and atgpx3-2 abi2-1 after exposure to different concentrations of ABA for 10 d. Values are means ± sd of three independent experiments (>120 seeds per point).
- Figure 6.
Redox Regulation of ATGPX3 and ABI2, Inactivation of PP2C Activities by ATGPX3, and Alterations of Gene Transcription in atgpx3 Mutants.
(A) In vitro analysis of the ATGPX3 and ABI2 redox states. GST-ATGPX3 and GST-ABI2 extracts from E. coli were exposed to H2O2 (5 mM) for 5 min and analyzed under nonreducing conditions as indicated.
(B) Redox states of ATGPX3 in planta. ATGPX3-FLAG was transiently expressed in Arabidopsis protoplasts. The protein was extracted from 106 protoplasts, and protein gel blot analysis was performed using anti-FLAG antibody. Lane 1, control (empty vector); lane 2, ATGPX-FLAG. The gel shown is representative of five independent experiments.
(C) ATGPX3 is required for the oxidation of ABI2 in vitro. To make reduced proteins, extracts from E. coli BL21 strains carrying GST-ATGPX3/ABI2 were incubated with anti-GST-bound Sepharose beads for 4 h under reducing conditions (2 mM DTT) at 4°C. The eluate containing expressed GST-ATGPX3 and GST-ABI2 was applied to analyze redox state. To make oxidative ATGPX3 and ABI2, H2O2 (5 mM) was added to purified reduced GST-ATGPX3 and GST-ABI2 under anaerobiosis. The reaction was stopped by N-ethylmaleimide (10 mM) after 10 min, and the ATGPX3 redox state was monitored by immunoblotting. The gel shown is a representative image from three independent experiments.
(D) ATGPX3 inactivated ABI2/1 activity. In vitro PP2C activity was assayed by measuring the remaining 32P in the substrate casein. The data are presented as relative PP2C activity from three independent experiments. Error bars indicate sd.
(E) Expression of ABA- and stress-responsive genes in the wild type and atgpx3 mutants. Total RNA was extracted from wild-type and atgpx3-1 and atgpx3-2 seedlings. Real-time PCR was performed in three independent experiments. Error bars indicate sd. An Actin2 primer was used in the PCR as an internal control.
- Figure 7.
Changes of Calcium Channel Activity in Guard Cells of ATGPX3-Deficient Mutant and Overexpression Plants.
(A) ABA (50 μM) failed to activate Ca2+ channel currents in the atgpx3-1 mutant but greatly increased Ca2+ channel currents of guard cells in ATGPX3 overexpression transgenic lines.
(B) Effects of ATGPX3 mutation and overexpression on calcium channel activity at –150 mV (n = 35 [wild type], 28 [wild type + ABA], 13 [atgpx3-1 + ABA], 7 [ATGPX3 overexpression + ABA], and 10 [complementation line + ABA]). Error bars indicate sd.
(C) Single-channel current of calcium from guard cells of an ATGPX3 overexpression plant.
(D) Dependence of channel open probability on pressure in the pipette. The open probability gradually increased with negative pressure. Values are means ± sd (n = 3 or 4).
- Figure 8.
Model Showing the Putative Signal Transduction Pathway Mediated by ATGPX3.
For details, see the text. Arrows indicate positive regulation, and open blocks indicate negative regulation.
Additional Files
Supplemental Data
Files in this Data Supplement:
- Supplemental Figure 1 - Response of Overexpression AtGPX3 to osmotic Stress.
- Supplemental Figure 2 - Responses of atgpx3 Mutants to Drought Stress.
- Supplemental Figure 3 - No Fluorescence Signal Was Measured When pSPYNE Was Cotransformed with the pSPYCE-ABI2 Vector.
- Supplemental Figure 4 - Analysis of the ATGPX3 and ABI2 Redox State in vitro.
